CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/530,605 filed on July 10, 2017, which is herein incorporated by reference in its entirety.

BACKGROUND OF DISCLOSE

[0002] Active components such as probiotics are microorganisms considered "generally recognized as safe" (GRAS) by the Food and Drug Administration. Probiotics can be consumed to promote various areas of health including, but not limited to, gastrointestinal health, immune health, nervous system health, and reproductive health. More specifically, probiotics have shown to be effective in: 1) alleviating intestinal disorders caused by constipation, diarrhea, antibiotics, chemotherapy, and food intolerance 2) enhancing innate immunity by modulating inflammation 3) actuating anti-tumoral and anti-cancer effects by inactivating or inhibiting carcinogenic compounds 4) producing neurotransmitters and activating neuropathways 5) maintaining epithelial and mucosal integrity and/or 6) decreasing the occurrence of vaginosis/vaginitis. Health benefits have mainly been demonstrated for specific probiotic strains of the following genera: Lactobacillus, Bifidobacterium, Saccharomyces, Enterococcus, Streptococcus,

Pediococcus, Leuconostoc, Bacillus, Escherichia coli.

[0003] Due to their perceived benefits, probiotics have increasingly been sold as a standalone product or included in supplements and food products. To confer health benefits, probiotics should be viable in large numbers after transit through the upper digestive tract and be able to multiply to exert their effects in the small intestine and colon. Commercial probiotics typically range from 10 ⁶ colony forming units (CFUs) to 10 ¹² CFUs of viable bacteria per serving.

Commercial probiotics are typically packaged in capsules, tablets, and powder form. Probiotic capsules and tablets are suitable for adolescents and adults. Probiotic powders are more appropriate for young children, seniors, and adults who have difficulty swallowing pills. SUMMARY OF THE DISCLOSURE

[0004] The present invention comprises of a mixture of biopolymers, prebiotics, sugars, plant based proteins, minerals, algae (Chlorella) powder, cyanobacteria (Spirulina) powder, and nanoparticles to create a packaging system for probiotics in the micron scale. The mixture can provide probiotic microencapsulation. The packaging can have many uses such as, but not limited to: increase active component survivability during freeze drying, protect the active components from the stomach acid and release the active components in the small intestine or colon, provide nourishment for the probiotics inside the microsphere, and protect the active components from antibiotics or bacteriocins. The average size of the microencapsulated probiotic can be between 1 micron and 200 microns (μιη) in diameter. The probiotic bacteria can be from the Lactobacillus genus or any probiotic genus. The microspheres can be manufactured on a commercial scale in a continuous closed system by way of in-situ cross-linking using a spray dryer. The powder microspheres can be stored in a food grade dissolvable capsule or pouch for easy incorporation in beverages.

[0005] According to at least one aspect of the disclosure, a drug delivery device includes a first dissolvable film and a second dissolvable film. The second dissolvable film is coupled with the first dissolvable film to form a volume between the first dissolvable film and the second dissolvable film. The volume can store probiotic microspheres and a polymer matrix

encapsulating the probiotic microspheres to protect the probiotic microspheres from at least one of an antibiotic and a stomach acid. The polymer matrix can include a sodium alginate gel and calcium carbonate.

[0006] In some implementations, the first dissolvable film and the second dissolvable film can include at least one of gellan gum or hydroxypropylmethyl cellulose. The first dissolvable film and the second dissolvable film can include nanocellulose fibers. The sodium alginate gel can be crosslinked with at least one of a Ca2+, Mg2+, or Zn2+.

[0007] In some implementations, the polymer matrix can be configured to release the probiotic microspheres within a small intestine of a patient. The polymer matrix can be configured to release the probiotic microspheres when exposed to a fluid with a pH above 7.5. The polymer matrix can include low viscosity sodium alginate. The polymer matrix can include 10% (w/v) maltodextrin. The device can include at least one of 10% (w/v) galactooligosaccharide/beta- glucan, 2% (w/v) low viscosity sodium alginate, 2% (v/v) probiotic slurry, or 1% (w/v) calcium carbonate.

[0008] In some implementations, the first dissolvable film and the second dissolvable film can include at least one of protein, casein, gelatin, soy protein, vegetable protein, zein, starch, shellac polyvinyl alcohol, cellulose, cellulose derivatives, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, alginate, agar, gellan gum, arabic gum, xanthan gum, guar gum, acacia gum, chitosan, pectin, or biopolymer hydrocolloid.

[0009] In some implementations, the first dissolvable film and the second dissolvable film can include at least one of a fruit extract, a vegetable extract, a puree, or a pomace. In some implementations, the first dissolvable film and the second dissolvable film can include a plasticizer, wherein the plasticizer is one of polyol, glycerol, sorbitol, sucrose, or corn syrup.

