Thermostable probiotic compositions and healthy food on their basis

FIELD: chemistry.

SUBSTANCE: invention relates to healthy food products. The invention offers probiotic granules with a core, comprising probiotic microorganisms and a substrate in which the above microorganisms are absorbed; there are at least three layers, comprising an inner oily layer coating the core, and the first and second outer layers. The first outer layer is an enteric coating layer, which contains a pH-sensitive polymer, selected from a group comprising alginic acid, ammonia alginate, sodium, potassium, magnesium or calcium alginates. The second outer layer is an outer heat resistant coating. A method of granule manufacturing and a granule containing food product are offered.

EFFECT: invention provides the probiotic granules designed to stand higher temperatures during food processing.

11 cl, 1 dwg, 5 tbl, 13 ex

 

Field of the invention

The present invention relates to products of a healthy food, in particular baked goods that contain probiotics. A method of manufacturing a product, which is subjected to heat treatment at least at one stage of manufacture, while maintaining sufficient living probiotic microorganisms.

Background of the invention

Probiotics are live microbial food supplements that beneficially affect the body through maintenance of natural intestinal flora through competition with harmful microorganisms in the gastrointestinal tract, through participation in beneficial metabolic processes and by enhancing the resistance of the host body to toxic substances. The number of organisms used in probiotic foods and examples are microbial genera Lactobacillus or Bifidobacterium. Probiotic organisms need to stay alive during the shelf life of the product, in order to be effective, and, in addition, they must stay alive all the way through the gastro-intestinal tract into the colon. Probiotic organisms usually include dairy products such as yogurt. There is a need in the introduction of beneficial microorganisms into other types of food�'s products for example, in bakery products. However, the main problem in the production of dietary bakery products is the heat treatment where the temperature is usually so high (exceeding 180°C) that the products are almost sterilized. In WO 94/00019 described method of manufacture of bakery products containing live microorganisms, including cooling of bakery products and the introduction of a suspension of living organisms. In WO 2009/069122 the same authors who developed the present invention, the described method of manufacture of bakery products, including the encapsulation of the granules probiotic, thereby increasing their stability. The object of the present invention is to provide a method of manufacture acceptable to food compositions containing probiotic microorganisms, and this composition is resistant to high temperatures.

Another object of the invention is to offer a bakery product containing viable micro-organisms in sufficient quantity.

Another object of the invention is to provide a method of manufacturing a probiotic bakery products without the need to introduce viable microorganisms in the bakery product after the baking process.

Additional task izobreteny� is to offer bakery products containing live probiotic microorganisms during the entire baking process.

Another object of the invention is to offer baked goods containing thermally stabilized probiotic composition.

Also object of the invention is to offer a probiotic bakery products with a long shelf life.

Other objectives and advantages of the present invention will be apparent during the description.

A brief summary of the invention

According to the present invention proposed a granule of a probiotic containing: (1) a core containing probiotic microorganisms and the substrate, which absorbed these microorganisms; (2) internal oil layer covering the said core; and (3) the first outer layer and second outer layer, covering the said core and said inner layer containing at least two different polymer. The specified substrate and the two different polymer can contain three polysaccharide, acceptable for food. The said core may further contain one or more than one auxiliary agent for specified microorganisms. Preferably, the agents contribute to the growth of these microorganisms and may contain prebiotic ve�society, such as oligosaccharides. Said inner oil layer may contain a substance selected from fatty acids, waxes, fats, oils and lipids. The first outer layer provides stability of these micro-organisms in the upper gastrointestinal tract, and the specified second outer layer increases the stability of these microorganisms in the specified core at an elevated temperature. The specified substrate contains one or more than one component selected from saccharides and additional agents. These agents are selected from a stabilizer, a chelating agent, a synergistic agent, antioxidant and pH regulator, and these saccharides preferably include prebiotic oligosaccharides.

The two outer layers in granules of the invention contain two different polymer; the polymer may have a fibrous or gelatinous nature. In one embodiment, at least one outer layer contains a fibrous polysaccharide, and at least one of the outer layers contains a gel-like polysaccharide. A probiotic granule according to the invention may contain additional layers, for example at least one intermediate layer located between said oil layer and the second outer layer. A probiotic granule according to the present invention contains �probioticeski microorganism, which preferably is a bacterial. The specified microorganism mainly includes the genera selected from Lactobacillus, Bifidobacterium, Bacillus, Escherichia, Streptococcus, Diacetylactis, and Saccharomyces, or a mixture thereof. Microorganisms represent in a preferred embodiment the probiotic bacteria.

According to the invention a method of manufacturing granules of a probiotic containing a core containing probiotic microorganisms and the substrate, which absorbed these microorganisms surrounded by the inner oil layer and two outer polymeric layers, including: (1) mixing the suspension of probiotic microorganisms with substrates based on cellulose and auxiliary agents for microorganisms with obtaining thus a mixture of core; (2) coating particles of said mixture kernel oil layer thus obtaining particles coated with oil; (3) the coating of these particles with the oil coating the first polymer layer, which provides stability of these micro-organisms in the upper gastrointestinal tract, thus obtaining particles coated with two layers; and (4) coating of the specified double-layer of particles of the second polymer layer, which increases the stability of microorganisms in the specified kernel in terms of baking. These microorganisms can VC�ucati one or more than one microbial strain, and they are mixed and absorbed in the microbiologically compatible polymer that is also acceptable and is approved for food, which is a polysaccharide, such as a substance on the basis of cellulose. In this mixture may be added substances that support the stability or growth of the above microorganisms. Preferably include means of supporting probiotics, known as prebiotics, such as maltodextrin, trehalose, and so on. At the stages of coating can be used in methods known in the art, including coating in a fluidized bed, spraying and so on. When you create a covering layer can be used solutions or suspensions, and powders and so on. These stages of the coating (2) to (4) lead to an increase in mass by an amount of from 10 to 100% relative to the mass of the nucleus, for example, from 15 to 50%. In a preferred embodiment of the method of producing granules of probiotic includes (1) mixing an aqueous suspension of probiotic microorganisms containing at least one genus selected from Lactobacillus, Bifidobacterium, Bacillus, Escherichia, Streptococcus, Diacetylactis, and Saccharomyces or mixtures thereof, with at least one polysaccharide and at least one oligosaccharide (an example is microcrystalline cellulose with maltodextrin and trehalose), to obtain the�them by way of the mixture for the core; (2) coating particles of said mixture kernel oil layer thus obtaining particles coated with oil; (3) the coating of these particles with the oil coating the first polymer layer and second polymer layer; where said two polymer layers are different and include at least two substances selected from cellulose, modified cellulose, polysaccharide and/or synthetic polymers and mixtures thereof.

Importantly, the invention relates to a probiotic composition comprising granules having a core containing probiotic microorganisms and the substrate, which absorbed these microorganisms surrounded by the inner oil layer and two outer polymer layers. Probiotic composition according to the invention shows a high resistance to high temperatures. Talking here about high resistance to elevated temperatures, high thermal stability, mean viability of probiotic microorganisms in the granules compared to free of microorganisms and, in particular, the viability of probiotic microorganisms in the granules, is added to a food product, compared to free microorganisms. In one aspect of the invention, the probiotic microorganisms in the core of a three-layer pellets can withstand the impact on �of ranula higher temperatures, than the ambient temperature. thermal stability of the probiotic composition according to the invention is sufficiently high to ensure that a portion of the initial bacterial load, added in a probiotic food product according to the invention, remains viable even after all the necessary stages of production. Such stages may include cakes.

According to the invention proposed a healthy food product or dietary Supplement containing probiotic composition as described above containing stable granules of probiotic. The specified product may preferably include bakery product, such as pastry or bread. The specified product may also include tuna, chocolate, fruit juices and dairy products.

According to the invention proposed a healthy food product containing flour confectionery, bread, flour, flour products, bakery products, frozen cakes, yoghurt, dairy products, chocolate, nectars, fruit juices, and tuna. A food product according to the invention, containing granules of probiotic, can be subjected to a higher temperature than the ambient temperature during the production process.

The above and other features and advantages of the invention will become more obvious benefit�autumnal to the following examples and on the basis of the accompanying graphic material, where:

The drawing shows a diagram of the multilayer capsule according to one of embodiments of the invention for inclusion in the healthy food; the encapsulation is designed to give probiotic microorganisms maximum heat resistance during the heating stage in the production process upon receipt of the specified food product, as well as high biological efficiency in the lower divisions of the LCD tract after leaving the stomach intact; white kernel contains probiotic microorganisms and the absorbent substrate; the first dark layer adjacent to the core, is an oil layer; a light layer adjacent to the oil layer is an acid-resistant layer; the dark layer, adjacent to the acid-resistant layer is an intermediate layer; and an external light layer is a heat-resistant layer.

