Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
  • A23P10/00—Shaping or working of foodstuffs characterised by the products
  • A23P10/20—Agglomerating; Granulating; Tabletting
  • A23P10/22—Agglomeration or granulation with pulverisation of solid particles, e.g. in a free-falling curtain
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
  • A23B2/00—Preservation of foods or foodstuffs, in general
  • A23B2/90—Preservation of foods or foodstuffs, in general by drying or kilning; Subsequent reconstitution
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
  • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
  • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/142—Amino acids; Derivatives thereof
  • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/158—Fatty acids; Fats; Products containing oils or fats
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/163—Sugars; Polysaccharides
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K40/00—Shaping or working-up of animal feeding-stuffs
  • A23K40/30—Shaping or working-up of animal feeding-stuffs by encapsulating; by coating
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
  • A23K50/42—Dry feed
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K50/00—Feeding-stuffs specially adapted for particular animals
  • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
  • A23P20/00—Coating of foodstuffs; Coatings therefor; Making laminated, multi-layered, stuffed or hollow foodstuffs
  • A23P20/10—Coating with edible coatings, e.g. with oils or fats
  • A23P20/11—Coating with compositions containing a majority of oils, fats, mono/diglycerides, fatty acids, mineral oils, waxes or paraffins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/10—Laxatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/12—Antidiarrhoeals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2300/00—Processes
  • A23V2300/10—Drying, dehydrating
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2400/00—Lactic or propionic acid bacteria
  • A23V2400/11—Lactobacillus
  • A23V2400/175—Rhamnosus
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2400/00—Lactic or propionic acid bacteria
  • A23V2400/51—Bifidobacterium
  • A23V2400/531—Lactis
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
  • Y02A40/81—Aquaculture, e.g. of fish
  • Y02A40/818—Alternative feeds for fish, e.g. in aquacultures

Definitions

• the disclosure relates generally to the fields of probiotics and food.
• the activity and long term stability of many biological materials may be affected by a number of environmental factors; for example, temperature, pH, the presence of water and oxygen or oxidizing or reducing agents.
• biological materials must be dried before or during mixing with other foodstuff ingredients. The drying process can often result in a significant loss in activity from mechanical, chemical, and osmotic stresses induced by the drying process. Loss of activity occurs at many distinct stages, including drying during initial manufacturing, feed preparation (high temperature and high pressure), transportation and long term storage (temperature and humid exposure), and after consumption and passage in the gastrointestinal (GI) track (exposure to low pH, proteolytic enzymes and bile salts).
• GI gastrointestinal
• Probiotic microorganisms are living microorganisms, which upon ingestion in certain numbers, exert health benefits beyond basic nutrition. The beneficial effects that probiotics may induce are numerous.
• probiotic microorganisms can be encapsulated in protective microenvironrnents.
• microencapsulation and enteric coating techniques involve applying a film forming substance, usually by spraying liquids containing sugars or proteins onto the dry probiotics (Ko and Ping WO 02/058735).
• coating the microencapsulated probiotics with moisture protecting layers is an expensive process, and generally several layers must be added, to avoid water entering the microcapsules. In addilion, it is extremely difficult to remove the added liquid in the coating substance without a corresponding decrease in shelf life.
• Various protective agents have been used in the art, with varying degrees of success. These include proteins, certain polymers, skim milk, glycerol, polysaccharides, oligosaccharides and disaccharides. Disaccharides, such as sucrose and trehalose, are particularly attractive cryoprotectants because they are actually help plants and microbial cells to remain in a state of suspended animation during periods of drought. Trehalose has been shown to be an effective protectant for a variety of biological materials, both in ambient air- drying and freeze-drying (Crowe et a!. 1998). However, there are some drawbacks associated with the use of sugars as the sole cryoprotectant.
