US 20160360777A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0360777A1 PENHAS (43) Pub. Date: Dec. 15, 2016

(54) COMPOSITION AND METHOD FOR A 6LX 9/50 (2006.01) IMPROVING STABILITY AND EXTENDING A 6LX 35/745 (2006.01) SHELF LIFE OF PROBOTC BACTERIA (52) U.S. Cl. AND FOOD PRODUCTS THEREOF CPC ...... A2.3L 33/135 (2016.08); A61K 35/745 (2013.01); A61K 9/4858 (2013.01); A61 K (71) Applicant: Adel PENHASI, Holon (IL) 9/5015 (2013.01); A61K 9/5031 (2013.01); A61K 9/5036 (2013.01); A61K 9/5073 (72) Inventor: Adel PENHASI, Holon (IL) (2013.01); A61K 9/5042 (2013.01); A61 K (73) Assignee: DeGama Berrier Ltd., Grand Cayman 2035/115 (2013.01) (KY) (57) ABSTRACT (21) Appl. No.: 15/246,941 A composition comprising probiotic bacteria, the composi (22) Filed: Aug. 25, 2016 tion comprising: (a) a core composition containing the probiotic bacteria and a stabilizer, wherein the total amount Related U.S. Application Data of probiotics in the mixture is from about 10% to about 90% by weight of the core composition; (b) an innermost coating (63) Continuation of application No. 13/991,911, filed on layer, layered on said core composition, comprising at least Aug. 6, 2013, filed as application No. PCT/IB2011/ one hydrophobic solid fat or fatty acid having a melting 055462 on Dec. 5, 2011. point lower than 60° C.; (c) an intermediate coating layer (60) Provisional application No. 61/419,885, filed on Dec. layered on said innermost coating layer, which when present 6, 2010. in an aqueous Solution in the amount of 0.1% weight/weight over the weight of the solution, has a surface tension lower Publication Classification than 60 mN/m, when measured at 25°C.; and (d) an outer coating layer, layered on said intermediate coating layer; (51) Int. C. wherein the composition is in the form of particles; food A2.3L 33/35 (2006.01) products containing the composition and methods of prepa 469/48 (2006.01) ration thereof. Patent Application Publication Dec. 15, 2016 Sheet 1 of 3 US 2016/0360777A1

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COMPOSITION AND METHOD FOR temperature and moisture, all of which are typically present IMPROVING STABILITY AND EXTENDING in the foodstuff itself and/or its manufacture. SHELF LIFE OF PROBOTC BACTERIA 0005. Many probiotics exhibit their beneficial effect AND FOOD PRODUCTS THEREOF mainly when they are alive. Hence, they need to survive the manufacturing process and shelf life of the food. Likewise, FIELD OF THE INVENTION upon consumption of the food, they should Survive adverse gastro-intestinal tract conditions, such as very low pH values 0001. The present invention relates generally to probiot present in the stomach, before reaching the proper location ics and food products containing Such and in particular, but in the intestine for colonization. Although many commercial not exclusively, to compositions containing stabilized pro probiotic products are available for animal and human biotics with improved stability and extended shelf life. consumptions, most of them lose their viability during the manufacture process, transport, storage and in the animal/ BACKGROUND OF THE INVENTION human GI tract. 0002 Probiotics are live microbial food supplements 0006 To compensate for such loss, an excessive quantity which beneficially affect the host by supporting naturally of probiotics is included in the product, in anticipation that occurring gut flora, by competing harmful microorganisms a portion will survive and reach their target. In addition to in the gastrointestinal tract, by assisting useful metabolic the questionable shelf-life viability of these products, such processes, and by strengthening the resistance of the host practices are certainly not cost-effective. Furthermore. Such organism against toxic Substances. The beneficial effects that practices may also lead to highly variable dosages of pro probiotics may induce are numerous. Some non-limiting biotic bacteria that actually reach their intended destination examples include the reduction of intolerance, the for colonization, which is also undesirable. inhibition of pathogenic bacteria and parasites, the reduction 0007 For protection of the bacteria, various formulations of diarrhea, activity against Helicobacter pylori, the preven have been devised, frequently incorporating protective tion of colon cancer, the improvement or prevention of agents such as proteins, certain polymers, skim milk, glyc constipation, the in situ production of vitamins, the modu erol, , and , lation of blood fats, and the modulation of host immune and the like. However, none of these formulations are able functions. In domesticated and aquatic animals they also can to protect properly against oxygen and moisture, nor are they improve growth, Survival and stress resistance associated able to provide Suitable protection against high temperatures with diseases and unfavorable culture conditions. Therefore, required for processing many foodstuffs. there is considerable interest in including probiotics into 0008 For example, the probiotic microorganisms can be human foodstuffs and into animal feed. encapsulated by enteric coating techniques involve applying 0003 Probiotic organisms are preferably alive and a film forming Substance, usually by spraying liquids con capable of activity until and including during ingestion, in taining enteric polymer and generally other additives such as order to be effective. Probiotic organisms are usually incor or proteins onto the dry probiotics (Ko and Ping WO porated into milk products, such as yogurts, which provide 02/058735). However, the enteric polymers film coating the some inherent stability and which do not require heat for probiotics during the microencapsulation process usually processing. However, it is difficult to incorporate the ben cannot function as a moisture protecting barrier and gener eficial microorganisms in other foodstuff types, for example ally several layers must be added, to avoid water entering the creams, biscuits fill-in, chocolate, sauces, mayonnaise and microcapsules. In addition, such polymers also cannot pro etc., especially those which optionally undergo heat treat vide an appropriate protection against oxygen upon very ment in at least one stage of their preparation. Heat treatment poor oxygen occlusion properties of Such polymers. is known to decrease viability of probiotic organisms and, if 0009 Giffard and Kendall (US 2005/0079244) disclose a sufficiently high or applied for a sufficiently long period of foodstuff in the form of a dried or semi-moist ready-to-eat time, will kill these organisms. kibble or powder mix, which contains a combination of a 0004. The activity and long term stability of probiotic probiotic, prebiotic and a coating of colostrum. Prior to bacteria may be affected by a number of environmental mixing in the food stuff, the probiotic is coated or encap factors; for example, temperature, pH, the presence of Sulated in a , fat, , protein or in a water/humidity and oxygen or oxidizing or reducing agents. matrix using standard encapsulation techniques. Similar to It is well known that, in an aqueous phase, probiotics the above disclosure, neither the encapsulating polymers nor instantly lose their activity during Storage at ambient tem the additives used in both core and coating layers have low peratures (AT). Generally, probiotic bacteria are dried before water vapor and oxygen transmission, and therefore the or during mixing with other foodstuff ingredients. The negative effects of water (humidity) and oxygen cannot be drying process can often result in a significant loss in activity avoided. from mechanical, chemical, and osmotic stresses induced by 0010. Accordingly, it has been proposed to dry sugar the drying process. Loss of activity may occur at many based probiotic systems by foam formation in a very thin different stages, including drying during initial manufactur layer (Bronshtein WO2005117962), or to use combinations ing, food preparation (upon exposure to high temperature, of Sugars with a polymeric gelling agent, such as alginate, high humidity, oxygen and high pressure), transportation , carboxymethylcellulose or carboxyethylcellulose. and long term storage (temperature, oxygen and humid Cavadini et al. (EPO 862 863) provide a cereal product exposure), and after consumption and passage in the gas comprising a gelatinized Starch matrix including a coating or trointestinal (GI) track (exposure to low pH, proteolytic a filling. The probiotic is included with the coating. Accord enzymes and bile salts). Manufacturing food or feedstuffs ing to that process, spray-dried probiotics are mixed with a with live cell organisms or probiotics is in particular chal carrier Substrate, which may be water, fat or a protein digest. lenging, because the probiotics are very sensitive to oxygen, The mixture is then sprayed onto the cereal product and the US 2016/0360777A1 Dec. 15, 2016 whole product is dried again. Re-hydrating of the already sition is then used as the filling of a confectionary product. dried bacteria and the additional coating/drying process is The subject matter described in that document avoids the costly and damaging to the bacteria. detrimental effects of water by embedding the probiotics in 0011 US 2005/0019417 A1 describes a method of pre fat or oil rich matrix. However, fat based coating and paring products containing moisture-sensitive living micro preserving materials alone do not withstand oxygen and long organisms including probiotics, comprising at least the steps term exposure to humid conditions. through which a Suspension of probiotics and a Sugar 0017 None of the above compositions provide a mixture polymer in water miscible solvent is sprayed onto a water that can effectively protect the probiotic in both drying soluble, gel-forming solid particles. By these means, the processes and long-term storage at ambient temperatures core composed of water soluble gel-forming solid particles and varying degrees of humidity. In addition, none of the may absorb solvent residues and provide protection to above compositions provide a mixture that can effectively probiotics placed onto said core. protect the probiotics against oxygen which is a main cause 0012 Kenneth and Liegh (U.S. Pat. No. 6,900, 173) for poor stability for long time in storage conditions causing describe the manufacturing of multivitamin protein and very limited shelf life. probiotic bar for promoting an anabolic state in a person. The dried probiotic bacteria are blended in sugar syrup and BRIEF SUMMARY OF THE INVENTION several other constituents, and the resultant mixture is then extruded and cut into bars. However, the document does not 0018 Many probiotics may be temperature, water and/or disclose any process or composition that will improve oxygen sensitive and thus Suffer from lack of an extended viability or long-term stability of probiotics in the nutritional shelf life. Therefore, they need protection during processing, bars and there is no indication that the bacteria even survive transporting and storage as well as during delivery to the the process. gastro intestinal tract to maintain viability. 