Detailed description of the invention

Now unexpectedly found that the probiotic microorganisms may be included in the composition of the cores of granules having at least three layers, thus obtaining probiotic composition of viable probiotic organisms even after baking, wherein the composition also remain stable during storage and is able to the introduction of viable microorganisms in the colon �donkey oral administration.

According to the invention proposed granular probiotics for use in dietary supplements for a healthy diet. In particular, the present invention relates to a method of producing bakery products, such as probiotic pastry. The suspension of probiotic microorganisms are mixed and granulated with a suitable substance carrier with the formation of particles constituting the core are 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-1; Lactobacillus acidophilus LAFTI® L10; Lactobacillus acidophilus LA-5; Lactobacillus acidophilus NCFM; Lactobacillus casei; Lactobacillus casei LAFTI® L26; Lactobacillus casei CRL431; Lactobacillus casei DN114-001 (Lactobacillus casei lmmunitas(s)/Defensis); Lactobacillus brevis; Lactobacillus bulgaricus; Lactobacillus gasseri; Lactobacillus paracasei; Lactobacillus casei F19; Lactobacillus casei Shirota; Lactobacillus paracasei St11 (NCC2461 or); Lactobacillus plantarum; Lactobacillus plantarum 299V; Lactobacillus reuteri ATTC 55730 (Lactobacillus reuteri SD2112); Lactobacillus rhamnosus; Lactobacillus salivarius; Lactobacillus delbrueckii; Lactobacillus fermentum; Lactococcus lactis; Lactococcus lactis L1A; Lactococcus lactis subsp; Lactococcus lactis Rosell-1058; Lactobacillus paracasei St11 (or NCC24610; Lactobacllus johnsonii La1 (corresponds to Lactobacillus LC1); Lactobacillus johnsonii La1 (corresponds to Lactobacillus LC1, Lactobacillus johnsonii NCC533); Lactobacillus rhamnosus Rosell-11; Lactobacillus acidophilus Rosell-52; Streptococcus thermophilus; Diacetylactis; Lactobacillus rhamnosus ATCC 53013 (open Gorbach and Goldin(=LGG)); Lactobacillus rhamnosus LB21; Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14; Lactobacillus acidophilus NCFM and Bifidobacterium bifidum BB-12; Saccharomyces cerevisiae; Saccharomyces cerevisiae (boulardii) lyo; and mixtures thereof.

For the granulation process can be used a suitable pellet mill or, alternatively, the fluidized bed. The drying process may include lyophilization. Granules probiotic according to the invention can have a wide range of sizes. Non-limiting examples of probiotic pellets according to the invention is an essentially spherical particle having an average diameter from about 0.1 to about 1000 microns. Not wanting to be limited by any theory, it is believed that mixing the probiotic microorganisms with microbiologically acceptable polymer, such as cellulose derivative, in the core of the particles to be coated with a triple layer of microbiologically acceptable substances leads to an increased heat resistance of a microorganism, where increased resistance may arise from a reduced thermal conductivity and stabilization of cells. Processed probiotic microorganisms according to the invention withstand heating during baking at the predetermined baking temperature and times� baking. I believe that domestic oil layer and first outer layer (layer enteric coating) to further protect the probiotic microorganisms during their passage through the upper gastrointestinal tract, making possible the release of probiotics in either the small intestine or in the colon, or both. The granular structure of the probiotic composition according to the invention provides a relatively high stability (viability of microbial cells) during storage prior to use in obtaining food, and in food products during storage. Moreover, this structure provides the desired release of viable microorganisms in the lower gastrointestinal tract of a subject, receiving foods healthy foods, such as bread for a healthy diet. In addition, the total positive effect can be further enhanced by the inclusion of a probiotic composition also oligosaccharides (called probiotics) that promote the growth of beneficial microorganism. Perhaps the first outer layer (the layer is stable in the gastro-intestinal tract coating, called the enteric layer coating) can be separated from the specified second outer layer (outer layer of heat resistant coating�) using an intermediate layer of inert coating to prevent any possible interaction between them.

Specified oil the inner layer may be selected from the group consisting of beeswax, Carnauba wax, Japan wax, bone wax, paraffin wax, Chinese wax, lanolin (wool wax), shellac wax, spermaceti, mircowave wax, candelilla wax, hydrogenated castor oil, Esparto wax, jojoba wax ouricury, rice bran wax, soy wax, certinaly waxes, montenego wax, ozocerite, peat waxes, microcrystalline wax, petrolatum, polyethylene waxes, waxes, Fischer-Tropsch, chemically modified waxes, substituted amide waxes, polymerized α-olefins, vegetable oils, hydrogenated vegetable oil, hydrogenated castor oil, fatty acids, esters of fatty acids, fatty alcohol, esterified fatty diols, hydroxylated fatty acids, stearic acid, sodium stearate, calcium stearate, magnesium stearate, palmitate, palmitoleate, hydroxyvalerate, Reutov long chain aliphatic alcohols, , phospholipids, lecithin, phosphatidylcholine.

The first outer layer may contain one or more pH-sensitive coating, commonly referred to in the art as "enteric coating" in soo�accordance with conventional methods to slow the release of probiotic microorganisms. Suitable pH-sensitive polymers include polymers which are relatively insoluble and impermeable at the pH of the stomach, but which are more soluble or degradable or permeable at the pH in the small intestine or the colon. Such pH-sensitive polymers include polyacrylamides, phthalates derivatives such as acid phthalates of carbohydrates, acetated amylose, acetated cellulose (cap), other phthalates of cellulose esters, phthalates ethers of cellulose, phthalate of hydroxypropyl cellulose (NRSR), the phthalate of hydroxypropylmethylcellulose (NRSR), the phthalate of hydroxypropylmethylcellulose (NRSR), succinate acetate hydroxypropylmethylcellulose (HPMCAS), methylcellulose phthalate (MCP), poly (vinyl acetate) phthalate (PVAcP), hydroptila poly (vinyl acetate), SAR sodium acid phthalate starch, acetatetreated cellulose (CAT), a copolymer of styrene and maleic acid dibutyl phthalate, the copolymer of styrene and polivinilatsetatftalat maleic acid, copolymers of styrene and maleic acid, polyacrylic acid derivatives, such as copolymers of acrylic acid and ester of acrylic acid, poly (methacrylic acid and its esters, copolymers of polyacrylic and methacrylic acids, shellac and copolymers of vinyl acetate and crotonic acid. Preferred pH-sensitive polymers include shell�, derivatives phthalate, CAT, HPMCAS, polyacrylic acid derivatives, particularly copolymers containing acrylic acid and at least one ester of acrylic acid, polymethylmethacrylate, random copolymers of acrylic acid and of esters of acrylic acid, and copolymers of vinyl acetate and crotonic acid, alginic acid and alginates such as ammonium alginate, sodium alginate, potassium, magnesium or calcium. A particularly preferred group of pH-sensitive polymers includes CAP, PVAcP, NRSR, HPMCAS, anionic acrylic copolymers of methacrylic acid and methylmethacrylate and osmopolitan containing acrylic acid and at least one ester of acrylic acid. Acetated pulp can be used as enteric coatings for encapsulated probiotic compositions of the invention to provide a delayed release probiotic microorganisms until the dosage form will not be released from the stomach. The ATS solution for coating may also contain one or more than one plasticizer such as diethyl, polyethylene glycol-400, triacetin, traceinternal, propylene glycol and others known in the art. The preferred plasticizers are diethyl and triacetin. The composition of ATS for the coating can also contain od�n or more than one emulsifier, such as Polysorbate-80.

Anionic acrylic copolymers of methacrylic acid and methylmethacrylate are also particularly useful substances for enteric coating to slow the release of probiotic microorganisms, as they do not take in the LCD tract position, distal to the stomach. Copolymers of this type are supplied by the company Rohm America, Inc. under the trade marks EUDRAGIT L and EUDRAGIT-S. EUDRAGIT L and EUDRAGIT S are anionic copolymers of methacrylic acid and methylmethacrylate. The ratio of free carboxyl groups and esters is approximately 1:1 in Eudragit-L and approximately 1:2 in Eudragit-S. can Also be used a mixture of Eudragit L and Eudragit-S. For these acrylic coating covering the polymer can be dissolved in an organic solvent or mixture of organic solvents or suspended in aqueous media. Suitable for this purpose solvents are acetone, isopropyl alcohol and methylene chloride. As a rule, it is advisable to include 5-20 wt.% plasticizer in the compositions of acrylic copolymers for coating.