• sugars for example, large amounts of sugars (often greater than 60% by weight) must be used to preserve the biological materials during the drying process. This is costly. More serious problems associated with the use of sugars include their readiness to form crystals when the material is dried below its freezing point, and the low glass transition temperature which causes instability of the preserved biological materials at high temperatures, and/or in humid environments. Further, high concentration of sugars reduces the solubility of other solutes in the system and at the same time renders the system extremely difficult to dry.
• the spray-dried bacteria are added to a composition comprising fats, fermented milk powder and saccharides. That composition is then used as the filling of a confectionary product.
• the subject matter described in thai document avoids the detrimental effects of water by embedding the probiotics in fat or oil rich matrix.
• fat based coating and preserving materials do not withstand long term exposure to humid conditions
• Giffard and Kendall disclose a foodstuff in the form of a dried or semi-moist ready-to-eat kibble or powder mix, which contains a combination of a probiotic, prebiotic and a coating of colostrum.
• the probiotic Prior to mixing in the food stuff, the probiotic is coated or encapsulated in a polysaccharide, fat, starch, protein or in a sugar matrix using standard encapsulation techniques. Similar to the above disclosure, the negative effects of water were avoided by embedding the probiotics in a matrix rich in fat or oil.
• Farber and Farber (WO 03/088755) describe an oral delivery system for functional ingredients uniformly dispersed in a matrix.
• the matrix components include a sugar, a carbohydrate, a hydrocolloid a polyhydric alcohol and a source of mono-or divalent cations.
• the delivery system is extruded or molded into a final shape with a moisture content of between 15% and 30% by weight.
• Porubcan discloses a formulation for protecting probiotic bacteria during passage through the stomach, whilst permitting their release in the intestine.
• the formulation has also a low water activity and correspondingly long shelf life.
• the capsule includes a water- free mixture of probiotic bacteria with monovalent alginate salts, and an enteric coating (e.g., gelatin or cellulose encapsulation). Upon contact with acidic environment, the outer shell of the capsule turned into a gel, which provides a protecting barrier against proton influx into the capsule core.
• McGrath and Mchale describe a method of delivering a microorganism to an animal.
• the micro-organism is suspended in a matrix of cross-linked alginate and cryopreservant (trehalose or lactose, or a combination of both).
• the matrix is then freeze or vacuum dried to form dry beads containing live probiotics with a shelf-life stability up to 6 months but only under refrigerated conditions.
• no description or direction was provided as to how probiotic bacteria are stabilized during manufacturing or for prolonged storage at room temperatures and high humidity conditions.
• compositions provide a mixture that can effectively protect the probiotic in both drying processes and long-term storage at elevated temperatures and varying degrees of humidity. Therefore, there is an urgent need for such a composition that can effectively protect the probiotic bacteria during manufacturing, long-term storage at elevated temperatures and humidity and during gastrointestinal passage. There is a need also for a drying process that is cost-effective and capable of entrapping and stabilizing probiotics in the protective mixture with minimal viability loss at the end of the entire operation. There is a need for a protective mixture that provides protection in the animal stomach while allowing the release of the probiotic along the intestinal tract. There is also a need for a protective mixture that contains only approved ingredients generally regarded as safe (GRAS), and is less costly than those presently being used.
• GRAS approved ingredients generally regarded as safe
• the subject matter described herein overcomes these needs and provides a composition and process for producing a composition that provides probiotic bacteria that are stable for long periods of time even at elevated temperatures and varying degrees of humidity. [0016] It is, in particular, a purpose of the present disclosure to describe viable probiotic cultures that are substantially stable at room temperature and high humidity conditions thereby obviate the need for refrigeration or storage under vacuum or oxygen free environment.
• the mixture comprises: (a) at least one sugar compound, where the total amount of sugar compound in the mixture is from about 10% to about 60% by weight of the mixture (b) proteins, where the total amount of proteins in the medium is from about 2% to about 20% by weight of the mixture and (c) polysaccharides, where the total amount of polysaccharides in the medium is from about 0.5% to about 5% by weight of the mixture.
• This aqueous protective mixture can be used in a multiplicity of preservation processes, including freezing, freeze-drying, spray-drying, vacuum-drying, or ambient air-drying, to provide a stable and preserved composition of probiotics.