[0010] According to at least one aspect of the disclosure, a composition can include probiotic microspheres and a polymer matrix encapsulating the probiotic microspheres to protect the probiotic microspheres from at least one of an antibiotic and a stomach acid. The polymer matrix can include a sodium alginate gel and calcium carbonate.

[0011] In some implementations, the sodium alginate gel is crosslinked with at least one of a Ca2+, Mg2+, or Zn2+. The polymer matrix can be configured to release the probiotic microspheres within a small intestine of a patient. The polymer matrix can be configured to release the probiotic microspheres when exposed to a fluid with a pH above 7.5. The

composition can include low viscosity sodium alginate. The composition can include 10% (w/v) maltodextrin. The composition can include 10% (w/v) galactooligosaccharide/beta-glucan, 2% (w/v) low viscosity sodium alginate, 2% (v/v) probiotic slurry, or 1% (w/v) calcium carbonate.

[0012] In some implementations, the polymer matrix can be configured to protect the probiotic microspheres in a freeze drying process. The polymer matrix can include at least one of pectin, cellulose, or a hydrocolloid polysaccharide. In some implementations, the polymer matrix can include at least one trehalose, maltodextrin, dextrose, or beta glucan. In some implementations, the composition can include a galacto-oligosaccharide or a fructo-oligosaccharide. In some implementations, the composition can include a protein derived from vegetables, peas, pumpkin, sacha inchi, quinoa, chlorella, or spirulina. The composition can include a bacteria from a geniuses Lactobacillus, Lactococcus, Bifidobacterium, Saccharomyces, Enterococcus,

Streptococcus, Pediococcus, Leuconostoc, Bacillus, or Escherichia coli. In some

implementations, the probiotic microspheres have a diameter between 50 microns and 200 microns.

BRIEF DESCRIPTIONS OF THE DRAWINGS:

[0013] The skilled artisan will understand that the figures, described herein, are for illustration purposes only. It is to be understood that in some instances various aspects of the described implementations may be shown exaggerated or enlarged to facilitate an understanding of the described implementations. In the drawings, like reference characters generally refer to like features, functionally similar and/or structurally similar elements throughout the various drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the teachings. The drawings are not intended to limit the scope of the present teachings in any way. The system and method may be better understood from the following illustrative description with reference to the following drawings in which:

[0014] FIG. 1 depicts an optical light microscopic image of wet probiotic microspheres in method 1 made of 2% low viscosity sodium alginate, 10% maltodextrin, 10% ProBiota™ beta- glucan/galactooligosaccharide, 1% calcium carbonate, and 2% probiotic slurry, according to one implementation of the disclosure.

[0015] FIG. 2 depicts an optical light microscopic image of wet probiotic microspheres in method 2 made of 2% low viscosity sodium alginate, 10% maltodextrin, 10% ProBiota™ beta- glucan/galactooligosaccharide, 1% nano calcium carbonate, and 2% probiotic slurry, according to one implementation of the disclosure.

[0016] FIG. 3 depicts an optical light microscopic image of wet probiotic microspheres in method 3 made of 2% low viscosity sodium alginate, 10% maltodextrin, 10% ProBiota™ beta- glucan/galactooligosaccharide, 1% calcium carbonate, 1% VegiDay™ plant-based protein powder (organic pea protein, organic pumpkin protein, organic sacha inchi protein, organic chia seed, organic spirulina, organic chlorella, organic quinoa powder), and 2% probiotic slurry, according to one implementation of the disclosure.

[0017] FIG. 4 depicts an optical light microscopic image of wet probiotic microspheres in method 4 made of 2% low viscosity sodium alginate, 10% maltodextrin, 10% ProBiota™ beta- glucan/galactooligosaccharide, 1% nano calcium carbonate, 1% VegiDay™ plant-based protein powder (organic pea protein, organic pumpkin protein, organic sacha inchi protein, organic chia seed, organic spirulina, organic chlorella, organic quinoa powder), and 2% probiotic slurry, according to one implementation of the disclosure.

[0018] FIG. 5 depicts a graph of the viable cell count (Logio CFU/mL) of non-encapsulated Lactobacillus rhamnosus GG cells versus microencapsulated Lactobacillus rhamnosus GG in methods 1 and 3 before and after freeze drying, according to one implementation of the disclosure.

[0019] FIG. 6 depicts a graph of the percent reduction of viable cell count (CFU/mL) of non- encapsulated Lactobacillus rhamnosus GG cells versus microencapsulated Lactobacillus rhamnosus GG in methods 1 and 3 after freeze drying, according to one implementation of the disclosure.

[0020] FIG. 7 depicts a graph of the viable cell count (Logio CFU/mL) of non-encapsulated Lactobacillus rhamnosus GG cells versus microencapsulated Lactobacillus rhamnosus GG in methods 1, 2, 3, and 4 following incubation in simulated gastric fluid and pepsin for 2 hours, according to one implementation of the disclosure.