Suitable plasticizers include polyethylene glycols, propylene glycols, diethyl, dibutyl phthalate, castor oil, and triacetin. Eudragit-L is preferred because it is relatively fast dissolves at pH environment of the intestine in Addition to the pH-sensitive polymers, listed above, coating for sustained release may consist of a mixture or combination of two or more pH-sensitive polymers, or may consist of mixtures of one or more than one pH-sensitive polymer and one or more than one pH-insensitive polymer. Adding a pH-insensitive polymer and a pH-sensitive polymer useful in regulating the duration of the delay or release rate of the probiotic microorganisms in the granules, spherical particles or pellets. For example, the delay can be extended by mixing a water-insoluble polymer with a pH-sensitive polymers, whereas the delay can be reduced by mixing water-soluble polymer with a pH-sensitive polymers. Preferred pH-insensitive water-insoluble polymers include esters of cellulose, cellulose ethers, polyacrylates, polyamides, polyesters and vinyl polymers. Preferred pH-insensitive water-soluble polymers include hydroxyethylamine cellulose derivatives, such as LDCs, NES and HPMC, polyvinyl acetate (PVA), polyethylene glycol (PEG), polyethylene oxide (PEO), copolymers of polyethylene glycol and polypropylene glycol (PEG/PPG) and water-soluble polyamides, polysaccharides and polyacrylates.

In such coatings may be included diffrent�e auxiliary substances, including emulsifiers, plasticizers, surfactants, fillers and buffer solutions. Finally, the polymer coating may be characterized as "quasi-enteric" in the sense that it remains essentially intact for a considerable period of time (e.g. one hour after the dosage form leaves the stomach, then becoming sufficiently permeable to probiotic microorganisms to ensure a gradual release of probiotic microorganisms by diffusion through the coating.

Perhaps, in the preparation according to the present invention, there is an intermediate layer between the enteric layer and the outer heat resistant layer. The intermediate layer coating composition according to the present invention substantially completely 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, thereby preventing any interaction between them. The intermediate layer coating according to any one of the embodiments of the present invention may and preferably contains a water-soluble polymer that includes polyvinyl, such as povidone (PVP: polyvinyle�Lydon), polyvinyl alcohol, copolymer of PVP and polyvinyl acetate, cross-linked polyvinyl, LDC (hydroxypropyl cellulose) (more preferably a low molecular weight), HPMC (hydroxypropyl methylcellulose) (more preferably a low molecular weight), CMC (carboxymethylcellulose) (more preferably a low molecular weight), ethylcellulose, MO (metilcellulose), MIXTURE (carboximetilzellulozu), NAES (hydroxyethyl cellulose), NEMS (hydroxyethylmethylcellulose), polyethylene oxide, acacia, dextrin, magnesium aluminosilicate, starch, polyacrylic acid, polyhydroxyethylmethacrylate (MEMA), polymethacrylates and their copolymers, gum, water soluble gum, polysaccharide, crosslinked polysaccharides, peptides or cross-linked peptide, protein or crosslinked proteins, gelatin or cross-linked gelatin, hydrolyzed gelatin or cross-linked hydrolyzed gelatin, collagen or cross-linked collagen, modified cellulose, polyacrylic acid or crosslinked polyacrylic acid and/or mixtures thereof, but is not limited.

This second outer layer, outer heat resistant coating may contain linear, branched or crosslinked polymers. They may be homopolymers, or copolymers, or graft copolymers, or block copolymers, taken in otdelno�and or mixture. Although they may be synthetic polymers, preferably such polymers can be natural polymers, such as polysaccharides, cross-linked polysaccharides, gums, modified polysaccharides, modified starch and modified cellulose. The polysaccharide may be selected from the group consisting of chitin, chitosan, dextran, pullulan, agar gum, gum Arabic, gum karaya gum, gum beans carob, tragacanth gum, carrageenan, gum ghatti, guar gum, xantanovy gum and scleroglucan, starches, dextrin and maltodextrin, hydrophilic colloids such as pectin, high detoxifaction and low detoxifaction. The composition can be used phosphatides, such as lecithin. Cross-linked polysaccharide may be selected from the group consisting of insoluble metal salts or cross-linked derivatives of alginate, pectin, xanthan gum, guar gum, tragacanth gums and gum beans carob, carrageenan, metal salts and their covalently crosslinked derivatives. The modified cellulose may be selected from the group consisting of cross-linked derivatives of hydroxypropyl cellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, methylcellulose, carboxymethylcellulose and salts metals and ka�of maximalizalasi. More preferably, such polymers can be cationic polymers. Examples of cationic polymers include cationic polyamines, cationic polyacrylamides, cationic polyethylenimine, cationic polyvinyl alcohol, which is a Quaternary salt of methyl chloride and grafted copolymer of poly(dimethylaminoethylacrylate)/polyvinyl alcohol or a Quaternary salt of metilsulfate and grafted copolymer of poly(dimethylaminoethylacrylate)/polyvinyl alcohol, a number of dry mixtures of PVA with N-(3-chloro-2-hydroxypropyl)-N,N,N-trimethylammoniumchloride supplied by Dow Chemical Company under the name QUAT.RTM.-188 containing varying amounts of water and NaOH, cationic polyvinylpyrrolidone, gelatin, polyvinylpyrrolidone, a copolymer of polyvinyl acetate and polyvinylpyrrolidone, a copolymer of polyvinyl alcohol and polyvinylpyrrolidone, polyethyleneimine, polyallylamine and its salts, polyvinyliden and its salts, condensation product of dicyandiamide with polyalkyleneglycol, condensation product of polyallylamine with dicyandiamide, the condensation product of dicyandiamide with formalin, additive polymer of epichlorohydrin-dialkylamino; polymer of diallyldimethylammoniumchloride ("DADMAC"), a copolymer of dimethylaminoethylmethacrylate and neutral methacrylic esters supplied by the company Rohm Pharma (Degusa) under the name Eudragit E, copolym�R diallyldimethylammoniumchloride-SO 2polyvinylimidazole, polyvinylpyrrolidone, a copolymer of vinylimidazole, polyamidine, chitosan, cationizing starch, cationic polysaccharides such as cationic guar and cationic hydroxypropanoic, polymers of , (2-methacryloyloxyethyl)trimethylsilyl chloride, polymers of dimethylaminoethylmethacrylate, polyvinyl alcohol with Quaternary ammonium salt in the side chain, cationic polyvinylformamide, cationic polyvinylacetate, cationic polyvinylformamide, cationic polyvinylacetate, poly(dimethylaminoethylmethacrylate) (DMAPMAM), poly(dimethylaminoethylacrylate), poly(), poly() (polyarts), poly() (polemarchus) and its salts, poly(vinylpyridine) and its salts, copolymers of dimethylamine with epichlorohydrin, a copolymer of dimethylamine with epichlorohydrin and Ethylenediamine, poly(amidoamine-epichlorohydrin), cationic starch, copolymers which contain N-vinylformamide, allylamine, diallyldimethylammoniumchloride, N-vinylacetate, N-vinylpyrrolidone, N-methyl-M-vinylformamide, N-methyl-N-vinylacetate, dimethylaminoethylmethacrylate, dimethylaminoethylacrylate, diethylaminoethylamine, or �of lore in the form of polymerized structural elements and, if necessary, split in the form of their salts, and combinations thereof, but is not limited to these. The chitosan may have a degree of deacetylation, varying from 80% to over 95%. Chitosan may also be possible to have a viscosity varying from 50 MPa·s to 800 MPa·S. Chitosan can possibly represent trimethylchitosan or quaternionic chitosan. The polymer can also possible to constitute polyglucosan, one of the components of chitosan. For example, the polymer can be a polymer (β-1,4)-D-glucosamine or polymer (β-1,4)-D-glucosamine and N-acetyl-D-glucosamine.

According to a preferred embodiment of the invention the probiotic microorganisms in the specified core granules are mixed with the substrate. The said substrate may include monosaccharides, such as referred to as trioses, including ceturies (dihydroxyacetone) and allotrios (glyceraldehyde), tetrose, such as katoteros (erythrulose), aldosterone (erythrose, threose) and setopenmode (an, xylulose), a pentose, such as aldopentose (ribose, arabinose, xylose, lyxose), deoxy sugars (deoxyribose) and ketohexose (psicose, fructose, sorbose, tagatose), hexose, such as aldohexose (alloza, altrose, glucose, mannose, gulose, idose, galactose, talose), the deoxy sugars (fucose, Fukuoka, rhamnose) and heptose, such as (sedoheptulose), and octose and nonose (it�the amino acid). The substrate may contain complex sugars, such as (1) disaccharides, such as sucrose, lactose, maltose, trehalose, turanose and cellobiose, (2) trisaccharide, such as raffinoses, melezitose and maltotriose, (3) tetrasaccharide, such as acarbose and stachyose, (4) other oligosaccharides, such as fructooligosaccharides (FOS), galactooligosaccharides (GOS) and mannan oligosaccharide (MOS), (5) polysaccharides, such as polysaccharides based on glucose/glucan, including glycogen, starch (amylose, amylopectin), cellulose, dextrin, dextran, beta-glucan (zymosan, lentinan, sizofiran) and maltodextrin, polysaccharides on the basis of fructose/fructan, including inulin, Levan beta-2-6, polysaccharides based on mannose (mannan), polysaccharides on the basis of galactose (galactan) and polysaccharides based on N-acetylglucosamine, including chitin. May include other polysaccharides including gums, such as gum Arabic (gum acacia).

