Patent Application: US-51986007-A

Abstract:
the disclosure relates to a probiotic delivery system that can be consumed as a snack - food or added to a food product . in particular , the disclosure describes a crisp and tasty treat that comprises viable probiotic microorganisms preserved in a vacuum dried matrix of sugars , proteins , and polysaccharides . the probiotic remain viable within the treat for a longer time without the need for additional moisture barrier coating . the probiotic also remain viable in the animal gastrointestinal tract .

Description:
the present disclosure relates to a food or feed product comprising viable microorganisms , a mixture that protects probiotics against high temperature , humidity and low ph , the use of the probiotic substance in a food product and a process for obtaining a probiotic substance to supplement food or feed products . the disclosure further relates to a food product comprising the probiotic substance . the subject matter described herein relates generally to a composition for preserving probiotic microorganisms , and to the production and drying methods of the substance . more specifically , the subject matter described herein includes a dry probiotic substance with long - term shelf life under high temperature and humid conditions . as used herein , each of the following terms has the meaning associated with it in this section . the term “ food product ” is intended to encompass any consumable matter of either plant or animal origin or of synthetic sources that contain a body of nutrients such as a carbohydrate , protein , fat vitamin , mineral , etc . the product is intended for the consumption by humans or by animals , such as domesticated animals , for example cattle , horses , pigs , sheep , goats , and the like . pets such as dogs , cats , rabbits , guinea pigs , mice , rats , birds ( for example chickens or parrots ), reptiles and fish ( for example salmon , tilapia or goldfish ) and crustaceans ( for example shrimp ). preferably , the subject matter described herein includes standard food products pelleted feeds , and pet food ( for example a snack bar , crunchy treat , cereal bar , snack , biscuit , pet chew , pet food , and pelleted or flaked feed for aquatic animals ). the word “ probiotic ” is intended to refer to any consumable microorganism owing to any beneficial effect it may have on its consumer . the term “ probiotic substance ” is a dry consumable substance in any shape or form that contains probiotics . more specifically , a probiotic substance comprises live probiotics embedded in a matrix of sugars , proteins and polysaccharides . hence , it may be a food product on its own . the term “ micro - matrix ” particles may assume any dry powder form of the probiotic substance . the micro - matrix particles may serve as a carrier for the probiotics and comprise a size from 10 micron up to 2000 micron . the term “ flake or treat ” is not intended to refer to specific form or shape of the probiotic substance . a flake or treat may assume any form obtained by molding shaping or slicing a hydrogel . for example , a flake or treat may have the form of a sphere , cube , pyramid , tablet , cereal or any complex three - dimensional form that comprise a size of at least 2 millimeters . for example , if the treats are used as a probiotic delivery system for pet - food , they may have the form of bones , rods , rings or other kibble forms . the word “ hydrogel ” may refer to any moist food - grade substance that has the property of a solid or viscous gel . a hydrogel , either anionic or cationic , can be formed by one or more hydrophilic polymers , polysaccharides , gums , resins , or hydrolyzed proteins , either alone or in combination , in which the microorganisms are disposed . preferably , the hydrogel compounds include agarose , alginate , chitosan or any other compound , which preferably can present characteristics of a solid gel . in order to prepare the probiotic substance as described herein , a single or a mixture of several micro - organisms may be selected . as a probiotic micro - organism , any micro - organism may be selected . preferably , a micro - organism exerting beneficial effects on health and welfare on humans or animals is used . examples of suitable probiotic micro - organisms include yeasts such as saccharomyces cereviseae , molds such as aspergillus , rhizopus , mucor , and penicillium , bacteria such as the genera bifidobacterium , clostridium , bacillus and lactobacillus . specific examples of suitable probiotic micro - organisms are : aspergillus niger , a . oryzae , bacillus coagulans , b . lentus , b , licheniformis , b , mesentericus , b . pumilus , b , subtilis , b . natto , bifidobacterium adolescentis , b . animalis , b . breve , b . bifidum , b . infantis , b , lactis , b . longum , b . pseudolongum , b . thermophilum , candida pintolepesii , clostridium butyricum , enterococcus cremoris , e . diacetylactis , efaecium , e . intermedins , e . lactis , e . muntdi , e . thermophilus , lactobacillus acidophilus , l . alimentarius , l . amylovorus , l . crispatus , l . brevis , l , case l , curvatus , l . cellobiosus , l . delbrueckii ss , bulgaricus , lfarciminis , l . fermentum , l . gasseri , l . helveticus , l . lactis , l . plantarum , l . johnsonii , l . reuteri , l . rhamnosus , l . sakei , and l . salivarius . the probiotic micro - organisms are preferably mixed in a concentrated wet paste form or frozen paste form ( for example a probiotic paste of & gt ; 10 % solids ) with the other protective substances . microorganisms may also be mixed , directly after fermentation , with the protective components described herein followed by hydrogel formation and a drying process thereafter . for example , probiotic micro - organisms are mixed with the protective materials such as a saccharide , for example trehalose , sucrose , lactose or maltodextrin , a protein , for example egg albumen , soy protein isolate or hydrolysate either alone or in combination and a polysaccharide , for example , agarose , alginate or chitosan either alone or in combination . a hydrogel is then formed in a desired shape and size or sliced after hardening the gel according to established procedures known to persons skilled in the art . if micro - matrix particles are required , then the hydrogel can be sliced or extruded and then dried using a variety of drying techniques , for example fluidized bed drying , freeze drying , air drying , convention oven drying or another adequate drying process . the dry probiotic substance is then ground and sieved to preferred sizes . if flakes or treats are required , then the molded or otherwise pre - shaped or sliced hydrogel is preferably dried in a vacuum drier or freeze drier at a temperature above the freezing point of the hydrogel . the pre - shaped dried flake or treat is then ready for packaging alone or in combination with other food products . preferably , the probiotic substance comprises significant amounts of the protective composition , in which the micro - organisms are embedded . preferably , the probiotic substance comprises , in percent by weight of total dry matter , 1 - 50 %, preferably 5 - 25 %, more preferably 10 - 20 % of probiotic microorganisms in the protective composition . in one embodiment , the probiotic substance described herein comprises 10 6 to 10 12 viable micro - organisms ( cfu ) per gram dry weight . preferably , it comprises 10 7 to 10 11 cfu per gram dry weight . in another embodiment , the dried probiotic substance is characterized by a water activity below 0 . 2 . preferably , the water activity is below 0 . 1 , for example , the water activity is in the range of 0 . 01 to 0 . 09 . the probiotic substance in accordance with a preferred embodiment comprises of sugar , proteins and polysaccharides . wherein the protein is selected from natural proteins including albumen , arginine / lysine polypeptide , collagen and hydrolyzed collagen , gelatin and hydrolyzed gelatin , glycoproteins , milk protein , casein , whey protein , serum albumin , meat , fish , seafood , poultry , egg proteins , silk , soybean , corn , peanut , cottonseed , sunflower , pea , wheat protein , wheat germ protein , gluten - protein , zein and any isolate or hydrolyzed of any vegetable protein , and the like . preferably , the polysaccharide components of the probiotic substance may be selected in a way that a formation of a solid gel is possible . generally , this may be achieved by cross linking the polysaccharide ( for example by mixing divalent cations with alginate or by cooling the gel ( for example agarose ). additional functional ingredients may be selected to provide further benefits to the probiotic substance described herein . for example fructo - oligosaccharides ( fos ) and polyfructoses , for example , inulin , pectin , 6 - glucans , resistant starches , for example high amylose starch , dextrans , acacia gum , guar and locust bean gum , agar , carrageenans , xanthan and maltodextrins , and mixtures thereof . additional functional ingredients may also comprise trace elements , minerals , vitamins , antioxidants , sterols , antioxidants and / or other functional molecules . however , the effect of any additional components on the protective characters of the probiotic substance should be evaluated first . examples of vitamins and / or antioxidants are carotenoids , such as lycopene , beta - carotene , lutein , xanthophylls , vitamin a , tocopherols , vitamin c , and mixtures thereof . since one of the objectives of the compositions and processes described herein is to add the probiotic substance to a food product , it is an advantage that the probiotic substance may be shaped like the desired food product . for example , if the probiotic substance is to be added to a pet food , the probiotic substance may be shaped like a pellet , kibble or bone . accordingly , if the probiotic substance is added