[0021] FIG. 8 depicts a graph of the percent reduction in cell count (CFU/mL) of non- encapsulated Lactobacillus rhamnosus GG cells versus microencapsulated Lactobacillus rhamnosus GG in methods 1, 2, 3, and 4 following incubation in simulated gastric buffer for 2 hours, according to one implementation of the disclosure. [0022] FIG. 9 depicts a graph of the viable cell count (Logio in CFU/mL) of non-encapsulated Lactobacillus rhamnosus GG cells versus microencapsulated Lactobacillus rhamnosus GG in methods 1 and 3 following incubation in simulated gastric fluid with pepsin and ampicillin antibiotics for 1 and 2 hours, according to one implementation of the disclosure.

[0023] FIG. 10 depicts a graph of the viable cell count (Logio CFU/mL) of non-encapsulated Lactobacillus rhamnosus GG cells versus microencapsulated Lactobacillus rhamnosus GG in method 1 following incubation in simulated gastric and small intestine buffers, according to one implementation of the disclosure.

[0024] FIG. 11 depicts a drawing of the continuous closed powder microsphere production system, according to one implementation of the disclosure.

[0025] FIG. 12 depicts a block diagram of an example method to form powdered, probiotic microspheres with the system illustrated in FIG. 11.

[0026] FIG. 13 depicts a drawing of the dissolvable capsule use for packaging powder microspheres, according to one implementation of the disclosure.

[0027] FIG. 14 depicts a drawing of the dissolvable pouch/pod use for packaging powder microspheres, according to one implementation of the disclosure.

DETAILED DESCRIPTION

[0028] The various concepts introduced above and discussed in greater detail below may be implemented in any of numerous ways, as the described concepts are not limited to any particular manner of implementation. Examples of specific implementations and applications are provided primarily for illustrative purposes.

[0029] Probiotics can be sensitive to various conditions, including but not limited to moisture, temperature, osmotic stress, pH and antibiotics. Packaging systems such as microencapsulation techniques used to encapsulate probiotics in protective matrixes, can be applied to enhance processing, storage, digestive stability, and targeted release of probiotics. Probiotics are commonly microencapsulated using emulsion and extrusion utilizing ionotropic gelation. In ionotropic gelation via emulsion and extrusion, the protective matrixes surrounding probiotics can be hydrocolloids such as alginate and pectin and crosslinked by divalent cations such as Ca ²⁺ , Zn ²⁺ Mg ²⁺ to form hydrogel microspheres. These microspheres are then further rinsed and dried via freeze drying (lyophilization) or fluid bed drying. These techniques are useful in creating protective matrixes surround probiotics.

[0030] During the course of production and consumption, probiotics can be inactivated in many ways. Probiotics can be converted into a powder form of minimal moisture content in order to be room temperature shelf stable for an extended period of time. One main technique of creating viable probiotic powder is freeze-drying or lyophilization. During the course of freezing the probiotic, ice crystals form and damage the cells and inactivate them. During consumption, the probiotics can be inactivated by the plethora of enzymes and extreme acidity of the stomach. Additionally, the small fraction of probiotics that make it past the gastric juices need

nourishment to survive and compete in the small intestine. The enzymes and bile acids in the small intestine also inactivate probiotics. Lastly, if a person or animal consumes antibiotics and probiotics at the same time or one-after-the-other, the antibiotics have detrimental effects on the survivability of probiotics and the host's microbiome.

[0031] To protect probiotics during freeze drying, cryoprotectants, such as sugars,

polysaccharides, proteins, and polymers can be used. To increase probiotic survival in the stomach, substances such as waxes, oils, and polymers have been used to encapsulate probiotics. Additionally, prebiotics and sugars can be added to provide nutrients for the probiotics while they are in the intestines. Additionally, probiotics can be protected from antibiotics by, for example, using enzymes that can break down antibiotics to protect the microflora from degradation but not necessarily applied to probiotics. Microspheres should be below 200 microns in diameter in order to be appropriate for the pediatric and senior populations for ease of swallow and no gritty mouth feel. The present solution can provide microencapsulation technology that is below 200 microns in size, can protect probiotics during freeze-drying, increase probiotic survival in the stomach, nourish probiotics with prebiotics within the microspheres, and increase probiotic survival in the presence of antibiotics in gastric conditions. Additionally, rather than providing wet probiotic microspheres, which are unsuitable for long term shelf storage over six months, the present solution can provide probiotic microspheres that are converted to powder form in order to maintain shelf storage over six months.

[0032] Active components such as probiotic powders can be stored in capsules, compressed tablets, sachets, stick packs, and bottles for the consumer. However, these product packaging may not suitable for senior citizens, children, and adults who have limited dexterity, motor control loss, and or difficulty swallowing pills. Dissolvable films manufactured into capsules, pouches, or pods, can allow for easy oral administration of powder probiotics or powder active components. The consumer will simply have to drop the dissolvable capsule, pouch, or pod into a beverage and consume the drink to obtain the daily dose of the active component or probiotic powder.