According to a preferred embodiment of the invention the probiotic microorganisms in the specified inner core is mixed with the substrate, which may further contain additional components. These components can be selected from chelating agents. Preferably the chelating agent is selected from the group consisting of antioxidants, of edetate dipotassium, edetate, denetria, edetate calcium-denetria, agetaway sour�s, fumaric acid, malic acid, maltol, edetate sodium, edetate, trinacria.

According to some embodiments of the present invention, the core further comprises as a chelator and synergistic agent (sequestrant). Not wanting to be limited by a single hypothesis or a theory, chelating agents and sekwestrantov can be differentiated, as indicated below. A chelating agent such as citric acid, is designed to assist in the chelation of trace quantities of metals, thereby contributing to the prevention of loss of the active ingredient(s), such as simvastatin, due to oxidation. Sequestrant, such as ascorbic acid, possibly and preferably has several hydroxyl and/or carboxyl groups, which can provide a supply of hydrogen atoms to regenerate the free radical inactivated antioxidant. Therefore sequestrant preferably acts as a supplier of hydrogen atoms to restore 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-dimethyltrimethylene-dipyrromethene), tocopherol-rich extract (natural vitamin E), α-tocopherol (synthetic�cue vitamin E), β-tocopherol, γ-tocopherol, δ-tocopherol, butylhydroquinone, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), propylgallate, octisalate, dodecylsulfate, tertiary butylhydroquinone (TBHQ), fumaric acid, malic acid, ascorbic acid (vitamin C), sodium ascorbate, calcium ascorbate, potassium ascorbate, ascorbyl palmitate and ascorbinsaeure. In the kernel can be included citric acid, sodium lactate, potassium lactate, calcium lactate, magnesium lactate, anoxemic, altarboy acid, erythorbate, sodium, erythropenia acid erythorbic sodium, ethoxyquin, glycine, guaiac gum, sodium citrate (sodium citrate odnozameshchenny, sodium citrate dibasic, sodium citrate trehzameshchenny), potassium citrate (potassium citrate odnozameshchenny, potassium citrate trehzameshchenny), lecithin, polyphosphate, tartaric acid, sodium tartrate (tartrate of sodium odnozameshchenny, sodium tartrate dibasic), potassium tartrate (potassium tartrate odnozameshchenny, dipotassium tartrate), tartrate of sodium, potassium, phosphoric acid, sodium phosphates (sodium phosphate odnozameshchenny, sodium phosphate dibasic, sodium phosphate trehzameshchenny), potassium phosphate (potassium phosphate odnozameshchenny, potassium phosphate dibasic, potassium phosphate trehzameshchenny), calcium ethylenediaminetetraacetate sodium (EDTA calcium-sodium), lactic acid, frigid�oxybutyrate and thiodipropionic acid. According to one preferred embodiment, the antioxidant is a VNA.

According to preferred embodiments of the present invention, the core further comprises a stabilizer. Preferably, the stabilizer may be a base material, which can increase the pH value of the aqueous solution or dispersion of the drug to at least about 6.8. Examples of such basic substances include antacids, such as alumosilicate magnesium, magnesium aluminosilicate, magnesium aluminate, anhydrous aluminum hydroxide, synthetic hydrotalcite, synthetic aluminum silicate, magnesium carbonate, precipitated calcium carbonate, magnesium oxide, aluminum hydroxide and sodium bicarbonate and mixtures thereof; and pH regulators, such as L-arginine, sodium phosphate, hydrophosphate, denetria, sodium dihydrogen phosphate, potassium phosphate, hydrophosphate of dipotassium, potassium dihydrogen phosphate, citrate, denetria, sodium succinate, ammonium chloride and sodium benzoate, and mixtures thereof, but is not limited to these. The base material may be selected from the group consisting of inorganic water-soluble or water-insoluble inorganic compounds. Examples of inorganic water-soluble basic substance include carbonates such as sodium carbonate or potassium or sodium bicarbonate, potassium bicarbonate, phosphates, selected from, for example, anhydrous�of dibasic sodium phosphate, potassium or calcium, trisodium phosphate, hydroxides of alkali metals selected from sodium hydroxide, potassium or lithium, and mixtures thereof, but is not limited to these. Sodium bicarbonate is primarily used to neutralize acid groups in the composition in the presence of moisture that may be absorbed on the particles of the composition during storage. Calcium carbonate has a buffer effect stored in the composition with no visible impact on the release of substance ingestion. Additionally it is established that the carbonates significantly stabilize the composition. Examples of water-insoluble inorganic basic substance include suitable alkaline compounds which are able to give the necessary basicity include some of pharmaceutically acceptable inorganic compounds usually used in antacid compositions, such as magnesium oxide, magnesium hydroxide or magnesium carbonate, magnesium bicarbonate, hydroxide or carbonate, aluminium or calcium, composite aluminium / magnesium compounds such as magnesium hydroxide and aluminum silicate compound, such as magnesium aluminosilicate (Veegum F), alumosilicate magnesium (Nesulin FH2), magnesium aluminosilicate (Nisulin A); and pharmaceutically acceptable salts of phosphoric acid, such as tricalcium phosphate; and mixtures thereof, but not limited to them.

The invention �AET the ability to produce different foods for a healthy diet, not sharing the stage of mixing and heating. There is the possibility, for example, making bread dough, containing granules of probiotic, avoiding any difficult stages of the introduction of the methods of the prior art. The weight ratio between the probiotic composition and the rest of the test may be, for example, 1:100.

Encapsulated probiotic microorganisms according to the present invention can be introduced into flour, flour products, bakery products, yogurt, tuna, frozen pastries, chocolate, hot drinks, and nectars fruit juices and other products during processing and/or manufacturing process may be exposed to higher temperatures than the ambient temperature (room).

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

EXAMPLES

td align="left"> Microcrystalline cellulose (MCC)
Example 1
Materials
Materials:Function:
Lactobacillus acidophilusProbiotic microorganisms
BifidobacteriumProbiotic microorganisms
The substrate core
MaltodextrinAuxiliary agent for microorganisms
TrehaloseAuxiliary agent for microorganisms
Gidrirovannoe vegetable oilAgent first layer coating
Ethylcellulose E100The polymer of the second coating layer
Sodium alginateThe polymer of the second coating layer and the heat resistant polymer
Calcium chlorideHeat-resistant component (hardener)

Method

1. Adsorption of microorganisms on the microcrystalline substratekernel

Lactobacillus acidophilus and Bifidobacterium absorbed on MKC-substrate based on the ratio 38:62 respectively. For this purpose, prepared a 30% suspension of microorganisms and maltodextrin and trehalose, water-based. The concentration of microorganisms in the suspension was approximately 15% (wt./mass.). The absorption was carried out at finite temperature less than 35°C.

2. The first coating layer using hydrogenated vegetable oil

�Yesenia coating was carried out using a device for coating in the fluidized bed on the basis of the method of the hot melt. For this purpose gidrirovannoe vegetable oil sprayed on MKC-substrate with adsorbed bacteria at 40°C To produce a 40% weight gain. The flow of the incoming air is regulated so that it was low.

3. The second coating is enteric coating

The coating was carried out using a solution of ethyl cellulose E100 and sodium alginate in the ratio of 85:15 respectively, in ethanol with a total solid concentration of 6% (wt./mass.). The ultimate goal of the coating process was to obtain the 20% weight gain due to the coating. The coating process was carried out using a device for coating in the fluidized bed at 40°C.

4. The third layer of the coating is heat resistant coating

Calcium alginate was used as the heat resistant resin for the third layer of the coating. First separately prepared aqueous solutions of sodium alginate (3% wt./mass.) and calcium chloride (5% wt./mass.). Then both solutions of sodium alginate and calcium chloride alternately sprayed on the microorganisms coated to obtain a weight gain of 20% (wt./mass.).