• the probiotic substance is stable for extended periods of time at superambient temperatures and/or relative humidity.
• the aqueous protective mixture containing the probiotics can be molded into a desirable shape or form and vacuum- dried to produce a crisp and tasty probiotic treat that can be added to food stuff or consumed on its own by humans or animals. [0018] Therefore, the present disclosure also describes a method for preparing a preserved probiotic substances containing the above-noted protective mixture.
• the probiotic substance is provided in a dry form that is substantially free of water.
• the probiotic substance maybe freeze-dried, vacuum dried or air dried, or otherwise dried by methods known in the art.
• the probiotic substance preferably comprises a protective mixture capable of maintaining the viability of the probiotic micro-organisms for extended periods of time in ambient temperature and humidity conditions.
• the sugar in the protective mixture is a disaccharide, most preferably trehalose or sucrose or lactose or a combination thereof.
• the protective mixture preferably comprises trehalose at 20% w/v to 60% w/v trehalose, preferably 20%, 30% or 40%.
• the protein in the protective mixture of the subject matter described herein is egg albumen or soy protein isolate or hydrolysale and a mixture thereof.
• the protective mixture preferably comprises proteins at 2% w/v to 20% w/v proteins, preferably 5%, 10% or 20%.
• the polysaccharides in the protective mixture described herein can form a firm gel or viscous solution with the other ingredients in the mixture, most preferably a combination of alginates with different viscosities, agarose, pectin or chitosan.
• the protective mixture preferably comprises alginates at 0.5% w/v to 10% w/v alginates, preferably 1%, 2% or 4%.
• probiotic substance described herein for the manufacture of a probiotic product or a probiotic food or feed product for the consumption by humans, domestic animals, aquatic animals and pets.
• the probiotic substance may be coated with a moisture barrier component.
• 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, a mixture of oil based substances.
• the subject matter described herein includes a probiotic product comprising a dry micro-matrix particle of the above mixture, wherein the micro-matrix particle comprises viable microorganisms and has a size between 10 and 2000 microns.
• the subject matter described herein includes a food product containing probiotic flakes or treats, wherein the flakes or treats are the probiotic substance described herein, characterized in that the flake or treat has a desirable shape and size between 2-50 millimeters.
• the subject matter described herein includes a process for obtaining micro-matrix particles, to supplement a food product with viable micro-organisms.
• the process comprises the steps of mixing micro-organisms concentrate and further protective components, forming viscous solution or hydrogel, drying the mixture by freeze-drying, spray-drying, vacuum-drying, or ambient air-drying, and, if necessary, grinding the dry material to obtain micro -matrix particles comprising a size between 50-2000 microns.
• the subject matter described herein includes a process for obtaining flakes or treats as supplement or stand-alone food product with viable microorganisms.
• the process comprises the steps of mixing micro-organisms concentrate and further protective components, Forming a hydrogel in a desirable shape and size and drying by vacuum-drying, to obtain crispy and tasty flakes or treats comprising a size between 2-50 millimeters.
• One major advantage of the subject matter described herein is that it provides a significant improvement over other drying methods of sugar based substances and production methods of stable probiotic micro-organisms in semi-dry and/or humid particulate foodstuffs.
• Another advantage of the subject matter described herein is that the drying process is easy to upscale and straightforward with no need of additional coating or several drying stages.
• the probiotic substance provides gastric protection and a release mechanism of the probiotics along the intestinal tract at their site of action.
• the probiotic substance provides a suitable delivery vehicle for further functional ingredients, in particular non-digestible sugars, natural proteins and prebiotic fibers, which in turn may improve the physico-chemical characteristics of the probiotic substance, as described herein.
• Figure 1 is a table that shows drying loss after vacuum drying of L. rhamnosns probiotic in the MicroMatrix product described in Example 1.