to breakfast - cereals , it may be shaped like cereals . or , the probiotic substance may be added to a snack as chips . additionally , the probiotic substance may be added with flavors that used to prepare the food product . in one embodiment , the probiotic substance may be coaled with a moisture barrier component . in principle , any food - grade substance having water repelling or impermeable properties may be selected . suitable moisture barriers may be , for example , waxes ( paraffin wax , beeswax , carnauba wax , candellila wax , microcrystalline wax , rice bran wax , shellac , lanolin , hydrogenated castor oil , jojoba oil ), fatty acids ( for example , oleic acid , stearic acid , palmitic acid , lauric acid ), monoglycerides , diglycerides and triglycerides ( for example , mct oil , triglycerides based on coconut / palm kernel oil ), vegetable oils and fats ( for example , rapeseed , sesame , cornseed , nut , cottonseed , peanut , sunflower , linseed , olive , soy bean , cocoa butter ), hydrogenated or hardened vegetable oils and fats , oils and fats of animal origin ( for example , beef , poultry , pork , for example , beef tallow , lard and fish oil ), hydrogenated or hardened oils and fats of animal origin , dairy fats ( for example , milkfat , butterfat ), proteins ( for example , gluten , zein , sodium and calcium casemate ), phospholipids ( for example , lecithin ), carbohydrates , for example , cellulose and cellulose derivatives ( for example , hydroxypropyl methylcellulose , ethylcellulose , methylcellulose , carboxymethyl cellulose ), carrageenans , sorbitan esters ( for example , mono - oleate , - palmitate , - stearate , trioleate ) and mineral oils and fats ( for example , paraffin ). the preparation of the probiotic substance after selection of ( he probiotic microorganism and further components of the protecting matrix may be performed in any suitable way . a few principle steps of preparation of the probiotic substance may usually comprise the steps ; concentrating the probiotic yield from a fermentor to a solid content of at least 10 % and cfu counts of at least 5 × 10 cfu / g paste , wet mixing of the probiotic concentrate with the other protective components , hydrogel formation in a desirable shape , drying and packaging . if probiotic micro matrix particles are required then a grinding and sieving steps are added . if additional moisture barrier coating is required then the dry probiotic substance is coated immediately after the completion of the drying step . most of these steps , for example “ wet mixing ” and “ drying ”, may be subdivided , for example “ mixing only few of the ingredients , drying them by one drying method , adding other ingredients to the mixture , mixing , and drying again with the same or different drying method . for example , a wet mix of the micro - organisms and further components , is prepared by mixing all components in water , the slurry is then poured into molds of a desired shape and let to harden by adding a cross - linking chemical or by cooling the slurry , or hardening the gel slurry first and then slicing , chopping or shaping the hydrogel to a desired shape . then the molded chopped , or shaped hydrogel may be dried to water activity ( a w ) below 0 . 2 , preferably below 0 . 1 . possible drying processes include air dryers or convection ovens , belt dryers , vacuum dryers , fluidized bed dryers , rotary dryers , just to mention a few . alternatively , all components can be added to the wet probiotic concentrate without the addition of more water . for example , trehalose is first mixed in a probiotic concentrate ( that contains about 10 - 20 % solids ), and then egg albumen or soy protein isolate and polysaccharides are added . additional amount of proteins may be added to obtain semi - dry powder that can be further granulated , dried and sieved to specific size range of probiotic granulated - matrix particles . the preparation of probiotic substances in a shape of crispy flakes or treats involves a vacuum drying process where the product temperature is set above the freezing temperature of the probiotic substance . in general , vacuum drying are performed in two steps . the first step involves moderately reduced pressure ( ca . 5000 mtor ) and high shelf temperatures ( ca . 5 - 50 ° c . ), whereas the second step involves lower pressures ( e . g ., higher vacuum such as not more than 100 mtor ) while maintaining higher shelf temperature ( up to about 50 ° c .). this process can be achieved using a programmable control system for vacuum pressure and product temperature . the vacuum and temperature conditions for the first drying step is adjusted empirically according to the size of the drier , heat transfer capacity , and the product load , but the goal is to keep the product above its freezing temperature while maximizing the water evaporation rate . in one embodiment , the temperature is initially maintained at about 40 ° c . for about 6 hours , until most of the free water evaporated from the material , followed by gradually increasing the vacuum from the initial set up of ca . 