Function:
Example 2
Materials
Ingredients:
Lactobacillus acidophilusProbiotic microorganisms
BifidobacteriumProbiotic microorganisms
Microcrystalline cellulose (MCC)The substrate core
MaltodextrinAuxiliary agent for microorganisms
TrehaloseAuxiliary agent for microorganisms
Gidrirovannoe vegetable oilAgent first layer coating
Sodium alginate high viscosityThe polymer of the second coating layer
ChitosanHeat resistant polymer
Hydrochloric acid (HCl)Agent for regulating the pH level

Method

1. Adsorption of microorganisms on the microcrystalline substrate core

Lactobacillus acidophilus and Bifidobacterium absorbed on MKC-substrate based on the ratio 38:62 respectively. For this purpose, prepared a 30% suspension of microorganisms and maltodextrin and trehalose, water-based. Concentration MICR�organisms in the suspension was approximately 15% (wt./mass.). The absorption was carried out at finite temperature less than 35°C to avoid effects on the microorganisms to high temperatures and thus high temperature damage.

2. The first coating layer using hydrogenated vegetable oil

The coating was carried out using a device for coating in the fluidized bed on the basis of the method of the hot melt. For this purpose gidrirovannoe vegetable oil sprayed on MKC-substrate with adsorbed microorganisms at 40°C To produce a 40% weight gain. The flow of the incoming air is regulated so that it was low.

3. The second coating is enteric coating

Sodium alginate was used as the enteric polymer. Prepared aqueous solution of sodium alginate (2% wt./mass.). A solution of sodium alginate sprayed on the microorganisms coated to obtain a weight gain of 15%.

4. The third layer of the coating is heat resistant coating

Chitosan was used as a heat-resistant resin for the third layer of the coating. First prepared an aqueous solution of chitosan (4% wt./mass.) with pH 5 using HCl. The resulting solution was sprayed on the microorganisms coated to obtain a weight gain of 20% (wt./mass.).

Example 3
Materials
Ingredients:Function:
Lactobacillus acidophilusProbiotic microorganisms
BifidobacteriumProbiotic microorganisms
Microcrystalline cellulose (MCC)The substrate core
MaltodextrinAuxiliary agent for microorganisms
TrehaloseAuxiliary agent for microorganisms
Gidrirovannoe vegetable oilAgent first layer coating
Sodium alginate of low viscosityThe polymer of the second coating layer
ChitosanHeat resistant polymer
Hydrochloric acid (HCl)Agent for regulating the pH level

Method

1. Adsorption of microorganisms on the microcrystalline substrate core

Lactobacillus acidophilus and Bifidobacterium absorbed at MCC-sub�waste from the ratio of 38:62 respectively. For this purpose, prepared a 30% suspension of bacteria and maltodextrin and trehalose, water-based. The concentration of bacteria in the suspension was approximately 15% (wt./mass.). The absorption was carried out at finite temperature less than 35°C, to avoid impacts on bacteria high temperatures and, thus, high-temperature damage.

2. The first coating layer using hydrogenated vegetable oil

The coating was carried out using a device for coating in the fluidized bed on the basis of the method of the hot melt. For this purpose gidrirovannoe vegetable oil sprayed on MKC-substrate with adsorbed microorganisms at 40°C To produce a 40% weight gain. The flow of the incoming air is regulated so that it was low.

3. The second coating is enteric coating

Sodium alginate was used as the enteric polymer. Prepared aqueous solution of sodium alginate (2% wt./mass.). A solution of sodium alginate sprayed on the bacteria coated to obtain a weight gain of 15%.

4. The third layer of the coating is heat resistant coating

Chitosan was used as a heat-resistant resin for the third layer of the coating. First prepared an aqueous solution of chitosan (4% wt./mass.) with pH 5 using HCl. Get�nny solution was sprayed on the microorganisms coated to obtain a weight gain of 20% (wt./mass.).

Example 4
Materials
Ingredients:Function:
Lactobacillus acidophilusProbiotic microorganisms
BifidobacteriumProbiotic microorganisms
Microcrystalline cellulose (MCC)The substrate core
MaltodextrinAuxiliary agent for microorganisms
TrehaloseAuxiliary agent for microorganisms
Saturated vegetable oilAgent first layer coating
Sodium alginate high viscosityThe polymer of the second coating layer
ChitosanHeat resistant polymer
Silicon dioxideSliding agent
Hydrochloric acid (HCl)Agent for regulating the pH level

Method

1. Adsorption of microorganisms on the microcrystalline substrate core

Lactobacillus acidophilus and Bifidobacterium absorbed on MKC-substrate based on the ratio 38:62 respectively. For this purpose, prepared a 30% suspension of microorganisms and maltodextrin and trehalose, water-based. The concentration of microorganisms in the suspension was approximately 15% (wt./mass.). The absorption was carried out at finite temperature less than 35°C to avoid effects on the microorganisms to high temperatures and thus high temperature damage.

2. The first coating layer using saturated vegetable oil

The coating was carried out using a device for coating in the fluidized bed on the basis of the method of the hot melt. For this purpose a saturated vegetable oil sprayed on MKC-substrate with adsorbed microorganisms at 40°C To produce a 40% weight gain. The flow of the incoming air is regulated so that it was low.

3. The second coating is enteric coating

Sodium alginate was used as the enteric polymer. Prepared aqueous solution of sodium alginate (2% wt./mass.). A solution of sodium alginate sprayed on the microorganisms coated to obtain a weight gain of 15%.

4. The third layer of the coating - thermal�ostoyae coating

Chitosan was used as a heat-resistant resin for the third layer of the coating. First prepared an aqueous solution of chitosan (4% wt./mass.) with pH 5 using HCl. Then after complete dissolution of chitosan was added silica (1% wt./mass.). The resulting solution was sprayed on the microorganisms coated to obtain a weight gain of 25% (wt./mass.).

Example 5
Materials
Ingredients:Function:
Lactobacillus acidophilusProbiotic microorganisms
BifidobacteriumProbiotic microorganisms
Microcrystalline cellulose (MCC)The substrate core
MaltodextrinAuxiliary agent for microorganisms
TrehaloseAuxiliary agent for microorganisms
Gidrirovannoe vegetable oilAgent first layer coating
Sodium alginate high�coy viscosity The polymer of the second coating layer
ChitosanHeat resistant polymer
Hydrochloric acid (HCl)Agent for regulating the pH level

Method

1. Adsorption of microorganisms on the microcrystalline substrate core

Lactobacillus acidophilus and Bifidobacterium absorbed on MKC-substrate based on the ratio 38:62 respectively. For this purpose, prepared a 30% suspension of microorganisms and maltodextrin and trehalose, water-based. The concentration of microorganisms in the suspension was approximately 15% (wt./mass.). The absorption was carried out at finite temperature less than 35°C to avoid effects on the microorganisms to high temperatures and thus high temperature damage.

2. The first coating layer using hydrogenated vegetable oil

The coating was carried out using a device for coating in the fluidized bed on the basis of the method of the hot melt. For this purpose gidrirovannoe vegetable oil sprayed on MKC-substrate with adsorbed microorganisms at 40°C To produce a 40% weight gain. The flow of the incoming air is regulated so that it was low.

3. The second coating is enteric �open & nbsp;

Sodium alginate was used as the enteric polymer. Prepared aqueous solution of sodium alginate (2% wt./mass.). A solution of sodium alginate sprayed on the microorganisms coated to obtain a weight gain of 15%.

4. The third layer of the coating is heat resistant coating

Chitosan was used as a heat-resistant resin for the third layer of the coating. First prepared an aqueous solution of chitosan (4% wt./mass.) with pH 5 using HCl. The resulting solution was sprayed on the microorganisms coated to obtain a weight gain of 30% (wt./mass.).

Example 6
Materials
Ingredients:Function:
Lactobacillus acidophilusProbiotic microorganisms
BifidobacteriumProbiotic microorganisms
Microcrystalline cellulose (MCC)The substrate core
MaltodextrinAuxiliary agent for microorganisms
TrehaloseWww�support agent for microorganisms
Gidrirovannoe vegetable oilAgent first layer coating
Sodium alginate high viscosityThe polymer of the second coating layer
ChitosanHeat resistant polymer
Hydrochloric acid (HCl)Agent for regulating the pH level

Method

1. Adsorption of microorganisms on the microcrystalline substrate core

Lactobacillus acidophilus and Bifidobacterium absorbed on MKC-substrate based on the ratio 38:62 respectively. For this purpose, prepared a 30% suspension of microorganisms and maltodextrin and trehalose, water-based. The concentration of microorganisms in the suspension was approximately 15% (wt./mass.). The absorption was carried out at finite temperature less than 35°C to avoid effects on the microorganisms to high temperatures and thus high temperature damage.

2. The first coating layer using hydrogenated vegetable oil

The coating was carried out using a device for coating in the fluidized bed on the basis of the method of the hot melt. For this purpose gidrirovannoe vegetable oil sprayed on MKC-substrate with abso�lirovannye microorganisms at 40°C To produce a 40% weight gain. The flow of the incoming air is regulated so that it was low.