• Figure 2 is a graph that illustrates storage stability (recovered cfu/g ⁇ colony- forming units per gram ⁇ versus storage time) of dry L. rhamnosus probiotic substance stored at 40° C and 33% relative humidity.
• Figure 3 is a bar chart that illustrates gastric stability (recovered cfu/g) of probiotic substances incubated in simulated gastric juice (pH-1.2) at 37° C for 2 hours.
• Figure 4 is an image of examples of probiotic "chips" produced as described herein.
• the disclosure relates to food products including a probiotic component.
• 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.
• 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).
• 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).
• 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.
• probiotic is intended to refer to any consumable microorganism owing to any beneficial effect it may have on its consumer.
• 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.
• 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.
• 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.
• 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.
• the hydrogel compounds include agarose, alginate, chitosan or any other compound, which preferably can present characteristics of a solid gel.
• a single or a mixture of several micro-organisms may be selected.
• any micro- organism may be selected.
• a micro-organism exerting beneficial effects on health and welfare on humans or animals is used.
• 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.
• 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.
• 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.
• the probiotic micro-organisms are preferably mixed in a concentrated wet paste form or frozen paste form (for example a probiotic paste of >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.
• probiotic micro-organisms are mixed with the protective materials such as a saccharide, for example trehalose, sucrose, lactose or maitodextrin, a protein, for example egg albumen, soy protein isolate or hydro lysate 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.
• 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.
• 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.
• 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.
• the probiotic substance comprises significant amounts of the protective composition, in which the micro-organisms are embedded.
• 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.
• the probiotic substance described herein comprises 10" to 10 viable micro-organisms (cfu) per gram dry weight. Preferably, it comprises 10 to 10 cm per gram dry weight.
• the dried probiotic substance is characterized by a water activity below 0.2.
• 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.
• 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.
• 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.
• fructo-oligosaccharides (FOS) and polyfructoses for example, inulin, pectin, ⁇ -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.
• vitamins and/or antioxidants are carotenoids, such as lycopene, beta-carotene, lutein, xanthophylls, vitamin A, tocopherols, vitamin C, and mixtures thereof.
• the probiotic substance may be shaped like the desired food product.
• the probiotic substance may be shaped like a pellet, kibble or bone.
• the probiotic substance may be added to breakfast-cereals, it may be shaped like cereals.
• the probiotic substance may be added to a snack as chips.
• the probiotic substance may be added with flavors that used to prepare the food product, [0060]
• the probiotic substance may be coaled with a moisture barrier component.
• 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
• 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 x 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.
• 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.
• 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, fiuidized bed dryers, rotary dryers, just to mention a few.
• all components can be added to the wet probiotic concentrate without the addition of more water.
• 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.
• vacuum drying are performed in two steps. The first step involves moderately reduced pressure (ca. 5000 mTOR) and high shelf temperatures (ca. 5- 5O 0 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 0 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.
• the temperature is initially maintained at about 40 0 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. [0067] 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.
• 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.
• 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.
• Example 1 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. [0074] Example 1
• trehalose Cargill Minneapolis, MN
• Soy protein isolate 10Og, Feam Natural Foods, Mequon, WI
• soy hydrolysate 20 g, Sigma- Aldrich, St. Louis, MO
• 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.
• probiotic micromatrix particles can be obtained by grinding and sieving already dried probiotic flakes or treats as described above.
• 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 ⁇ 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 ⁇ 10 y /g dry micromatrix. This represents a loss of 0.28 log of probiotic activity during the manufacturing process (as illustrated in Figure 1).
• 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 0 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. Figure 2 shows that the protective mixture provided a significant protection to that of trehalose alone dried bacteria.
• 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 probiolic micromatrix particles in a drum tumbler.
• the pellets were first sprayed with about 5% of fat-based moisture barrier (a mixture of 40% chicken fal, 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 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 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).
• 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 glucono lactone 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 x 10 inch) and placed in a freeze drier and a drying procedure applied as described in Example#l. 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
• Figure 4 shows an example of probiotic chips containing 10
• 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.

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