5000 mtor up to 100 mtor , then maintaining these drying conditions until the water activity of the probiotic substance is sufficiently low ( see values given above ). following this protocol , the drying procedure is completed within 24 hours without substantially compromising the probiotic viability . the large surface area of the shaped , sliced or chopped hydrogel greatly increases evaporation rate without the need to “ boil ” or foam the product in thin layers as indicated by other disclosures , thus eliminating inconsistent drying conditions and splattering of the foamed product solution within the vacuum chamber . additionally , the disclosed composition and method of drying enable higher loading capacity of product in the vacuum or freeze drier as compared to other drying methods of materials with high sugar contents ( i . e ., foam formation ). the flake or treat may have the shapes as indicated above and be of any suitable , adequate or desired form . for example , they may have the form of spheres , cubes , pyramids , tablets , long tubes or any complex three - dimensional form . furthermore they may have a form that corresponds to the food product to which they are added . for example , if the treats are added to a pet food for dogs , they may have the form of bones , animals , cats or other forms that fit with the food product . in still another embodiment , the dried probiotic substance may be coated to further protect the micro - organism from deleterious effect of subsequent absorption of water during the shelf - life of the food product . the coating may be done by any suitable coating technique , for example , spraying , melt or solvent coating equipment , fluidized bed coater , drum coater or pan coater , just to mention a few . the amount of coating depends on the size and form of the probiotic substance . generally , the amount of coating compound is higher for smaller size particles . accordingly , the amount of the coating compound for probiotic micro - matrix or granulated matrix particles is from 10 to 50 %, more preferably 20 to 40 % of the total weight of the coated probiotic particles . the amount of the coating compound for flake or treat shapes probiotic substance is from 5 to 30 %, more preferably 10 to 20 % of the total weight of the coated probiotic treat . it is also understood that , for the purposes described herein , the coating process can result in a single layer of one compound or a mixture of compounds or to multiple layers of one or more compounds . in a further embodiment , the probiotic substance provides gastric protection to the probiotic microorganisms . while the sugar component in the protective matrix immediately dissolves upon intact with the animal stomach juice , the polysaccharide protein matrix retains its form in the acid environment , thereby protecting the embedded microorganisms from digestive incursions . the matrix , however , slowly disintegrate in the alkali - phosphate environment of the gut and hence liberating the intact probiotic microorganisms to colonize the animal gut . in additional embodiment , the probiotic product may be used exclusively as an entire food product , for example as a treat or a supplement , or added and mixed with a food product , or be used in its powder form to coat an existing food product , for example for top - coating pelleted or extruded feeds . the following examples are given by way of illustration only and in no way should be construed as limiting the subject matter of the present application . the subject matter of this disclosure is now described with reference to the following examples . these examples are provided for the purpose of illustration only , and the subject matter is not limited to these examples , but rather encompasses all variations which are evident as a result of the teaching provided herein . 300 g of trehalose ( cargill minneapolis , minn .) was added to 1000 ml water and allowed to completely dissolve . soy protein isolate ( 100 g , feam natural foods , mequon , wis .) and soy hydrolysate ( 20 g , sigma - aldrich , st . louis , mo .) were added under vigorous mixing using a standard household blender . sodium alginate ( 20 g ) was then mixed into the slurry and allowed to cool down to room temperature . lactobacillus paracasei ( 100 g frozen concentrate direct from fermentation harvest ) was then added to the slurry under vigorous mixing until a smooth and uniform thick gel was achieved . the composition of the hydrogel is provided in table 1 . five grams of calcium phosphate dibasic was mixed in the basic formula followed by 5 g of gluconolactone and the slurry was allowed to harden ( solid hydrogel ) at room temperature over the next 4 hours . the firm gel was sliced to thin and long leafs , through cheese grinder . the thin leafs were loaded on a