3. The second coating is enteric coating

Sodium alginate was used as the enteric polymer. Prepared aqueous solution of sodium alginate (2% wt./mass.). A solution of sodium alginate sprayed on the microorganisms coated to obtain mass gain by 25%.

4. The third layer of the coating is heat resistant coating

Chitosan was used as a heat-resistant resin for the third layer of the coating. First prepared an aqueous solution of chitosan (4% wt./mass.) with pH 5 using HCl. The resulting solution was sprayed on the microorganisms coated to obtain a weight gain of 20% (wt./mass.).

Example 7

Encapsulated granules with probiotic microorganisms were tested for resistance. Accordingly, the received encapsulated granules with microorganisms from Example 6 was maintained at 85°C for 45 minutes. Then determined the number of colony forming units per g (CFU/g) using the following method of calculation.

Counting method Lactobacillus acidophilus and Lactobacillus bifidus: 10 g of sample was suspended in 90 ml of phosphate buffer and placed in a Stomacher homogenizer for 10 minutes. Then the resulting suspension was stirred for 90 minutes. The mixture was then serially (tenfold) was diluted, and finally vlive�and in the appropriate medium for the cultivation on Petri dishes. For Lactobacillus acidophilus and bifidobacteria used nutrient medium Mrs (Moser-Cattail-Sharpe), containing either cysteine or maltose, respectively. Received the cups then were incubated for 3 days under anaerobic conditions. At the end of the microorganisms were counted and, accordingly, the expected value of CFU/g.

Results:
Lactobacillus acidophilusBifidobacterium bifidum
Without the coating before the coating process* (initial CFU/g)3,6×10107,2×109
After coating** (CFU/g)of 1.6×107of 1.2×107
After heating*** (CFU/g)of 1.4×107of 5.4×106
*The weight ratio between the two types of microorganisms in the final product is 1:1.
**The mixture of microorganisms is 10% (mass./mass.) from the final product.
***The process napr�tion was carried out at 80°C for 45 minutes.

Example 8

Probiotic cookies

This probiotic biscuits consists of 0.3 g of filler and 30 g of biscuits. Filler: the following ingredients are mixed at room temperature (percentages are by mass percent, based on total weight of the filler): cookie recipe: 1 part sugar, 2 parts of margarine, 3 parts flour, mixed with 1% probiotic powder.

Viability of microorganisms

The maximum temperature was approximately 200°C, applicable for up to 4.5 minutes, which corresponds to the majority of industrial processes of making cookies. About 50% of living microorganisms was maintained after baking.

Example 9

Probiotic bread

This probiotic bread consists of 0.3 g of filler and 30 g of bread. Viability of microorganisms

The level of viability of the microorganisms obtained by simulation of the process ranged from 50% to 80%. Were received from 83% of living organisms in 10 minutes baking at 200°C at an initial quantity of 109microorganisms per gram.

Example 10

Evaluation of heat resistance of encapsulated probiotic microorganisms according to the present invention in the dry state

Purpose

Evaluation of heat resistance and viability of encapsulated probiotic microorganisms in comparison with probiotic�and microorganisms without coating using the methods on the basis of the present invention in the dry state.

Summary

Both encapsulated and non-encapsulated probiotic organisms (L. Acidophillus and Bifidobacteria) were placed in an oven, pre-heated to 80°C, or for 30 minutes or for 45 minutes. Then probiotics was taken out and analyzed SOME numbers to determine the viability of microencapsulated microorganisms in comparison with unencapsulated. The results show that the impact on non-encapsulated probiotics such temperature conditions can be catastrophic when it cannot be calculated, the number of CFU/g, which indicates that there was a complete destruction of the non-encapsulated microorganisms. In contrast, encapsulated probiotics, obtained according to the microencapsulation process of the present invention did not exhibit a significant reduction in viability in the analysis in the conditions of such heat treatment. Based on these results we can conclude that the microencapsulation process using a multilayer film according to the present invention provides a heat resistance of probiotics in the conditions described above.

Materials

2 grams of a mixture of probiotic microorganisms coated (L. Acidophillus and Bifidobacteria) according to the present invention. The composition of the coating layers made�flax in table 1.

2 grams of a mixture of uncoated bacteria (L. Acidophillus and Bifidobacteria).

Method

The process of microencapsulation was carried out according to the basic Protocol of the process of production batch number RDEN 904051 and RDEN 904051.

The third layer coating
Table 1
Components at different stages of the process of microencapsulation
IngredientsStage
Microcrystalline cellulose (MCC)Granular inner core
Dehydrat trehaloseGranular inner core
Maltodextrin DE15Granular inner core
Lactobacillus acidophilusGranular inner core
BifidobacteriumGranular inner core
Gidrirovannoe vegetable oil (HVO)The first layer coating
Sodium alginate high densityThe second coating
Chitosan

The test of heat

As microencapsulated and non-encapsulated (control) probiotics were placed in an oven, pre-heated to 80°C for either 30 minutes or 45 minutes.

Analysis of the number of CFU

Analyses of the number of CFU was performed for bacteria before and after the heating process using the method described below:

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

2) the Stomacher homogenizer for 10 minutes;

3) mixing of samples within 90 minutes.

4) a tenfold dilution;

5) deep sowing;

6) of Lactobacillus acidophilus for the use of the CRM environment with cysteine;

7) for bifidobacteria using CRM environment with maltose instead of lactose;

8) incubation for 3 days under anaerobic conditions;

9) counting of bacteria and calculate the number of CFU/g Method described in detail elsewhere (K. S. G. de Lima et al./LWT - Food Science and Technology 42 (2009), 491-494).

The encapsulated probiotic bacteria was first destroyed by a multilayered sheath surrounding microorganisms, using a pestle and mortar and then used the above method of counting SOMETHING.

Results

Table 2
The influence of the process inkapsulirovanie� on the number of CFU
LactobacillusBifidobacterium
acidophilus (CFU/g)bifidum (CFU/g)
Non-encapsulated microorganisms*
(source pure microorganisms)
3,6×10107,2×109
After coating
(microencapsulatedof 1.6×107of 1.2×107
microorganisms)**
*the weight ratio between the two types of microorganisms in the final product is 1:1.
** The mixture of microorganisms is 10% (mass./mass.) from the final product.

Table 3
Influence of heat treatment on the viability of probiotic microorganisms in the dry state
Lactobacillus acidophilus (TO�/g) Bifidobacterium bifidum (CFU/g)
Encapsulated microorganisms after heat treatment, the dry conditions (80°C, 30 minutes)1,0×1078,6×106
Encapsulated microorganisms after heat treatment, the dry conditions (80°C, 45 min)of 1.4×107of 5.4×106
Nemikrocelularni microorganisms (80°C, 30 minutes)00

Conclusion

Based on the results of Table 3, we can conclude that the microencapsulation process using the process of deposition of multilayer coatings according to the present invention provides a heat resistance of probiotics in dry condition.

Example 11

Evaluation of heat resistance of encapsulated probiotic microorganisms according to the present invention in terms of poloniecki

Purpose

Evaluation of heat resistance and viability of encapsulated probiotic microorganisms using the methods according to the present invention in comparison with probiotic microorganisms without coverage in terms of poloniecki.

Summary

As encapsulated and pincap�lirovannye probiotic bacteria (L. Acidophillus and Bifidobacteria) were mixed with the ingredients of white bread and subjected to baking at 180°C, 70% humidity for 40 minutes. To facilitate the extraction of microorganisms from baked dough, and encapsulated and non-encapsulated microorganisms introduced into the dough using two different methods, called "serpyanka" and "ravioli". Accordingly, the microorganisms were added to the dough or indirectly when using serpyanka to isolate microorganisms from a test (experiment I method serpyanka), either directly by creating a separate pocket (experiment II method ravioli) made from the same dough, already containing the microorganisms. According to the method serpyanka microorganisms or pre-cast into serpyanka, which is then injected into the dough before baking (experiment Ia) or microorganisms were placed on a thin piece serpyanka, which were pre-injected into the dough through the formation of a small depression in the center portions of the dough and fill it with a thin piece serpyanka (experiment Ib). According to the method of "ravioli" first formed a small pocket of dough like ravioli, which was placed 2 grams of a mixture of microorganisms with the coating, and closed. The pocket is then placed in the center portions of the test. Using these methods you can also eliminate�tit adhesion test to microorganisms after the baking process. It is important to prevent sticking of the dough to microorganisms, because in this experiment the dough can create a mechanical obstacle for the amount of crushing force in the crushing process, acting as a "shock absorber". This way you can ensure that the coating is completely destroyed during the crushing process prior to the analysis, the number of CFU. After baking, the microorganisms were extracted and determined the number of CFU/g for each strain of microorganisms as encapsulated and non-encapsulated microorganisms.