[0033] Consumers are increasingly buying products incorporated with probiotics believed to provide health benefits. With 269 million antibiotic prescriptions given in 2017 in the United States, a microencapsulation technology that can deliver active components such as probiotics past the stomach and to the small intestine and protect the active components from antibiotics would be desirable. A production system that can commercially scale microspheres into powder form would be also desirable in order to meet consumer demands. And a food grade dissolvable capsule or pouch that can package the microsphere powder for convenient incorporation in beverages for administration is also advantageous.

[0034] The present invention relates to microspheres suitable for administering a composition to humans and animals, the commercially scalable production method, and the end consumer product packaging. The microspheres can include compositions that can include a mixture made of probiotics, biopolymers, prebiotics, sugars, plant based proteins, minerals, algae (Chlorella) powder, cyanobacteria (Spirulina) powder, and/or nanoparticles to create a packaging system for probiotics in the micron scale (e.g., probiotic microencapsulation).

[0035] The present solution can provide a micro packaging system that has many uses for the human and animal. For example, the system can increase active component survivability during freeze drying, protect the active components from the stomach acid and release the active components in the small intestine or colon, provide nourishment for the probiotics inside the microsphere, and protect the active components from antibiotics or bacteriocins. The average size of the microencapsulated probiotic is between 1 micron and 200 microns (μιη) in diameter.

A. ACTIVE COMPONENTS:

[0036] The microspheres described herein can include active components. The active components can be nutraceutical, pharmaceutical, biologies, biopharmaceutical components, bacteria derived products, or any combination thereof. The active components can be probiotics. The active components can be referred to as drugs. The probiotics can be in a liquid or powder form of any of the following microbial genus: Lactobacillus, Lactococcus, Bifidobacterium, Saccharomyces, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Bacillus, and

Escherichia coli.

B. MICROENCAPSULATION PACKAGING SYSTEM COMPOSITION:

[0037] The microencapsulation packaging system can include biopolymers such as, but not limited to, alginate and minerals such as calcium carbonate. The sodium alginate and calcium carbonate can create a polymer matrix that can microencapsulate the probiotics into probiotic microspheres and protect the probiotics from the low pH of the stomach acid and the presence of antibiotics. Sodium alginate is an insoluble hydrocolloid, anionic biopolymer derived from natural sources such as algae. It can be biocompatible, low in toxicity, low in cost, and considered a food item and GRAS by the FDA. It is undigested by the human and animal host and can be digested by the microflora inside the intestinal tract. Sodium alginate can be crosslinked and gelled by divalent cations such as Ca ²⁺ , Mg ²⁺ , and Zn ²⁺ .

[0038] Sodium alginate gels can shrink in low pH conditions and swell and dissolve in higher pH conditions, making it a suitable gastric acid resistant polymer. Since alginate is an anionic polymer, in acidic conditions the carboxyl groups of alginate are not ionized at pHs lower than the pKa of alginate. Any active component release will be associated with diffusion based on the pore size of the alginate gels. In neutral and basic conditions, the anionic alginate polymer becomes ionized and swells. Any active component release will be dependent on the swelling of the beads or the erosion of the polymer matrix. The higher the concentration of alginate in the composition, the lower the release rate of active components. Since sodium alginate gels are porous and also dissociate in the presence of monovalent cations such as Na , the addition of calcium carbonate (CaC0 ₃ ) within the sodium alginate gel can increases the gel's strength. In acidic conditions, the Ca ²⁺ dissociates from the CaC0 ₃ . The Ca ²⁺ then crosslinks with the sodium alginate to further crosslink and strengthen the gel. Thus, the addition of CaC0 ₃ in the sodium alginate gel can further protect the probiotics from the stomach acid and antibiotics. In the small intestine, the pH starts to increase to a pH of 7.5. The high pH of the small intestine can cause the sodium alginate to swell, slowly erode, and release the probiotics throughout the small intestine as it transits through the gastrointestinal tract.

[0039] Sodium alginate polymers can be low molecular weight or high molecular weight. In some implementations, the lower the molecular weight of the polymer, the lower the viscosity of the polymer solution. In some implementations, higher molecular weight polymers can have increasing gel strength but the higher viscosity makes the polymer difficult to process. In highly viscous polymer solutions, high shear mixing must be used and can damage proteins or cells in the polymer solution. In some implementations, the low viscosity sodium alginate is employed in the microspheres of the present disclosure.

[0040] The addition of sugars such as maltodextrin, PROBIOTA™ prebiotics such as galacto- oligosaccharides (GOS), and yeast based beta-glucans, and VEGIDAY™ plant based protein powders within the microencapsulation packaging system can serve a number of functions. For example, one function is to provide increased stabilization and protection for probiotics during freeze-drying, thus increasing probiotic viability. Another function is to buffer the microspheres from the low pH and enzymes of the stomach acid. The other function is to provide nourishment for the probiotics so that they can multiply and adhere to intestinal epithelial cells as the microencapsulated probiotics transit through the small intestine.