tray ( 13 × 10 inch ) and placed in a freeze drier ( virtis advantage , virtis , gardiner , n . y .). the condenser was set to − 70 ° c . and shelf temperature was set to + 40 ° c . the vacuum was then initiated and controlled at about 5000 mtor with an external vacuum controller ( thyr - cont , electronic , gmbh ). the wet product temperature fell to and stabilized at about − 5 to 0 ° c . the chamber atmospheric pressure was then gradually decreased as the product temperature started to warm up ( measured by a pair of temperature sensors plugged in the wet material ), until rail vacuum pressure of 100 mtor was established . over this time period of increasing vacuum , the product temperature was carefully maintained between − 5 ° c . and + 5 ° c . twenty four hours after establishing full vacuum , the dried product was taken out of the freeze drier . the water activity ( a w ) of the probiotic substance after the drying protocol was 0 . 05 ( measured by hygropalm awl , rotonic huntington , n . y .). the above basic formulation slurry was poured into muffin plates having molds of small hearts or stars shapes and allowed to harden ( solid hydrogel ) at room temperature over the next 4 hours . the plates containing the molded hydrogel was placed in a freeze drier and allowed to dry as described above . the above basic formula was extruded or dripped into a 1000 ml bath ( held at 0 - 5 ° c .) containing 5 g cacl 2 and 300 g trehalose using a syringe equipped with 18 g needle . the cacl 2 bath was gently stirred while injecting the slurry . the matrix strings or drops were allowed to cross - link for 30 minutes and then harvested and blotted on paper towel . the strings or drops were first dried in a convection oven at 35 degree c . until water activity of the material reduced to 0 . 5 then they were transferred to a freeze drier for final drying of about 24 hours . the dry drops or strings ( aw = 0 . 06 ) then ground to fine powder using standard coffee grinder and sieved through 50 - 250 micron screens . alternatively , probiotic micromatrix particles can be obtained by grinding and sieving already dried probiotic flakes or treats as described above . 600 g of trehalose ( cargill minneapolis , minn .) was added to 1200 ml of concentrated paste of lactobacillus acidophilus ( 20 % solid concentrate direct from fermentation harvest ) and allowed to completely dissolve . soy protein isolate ( 200 g , fearn natural foods , mequon , wis .) and soy hydrolysate ( 20 g , sigma - aldrich , st . louis , mo .) were added under vigorous mixing using a standard household mixer . sodium alginate ( 40 g ) was then mixed into the slurry and allowed to cool down to room temperature . after a smooth , thick and uniform gel was achieved , 5 g of calcium carbonate was added . immediately after , 1000 g of egg white ( sigma - aldrich , st . louis , mo .) was slowly added under vigorous mixing until a semi - moist free flowing powder was obtained . the granulated probiotic powder was dried in a convection oven at 40 degree c . for 2 h followed by a vacuum drying for 24 h to obtain dry ( aw & lt ; 0 . 06 ) granulated matrix particles . the granulated particles can be sieved to specific size range through a series of 50 - 500 micron sieves . the composition of the probiotic granulated matrix particles is provided in table 2 . l . rhamnosus micromatrix particles were produced as described above . cfu counts of concentrated l . rhamnosus after harvesting from a fermentor and centrifuging were 5 χ 10 10 / g paste and solid content was 24 %. after mixing the probiotic with all protective components , forming hydrogel , chopping the hydrogel to small threads , drying in a freeze - drier for 24 h , grinding and sieving to particle size between 50 - 250 micron , the cfu counts were 9 . 4 χ 10 9 / g dry micromatrix . this represents a loss of 0 . 28 log of probiotic activity during the manufacturing process ( as illustrated in fig1 ). stability of l . rhamnosus in 40 ° c . at 33 % relative humidity ( rh ). l . rhamnosus probiotic micromatrix particles were prepared and dried as described above . the dried probiotic particles were placed in temperature and humidity control incubators set at 40 ° c . and 33 % relative humidity for 4 weeks . viabilities of bacteria dried in only in 10 % trehalose solution or in the protective mixture described herein were measured on a weekly interval . fig2 shows that the protective mixture provided a significant protection to that of trehalose alone dried bacteria . stability of l . rhamnosus micromatrix particles in simulated gastric juices micromatrix particles ( 50 - 250 micron ) containing either l . rhamnosus ( lgg ), l . acidophilus ( la5 ) or l . paracase ( lpc ) were prepared and dried as described above . the micromatrix particles were then exposed for 2 hours to simulated gastric juice ( full stomach — 12 % non fat skim milk , 2 % glucose , 1 % yeast extract and 0 . 05 % cysteine ; ph 2 ; or empty stomach — 0 . 