0013 US 2004/0175389 (Porubcan) discloses a formu 0019. Therefore, there is an urgent need for a composi lation for protecting probiotic bacteria during passage tion that can effectively protect the probiotic bacteria during through the stomach, whilst permitting their release in the manufacturing, long-term storage at ambient temperature, intestine. The formulation has also a low water activity and humidity and oxygen and during gastrointestinal passage. correspondingly long shelf life. The capsule includes a There is a need also for a preparation process that is water-free mixture of probiotic bacteria with monovalent cost-effective and capable of entrapping and stabilizing alginate Salts, and an enteric coating (e.g., gelatin or cellu probiotics in the protective mixture with minimal viability lose encapsulation). Upon contact with acidic environment, loss at the end of the entire operation. There is a need for a the outer shell of the capsule turned into a gel, which protective mixture that provides protection in the stomach provides a protecting barrier against proton influx into the while allowing the release of the probiotic along the intes capsule core. However, this composition is only useful for tinal tract. There is also a need for a protective mixture that tablets and capsules Subjected to storage conditions of very contains only approved ingredients generally regarded as low water activity and further require storage in nitrogen safe (GRAS), and is less costly than those presently being flushed or vacuum-sealed containers. used. 0014 WO 03/088755 (Farber and Farber) describes an 0020. The present invention, in at least some embodi oral delivery system for functional ingredients uniformly ments, overcomes these drawbacks of the background art dispersed in a matrix. The matrix components include a and provides a solution to these needs, by providing a Sugar, a , a hydrocolloid, a polyhydric alcohol composition and process for producing a composition for and a source of mono- or divalent cations. The delivery probiotic bacteria that are stable during processing, and for system is extruded or molded into a final shape with a long periods of time at ambient temperatures and varying moisture content of between 15% and 30% by weight. This degrees of humidity. type of matrix provides very little protection to the probi 0021. In at least some embodiments, the present inven otics; the little protection that is provided requires refriger tion provides a fast and cost effective preparation process ated conditions, which are not suitable or desirable for many and protection in food products (foodstuffs). As described foodstuffs. No description or direction was provided as to herein, the terms “food” and “foodstuffs' may be understood how probiotic bacteria are stabilized during manufacturing to encompass any type of nutritional product that is Suitable or for prolonged storage at room temperatures. for mammals, including but not limited to humans. 0015 McGrath and Mchale (EP 13.82241) describe a 0022. The present invention, in at least some embodi method of delivering a microorganism to an animal. The ments, provides a composition comprising probiotic bacteria micro-organism is suspended in a matrix of cross-linked that is heat, oxygen and humidity resistant and that is alginate and cryopreservant ( or lactose, or a com suitable for a food product, wherein the composition is in the bination of both). The matrix is then freeze or vacuum dried form of particles, the composition comprising: (a) a core to form dry beads containing live probiotics with a shelf-life composition containing the probiotic bacteria and a stabi stability up to 6 months but only under refrigerated condi lizer, wherein the total amount of probiotics in the mixture tions. Here again, no description or direction was provided is from about 10% to about 90% by weight of the core as to how probiotic bacteria are stabilized during manufac composition; (b) an innermost coating layer, layered on said turing or for prolonged storage at room temperatures and core composition, comprising at least one hydrophobic solid high humidity conditions. fat or fatty acid having a melting point lower than 60° C.; (c) 0016 Ubbink et al. (US 2005/0153018) disclose the an intermediate coating layer layered on said innermost preservation of lactic acid bacteria in moist food. The coating layer, which when present in an aqueous Solution in spray-dried bacteria are added to a composition comprising the amount of 0.1% weight/weight over the weight of the fats, fermented milk powder and saccharides. That compo solution, has a surface tension lower than 60 mN/m, when US 2016/0360777A1 Dec. 15, 2016 measured at 25°C.; and (d) an outer coating layer, layered after they are added to a food product. An example of high on said intermediate coating layer. temperature to be resisted is a tempering step during prepa 0023 The stabilizer may optionally comprise any type of ration of chocolate, or mixing within cream compositions. oxygen scavenger, including but not limited to those con 0031. The present invention, in at least some embodi taining L-cysteine base or hydrochloride, of which other ments, relates to a process for the preparation of a food examples are listed herein. product containing the stabilized composition, in which the 0024. The core composition may also optionally com preparation process may optionally comprise a heating step, prise at least one Sugar compound including but not limited the product containing active probiotic bacteria, the method to , trehalose, lactose, , , fruc comprising i) preparing stabilized probiotic composition tose and the like, of which other examples are provided particles as described herein; ii) admixing said stabilized herein. Disaccharides, such as Sucrose and trehalose, are probiotic particles into a semi-final product; iii) heating the attractive as protective agents within the core because they mixture of said probiotic particles and semi-final product at are actually help plants and microbial cells to remain in a a predetermined temperature and for a predetermined time state of Suspended animation during periods of drought. period; and vi) completion of said semi-final product con Trehalose has been shown to be an effective protectant for a taining said stabilized probiotic particles by cooling down variety of biological materials, both in ambient air-drying said mixture, thereby obtaining said final product containing and freeze-drying. stabilized active probiotic bacteria showing high stability 0025. The core composition may also optionally com during the storage and shelf life of the final product. The prise one or more other food grade ingredients, including but term "semi-final product describes a stage in the prepara not limited to a filler, a surfactant and binder, of which tion of a food product as is known in the art, in which said various non-limiting examples are provided herein. food product does not yet contain all the components or has 0026. The innermost coating layer may optionally com not yet passed all the preparation steps, and hence is not yet prise at least one hydrophobic Solid fat or fatty acid having ready for the consumption. a melting point lower than 50° C. and preferably higher than 0032. The present invention, in at least some embodi 25°C. The melting point is optionally preferably lower than ments, provides stabilized probiotic particles for admixing 45° C. and higher than 30° C., and is optionally and most to a food product, resistant to ambient temperature, humidity preferably lower than 40° C. and higher than 35° C. The and oxygen, which can be prepared according to a variety of innermost coating layer may optionally form a stable hydro processes, including but not limited to hot melt processes, phobic matrix which embeds the core composition within wet granulation or dry granulation. If the initial particle size and/or forms a film around the probiotic core composition. of probiotics and other excipients included in the core 0027. The intermediate coating layer, which when pres composition is sufficient to enable the coating of the first ent in an aqueous solution in the amount of 0.1% weight/ coating layer, no granulation process is required. weight over the weight of the Solution, optionally has a 0033. A food product according to various embodiments surface tension lower than 50 mN/m and preferably lower of the present invention may be a product optionally having than 45 mN/m when measured at 25° C. the form of a suspension, emulsion, or paste, as non-limiting 0028. The outer coating layer optionally comprises a examples. Non-limiting examples of Such food products polymer having an oxygen transmission rate of less than according to various embodiments of the present invention 1000 cc/m/24 hr, preferably less than 500 cc/m/24 hr and include but are not limited to creams, biscuits creams, most preferably less than 100 cc/m/24 hr measured at biscuit fill-in, chocolates, sauces, cheese, mayonnaise and standard test conditions (which may for example be 73° F. etc., in which the product is a health food product comprising (23° C.) and 0% RH). The polymer also optionally has a probiotic bacteria which are stabilized as described above water vapor transmission rate of less than 400 g/m/day, for long term storage and shelf life. preferably less than 350 g/m/day and most preferably less 0034. The present invention, in at least some embodi than 300 g/m/day. ments, relates to healthy food beneficially affecting the 0029. The composition may optionally further comprise consumers intestinal microbial balance, wherein said heat an additional humidity barrier coating layer, layered on the resistance and heat-processability are ensured by coating outer coating layer, for preventing further humidity penetra probiotic cores by layers which limit the transmission of tion. The composition may optionally further comprise an heat, oxygen and humidity to the probiotic bacteria and so enteric polymer, layered on the humidity barrier coating increase their resistance during preparation process and layer, which may further provide protection against Such storage and thus extend the shelf life. By “consumer it is destructive characteristics of the gastrointestinal tract as low meant any mammal consuming the product, including but pH values and proteolytic enzymes. not limited to humans. 0030 The resultant composition is in a solid form which BRIEF DESCRIPTION OF THE DRAWINGS may optionally be any solid particulate form as described herein, including but not limited to granules, powder and the 0035. The above and other characteristics and advantages like. The stabilized composition is suitable for admixing/ of the invention will be more readily apparent through the adding to food products including but not limited to choco following examples, and with reference to the appended late, cheese, creams, sauces, mayonnaise and biscuit fill-in. drawing, wherein: The stabilized probiotic bacteria within the protective com 0036 FIG. 1. shows a schematic diagram of a multiple position maintain their viability during manufacturing or layered capsule according to one embodiment of the inven preparation processes that involve exposure to high tem tion; perature, humidity and oxygen. The stabilized bacteria 0037 FIG. 2 shows a schematic diagram of a multiple maintain their viability during storage conditions at ambient layered capsule according to one embodiment of the inven temperatures, in a humid oxygenated environment, even tion. US 2016/0360777A1 Dec. 15, 2016