The results of the analysis here clearly show that the microorganisms without coverage are unable to survive in the conditions of baking, whereas encapsulated organisms showed resistance during the baking process and the high value of viability after the baking process.

Materials

3 cups flour

10 grams of yeast

2 tablespoons olive oil

1/8 teaspoon salt

water

2 grams of a mixture of probiotic microorganisms coated (L. Acidophillus and Bifidobacteria)

2 grams of a mixture of uncoated bacteria (L. Acidophillus and Bifidobacteria)

Methods

The baking process

Mixed all the ingredients for baking bread, and after a few minutes of kneading the dough is left to rise. The dough is then divided into separate portions. Microorganisms were introduced into the portions of the test using d�uh different methods "serpyanka" and "ravioli", which is described below (commented in experiment I and experiment II, respectively).

Experiment I - method "serpyanka"

Experiment Ia - As encapsulated and non-encapsulated microorganisms introduced into the dough when they were previously enclosed in the serpyanka". 2 g of either encapsulated or non-encapsulated microorganisms were placed in the middle of each piece of pastry.

Experiment Ib - 2 g of encapsulated microorganisms were placed on the surface of a thin piece of serpyanka, which were pre-injected into the middle portions of the test by the formation of the recess and fill it with a thin piece serpyanka. The recess is then closed remaining dough.

Experiment II method "ravioli"

First formed a small pocket of dough like ravioli, which was placed 2 g of a mixture of encapsulated microorganisms, and closed. The pocket is then placed in the center portions of the test.

The dough was left to rise for a further 15 minutes.

The baking process was performed at 180°C for 40 minutes.

On the bottom shelf of the oven put a metal tray with 1/2 liter of water for creating humidity inside the oven before putting the loaves into the oven. Created inside the furnace humidity was measured before putting the loaves into the oven. To achieve the optimum level�I moisture when baking loaves of bread put into the oven, when the humidity reached values between 60 and 70%. Once the portions of dough were baked, microorganisms easily extracted and passed on to the analysis of the number of CFU.

Analysis of the number of CFU

Analyses of the number of CFU was performed for microorganisms before and after the baking process using the method described below:

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

2) the Stomacher homogenizer for 10 minutes;

3) mixing of samples within 90 minutes.

4) a tenfold dilution;

5) deep sowing;

6) of Lactobacillus acidophilus for the use of the CRM environment with cysteine;

7) for bifidobacteria using CRM environment with maltose instead of lactose;

8) incubation for 3 days under anaerobic conditions;

9) counting of microorganisms and calculation of the number of CFU/g.

The method is described in detail in other sources (K. S. G. de Lima et al./LWT - Food Science and Technology 42 (2009), 491-494).

The encapsulated probiotic microorganisms are first destroyed the multilayer sheath surrounding microorganisms using a mortar and pestle, and then used the above method of counting SOMETHING.

Results

Table 4
The number of CFU/g of encapsulated and non-encapsulated microorganisms before and after condition of baking
L. acidophilusBifidobacteria
Encapsulated probiotic bacteria before baking5×1055×105
Unencapsulated probiotic bacteria before baking5×1055×105
Encapsulated probiotic bacteria after baking - experiment Ia5×105of 3.1×105
Unencapsulated probiotic bacteria after baking - experiment Ia00
Encapsulated probiotic bacteria after baking - experiment Ib1×1051×105
Encapsulated probiotic bacteria after baking - experiment II1×1051×105

Conclusion

The results obtained above show that the encapsulated probiotic microorganisms using the methods according to this from�bretania are stable to heat when baking under the influence of humidity, present in the dough during the baking process.

Example 12

Evaluation of heat resistance of encapsulated probiotic microorganisms according to the present invention in full baking

Purpose

Evaluation of heat resistance and viability of encapsulated probiotic microorganisms using the methods according to the present invention in full baking using industrial method. This study is intended to demonstrate the suitability of the concept of encapsulated probiotics according to the present invention, is resistant to the baking process, wherein they are subjected to shear stresses, humidity and heat.

A brief overview of the

The purpose of this study was to evaluate the stability of encapsulated probiotics according to the present invention in the baking process used in the industry. In accordance with this encapsulated probiotics were directly added to the dough, first subjected to shear stresses during mixing and then heat and humidity in the baking process. This process is designed to simulate the baking process, which is carried out in an industrial method. For this purpose, encapsulated probiotics according to the present invention was added directly to Vacu and other ingredients to which was then added water (direct method add), and then kneaded and baked. Accordingly, the encapsulated probiotics were added directly to the ingredients for the dough and then homogeneously distributed in the dough by kneading, influence them moist environment during preparation of the dough followed by exposure to heat in the baking process. After the baking process was carried out analysis of the number of CFU to determine the viability of encapsulated microorganisms. The results of the analysis here clearly showed that the encapsulated microorganisms show high value of viability after the baking process. Therefore, we can conclude that the encapsulated probiotics according to the present invention is definitely resistant to the damp environment under conditions of strong shear, existing at the time of kneading the dough, and also to heat in the baking process.

Materials

Ingredients for bread:

white flour: 231 grams;

olive oil: 18,7 grams;

salt: 2 gram;

yeast: 5 gram;

encapsulated probiotics: 2 gram;

the dough before baking: 398,7 gram;

the bread after baking: is 364.5 grams.

General procedure for the baking process

Encapsulated probiotic microorganisms L. Addophillus and Bifidobacteria homogeneously mixed with� all the other ingredients for baking bread (white bread). Added water and then kneading the dough. The resulting dough is then baked at 180°C, 70% humidity for 40 minutes. This process was as follows:

Equipment

Mixer Kenwood VAT for 5 litres.

Preparation of the dough

Put the flour, yeast and encapsulated microorganisms in the tank for mixing.

Mix together all the ingredients.

Add the butter and salt.

Gradually add the water until the flour until the mixture forms a thick batter.

Leave the mixer included for kneading the dough for 10 minutes.

Turn off the mixer and leave the dough to rest covered with a VAT, and to rise for 30 minutes.

Turn on the mixer for a few seconds to "drop" the dough.

Baking procedure

First, the furnace is pre-heated to 180°C, then placed in a dough. Baking was performed at 180°C for 40 minutes. A metal pan containing 1/2 litre of water, were placed on the bottom shelf of the oven to create appropriate humidity (relative humidity about 70%) inside the oven before placing the dough. Created inside the furnace humidity was measured before baking. The dough was placed in a baking dish and baked for 40 minutes (180°C and a relative humidity of 70%). At the end of baking again checked the humidity.

Baking conditions

Humidity before baking: 70% (relative humidity). In�agnosti after baking: 70% (RH).

The duration of baking: 40 minutes.

After baking, the sample baked bread passed to determine the number of CFU/g of encapsulated microorganisms.

Analysis of the number of CFU

Analyses of the number of CFU was performed for encapsulated probiotics after the baking process using the method of determining SOMETHING that is described below:

1) took 20 g of the sample (baked bread), to which was added 90 ml of sterile phosphate buffer;

2) this mixture is then destroyed using a mortar and pestle for several minutes;

3) added an additional 160 ml of sterile phosphate buffer in a disposable sterile bag Stomacher homogenizer.

This mixture is then homogenized for 2 minutes using a Stomacher homogenizer.

Analysis of the number of CFU/g was performed using the following standard procedure:

1) a tenfold dilution;

2) deep sowing;

3) of Lactobacillus acidophilus for the use of the CRM environment with cysteine;

4) for bifidobacteria using CRM environment with maltose instead of lactose;

5) incubation for 3 days under anaerobic conditions;

6) counting of microorganisms and calculation of the number of CFU/g.

The method is described in detail in other sources (K. S. G. de Lima et al./LWT - Food Science and Technology 42 (2009), 491-494).

Results

The results of SOME analysis before and after baking are summarized in Table 5. �esult analysis here clearly show what encapsulated organisms showed resistance in the full process of baking and the high value of viability after the baking process.

Table 5
The number of CFU/g of encapsulated probiotics in full baking
L. acidophilusBifidobacteria
Encapsulated probiotic bacteria before baking5×1055×105
Encapsulated probiotic bacteria after bakingof 1.4×105of 1.3×105

Conclusion

Encapsulated probiotics according to the present invention is stable to heat when baking during industrial manufacture, when encapsulated probiotics directly add all the ingredients and then subjected to the process of mixing with the moisture present in the dough, and then heat in the baking process. These discoveries clearly show that drugs with the coatings of the invention provide probiotics necessary protection to vyd�to laugh all the stages of production of bakery products, including shear stress during mixing, relatively high humidity and heat during baking.