[0041] Freeze drying, also known as lyophilization, is a process of removing water from materials for preservation or extending shelf life. Freeze drying works by freezing the material, then lowering the pressure, then removing ice by sublimation. When used to dry probiotic and other cells, large ice crystals may form during the freezing phase. The large crystals may break the cell walls and denature cell proteins. During the drying phase, a vacuum is used to sublime the ice, further stressing the sensitive cell membranes. To prevent this, the composition can include protective solutes and additives that can be added to the probiotics during the freeze drying process.

[0042] For example, protective solutes and additives can include maltodextrin, which is a polysaccharide extracted from botanical sources and industrially produced by enzymatic or acid hydrolysis of starch. Maltodextrin is considered GRAS by the FDA and is widely used in food, beverage, dietetic, medical food, and pharmaceutical table and powder products. Most maltodextrin are fully soluble in water and can act as a gelling or freeze control by depressing the freezing point of the solution. Moreover, maltodextrins can protect proteins and cells from denaturation. With an energy value of approximately 16 kJ/g (4 kcal/g), digestible maltodextrin can be considered a good source of energy.

[0043] In some implementations, the maltodextrin can be combined with other sugars and proteins to provide increased protection when compared to maltodextrin alone. In some implementations, proteins, such as, but not limited to whey protein, soy protein, skim milk, and bovine serum albumin can be combined with sugars such as maltodextrin and glucose during the freeze drying process. In some implementations, the protein can be VEGIDAY™, which is a plant based protein.

[0044] In some implementations, the composition can include VEGIDAY™. VEGIDAY™ is a plant based protein and is a mixture of organic proteins. For example, VEGIDAY™ can include organic pea protein, organic pumpkin protein, organic sacha inchi protein, organic chia seed, organic spirulina, organic chlorella, and organic quinoa powder. In some implementations, protein can buffer the stomach acid and increase probiotic survival in the gastrointestinal tract. Thus, the addition of VEGIDAY™ plant based protein (or other source of protein) may further protect the microencapsulated probiotics from the harsh conditions of the stomach. Pea protein can be an effective functional additive and a good substitute for soy protein because of its low cost, availability, and high nutritional value with essential vitamins and minerals. Additionally, pea protein products have been associated with health benefits such as reduction of cancers, HDL cholesterol, heart disease, and anti-inflammatory effect. Pumpkin, sacha inchi, chia seed, and quinoa protein powder all have high nutritional values ranging from minerals such as magnesium copper, and zinc to fat soluble antioxidants such as tocopherols and carotenoids and plant based omega 3 fats. In some implementations, the composition can include tuna-based omega 3 fats microencapsulated with probiotics, which can significantly increase the survival and intestinal wall adherence of probiotics in the gastrointestinal tract and increased oxidative stability during spray drying and freeze drying. In some implementations, the addition of pumpkin, sacha inchi, chia seed, and quinoa protein powders rich in vitamins and plant based omega 3 fats in composition can reduce oxidation during processing and also significantly increase probiotic survival and intestinal wall adherence in the animal host. The two algae based proteins, chlorella and spirulina are also nutritionally dense with benefits. Chlorella is a "true plant" algae and contains about 45% protein, 20% fat, 20% carbohydrates, 5% fiber, and 10% minerals and vitamins, and 20 amino acids. Spirulina contains between 55% and 77% protein, antioxidant enzymes, vitamins, and 22 amino acids. In some implementations, the compound can include chlorella and/or spirulina. With high protein content, antioxidative enzymes, and vitamins, both chlorella and spirulina may enhance probiotic survival during processing and provide nutrients for the probiotics to multiply and grow.

[0045] In some implementations, the compositions can include PROBIOTA™ prebiotics galacto-oligosaccharides (GOS) and yeast based beta-glucans to promote the growth of probiotics within the microencapsulation system and assist in probiotic adhesion to the intestinal cell wall. Prebiotics can be non-digestible food ingredients that exert beneficial health effects in the host by modulating and stimulating growth of microorganisms in the intestines. Prebiotics are generally not absorbed in the stomach, are resistant to stomach acids, bile salts and hydrolyzing enzymes in the intestine, therefore they have the capacity to buffer the microencapsulated probiotics from the harsh external environment. Moreover, prebiotics are fermentable by intestinal microflora into short chain fatty acids. GOS and beta-glucan can be growth substrates for probiotics such as Lactobacillus and Bifidobacterium. The composition can include

PROBIOTA™ prebiotics galacto-oligosaccharides (GOS) and yeast based beta-glucan to enhance the microencapsulation system on numerous fronts.