0038 FIG. 3 shows a schematic diagram of a contact tion. Label “1” indicates the core, which comprises probiotic angle (0) which is formed when a liquid does not completely bacteria and a substrate Such as stabilizer and optionally also spread on a substrate (usually a Solid). a Sugar and one or more other ingredients as indicated herein. Label “2 indicates the first layer adjacent to the DETAILED DESCRIPTION OF PREFERRED core, which is the inner first sealing layer comprising a EMBODIMENTS hydrophobic material, such as a solid fat for example. Label '3' indicates the intermediate layer adjacent to said inner 0039. It has now been found that probiotic bacteria may layer, binding the outer layer to the inner layer. Label “4” be surprisingly efficiently stabilized for use in a food prepa indicates the outer layer adjacent to said intermediate layer, ration process by a unique combination of coating layers providing protection against oxygen and humidity (water having unique arrangement order. The bacteria were formu vapor), and thus also supporting longer storage stability and lated in a core coated with coating layers, thereby obtaining shelf life at ambient temperature. probiotic compositions providing viable probiotic organisms 0043 FIG. 2 shows a schematic diagram of a multiple even after a prolonged time of storage at ambient tempera layered capsule according to another embodiment of the ture at high humidity, the composition being further stable invention. As shown, the numbered labels indicate the on storage and shelf life of the food stuff containing the components of the composition. Label “1” indicates mul protected probiotics according to the present invention and tiple cores featuring probiotic bacteria and other possible capable of administering viable bacteria to the gastrointes excipients which may be included in the cores as described tinal tracts after the oral administration. herein. Label “2 indicates a first fat coating layer, which is 0040. The present invention, in at least some embodi the innermost coating layer comprising at least one hydro ments, provides a probiotic composition in particulate form phobic solid fat or fatty acid having a melting point lower to be used as healthy food additives. The present invention than 60° C. and preferably higher than 25°C., forming a is particularly directed to a process for the preparation of stable hydrophobic matrix which in which the probiotic protected probiotics against oxygen and humidity (water cores are embedded. Label '3' indicates an intermediate vapor) for incorporating into foodstuffs such as creams, layer, for which the aqueous solution of 0.1% has a surface biscuits creams, biscuit fill-in, chocolates, sauces, cheese, tension lower than 60 mN/m. Label “4” indicates an outer mayonnaise and etc. layer, comprising a polymer having an oxygen transmission 0041. In a preferred embodiment of the invention, said rate of less than 1000 cc/m2/24 hr. probiotic bacteria comprise at least one Bifidobacterium 0044 FIG. 3 shows a schematic diagram of a contact animalis lactis. The stabilized probiotic core granule or core angle (0) which is formed when a liquid does not completely mixing according to the invention is a coated granule, spread on a Substrate (usually a solid). It is a direct measure comprising at least three layered phases, for example a core of interactions taking place between the participating and three coats, or a core and three or more coats. Usually, phases. The contact angle is determined by drawing a one of the coats is hydrophobic solid fat contributing mainly tangent at the contact where the liquid and solid intersect. to prevention of water or humidity penetration into the core Contact angle is geometrically defined as the angle on the during the coating of the outer layer or during later stages. liquid side of the tangential line drawn through the three At least one other coat is an outer coating which is respon phase boundary where a liquid, gas and Solid intersect, or sible for preventing transmission of humidity and oxygen two immiscible liquids and solid intersect. into the core during the storage and shelf life, while the intermediate coating layer is present between these two Probiotics-Containing Particles coating layers and is responsible for providing binding and adhesion of the previous coats to each other wherein said 0045. According to a preferred embodiment of the inven intermediate coat may further provide oxygen and/or humid tion, the probiotic bacteria in said inner core are mixed with ity resistance to the core. Such an intermediate coating may a stabilizer, optionally and preferably with at least one Sugar also optionally comprise a layer that contributes signifi and/or at least one or polysaccharides (as a cantly to oxygen resistance, and also optionally provides a Supplemental agent for the bacteria), and optionally other barrier against water or humidity penetration into the core; food grade additives such as fillers, binders, Surfactant, and however, the stabilized probiotic granule of the invention so forth. may comprise more layers that contribute to the stability 0046 Examples of probiotic bacteria include but are not process of the bacteria, as well as to their stability during limited to Bacillus coagulans GBI-30, 6086, Bacillus sub storing said food and during safe delivery of the bacteria to tilis var natt, Bifidobacterium LAFTIR B94, Bifidobacte the intestines. rium sp. LAFTI B94, Bifidobacterium bifidum, Bifidobacte rium bifidum rosell-71, Bifidobacterium breve, Schematic Description of the Composition According to at Bifidobacterium breve Rosell-70, Bifidobacterium infantis, Least Some Embodiments of the Present Invention Bifidobacterium lactis, Bifidobacterium longum, Bifidobac terium longum Rose-175, Bifidobacterium animalis, Bifido 0042 FIG. 1 shows a schematic diagram of a multiple bacterium animalis subsp. lactis BB-12, Bifidobacterium layered capsule according to a non-limiting, illustrative animalis subsp. lactis HNO19, Bifidobacterium infantis embodiment of the invention, to be provided (for example) 35624, Escherichia coli M-17, Escherichia coli Nissle 1917, through food; the encapsulation is designed to provide Lactobacillus acidophilus, Lactobacillus acidophilus probiotic bacteria with maximum stability during storage LAFTIR) L10, Lactobacillus acidophilus LAFTI L10, Lac and shelf life at ambient temperature by providing protection tobacillus casei LAFTIR) L26, Lactobacillus casei LAFTI against oxygen and humidity during manufacturing process L26, Lactobacillus brevis, Lactobacillus bulgaricus, Lacto or preparation process as well as storage. As shown, the bacillus casei, Lactobacillus gasseri, Lactobacillus paraca numbered labels indicate the components of the composi sei, Lactobacillus plantarum, Lactobacillus reuteri ATTC US 2016/0360777A1 Dec. 15, 2016

55730 (Lactobacillus reuteri SD2112), Lactobacillus rham C), Sodium ascorbate, calcium ascorbate, potassium ascor nosus, Lactobacillus salivarius, Lactobacillus delbrueckii, bate, ascorbyl palmitate, and ascorbyl Stearate. According to Lactobacillus fermentum, Lactococcus lactis, Lactococcus some embodiments of the present invention, the core further lactis subsp. Lactococcus lactis Rosell-1058, Lactobacillus comprises both a stabilizer and an antioxidant. Without paracasei St11 (or NCC24611 Lactobacillus fortis Nestlé, wishing to be limited by a single hypothesis or theory, Lactobacillus johnsonii Lal (Lactobacillus LC1, Lactoba stabilizing agents and antioxidants may optionally be dif cillus johnsonii NCC533) Nestlé, Lactobacillus rhamnosus ferentiated. According to one preferred embodiment, the Rosell-11, Lactobacillus acidophilus Rosell-52, Streptococ antioxidant is L-cysteine hydrochloride or L-cysteine base. cus thermophilus, Diacetylactis, Saccharomyces cerevisiae, 0052 Filler and Binder: and a mixture thereof. 0053 According to some embodiments of the present 0047 Sugar: invention, the core further comprises both filler and binder. 0048. According to a preferred embodiment of the inven Examples of fillers include but are not limited to, for tion, the probiotic bacteria in the core composition are mixed example, microcrystalline , a Sugar, Such as lactose, with a Sugar. The Sugar preferably comprises at least one , galactose, , or Sucrose; dicalcium phos material that is also a Supplemental agent for the probiotic phate: Sugar alcohols such as Sorbitol, manitol, mantitol, bacteria. It should be noted that optionally the sugar is the lactitol. Xylitol, isomalt, erythritol, and hydrogenated Starch Supplemental agent itself. A Supplemental agent as used hydrolysates; corn starch; and potato starch; and/or a mix herein refers to an agent with a nutritional and/or protective ture thereof. More preferably, the filler is lactose. Examples role, for example. of binders include Povidone (PVP: polyvinyl pyrrolidone), 0049. The sugar may optionally comprise any one or Copovidone (copolymer of vinyl pyrrolidone and vinyl more of the following: Such as acetate), polyvinyl alcohol, low molecular weight HPC including ketotriose () and aldotriose (hydroxypropyl cellulose), low molecular weight HPMC (), such as ketotetrose (), (hydroxypropyl methylcellulose), low molecular weight aldotetroses (, ) and ketopentose (, hydroxymethyl cellulose (MC), low molecular weight ), such as aldopentose (, , Sodium carboxy methyl cellulose, low molecular weight , ), () and ketohexose hydroxyethylcellulose, low molecular weight hydroxymeth (, fructose, , ), such as aldo ylcellulose, cellulose acetate, gelatin, hydrolyzed gelatin, (, , glucose, , , , polyethylene oxide, acacia, , starch, and water galactose, ), deoxy Sugar (, fuculose, ) soluble polyacrylates and polymethacrylates, low molecular and such as (), and and weight ethylcellulose or a mixture thereof. More preferably, (). The Substrate may comprise multiple the binder is low molecular weight HPMC. When using a hot saccharides such as 1) disaccharides, such as Sucrose, lac melt for making granulation one or more of the following, tose, , trehalose, , and , 2) trisac having a melting point below 60° C. can be used as a binder: charides Such as , and , 3) a solid fat, fatty acid, a wax or a polyethylene glycol (PEG). such as and , 4) other For this purpose first the binder is melted and then sprayed oligosaccharides such as (FOS), onto the dry mixture of powders which is at a temperature (GOS) and mannan-oligosaccha which is well below the melting point of the binder. rides (MOS), 5) polysaccharides such as glucose-based 0054 Surfactant: polysaccharides/ including starch (, 0055 According to some embodiments of the present ), cellulose, dextrin, , beta-glucan (Zy invention, the core further comprises a probiotics friendly mosan, , sizofiran), and maltodextrin, fructose-based Surfactant Such as nonionic Surfactants. Examples of non polysaccharides/ including , levan beta 2-6, ionic Surfactant include but are not limited to, for example, mannose-based polysaccharides (mannan), galactose-based tween 80 (polysorbate 80, Polyoxyethylene (20) sorbitan polysaccharides (), and N-acetylglucosamine-based monooleate), tween 20 (polysorbate 20, Polyoxyethylene polysaccharides including . Other polysaccharides (20) sorbitan monolaurate), Tween 85 (Polyoxyethylene may be included, including but not limited to gums such as sorbitan trioleate), glycereth-2-cocoate (Levenol R. C-421), arabic gum (gum acacia). glycereth-6-cocoate (LevenolR) F-200), glycereth-7-cocoate 0050 Stabilizer and Antioxidant (Oxygen Scavenger): (LevenolR C-301), glycereth-17-cocoate (LevenolR C-201) 0051. According to a preferred embodiment of the inven or a mixture thereof. tion, the probiotic bacteria in said inner core are mixed with 0056 First Coating Layer: a stabilizer which may be selected from the group consisting 0057 According to a preferred embodiment of the inven of dipotassium edetate, disodium edetate, edetate calcium tion particles of said core mixture are coated with an inner disodium, edetic acid, fumaric acid, malic acid, maltol, coating layer comprising a hydrophobic solid fat or fatty Sodium edetate, trisodium edetate. According to preferred acid or a wax having a melting point below 50° C., forming embodiments of the present invention, the core further a stable hydrophobic film or matrix which embeds the comprises an antioxidant. Preferably, the antioxidant is probiotic, preventing or reducing the penetration of water or selected from the group consisting of L-cysteine hydrochlo humidity into said core during the further coating processes. ride, L-cysteine base, 4.4 (2.3 dimethyl tetramethylene dipy As used herein the term fats consist of a wide group of rocatechol), tocopherol-rich extract (natural vitamin E), hydrophobic compounds that are generally soluble in C-tocopherol (synthetic Vitamin E), B-tocopherol, Y-tocoph organic solvents and largely insoluble in water. Chemically, erol, 8-tocopherol, butylhydroxinon, butyl hydroxyanisole fats are generally triesters of glycerol and fatty acids. Fats (BHA), butyl hydroxytoluene (BHT), propyl gallate, octyl may be either Solid or liquid at room temperature, depending gallate, dodecyl gallate, tertiary butylhydroquinone on their structure and composition. Although the words (TBHQ), fumaric acid, malic acid, ascorbic acid (Vitamin “oils”, “fats, and “lipids” are all used to refer to fats, “oils” US 2016/0360777A1 Dec. 15, 2016