32 % pepsin , 0 . 2 % sodium chloride , ph 1 . 2 ). bacterial viabilities were recorded before and after the exposure to the simulated gastric juices . fig3 demonstrate a significant protection of the probiotic bacteria in the micromatrix substance in simulated gastric conditions . probiotic substances were coated with a fat - based moisture barrier ( a mixture of 20 % jojoba oil , 70 % cocoa butter and 10 % beeswax ) in a drum tumbler at a temperature of 40 degrees c . the moisture barrier was sprayed on using a spraying nozzle while the dry material is agitated in the drum tumbler to ensure homogeneous coating . the total amount of coating was about 20 % ( of the uncoated probiotic substances ) for probiotic flakes and treats , and 40 - 50 % for probiotic micromatrix particles . pet food for dogs that is commercially available was first dried in a convection oven to a water activity of 0 . 1 , and then coated with probiotic micromatrix particles in a drum tumbler . the pellets were first sprayed with about 5 % of fat - based moisture barrier ( a mixture of 40 % chicken fat , 40 % cocoa butter and 20 % beeswax ), then mixed with the micro matrix particles ( usually 0 . 1 - 0 . 5 % of total pet food that provides a dosage of 10 8 cfu / g ) and finally sprayed with additional coat of the fat - based moisture barrier . the total amount of coating was about 15 % ( of the pet food ). coating time was about 30 minutes . pelleted feed for fish was prepared with a mixture of several probiotics . probiotic micromatrix particles containing a mixture of l . rhamnosus , l . acidophilus and bifidobacterium lactis ( dsm 20215 ) were prepared as described in example 1 . fish feed that is commercially available was first dried in a convection oven to a water activity of 0 . 1 , and then coaled with probiotics micromatrix particles in a drum tumbler . the pellets were first sprayed with about 5 % of fat - based moisture barrier ( a mixture of 40 % fish oil , 40 % cocoa butter and 20 % beeswax ), then mixed with the micro matrix particles ( usually 0 . 1 - 0 . 5 % of total fish feed that provides a dosage of 10 7 cfu / g ) and finally sprayed with additional coat of the fat - based moisture barrier . the total amount of coating was about 10 % ( of the fish feed ). preparation of probiotic “ chips ” ( as shown in fig4 ) probiotic chips were prepared with a mixture of l . rhamnosus and l . acidophilus as described in example 1 . the basic formula was added with about 1 - 2 % of probiotic pastes instead of the standard 10 - 20 % load . five gram of calcium phosphate dibasic was mixed in the basic formula followed by 5 g of gluconolactone and the slurry was pored into long plastic tubes of 1 - inch diameter and allowed to harden ( solid hydrogel ) at room temperature over the next 4 hours . the firm “ hot - dog ” shaped gel was sliced to thin discs , loaded on a tray ( 13 × 10 inch ) and placed in a freeze drier and a drying procedure applied as described in example # 1 . the dried probiotic chips product was taken out of the freeze drier and packed under nitrogen in small aluminum foiled packs that provide tasty and crispy treat containing an effective dose of 10 8 cfu / per serving . fig4 shows an example of probiotic chips containing 10 8 cfu / serving of rhamnosus produced as described herein . breakfast cereal that is commercially available was first dried in a convection oven to a water activity of 0 . 1 . probiotic chips containing a standard 10 - 20 % load of probiotics were produced and further coated with a moisture barrier as described in examples 1 and 4 . the cereals were then mixed with 0 . 1 - 0 . 5 % probiotic chips ( to provide a dosage of 10 8 cfu / g ). alternative delivery forms of probiotic matrix substances in the lid of a container or a bottle of beverage drink in general , all forms of the probiotic substances produced as in example 1 or example 4 work similarly well , confirming the high versatility of usage and application of the materials . the probiotic substances produced as described herein can be provided in small bags or paper “ stick ” packaging or in any other manner along with an edible food product . the dry micromatrix particles or chips may also be provided in the lid of a yogurt container or a bottle of beverage drink to be mixed with the liquid food product for consumption . bronshtein , v . 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( us 2005 / 0153018 ). probiotic delivery system ( 2005 ). the disclosure of every patent , patent application , and publication cited herein is hereby incorporated herein by reference in its entirety . while this subject matter has been disclosed with reference to specific embodiments , it is apparent that other embodiments and variations can be devised by others skilled in the art without departing from the true spirit and scope of the subject matter described herein . the appended claims include all such embodiments and equivalent variations .