C. MICROENCAPSULATION PACKAGING SYSTEM SMALL SCALE PRODUCTION:

[0046] The microencapsulation packaging system of the present disclosure can form probiotic microspheres that can include probiotics, biopolymers, prebiotics, sugars, plant based proteins, minerals, algae (chlorella) powder, and cyanobacteria (spirulina) powder. The probiotic microspheres can be formed by anionotropic gelation technique via water-in-oil emulsion.

Emulsions can include two immiscible liquids, one of which is the dispersed phase and the other the continuous phase. The dispersed phase can be water-based, the continuous phase can be oil- based, and vice versa. The dispersed phase forms droplets within the continuous phase. In some implementations, the composition of the probiotic microspheres is the water-based dispersed phase that is emulsified in the continuous an oil-based phase. In the oil based continuous phase, the water based dispersed phase can form spherical droplets. The size of the droplets can depend on the degree of mixing. For example, the higher the degree of mixing, the smaller the size of the droplets. Emulsifiers may be added to the oil phase to increase stabilization of the water droplets so that the droplets can remain dispersed and the solution does not separate into two layers.

Emulsifiers can include a hydrophilic head and hydrophobic tail. Types of emulsifiers can include lecithins, mono and diglycerides, monoglyceride derivatives, and fatty acid derivatives. In some implementations, the emulsifier may be Tween 80, a nonionic surfactant commonly used in food. A divalent cationic solution is added to the water-in-oil emulsion to cross-link and gel the microspherical droplets. The resulting probiotic microspheres may be left in an aqueous hydrogel state or further processed into a powder form via freeze drying.

[0047] In some implementations, the microencapsulation packaging (e.g., the microspheres) can be manufactured by ionotropic gelation via extrusion into a cationic solution. In this technique, aqueous alginate solution is extruded through an orifice forming droplets. The size of the orifice determines the size of the droplets. The smaller the orifice, the smaller the droplets. The droplets can be added into a cationic divalent solution to form gelled microspheres. These microspheres can be then further rinsed and dried via freeze drying (lyophilization) or fluid bed drying.

[0048] In another technique the aqueous alginate composition can include calcium carbonate and the weak acid glucono-delta-lactone to create an in-situ release of cross-linking calcium ions. This technique can be called in-situ cross-linking. In this technique, the final composition is spray dried into a powder. The high heat from spray drying increases the dissolution of glucono- delta-lactone which increases the acidity of the composition. The increased acidity of the composition causes the Ca to dissociate from calcium carbonate and cross-link the alginate polymer. The powder microspheres from this spray drying technique may be further coated in a fluid bed dryer or spray dryer.

D. MICROENCAPSULATION PACKAGING SYSTEM SCALABLE PRODUCTION:

[0049] In some implementations, the microencapsulation techniques described herein can be used to manufacture small batches of microspheres and can useful in creating protective matrices around probiotics. In some implementations, the techniques described herein can be used for continuous, closed system method to generate large batches. In some implementations, small batches can only be produced in batches, require multiple steps, are in an open system and are difficult and costly to scale up for commercial production. A continuous closed system incorporating in-situ cross-linking and a spray dryer may circumvent the disadvantages of small batch techniques. In a closed system, the probiotic slurry, microencapsulation formulation, and weakly acidic solution are individually stored in separate holding tanks or bioreactors.

[0050] Separation of the materials can prevent premature gelation. Each of the holding tanks are connected via pipes. The solutions from the probiotic slurry, microencapsulation formulation, and weakly acidic solution can be mixed using in-line static/motorized mixers within the pipes. The final mixture can be pumped to a spray dryer to produce powdered probiotic microspheres. Probiotics, microencapsulation formulation, and weak acid can be continually added to the respective tanks to create a continuous process. Since the mixing of each components is done inside the pipes using static or motorized mixers, the system can be a closed system that can be made sterile and unexposed to the outside environment. In some implementations, the pipes can be kept cooled to prevent premature gelation.

[0051] In some implementations, the resulting microspheres (in their powder or other form) produced from any of the methods and techniques described herein may further be packaged in capsules, tablets, sachets, stick packs, bottles, and more preferably food grade dissolvable capsules/pouches/pods for easy oral administration.

E. DISSOLVABLE POWDER MICROSPHERE PRODUCT PACKAGING: [0052] The active components (e.g., the probiotics) can be stored in capsules, compressed tablets, sachets, stick packs, and bottles for the consumer. Senior citizens, children, and adults (and others) who have difficulty swallowing pills often time wish to incorporate the probiotic powder into beverages for easier oral administration. To incorporate and dissolve probiotics into water or beverages, consumers must either break the capsules in half and then add it to the liquid, vigorously shake the beverage with the compressed tablets in the liquid, individually open sachets or stick packs and pour the contents into the liquid or scoop out the contents from the bottle with a spoon and add it to the liquid. These methods of incorporating probiotics into beverages are easy for the average healthy adult. However, for individuals with motor-control loss or senior citizens and children who do not have the dexterity and strength to perform these tasks, incorporating probiotics powder into beverages can be a challenge.