is usually used to refer to fats that are liquids at normal room especially by Surfactants, will lower Surface tension and temperature, while “fats” is usually used to refer to fats that lower Surface free energy. Some Surface tension values of are solids at normal room temperature. "Lipids” is used to common liquids and solvents are shown in the following refer to both liquid and solid fats, along with other related table, Table 1. substances. The word “oil is used for any substance that does not mix with water and has a greasy feel. Such as TABLE 1. petroleum (or crude oil) and heating oil, regardless of its chemical structure. Examples of fats according to the present Surface tension values invention include but are not limited to Fats as described Substance Y (mN/m) Y (mN/m) Y (mN/m) above, fatty acids, fatty acid esters, fatty acid triesters, slats Water 72.8 51.0 21.8 of fatty acids such as aluminum, sodium, potassium and Glycerol 64 30 34 magnesium, fatty alcohols, phospholipids, Solid lipids, Ethylene glycol 48 19 29 waxes, and a combination thereof having a melting point Dimethyl sulfoxide 44 8 36 lower than 50° C. and higher than 25°C., preferably lower Benzyl alcohol 39 11.4 28.6 than 45° C. and higher than 30° C. and most preferably Toluene 28.4 2.3 26.10 Hexane 18.4 18.4 lower than 40° C. and higher than 35° C. forming a stable Acetone 23.7 23.7 hydrophobic film or matrix which embeds the probiotic or Chloroform 27.15 27.15 forms film around the probiotics core particles. The solid fat Diiodomethane SO.8 SO.8 is optionally and preferably at least one of lauric acid, hydrogenated coconut oil, cacao butter and a combination 0063. The adhesion and uniformity of a film are also thereof. influenced by the forces which act between the coating 0058 According to another preferred embodiment of the formulation which is in a solution form and the core surface invention particles of said core mixture are granulated by of the film coated surface. Therefore, coating formulations said hydrophobic solid fat or fatty acid using a hot melt for certain core Surface can be optimized via determination granulation process. In this case a melt of said hydrophobic of wetting behavior, the measure of which is the contact or Solid fat or fatty acid is used for granulation of said core wetting angle. This is the angle that forms between a liquid particles therefore, said hydrophobic solid fat or fatty acid droplet and the surface of the solid body to which it is constitutes a matrix in which said core particles are embed applied. ded thus said hydrophobic solid fat or fatty acid functions as 0064. When a liquid does not completely spread on a both binder for granulation and first coating layer of said Substrate (usually a solid) a contact angle (0) is formed core particles. In other words said hot melt of said hydro which is geometrically defined as the angle on the liquid side phobic solid fat or fatty acid may simultaneously constitute, of the tangential line drawn through the three phase bound by hot melt granulation process, both core binder as well as ary where a liquid, gas and solid intersect, or two immiscible the first inner coating layer. liquids and Solid intersect. 0059 Intermediate Coating Layer: 0065. It is a direct measure of interactions taking place 0060 According to a preferred embodiment of the inven between the participating phases. The contact angle is deter tion, particles of said core mixture coated with said inner mined by drawing a tangent at the contact where the liquid coating layer are coated with an intermediate coating layer and solid intersect. whose the aqueous Solution of 0.1% has a Surface tension 0066. The contact angle is small when the core surface is lower than 60 mN/m, preferably lower than 50 mN/m and evenly wetted by spreading droplets. If the liquid droplet most preferably lower than 45 mN/m measured at 25°C. for forms a defined angle, the size of the contact angle is adjusting Surface tension for further coating with outer described by the Young-Dupre equation; coating layer. 0061 Surface tension (ST) is a property of the surface of a liquid that allows it to resist an external force. In the other 0=Contact angle word Surface tension is the measurement of the cohesive Ys Surface tension of the Solid body (excess) energy present at a gas/liquid interface. The mol Y. Surface tension of the liquid ecules of a liquid attract each other. The interactions of a Ys, interfacial tension between liquid and solid body (can molecule in the bulk of a liquid are balanced by an equally not be measured directly) attractive force in all directions. Molecules on the surface of 0067. With the aid of this equation it is possible to a liquid experience an imbalance of forces as indicated estimate the Surface tension of a solid body by measuring the below. The net effect of this situation is the presence of free relevant contact angles. If one measured them with liquid of energy at the Surface. The excess energy is called Surface varying Surface tension and plots their cosines as a function free energy and can be quantified as a measurement of of the Surface tension of the liquids, the result is a straight energy/area. It is also possible to describe this situation as line. The abscissa value of the intersection of the straight line having a line tension or Surface tension, which is quantified with cos 0-1 is referred to as the critical surface tension of as a force/length measurement. The common units for wetting Y. A liquid with a Surface tension Smaller than Y, Surface tension are dynes/cm or mN/m. These units are wets the Solid in question. equivalent. 0068. The wetting or contact angle can be measured with 0062 Polar liquids, such as water, have strong intermo ease by means of telescopic goniometers (e.g. LuW Wetta lecular interactions and thus high Surface tensions. Any bility Tester by AB Lorentzenu. Wettre, S-10028 Stockholm factor which decreases the strength of this interaction will 49). In many cases, the quantity Y does not suffice to lower Surface tension. Thus an increase in the temperature of characterize polymer Surfaces since it depends amongst this system will lower Surface tension. Any contamination, other factors on the polar character of the test liquids. This US 2016/0360777A1 Dec. 15, 2016 method can, however, be improved by dividing Y into non (0075. The following table, Table 2, shows for example polar party" (caused by dispersion forces) and a polar part the surface tension of the solution of some water soluble Y (caused by dipolar interactions and hydrogen bonds). polymers. The surface tension was measured at 25°C., 0.1% aqueous solution of the polymers.

TABLE 2 Y Surface tension of the test liquid surface tension values of selected polymers Ys surface tension of the solid body Ys and Ys' can be determined by means of the following Polymer Surface Tension mNim equation: Sodium Carboxymethylcellulose (Na-CMC) 71.O Hydroxyethyl cellulose (HEC) 66.8 Hydroxypropyl cellulose (HPC) 43.6 I0069. If 1+(cos 0/2)(Y/Vy,') is plotted against V(Y, -Y.")/ Hydroxypropyl methyl cellulose (HPMC) 46-51 Y,", straight lines are obtained, from the slops and ordinate Hydroxymethyl cellulose (HMC) 50-55 intercepts of which Y and Y can be determined thus YS calculated. 0076 Non-limiting examples of polymers which may be 0070 Y, and Ys are approximately, but not exactly, the used as intermediate coating layer include Hydroxypropyl same. Since the measurement is also influenced by irregu methylcellulose (HPMC), Hydroxypropylethylcellulose larities of the polymer Surfaces, one cannot obtain the ture (HPEC), Hydroxypropylcellulose (HPC), hydroxypropyl contact angle 0 but rather the quantity 0". Both quantities are ethylcellulose (HPEC), hydroxymethylpropylcellulose linked by the relationship; (HMPC), ethylhydroxyethylcellulose (EHEC) (Ethulose), Roughness factor r=cos 0'cos 0 hydroxyethylmethylcellulose (NEMC), hydroxymethyleth ylcellulose (HMEC), propylhydroxyethylcellulose (PHEC), 0071. The lower the surface tension of the coating for methylhydroxyethylcellulose (MHEC), hydrophobically mulation against that of the core surface, the better the modified hydroxyethylcellulose (NEXTON), carboxymeth droplets will spread on the surface. If formulations with ylhydroxyethylcellulose (CMHEC), Methylcellulose, Ethyl organic solvents are used, which wet the surface very well cellulose, water Soluble vinyl acetate copolymers, gums, the contact angle will be close to Zero. The Surface tensions polysaccharides such as alginic acid and alginates Such as of such formulations are then about 20 to 30 mN/m. Aque ammonia alginate, sodium alginate, potassium alginate, pH ous coating dispersion of some polymer like EUDRAGITL sensitive polymers for example enteric polymers including 30 D type shows low surface tension in the range of 40 to phthalate derivatives such as acid phthalate of carbohy 45 mN/m. drates, amylose acetate phthalate, cellulose acetate phthalate 0072 Contact angle measurements may optionally pro (CAP), other cellulose ester phthalates, cellulose ether vide the following information, which may be useful for phthalates, hydroxypropylcellulose phthalate (NPCP), selecting coating materials according to at least some hydroxypropylethylcellulose phthalate (HPECP), hydroxy embodiments of the present invention (without wishing to be proply methylcellulose phthalate (HPMCP), hydroxypr limited by a closed list): oplymethylcellulose acetate succinate (HPMCAS), methyl 1. Smaller contact angles give Smoother film coatings cellulose phthalate (MCP), polyvinyl acetate phthalate 2. The contact angle becomes Smaller with decreasing (PVAcP), polyvinyl acetate hydrogen phthalate, sodium porosity and film former concentration. CAP, starch acid phthalate, cellulose acetate trimellitate 3. Solvents with high boiling point and high dielectric (CAT), styrene-maleic acid dibutyl phthalate copolymer, constant reduce the contact angle. styrene-maleic acid/polyvinylacetate phthalate copolymer, 4. The higher the critical surface tension of core, the better styrene and maleic acid copolymers, polyacrylic