Example 13
Materials:
IngredientsFunction
Lactobacillus acidophilusProbiotic microorganisms
BifidobacteriumProbiotic microorganisms
Microcrystalline cellulose (MCC)The substrate core
MaltodextrinAuxiliary agent for microorganisms
TrehaloseAuxiliary agent for microorganisms
Stearic acidAgent first oil coating layer
Sodium alginate high viscosityThe polymer of the second coating layer
ChitosanHeat resistant polymer
Hydrochloric acid (HCl)Agent for regulating the pH level

Method

1. Adsorption of microorganisms on the microcrystalline substrate core

Lactobacillus acidophilus and Bifidobacterium absorbed on MKC-substrate based on the ratio 38:62 respectively. For this purpose, prepared a 30% suspension of microorganisms and maltodextrin and trehalose, water-based. The concentration of microorganisms in the suspension was approximately 15% (wt./mass.). The absorption was carried out at finite temperature less than 35°C to avoid effects on the microorganisms to high temperatures and thus high temperature damage.

2. The first coating layer using stearic acid

The coating was carried out using a device for coating in the fluidized bed on the basis of the method of the hot melt. For this purpose, stearic acid sprayed on MKC-substrate with adsorbed microorganisms at 40°C To produce a 40% weight gain. The flow of the incoming air is regulated so that it was low.

3. The second coating is enteric coating

Sodium alginate was used as the enteric polymer. Prepared aqueous solution of sodium alginate (2% wt./mass.). A solution of sodium alginate sprayed on the microorganisms coated to obtain a weight gain of 15%.

4. The third layer of the coating is warm�resistant coating

Chitosan was used as a heat-resistant resin for the third layer of the coating. First prepared an aqueous solution of chitosan (4% wt./mass.) with pH 5 using HCl. The resulting solution was sprayed on the microorganisms coated to obtain a weight gain of 30% (wt./mass.).

Although this invention is described in aspect to some specific examples, many modifications and changes. Therefore, it is clear that in the scope of the attached claims of the invention the invention may be implemented otherwise than specifically described.

1. Granule probiotics containing:
1) a core containing probiotic microorganisms and the substrate, which absorbed these microorganisms; and
at least three layers, including
2) internal oil layer covering the said core; and
3) the first outer layer and second outer layer, covering the said core and said inner layer, where the first outer layer is a layer of enteric coatings containing a pH-sensitive polymer is selected from the group comprising alginic acid, ammonium alginate, sodium alginate, potassium, magnesium or calcium; and the specified second outer layer is an outer layer of heat resistant coatings comprising at least one polymer selected from the group include�her polysaccharides, crosslinked polysaccharides, gums, modified polysaccharides, modified starch, modified cellulose, chitin, chitosan, dextran, pullulan, agar gum, gum Arabic, gum karaya gum, gum beans carob, tragacanth gum, carrageenans, gum ghatti, guar gum, xanthan gum, scleroglucan, starches, dextrin, maltodextrin, pectin, high detoxifaction, low detoxifaction, lecithin, insoluble metal salts, cross-linked derivatives of alginate, pectin, xanthan gum, guar gum, tragacanth gum, gum beans carob, carrageenan, their metal salts and their covalently cross-linked derivatives, cross-linked hydroxypropyl cellulose derivative, hydroxypropylmethylcellulose, hydroxyethylcellulose, methylcellulose, carboxymethylcellulose, and metal salts and carboxymethyl cellulose, cationic polymers, cationic polyamines, cationic polyacrylamide, cationic polyethyleneimine, cationic polyvinyl alcohol, Quaternary salt of methyl chloride and grafted copolymer of poly(dimethylaminoethylacrylate)/polyvinyl alcohol or a Quaternary salt of metilsulfate and grafted copolymer of poly(dimethylaminoethylacrylate)/polyvinyl alcohol, a number of dry mixtures of PVA (polyvinyl acetate) with N-(3-chloro-2-hydroxyprop)-N,N,N-trimethylammonio�IDOM, cationic polyvinylpyrrolidone, gelatin, polyvinylpyrrolidone, a copolymer of polyvinyl acetate and polyvinylpyrrolidone, a copolymer of polyvinyl alcohol and polyvinylpyrrolidone, polyethyleneimine, polyallylamine and its salts, polyvinyliden and its salts, condensation product of dicyandiamide with polyalkyleneglycol, condensation product of polyallylamine with dicyandiamide, the condensation product of dicyandiamide with formalin, additive polymer of epichlorohydrin-dialkylamino, the polymer diallyldimethylammoniumchloride ("DADMAC"), a copolymer of dimethylaminoethylmethacrylate and neutral methacrylic esters, the copolymer diallyldimethylammoniumchloride-SO2polyvinylimidazole, polyvinylpyrrolidone, a copolymer of vinylimidazole, polyamidine, chitosan, cationizing starch, cationic polysaccharides, cationic guar and cationic hydroxypropanoic, polymers of , (2-methacryloyloxyethyl)trimethylsilyl chloride, polymers of dimethylaminoethylmethacrylate, polyvinyl alcohol with Quaternary ammonium salt in the side chain, cationic polyvinylformamide, cationic polyvinylacetate, cationic polyvinylformamide, cationic polyvinylacetate, poly(dimethylaminoethylmethacrylate) (DMAPMAM), poly(dimethylaminoethylacrylate), poly(), poly(acrylamide�trimethylammoniumchloride) (polyarts), poly() (polemarchus) and its salts, poly(vinylpyridine) and its salts, copolymers of dimethylamine with epichlorohydrin, a copolymer of dimethylamine with epichlorohydrin and Ethylenediamine, poly(amidoamine-epichlorohydrin), cationic starch, copolymers which contain N-vinylformamide, allylamine, diallyldimethylammoniumchloride, N-vinylacetate, N-vinylpyrrolidone, N-methyl-N-vinylformamide, N-methyl-N-vinylacetate, dimethylaminoethylmethacrylate, dimethylaminoethylacrylate, diethylaminoethylamine, or in the form of polymerized structural elements and in split form and their salts, and combinations thereof, polyglucosan, the polymer (β-1,4)-D-glucosamine or polymer (β-1,4)-D-glucosamine and N-acetyl-D-glucosamine.

2. Granule probiotics according to claim 1, where said probiotic organisms in the three-layer core of the pellets can withstand the impact of the pellets higher temperatures than the ambient temperature.

3. Granule probiotics according to claim 1, where said probiotic organisms absorbed in the substrate containing one or more than one component selected from saccharides and additional agents.

4. Granule probiotics according to claim 3, where said agents selected from a stabilizer, a chelating agent, a synergistic agent�, antioxidant and pH regulator and where these saccharides include prebiotic oligosaccharides.

5. Granule probiotics according to claim 1, further comprising at least one intermediate layer located between said oil layer and the second outer layer.

6. Granule probiotics according to any one of claims. 1-5, where these microorganisms include a genus selected from Lactobacillus, Bifidobacterium, Bacillus, Escherichia, Streptococcus, Diacetylactis, and Saccharomyces or mixtures thereof.

7. A method of manufacturing granules according to claim 1, comprising mixing a suspension of probiotic microorganisms with substrates based on cellulose and auxiliary agents for microorganisms with obtaining thus a mixture of core; coating said mixture of particles for the core of the oil layer thus obtaining particles with the oil coating; coating these particles with the oil coating the first polymer layer, which provides the stability of these micro-organisms in the upper gastrointestinal tract, thus obtaining particles coated with two layers; and a coating of the specified double-layer of particles of the second polymer layer, which increases the stability of microorganisms in the specified kernel in terms of baking.

8. A method according to claim 7, where each of the steps of coating leads to an increase in mass on the value from 10 to 100% relative to the weight of the specified kernel.

9. A method of manufacturing granules according to claim 1, including:
1) mixing an aqueous suspension of probiotic microorganisms comprising at least one genus selected from Lactobacillus, Bifidobacterium, Bacillus, Escherichia, Streptococcus, Diacetylactis, and Saccharomyces or mixtures thereof, with at least one polysaccharide and at least one oligosaccharide with obtaining thus a mixture of core;
2) coating particles of said mixture kernel oil layer thus obtaining particles coated with oil;
3) coating of these particles with the oil coating the first polysaccharide layer and a second polysaccharide layer;
where the two polysaccharide layer are different and contain at least two components made of cellulose, alginate, chitosan or mixtures thereof.

10. A food product or dietary Supplement containing granule probiotics according to any one of claims. 1-6.

11. A food product according to claim 10 selected from the group consisting of pastry; bread, flour, flour products, bakery products, frozen cakes, yoghurt, dairy products, chocolate, nectars, fruit juice, and tuna.



 

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