[0053] In some implementations, the probiotics or microspheres can be incorporated into dissolvable films. The dissolvable films can be food grade dissolvable films that are thin, elastic sheets of polymer. The dissolvable films can be used in manufacturing ingestible capsules, pouches, and pods to package probiotic powders. The probiotic powders (via the dissolvable film) can then be added to liquids and beverages. Once in the beverage, the film packaging the probiotic powder quickly dissolves in the liquid and releases the probiotic powder into the beverage. The consumer can then drink the beverage incorporated with the probiotic powder for oral administration.

[0054] Food grade dissolvable films can be made from food grade proteins, polysaccharides, and polymers. For example, the dissolvable films can include whey protein, casein, gelatin, soy protein, vegetable protein, zein, starch, shellac polyvinyl alcohol, cellulose, cellulose derivatives, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, alginate, agar, gellan gum, arabic gum, xanthan gum, guar gum, acacia gum, chitosan, pectin, any biopolymer hydrocolloid, and any combination thereof. In some implementations, the dissolvable films can include fruits and vegetable extracts, purees, and pomaces.

[0055] In some implementations, the dissolvable films can include food grade plasticizers such as polyols, glycerols, sorbitol, sucrose, and corn syrup may improve film characteristics. The film characteristics that can be improved by the plasticizer can include reduced brittleness of the dissolvable film, improved microstructure, crystallinity, solubility in water, moisture absorption, water vapor permeability, optical, and mechanical properties. In some implementations, nano- sized particles and polymers such as nano-cellulose can be added to the dissolvable film to improve moisture barriers and mechanical strength.

[0056] The dissolvable films can be manufactured using solvent casting, semisolid casting, hot melt extrusion, rolling, or any combination thereof. In solvent casting, water soluble polymers and additives are dissolved in water. The solution can be casted into molds and allowed to dry. The film is peeled from the mold and used or stored for future use. In semisolid casting, water soluble polymers are dissolved in water to create a gel. The gel is then cast through ribbons using heat controlled drums to form a thin film. In hot melt extrusion, the polymer and additives are melted to the appropriate temperature and shaped into films using dies. In the rolling method the water soluble polymer solution is rolled on a carrier. The film is dried on the rollers and cut into the desired shapes.

F. PREPARATIONS OF MICROSPHERES:

[0057] Probiotic microspheres were prepared using the water-in-oil emulsion method. Four probiotic microsphere compositions were made according to the descriptions in Table 1 as follow:

Table 1

Materials Method 1 Method 2 Method 3 Method 4

Maltodextrin(g) 1 1 1 1

Probiota™ GOS/Beta- 1 1 1 1

glucan(g)

Low Viscosity Sodium 0.2 0.2 0.2 0.2

Alginate (Alginic Acid

Sodium Salt from brown

algae 4-12 cP, l% in H20

(25°C)) (g)

VegiDay™ protein(g) 0.1 0.1 Calcium Carbonate(g) 0.1 0.1

Nano Calcium Carbonate 0.1 0.1

(15-40nm) (g)

Water Q.S. (mL) 8 8 8 8

Probiotic Slurry (mL) 2 2 2 2

Total (mL) 10 10 10 10

[0058] In method 1, lactic acid bacteria were grown for 24 hours in 50mL of MRS Broth at 37°C. The overnight culture was spun down at 4000 rpm for 5 minutes and washed 3 times with a 0.9% saline solution. The probiotic pellet was approximately lg. The lactic acid bacteria were resuspended in 2mL of 0.9% saline solution to create a probiotic slurry. To create a polymer matrix solution, 5mL of sterile distilled water was first heated to 60°C. Then 0.2g of low viscosity sodium alginate, lg of maltodextrin, lg of galactooligosacchride/beta-glucan, and O. lg of calcium carbonate were added. The polymer solution was then Q.S. to 8mL using sterile distilled water. The polymer solution was mixed by vortexing. The polymer solution was then cooled to room temperature. 2mL of the probiotic slurry was then added to the polymer solution for a final concentration of 20% (v/v). The probiotic-polymer slurry was then mixed until homogenous. The probiotic-polymer slurry had the composition of 10% (w/v) maltodextrin, 10% (w/v) galactooligosaccharide/beta-glucan, 2% (w/v) low viscosity alginate, 2% (v/v) probiotic slurry, and 1% (w/v) calcium carbonate. To form the microspheres, lOmL of the probiotic- polymer slurry was then slowly added to 50mL of heat sterilized vegetable oil solution comprising of 99.8% (v/v) vegetable oil and 0.2% (w/v) Tween 80 under stirring at 500 rpm. The water-in-oil emulsion solution was allowed to stir for up to 10 minutes. 40mL of 0.1M CaCl ₂ solution was quickly added to the water-in-oil emulsion solution. The solution was allowed to stir for another 15 minutes. After 15 minutes, the stirring was stopped and the solution was allowed to settle for 30 mins at 4°C. The probiotic microspheres settled to the bottom and was harvested from the top oil layer. The probiotic microspheres were spun down at 350xg for 5 minutes and rinsed 3 times with a 0.9% saline solution. The probiotic microspheres were stored in a solution including 0.1% peptone and 0.9% saline at pH 7 in a 4°C refrigerator. The probiotic microspheres ranged in size 1 to 200 microns with an average size of 100 microns. FIG. 1 illustrates the probiotic microspheres 50 formed with this method. In some implementations, the probiotic microspheres have a diameter between about 50 microns and about 300 microns, between about 50 microns and about 250 microns, between about 50 microns and about 200 microns, between about 50 microns and about 150 microns, or between about 50 microns and about 100 microns.