acid deriva the adhesion of the film to the core. tives such as acrylic acid and acrylic ester copolymers, 5. The smaller the contact angle, the better the adhesion of polymethacrylic acid and esters thereof, polyacrylic and the film to the core. methacrylic acid copolymers, shellac, and vinyl acetate and 0073. The critical surface tension of said core or granules crotonic acid copolymers. Preferred pH-sensitive polymers coated with a hydrophobic solid fat is essentially very low. include shellac, phthalate derivatives, CAT, HPMCAS, Therefore for providing better spreading and thus better polyacrylic acid derivatives, particularly copolymers com adhesion of the outer coating layer film to the core, accord prising acrylic acid and at least one acrylic acid ester, ing to at least some embodiments it is desirable to reduce the EudragitTM S (poly(methacrylic acid, methyl methacrylate) surface free energy at the interface between the surface of 1:2); Eudragit L100TM (poly(methacrylic acid, methyl meth the fat coated core/granules and the solution of the outer acrylate)1:1); Eudragit L30DTM, (poly(methacrylic acid, coating layer polymer. ethyl acrylate)1:1); and (Eudragit L100-55) (poly(meth 0074 According to a preferred embodiment of the pres acrylic acid, ethyl acrylate) 1:1) (EudragitTML is an anionic ent invention, particles of said core mixture coated with said polymer synthesized from methacrylic acid and methacrylic hydrophobic solid fat (inner coating layer) are coated with acid methyl ester), polymethyl methacrylate blended with an intermediate coating layer whose the aqueous solution of acrylic acid and acrylic ester copolymers, alginic acid and 0.1% has a surface tension lower than 60 mN/m, preferably alginates Such as ammonia alginate, Sodium, potassium, lower than 50 mN/m and most preferably lower than 45 magnesium or calcium alginate, vinyl acetate copolymers, mN/m measured at 25° C. for reducing the surface free polyvinyl acetate 30D (30% dispersion in water), a poly energy at the interface between the surface of the fat coated (dimethylaminoethylacrylate) which is a neutral methacrylic core/granules and the solution of the outer coating layer ester available from Rohm Pharma (Degusa) under the name polymer. “Eudragit ETM, a copolymer of methylmethacrylate and US 2016/0360777A1 Dec. 15, 2016

ethylacrylate with small portion of trimethylammonioethyl I0084 Another assumption inherent to the calculation of methacrylate chloride (Eudragit RL, Eudragit RS), a copo permeability is its independence from film thickness. This lymer of methylmethacrylate and ethylacrylate (Eudragit assumption is not true for hydrophilic films. Because of that NE 30D), Zein, shellac, gums, polysaccharides and or the experimentally determined water vapor permeability of combination thereof. most films applied only to the specific water vapor gradients used during the testing and for the specific thickness of the Outer Layer tested specimens, it has been proposed the use of the terms “Effective Permeability” or “Apparent Permeability.” 0077 According to at least some embodiments of the I0085 Moisture transport mechanism through a compos present invention, the composition further comprises an ite depends upon the material and environmental conditions. outer coating layer comprising a polymer having oxygen Permeability has two different features in case of compos transmission rate of less than 1000 cc/m/24 hr, preferably ites. First, in non-porous membranes, permeation can occur less than 500 cc/m/24 hr and most preferably less than 100 by Solution and diffusion; and the other, simultaneous per cc/m/24 hr measured at standard test conditions i.e. 73° F. meation through open pores is possible in porous membrane. (23°C.) and 0% RH, and a water vapor transmission rate of I0086. There are various methods of measuring perme less than 400 g/m/day, preferably less than 350 g/m/day ability. Weight loss measurements are of importance to and most preferably less than 300 g/m/day coats said water determine permeability characteristics. Water vapor perme sealed coated particles having an adjusted Surface for reduc ability is usually determined by direct weighing because, ing or preventing the transmission of oxygen and humidity despite its inherent problems, mainly related to water prop into the core thereby obtaining a multiple-layered particle erties such as high solubility and cluster formation within containing probiotics demonstrating improved Stability the polymer and tendency to plasticize the polymer matrix, against both humidity as well as oxygen. Various Suitable it is simple and relatively reliable method. The major examples of such polymers are described below. disadvantage of this method resides in its weakness to provide information for a kinetic profile, when such a Water Vapor Permeability (WVP) of Films response is required. 0078. The water vapor permeability is one of the most 0087 Another measurement method is based on the important properties of said outer layer coating films mainly standard described in ASTM E96-80 (standard test method because of the importance of the role of water in deteriora procedure for water vapor permeability). According to this tive reactions. method water vapor permeability is determined gravimetri 0079 Water acts as a solvent or carrier and causes texture cally and generally, the applied procedures are nearly the degradation, chemical and enzymatic reactions, and thus is same in many research papers that are related with this destructive of probiotics. Also the water activity of foods is purpose. In this procedure firstly, the test film is sealed to a an important parameter in relation with the shelf-life of the glass permeation cell which contain anhydrous calcium food and food-containing probiotics. In low-moisture foods chloride (CaCl2), or silica gel (Relative vapor pressure; and probiotics, low levels of water activity must be main RVP=0) and then the cell is placed in the desiccators tained to minimize the deteriorative chemical and enzymatic maintained at specific relative humidity and temperature reactions and to prevent the texture degradation. The com (generally 300 C, 22% RH) with magnesium nitrate or position of film forming materials (hydrophilic and hydro potassium acetate. Permeation cells are continuously phobic character), temperature and relative humidity of the weighed and recorded, and the water vapor that transferred environment affect the water vapor permeability of the films. through the film and absorbed by the desiccant are deter 0080 When considering a suitable barrier in foods-con mined by measuring the weight gain. Changes in weight of taining probiotics the barrier properties of the films are the cell were plotted as a function of time. When the important parameters. relationship between weight gain (Aw) and time (At) is 0081 Polysaccharide films and coatings are generally linear, the slope of the plot is used to calculate the water good barriers against Oxygen and carbon dioxide and have vapor transmission rate (WVTR) and water vapor perme good mechanical properties but their barrier property against ability (WVP). Slope is calculated by linear regression and water vapor is poor because of the their hydrophilic char correlation coefficient (r2) 0.99). acter. I0088. The WVTR is calculated from the slope (Aw/At) of 0082 To add an extra hydrophobic component e.g. a lipid the straight line divided by the test area (A), (g S-1 m-2); (waxes, fatty acids) in the film and produce a composite film is one way to achieve a better water vapor barrier. Here the where Aw/At=transfer rate, amount of moisture loss per unit lipid component serves as the barrier against water vapor. By of time (g's-1); adding lipid, the hydrophobicity of the film is increased and A area exposed to moisture transfer (m2) as a result of this case water vapor barrier property of the film increases. I0089. The WVP (kg Pa-1 s-1 m-1) is calculated as: I0083 Water Vapor Permeability of a film is a constant that should be independent of the driving force on the water where S=Saturation vapor pressure (Pa) of water at test vapor transmission. When a film is under different water temperature, R1 =RVP (relative vapor pressure) in the des vapor pressure gradients (at the same temperature), the flow iccator, R2=RVP in the permeation cell, and d=film thick of water vapor through the film differs, but their calculated ness (m). At least three replicates of each film should be permeability should be the same. This behavior does not tested for WVP and all films should be equilibrated with happen with hydrophilic films where water molecules inter specific RH before permeability determination. act with polar groups in the film structure causing plastici 0090 The water vapor permeability can also be calcu Zation or Swelling. lated from the WVTR as follows: US 2016/0360777A1 Dec. 15, 2016

TABLE 3 L-film thickness (m); Ap-water vapor pressure gradient between the two sides of the film (Pa); P film permeability OTR and WVTR values (gm-2s-1 Pa-1). Oxygen Transmission Water vapor 0091. The rate of permeation is generally expressed by Film Forming rate, Transmission rate, the permeability (P) rather than by a diffusion coefficient (D) Polymer Cm3/m2/atm O2 day g/m2/day and the solubility (S) of the penetrant in the film. When there HPC, Klucel EF Medium Low 776 126 is no interaction between the water vapor and film, these CMC, Aqualon or Low Low laws can apply for homogeneous materials. Then, perme Blanose 7L 18 228 ability follows a solution—diffusion model as; HEC, Natrosol 250L Low Medium 33 360 HPMC 5cps High High where D is the diffusion coefficient and the S is the slope of 318O 420 the Sorption isotherm and is constant for the linear Sorption isotherm. The diffusion coefficient describes the movement 0098. Non-limiting examples of suitable outer layer coat of permeant molecule through a polymer, and thus repre ing polymer include water-soluble, hydrophilic polymers, sents a kinetic property of the polymer-permeant system. such as, for example, polyvinyl alcohol (PVA), Povidone 0092. As a result of the hydrophilic characteristics of (PVP: polyvinyl pyrrolidone), Copovidone (copolymer of polysaccharide films, the water vapor permeability of films vinyl pyrrolidone and vinyl acetate), Kollicoat Protect is related to their thickness. The permeability values increase (BASF) which is a mixture of Kollicoat IR (a polyvinyl with the increasing thickness of the films. alcohol (PVA)-polyethylene glycol (PEG) graft copolymer) 0093. Thickness of films and the molecular weight (MW) and polyvinyl alcohol (PVA), Opadry AMB (Colorcon) of the film forming polymers may also affect both water which is a mixture based on PVA, Aquarius MG which is a vapor permeability (WVP) and oxygen permeability (OP) of cellulosic-based polymer containing natural wax, lecithin, the films. and talc, low molecular weight HPC (hydroxy propyl cellulose), low molecular weight HEC (hydroxyethyl Oxygen Transmission Determination (OTR) cellulose), low molecular weight carboxy methyl cellulose 0094. Oxygen transmission rate is the steady state rate at such as 7LF or 7L2P, or a mixture thereof. In some cases which oxygen gas permeates through a film at specified mixture of water soluble polymers with insoluble agents conditions of temperature and relative humidity. Values are Such as waxes, fats, fattu acids, and etc. may be of benefit. expressed in cc/100 in2/24 hr in US standard units and 0099 More preferably the outer coating polymers are cc/m2/24 hr in metric (or SI) units. carboxy methyl cellulose such as 7LF or 7L2P, low molecu 0095 To assess the protective function which the coating lar weight of hydroxyethyl cellulose (HEC) and low molecu performs on the core, its gas permeability is measured. lar weight HPC (hydroxypropyl cellulose) According to at least Some embodiments of the present invention, the most critical gas for improved stability of the Exterior Coating Layer probiotic bacteria is oxygen. It is well known that probiotic bacteria are anaerobic microorganism where their vitality 0100. According to at least some embodiments of the may significantly be reduced upon exposing to oxygen. present invention, the composition comprises an exterior Therefore for providing long term stability and receiving an coating layer comprising a polymer having a water vapor extended shelf life for probiotic bacteria the outer layer transmission rate of less than 400 g/m/day, preferably less preferably provides a significant oxygen barrier. than 300 g/m/day and most preferably less than 200 g/m/ 0096. The gas permeability, q, (ml/m2-day atm) (DIN day, which coats said oxygen and humidity sealed coated 53380) is defined as the volume of a gas converted to 0°C. particles for further reducing or preventing the transmission and 760 torr which permeates 1 m2 of the film to be tested of humidity into the core thereby obtaining a multiple within one day at a specific temperature and pressure layered particle containing probiotics demonstrating gradient. It is therefore calculated according to the following improved Stability against humidity as well. formula: 0101. Non-limiting examples of polymers that are suit q = {TP/IPTA (P-P):249-(Ax/At):104 able for the exterior coating layer include water-soluble, hydrophilic polymers, such as, for example, polyvinyl alco P normal pressure in atm hol (PVA), Povidone (PVP: polyvinyl pyrrollidone), Cop T-normal temperature in K ovidone (copolymer of vinyl pyrrolidone and vinyl acetate), T=experimental temperature in K Kollicoat Protect (BASF) which is a mixture of Kollicoat IR A sample area in m2 (a polyvinyl alcohol (PVA)-polyethylene glycol (PEG) graft T=time interval in hrs between two measurements copolymer) and polyvinyl alcohol (PVA), Opadry AMB Patmospheric pressure in atm (Colorcon) which is a mixture based on PVA, Aquarius MG which is a cellulosic-based polymer containing natural wax, P. pressure in test chamber between sample and mercury lecithin, xanthan gum and talc, low molecular weight HPC thread (hydroxypropyl cellulose), low molecular weight carboxy Q cross section of capillaries in cm methyl cellulose such as 7LF or 7L2P, or a mixture thereof. AX/At=sink rate of the mercury thread in cm/hr In some cases a mixture of water soluble polymers with 0097. The following table, Table 3, shows OTR and insoluble agents such as waxes, fats, fatty acids, and so forth, WVTR of some water soluble polymers for example. may be of benefit. US 2016/0360777A1 Dec. 15, 2016

0102 More preferably the exterior coating polymers are particulate matter, including but not limited to dry mix, wet polyvinyl alcohol, Kollicoat Protect (BASF) which is a granulation, dry granulation or hot melt (optionally in the mixture of Kollicoat IR (a polyvinyl alcohol (PVA)-poly form of a granulation process). ethylene glycol (PEG) graft copolymer) and polyvinyl alco 0107 The core composition, prepared according to one hol (PVA), Opadry AMB (Colorcon) which is a mixture of the above processes, contains at least the probiotic based on PVA, low molecular weight HPC (hydroxypropyl bacteria and a stabilizer, wherein the total amount of pro cellulose) and Aquarius MG which is a cellulosic-based biotics in the mixture is from about 10% to about 90% by polymer containing natural wax. These polymers provide weight of the core composition. Superior barrier properties against water vapor/humidity 0108. The stabilizer may optionally comprise any type of penetration into the core material. oxygen scavenger, including but not limited to those con taining L-cysteine base or hydrochloride, of which other Enteric Coating Layer examples are listed herein. 0103) According to preferred embodiments of the present 0109 The core composition may also optionally com invention the core particles are further optionally coated by prise at least one Sugar compound including but not limited an enteric polymer which may further provide protection to maltodextrin, trehalose, lactose, galactose, Sucrose, fruc against destructive conditions present in the gastrointestinal tose and the like, of which other examples are provided tract for example. herein. Disaccharides, such as Sucrose and trehalose, are 0104. Non-limiting examples of suitable enteric poly attractive as protective agents within the core because they mers include pH-sensitive polymers, acid phthalate of car are actually help plants and microbial cells to remain in a bohydrates, amylose acetate phthalate, cellulose acetate state of Suspended animation during periods of drought. phthalate (CAP), other cellulose ester phthalates, cellulose Trehalose has been shown to be an effective protectant for a ether phthalates, hydroxypropylcellulose phthalate (NPCP), variety of biological materials, both in ambient air-drying hydroxypropylethylcellulose phthalate (HPECP), hydroxy and freeze-drying. proply methylcellulose phthalate (HPMCP), hydroxypr 0110. The core composition may also optionally com oply methylcellulose acetate succinate (HPMCAS), methyl prise one or more other food grade ingredients, including but cellulose phthalate (MCP), polyvinyl acetate phthalate not limited to a filler, a surfactant and binder, of which (PVAcP), polyvinyl acetate hydrogen phthalate, sodium various non-limiting examples are provided herein. CAP, starch acid phthalate, cellulose acetate trimellitate 0111. These various ingredients may optionally be added (CAT), styrene and maleic acid copolymers, styrene-maleic sequentially during the core preparation process or alterna acid dibutyl phthalate copolymer, styrene-maleic acid/poly tively may optionally be added together in any Suitable vinylacetate phthalate copolymer, polyacrylic acid deriva combination. tives such as acrylic acid and acrylic ester copolymers, 0112. Once the core composition has been formed, it is polymethacrylic acid and esters thereof, polyacrylic and coated with an innermost coating layer, layered on said core methacrylic acid copolymers, polyacrylic acid derivatives composition, comprising at least one hydrophobic Solid fat Such as particularly copolymers comprising acrylic acid and or fatty acid having a melting point lower than 60° C. at least one acrylic acid ester, Eudragit STM (poly(meth 0113. The innermost coating layer may optionally com acrylic acid, methyl methacrylate) 1:2); Eudragit LTM which prise at least one hydrophobic Solid fat or fatty acid having is an anionic polymer synthesized from methacrylic acid and a melting point lower than 50° C. and preferably higher than methacrylic acid methyl ester), Eudragit L100TM (poly 25°C. The melting point is optionally preferably lower than (methacrylic acid, methyl methacrylate)1:1); Eudragit 45° C. and higher than 30° C., and is optionally and most L3ODTM, (poly(methacrylic acid, ethyl acrylate)1:1); and preferably lower than 40° C. and higher than 35° C. The Eudragit L100-55TM (poly(methacrylic acid, ethyl acrylate) innermost coating layer may optionally form a stable hydro 1:1), polymethyl methacrylate blended with acrylic acid and phobic matrix which embeds the core composition within acrylic ester copolymers, alginic acid and alginates such as and/or forms a film around the probiotic core composition. ammonia alginate, Sodium, potassium, magnesium or cal 0114. Next, the intermediate coating layer is layered over cium alginate. the innermost coating layer. As previously described with regard to the intermediate coating layer, when present in an Process for Preparation of a Stabilized Probiotic aqueous solution in the amount of 0.1% weight/weight over Composition According to at Least Some Embodiments of the weight of the solution, the coating layer material has a the Present Invention surface tension lower than 60 mN/m, when measured at 25° C. The intermediate coating layer, which when present in an 0105. The present invention provides a process for the aqueous solution in the amount of 0.1% weight/weight over preparation of heat, oxygen and humidity resisting probiotic the weight of the solution, optionally has a Surface tension bacteria for a food product. In an embodiment of the lower than 50 mN/m and preferably lower than 45 mN/m invention, said stabilized probiotic particles are added to a when measured at 25° C. The intermediate coating layer food product such as Such as creams, baked goods, biscuit optionally comprises at least one plasticizer selected from creams or fill-in material, chocolates, sauces, cheese, may the group consisting of polyethylene glycol (PEG), triethyl onnaise and so forth. citrate and triacetin. 0106. In a preferred embodiment, the preferred process of 0115) Next an outer coating layer is layered over the the invention comprises preparation of a core composition in intermediate coating layer. The outer coating layer option form of Solid particulate matter containing probiotic bacte ally comprises a polymer having an oxygen transmission ria, followed by layering of various coating layers on the rate of less than 1000 cc/m/24 hr, preferably less than 500 particulate core composition. The core composition may cc/m/24 hr and most preferably less than 100 cc/m/24 hr optionally be prepared by any suitable method for preparing measured at Standard test conditions (which may for US 2016/0360777A1 Dec. 15, 2016

example be 73° F (23°C.) and 0% RH). The polymer also I0123. The water sealed coated particles are coated with optionally has a water vapor transmission rate of less than an intermediate coating layer for adjusting Surface tension 400 g/m/day, preferably less than 350 g/m/day and most thereby obtaining water sealed coated particles having an preferably less than 300 g/m/day. The intermediate coating adjusted Surface tension. layer acts as a binder or “glue” to bind the outer coating layer 0.124. The water sealed coated particles having an to the innermost coating layer. adjusted Surface tension are coated with an outer coating 0116. The composition may optionally further comprise layer for reducing transmission of oxygen and humidity into an additional humidity barrier coating layer, layered on the the core obtaining oxygen and humidity sealed coated outer coating layer, for preventing further humidity penetra particles. tion. If present, this additional humidity barrier coating layer 0.125. The oxygen and humidity sealed coated particles is layered over the outer coating layer. are coated with an exterior coating layer for reducing 0117 The composition may optionally further comprise transmission of humidity into the core, thereby obtaining a an enteric polymer, layered on the humidity barrier coating multiple-layered particle containing probiotics showing layer, which may further provide protection against Such Superior stability against Oxygen and humidity, and hence destructive characteristics of the gastrointestinal tract as low having higher viability and vitality. pH values and proteolytic enzymes. The enteric polymer 0.126 Granulation as described herein may optionally be optionally comprises at least one plasticizer selected from performed with fluidized bed technology such as Glatt or a the group consisting of polyethylene glycol (PEG), triethyl Glatt turbo jet, or an Innojet coater/granulator, or a Huttlin coater/granulator, or a GranuleX. citrate and triacetin. I0127. In any case the resulting probiotic composition 0118. It should be noted that by “layering it is meant any according to the above processes may optionally be intro Suitable process for adding the coating layer to the compo duced to a food product which may also undergo a heating sition including but not limited to spraying, dipping, sprin step during its preparation process. Alternatively the above kling and the like. Upon the addition of the layers to the core resulting probiotic composition can be added to a food composition, particles are formed with three, four or more product which may not undergo a heating step during its coating layers. preparation process. 0119 Optionally the following combination of ingredi ents may be applied as a non-limiting example: the at least Example 1 one Sugar may comprise, lactose, galactose or a mixture thereof, said at least one oligosaccharide or polysaccharides Preparation and Testing of an Exemplary may comprise, galactan, maltodextrin, and trehalose, said Formulation stabilizer comprises L-cysteine base, said Surfactant com prises tween 80 (polysorbate 80, Polyoxyethylene (20) sor I0128 Preparation of an exemplary, illustrative formula bitan monooleate), said filler comprises lactose DC and/or tion, described herein as Formula I, was performed as microcrystalline cellulose, said binder comprises hydroxy described below. Tests performed on Formula I are also propylmethylcelluloses, said hydrophobic solid fat or fatty described below. acid comprises lauric acid and/or cacao butter said inner I0129. For the preparation of Formula I, trehalose dihy coating layer may comprise lauric acid and/or cacao butter, drate 160 g, Maltodextrin DE 15 314 g. L-Cystein-HCl said intermediate coating layer polymer may comprise alg Monohydrate 6 g and the bacteria BB12 (Bifidobacteria inic acid or Sodium alginate, said outer coating layer com BB12) 120 g were loaded into Innojet ventilus machine to prises carboxymethylcellulose (CMC) 7LFPH and/or car receive a dry blend. Lauric acid 270 g was melted at 50° C. boxymethylcellulose (CMC) 7L2P said plasticizer is using a heating plate while stirring. Then hot melt of lauric polyethylene glycol (PEG) 400 and/or triacetin and said acid was sprayed onto the above dry blend under an inert exterior coating layer comprises hydroxypropyl cellulose. atmosphere using nitrogen. The temperatures of pump head, liquid, and spray pressure were set at 60° C. Core particles 0120 According to another non-limiting embodiment of were therefore formed, based on a hot melt granulation the present invention, there is provided another process of process. manufacturing probiotic bacteria in a stabilized composition 0.130 Next, the various layers were coated over the core as follows. First, the core material is prepared as a mixture, particles as follows. For the first (innermost) layer, lauric with probiotic bacteria, a stabilizer, at least one Sugar and at acid 315 g was melted at 50° C. using a heating plate while least one oligosaccharide, and optionally other food grade stirring. Then hot melt of lauric acid was sprayed onto the additives such as fillers, Surfactant, binders, antioxidant, and above granulates under an inert atmosphere using nitrogen etc., thereby obtaining a core mixture. and Such spraying parameters to obtain a film coat as a first 0121 The core mixture is then wet granulated using a sealing layer. Then Na-alginate (25.2 g) solution (2% w/w in binder solution in purified water, or purified water under purified water) was sprayed onto the above resulting gran either air or nitrogen environment; alternatively, the core ules (400 g) to obtain Na-alginate coated granules. 320 g of mixture is prepared using a hot melt granulation by using the above resulting Na-alginate coated granules were melt of a hydrophobic solid fat or fatty acid having a melting reloaded into the Innojet ventilus machine and additional point below 50° C. In any case, the granulation process portion of Na-alginate (58.97 g) solution (2% w/w in puri results in granulated core particles. fied water) was sprayed to obtain finally 16.4% W/W 0122) The particles of said core composition are coated Na-alginate in the formulation. Then 290 g of the above with an inner coating layer comprising a hydrophobic solid resulting Na-alginate coated granules were reloaded into the fat or fatty acid for preventing or reducing the penetration of Innojet ventilus machine and the aqueous solution (5% w/w) water or humidity into said core, thereby obtaining water of Na-carboxy methyl cellulose (Na-CMC) (72.92 g) and sealed coated particles. 19.98g polyethylene glycol (PEG 400) (PEG 400/Na-CMC, US 2016/0360777A1 Dec. 15, 2016

20% w/w) was sprayed onto the above resulting Na-alginate (O140 MRS: Composition of MRS Agar (Purchased ready coated granules to obtain Na-CMC coated granules. Then to use from Difco 288210 or equivalents): 250 g of the above resulting Na-CMC coated granules were reloaded into the Innojet ventilus machine and additional Polypeptone 10 g/l portion of aqueous solution (5% w/w) of Na-CMC (61.70 g) Meat Extract 10 g/l and 15.4 g PEG 400 (PEG 400/Na-CMC, 20% w/w) was Yeast Extract 5 g/l sprayed to obtain finally 29.32% W/W Na-CMC+PEG 400 Dextrose 20 g/l in the final product. Tween 80 (polysorbate 80, 1.0 g/l Polyoxyethylene (20) 0131 The final product, particles of Formula I, was dried sorbitan monooleate) and kept in a double sealed polyethylene bag with a proper Dipotassium phosphate 2 g/l desiccant under refrigeration. It should be noted that various Sodium acetate 5 g/l Ammonium citrate 2 g/l samples were taken from the above stages of preparation and Magnesium sulfate 0.1 g/l were tested as described below. Manganese Sulfate 0.05 g/1 Bacteriological Agar 15 g/l Colony Forming Units Total Count Testing Method for Encapsulated Bifidobacteria-BB12 in Formula I 0141 Cysteine Hydrochloride (10% Solution) was pre 0132) The following testing method was used for the pared as follows. 10 grams of L-Cysteine was added to a determination of colony forming units-total count (CFU) for clean 200 ml flask, with deionized water added to reach 100 encapsulated Bifidobacteria-BB12, prepared in Formula I as ml volume. The L-cysteine was dissolved and filter sterilized described above. This method is based on the dissolution of using a 0.45 micron filtration unit. the coating layers of the encapsulated probiotics to liberate 0142. The following procedure was used to test the the probiotics prior to performing CFU test. protective effect of the composition of Formula I, to protect bacteria during storage and simulated release in the gastro 0133. In this procedure both hydration and dissolution intestinal tract, and to deliver live, viable bacteria to the site processes are applied to the coated particles, simulating the of colonization in the intestine. As used below, the term gastrointestinal tract and hence simulating the effect of “sample” refers to the composition of Formula I. ingesting the coated particles. As a result, the free bacterial 0.143 About 20 g of the sample was crushed using a cells are released. A medium that incorporates various mortar and pestle for about 120 seconds, until the sample selective agents is used to aid in the recovery of specific was homogeneously and finely crushed. 10 gram of the species and incubation in anaerobic conditions for increased prepared crushed probiotic sample was placed into a sterile recovery of the organisms. This method uses MRS agar with stomacher bag. 99 mL of warm phosphate buffer was added the additive Cysteine Hydrochloride as an oxygen scaven to the bag, warmed in the water-bath at 48°C. ger. 0144. The powder was dissolved uniformly and was then 0134. The samples were plated in duplicates and average placed for 15 min (hydration time) in incubator (37° C.). counts are reported (CFU/g). Next, 2 mL of Tween 85 (Polyoxyethylene sorbitan trioleate) 0135 The equipment included a sterile sampling device: in hydrated mixture was added. The bag was kept in the 0.45 Micron Filtration Unit; Colony counter; Sterile Petri water bath for 2 minutes, followed by application of the Dishes; Sterile pipettes; Incubator 37+/-2 C.; Water bath stomacher at 250 RPM for 2 min and incubation again in the 45+/-2°C. for media tempering; Gaspak Anaerobic System: water bath for 1 min. The stomacher was again applied at jar, or plastic chamber, anaerobic packets, and anaerobic 250 RPM for 1 min. The resultant solution was serially indicator, Autoclave; Mortar and pestle; Stomacher, Ana diluted. lytical Balance; and Microscope slides. 0145 1 ml from appropriate dilutions was plated on appropriately labeled sterile Petri plates, followed by pour Media: ing MRS agar at a temperature of about 48°C. For each 100 ml of MRS agar, 0.5 mL offilter sterilized 10% L-Cysteine 0136. The following media and reagents were used: MRS Hydrochloride was added before pouring. Plates were pre Broth, Difco #288130 (500 g); MRS Agar, Bacto Agar pared in duplicate. #21401 (484 g); L-Cysteine Hydrochloride, J.T. baker 0146 The plates were incubated anaerobically at 37° C. #G121-05; Tween-85 (Polyoxyethylene sorbitan trioleate), for 48-72 hours, followed by colony counting. I0137 Sigma #P4634 (500 mL); Butterfield's Buffered 0147 The following quantitative procedures were fol Phosphate Diluent (BUT) MB 101. lowed to count the colonies. 0138 MRS Broth (55 g/L) was prepared as follows. In a 0148 Colonies were counted on the MRS-cysteine 1000 ml flask, the following ingredients were placed and plates, after selecting for those plates having 25-400 colo mixed: 500 ml deionized water; 27.5 grams of dehydrated nies, and the number was recorded as the Probiotic viable MRS Broth: 1 ml Tween-80 (polysorbate 80, Polyoxyeth cell count per gram (CFU/g), taking into account the dilution ylene (20) sorbitan monooleate) (before sterilization). The factor of the plate counted. The counts from each plate were broth was adjusted to a pH of 6.5+/-0.2 at 25°C. averaged at a given dilution for the total viable count per 0139 MRS Agar (70 g/L) was prepared as follows. In a gram (i.e. CFU/g). 1000 ml flask: the following ingredients were placed and mixed: 500 ml deionized water; 35 grams of dehydrated Results MRS Broth, followed by adjustment of the pH to 6.5+/-0.2 014.9 The results of the test from different stages of the (a 25°C. Filter Sterilized L-Cysteine was added to the agar process for preparing the composition of Formula I are after sterilization. shown as CFU/g in the following table, Table 4. US 2016/0360777A1 Dec. 15, 2016

TABLE 4 3. The composition of claim 1, wherein said stabilizer comprises a material selected from the group consisting of Probiotic bacteria viability after various stages of preparation dipotassium edetate, disodium edetate, edetate calcium diso Sample CFU/g dium, edetic acid, fumaric acid, malic acid, maltol, Sodium edetate, and trisodium edetate. Hot melt granulate of BB12 1.42 x 10 exp 13 Na-alginate coated granulate of BB12 2.19 x 10 exp 13 4. The composition of claim 1, wherein said core com CMC coated granulate of BB12 5.42 x 10 exp 12 position further comprises at least one Sugar compound, 1.29 x 10 exp 13 wherein said at least one Sugar compound comprises one or more of a , a , a , a 0150. As can be seen from these results, fully coated , a higher oligosaccharide, or a polysaccha particles provided the best protection to the bacteria, albeit ride, or a combination thereof, wherein said monosaccharide with some variability in the results. comprises one or more of ketotriose (dihydroxyacetone), 0151. It is appreciated that certain features of the inven aldotriose (glyceraldehyde), ketotetrose (erythrulose), eryth tion, which are, for clarity, described in the context of rose, threose, ribulose, Xylulose, ribose, arabinose, Xylose, separate embodiments, may also be provided in combination lyxose, deoxyribose, psicose, fructose, Sorbose, tagatose, in a single embodiment. Conversely, various features of the allose, altrose, glucose, mannose, gulose, idose, galactose, invention, which are, for brevity, described in the context of talose, fucose, fuculose, rhamnose, Sedoheptulose, or a single embodiment, may also be provided separately or in neuraminic acid, or a combination thereof, wherein said any Suitable Subcombination. disaccharide comprises one or more of Sucrose, lactose, 0152 Although the invention has been described in con maltose, trehalose, turanose, or cellobiose, or a combination junction with specific embodiments thereof, it is evident that thereof, wherein said trisaccharide comprises one or more of many alternatives, modifications and variations will be raffinose, melezitose or maltotriose, or a combination apparent to those skilled in the art. Accordingly, it is thereof, wherein said tetrasaccharide comprises one or more intended to embrace all such alternatives, modifications and of acarbose or stachyose, or a combination thereof, wherein variations that fall within the spirit and broad scope of the said higher oligosaccharide comprises one or more of fruc appended claims. All publications, patents and patent appli tooligosaccharide (FOS), galactooligosaccharides (GOS) or cations mentioned in this specification are herein incorpo mannan-oligosaccharides (MOS), or a combination thereof. rated in their entirety by reference into the specification, to wherein said polysaccharide comprises one or more of the same extent as if each individual publication, patent or patent application was specifically and individually indi glucose-based polysaccharides/glucan cellulose, dextrin, cated to be incorporated herein by reference. In addition, dextran, beta-glucan, maltodextrin, fructose-based polysac citation or identification of any reference in this application charides/fructan, levan beta 2-6, mannose-based polysac shall not be construed as an admission that such reference is charides, galactose-based polysaccharides, N-acetylglu cosamine-based polysaccharides, or a gum, or a combination available as prior art to the present invention. thereof, or wherein said polysaccharide comprises one or What is claimed is: more of amylase, amylopectin, , lentinan, sizofiran, 1. A composition comprising probiotic bacteria, the com inulin, mannan, galactan, chitin, arabic gum, or gum acacia, position comprising: (a) a core composition containing the or a combination thereof. probiotic bacteria and a stabilizer, wherein the total amount 5. The composition of claim 1, wherein said core further of probiotics in the mixture is from about 10% to about 90% comprises one or more of a filler, a Surfactant or binder, or by weight of the core composition; (b) an innermost coating a combination thereof; wherein said filler comprises one or layer, layered on said core composition, comprising at least more of microcrystalline cellulose, a Sugar, dicalcium phos one hydrophobic solid fat, fatty acid or a wax having a phate: Sugar alcohols, hydrogenated Starch hydrolysates; melting point lower than 60° C., or a combination thereof; corn starch; and potato starch; and/or a mixture thereof. (c) an intermediate coating layer layered on said innermost wherein said binder comprises one or more of Povidone coating layer, which when present in an aqueous Solution in (PVP: polyvinyl pyrrolidone), Copovidone (copolymer of the amount of 0.1% weight/weight over the weight of the vinyl pyrrolidone and vinyl acetate), polyvinyl alcohol, low solution, has a surface tension lower than 60 mN/m, when molecular weight HPC (hydroxypropyl cellulose), low measured at 25°C.; and (d) an outer coating layer, layered molecular weight HPMC (hydroxypropyl methylcellulose), on said intermediate coating layer, wherein the composition low molecular weight hydroxymethyl cellulose (MC), low is in the form of particles. molecular weight sodium carboxy methyl cellulose, low 2. The composition of claim 1, wherein said stabilizer molecular weight hydroxyethylcellulose, low molecular comprises an oxygen Scavenger wherein said oxygen Scav weight hydroxymethylcellulose, cellulose acetate, gelatin, enger comprises one or more of L-cysteine hydrochloride, hydrolyzed gelatin, polyethylene oxide, acacia, dextrin, L-cysteine base, 4.4 (2.3 dimethyl tetramethylene dipyro starch, water Soluble polyacrylates and/or polymethacry catechol), tocopherol-rich extract (natural vitamin E), a-to lates, low molecular weight ethylcellulose, hydrophobic copherol (synthetic Vitamin E), P-tocopherol, y-tocopherol, Solid fat or fatty acid or a wax or a polyethylene glycol 8-tocopherol, butylhydroxinon, butyl hydroxyanisole having a melting point lower than 60° C. or a mixture (BHA), butyl hydroxytoluene (BHT), propyl gallate, octyl thereof; or wherein said surfactant comprises tween 80 gallate, dodecyl gallate, tertiary butylhydroquinone (polysorbate 80, Polyoxyethylene (20) sorbitan (TBHQ), fumaric acid, malic acid, ascorbic acid (Vitamin monooleate), tween 20 (polysorbate 20, Polyoxyethylene C), sodium ascorbate, calcium ascorbate, potassium ascor (20) sorbitan monolaurate), tween 85 (Polyoxyethylene sor bate, ascorbyl palmitate, or ascorbyl Stearate. bitan trioleate) glycereth-2-cocoate (Levenol R. C-421), US 2016/0360777A1 Dec. 15, 2016

glycereth-6-cocoate (LevenolR) F-200), glycereth-7-cocoate 13. The composition of claim 12, wherein said interme (LevenolR C-301), glycereth-17-cocoate (LevenolR C-201) diate coating layer comprises a polymer, comprising one or or a mixture thereof. more of Hyroxypropylmethylcellulose (HPMC), Hydroxy 6. The composition of claim 1, wherein said probiotic propylethylcellulose (HPEC), Hydroxypropylcellulose bacteria comprises one or more of Bacillus coagulans GBI (HPC), hydroxypropylethylcellulose (HPEC), hydroxym 30, 6086, Bacillus subtilis var natt, Bifidobacterium ethylpropylcellulose (HMPC), ethylhydroxyethylcellulose LAFTIR B94, Bifidobacterium sp. LAFTI B94, Bifidobac (EHEC) (Ethulose), hydroxyethylmethylcellulose (HEMC), terium bifidum, Bifidobacterium bifidum rosell-71, Bifido hydroxymethylethylcellulose (HMEC), propylhydroxyeth bacterium breve, Bifidobacterium breve Rosell-70, Bifido ylcellulose (PHEC), methylhydroxyethylcellulose (MHEC), bacterium infantis, Bifidobacterium lactis, Bifidobacterium hydrophobically modified hydroxyethylcellulose (NEX longum, Bifidobacterium longum Rosell-I 75, Bifidobacte rium animalis, Bifidobacterium animalis Subsp. lactis TON), carboxymethylhydroxyethylcellulose (CMHEC), BB-12, Bifidobacterium animalis subsp. lactis HNO19, Bifi Methylcellulose, Ethylcellulose, water soluble vinyl acetate dobacterium infantis 35624, Escherichia coli M-17, copolymers, gums, polysaccharides such as alginic acid and Escherichia coli Nissie 1917, Lactobacillus acidophilus, alginates Such as ammonia alginate, sodium alginate, potas Lactobacillus acidophilus LAFTIR LI 0, Lactobacillus aci sium alginate, acid phthalate of , amylase dophilus LAFTI LI 0, Lactobacillus casei LAFTIR) L26, acetate phthalate, cellulose acetate phthalate (CAP), cellu Lactobacillus casei LAFTI L26, Lactobacillus brevis, Lac lose ester phthalates, cellulose ether phthalates, hydroxy tobacillus bulgaricus, Lactobacillus casei, Lactobacillus propylcellulose phthalate (HPCP), hydroxypropylethylcel gasseri, Lactobacillus paracasei, Lactobacillus plantarum, lulose phthalate (HPECP), hydroxyproplymethylcellulose Lactobacillus reuteri ATTC 55730 (Lactobacillus reuteri phthalate (HPMCP), hydroxyproply methylcellulose acetate SD2112), Lactobacillus rhamnosus, Lactobacillus sali succinate (HPMCAS), methylcellulose phthalate (MCP), varius, Lactobacillus delbrueckii, Lactobacillus fermentum, polyvinyl acetate phthalate (PVAcP), polyvinyl acetate Lactococcus lactis, Lactococcus lactis Subsp. Lactococcus hydrogen phthalate, sodium CAP, starch acid phthalate, lactis Rosell-I 058, Lactobacillus paracasei Stil 1 (or cellulose acetate trimellitate (CAT), styrene-maleic acid NCC2461 J Lactobacillus fortis Nestle, Lactobacillus john dibutyl phthalate copolymer, styrene-maleic acid/polyviny sonii Lal (Lactobacillus LC.J. Lactobacillus johnsonii lacetate phthalate copolymer, styrene and maleic acid copo NCC533) Nestle, Lactobacillus rhamnosus Rosell-11, Lac lymers, or polyacrylic acid derivatives or a combination tobacillus acidophilus Rosell-52, Streptococcus thermophi thereof. lus, Diacetylactis, Saccharomyces cerevisiae, and a mixture thereof. 14. The composition of claim 13, wherein said polymer 7. The composition of claim 1, wherein said innermost comprises one or more of shellac, phthalate derivatives, coating layer comprises at least one hydrophobic solid fat or CAT, HPMCAS, polyacrylic acid derivatives, copolymers fatty acid having a melting point lower than 50° C. comprising acrylic acid and at least one acrylic acid ester, 8. The composition of claim 7, wherein said melting point EudragitTM S (poly(methacrylic acid, methyl methacrylate) is higher than 25°C. 1:2); Eudragit L1OOTM (poly(methacrylic acid, methyl 9. The composition of claim 7, wherein said innermost methacrylate) 1:1); Eudragit L30DTM, (poly(methacrylic coating layer comprises fatty acids, fatty acid esters, fatty acid, ethyl acrylate) 1:1); and (Eudragit L100-55) (poly acid triesters; aluminum, Sodium, potassium and magnesium (methacrylic acid, ethyl acrylate) 1:1) (EudragitTM L is an salts of fatty acids; fatty alcohols, phospholipids, Solid fats, anionic polymer synthesized from methacrylic acid and waxes, and a combination thereof. methacrylic acid methyl ester), polymethyl methacrylate 10. The composition of claim 9, wherein the innermost blended with acrylic acid and acrylic ester copolymers, coating layer comprises a Solid fat comprising one or more alginic acid, ammonia alginate, Sodium, potassium, magne of lauric acid, hydrogenated coconut oil, cacao butter or a sium or calcium alginate, vinyl acetate copolymers, polyvi combination thereof. nyl acetate 30D (30% dispersion in water), a neutral meth 11. The composition of claim 1, wherein said innermost acrylic ester comprising poly(dimethylaminoethylacrylate) coating layer forms a stable hydrophobic matrix which (“Eudragit ETM), a copolymer of methylmethacrylate and embeds the core composition within and/or forms a film ethylacrylate with trimethylammonioethyl methacrylate around the probiotic core composition; and optionally chloride, a copolymer of methylmethacrylate and ethylacry wherein said core comprises a plurality of cores and said late, Zein, shellac, gums, or polysaccharides, or a combina plurality of cores is embedded in said stable hydrophobic tion thereof. matrix. 12. The composition of claim 1, wherein said intermediate 15. The composition of claim 14, wherein said outer coating layer, when present in an aqueous solution in the coating layer comprises a polymer having an oxygen trans amount of 0.1% weight/weight over the weight of the mission rate of less than 1000 cc/m/24 hr, measured at solution, has a surface tension lower than 50 mN/m when standard test conditions. measured at 25° C. k k k k k