[0059] In method 2, a lactic acid bacteria were grown for 24 hours in 50mL of MRS Broth at 37°C. The overnight culture was spun down at 4000 rpm for 5 minutes and washed 3 times with a 0.9% saline solution. The probiotic pellet was approximately lg. The lactic acid bacteria were resuspended in 2mL of 0.9% saline solution to create a probiotic slurry. To create a polymer matrix solution, 5mL of sterile distilled water was first heated to 60°C. Then 0.2g of low viscosity sodium alginate, lg of maltodextrin, lg of galactooligosacchride/beta-glucan, and O. lg of nano calcium carbonate (15-40nm in diameter) were added. The polymer solution was then Q.S. to 8mL using sterile distilled water. The polymer solution was mixed by vortexing. The polymer solution was then cooled to room temperature. 2mL of the probiotic slurry was then added to the polymer solution for a final concentration of 20% (v/v). The probiotic-polymer slurry was then mixed until homogenous. The probiotic-polymer slurry had the composition of 10%) (w/v/) maltodextrin, 10% (w/v) galactooligosaccharide/beta-glucan, 2% (w/v) low viscosity alginate, 2% (v/v) probiotic slurry, and 1% (w/v) nano calcium carbonate. To form the microspheres, lOmL of the probiotic-polymer slurry was then slowly added to 50mL of a heat sterilized vegetable oil solution comprising of 99.8% (v/v) vegetable oil and 0.2% (v/v) Tween 80 under stirring at 500 rpm. The water-in-oil emulsion solution was allowed to stir for up to 10 minutes. 40mL of 0.1M CaCl ₂ solution was quickly added to the water-in-oil emulsion solution. The solution was allowed to stir for another 15 minutes. After 15 minutes, the stirring was stopped, and the solution was allowed to settle for 30 mins at 4°C. The probiotic microspheres settled to the bottom and was harvested from the top oil layer. The probiotic microspheres were spun down at 350xg for 5 minutes and rinsed 3 times with a 0.9% saline solution. The probiotic microspheres were stored in a solution including 0.1% peptone and 0.9% saline at pH 7 in a 4°C refrigerator. The probiotic microspheres ranged in size 1 to 200 microns with an average size of 100 microns. FIG. 2 illustrates example probiotic microspheres 50 generated using method 2. [0060] In method 3, a lactic acid bacteria were grown for 24 hours in 50mL of MRS Broth at 37°C. The overnight culture was spun down at 4000 rpm for 5 minutes and washed 3 times with a 0.9% saline solution. The probiotic pellet was approximately lg. The lactic acid bacteria were resuspended in 2mL of 0.9% saline solution to create a probiotic slurry. To create a polymer matrix solution, 5mL of sterile distilled water was first heated to 60°C. Then 0.2g of low viscosity sodium alginate, lg of maltodextrin, lg of galactooligosacchride/beta-glucan, 0.1 g of VEGIDAY™ plant-based protein powder (organic pea protein, organic pumpkin protein, organic sacha inchi protein, organic chia seed, organic spirulina, organic chlorella, organic quinoa powder), and O. lg of calcium carbonate were added. The polymer solution was then Q.S. to 8mL using sterile distilled water. The polymer solution was mixed by vortexing. The polymer solution was then cooled to room temperature. 2mL of the probiotic slurry was then added to the polymer solution for a final concentration of 20% (v/v). The probiotic-polymer slurry was then mixed until homogenous. The probiotic-polymer slurry had the composition of 10% (w/v) maltodextrin, 10%) (w/v) galactooligosaccharide/beta-glucan, 2% (w/v) low viscosity alginate, 2% probiotic slurry (v/v), 1% (w/v) VEGIDAY™ plant-based protein powder and 1% (w/v) calcium carbonate. To form the microspheres, lOmL of the probiotic-polymer slurry was then slowly added to 50mL of a heat sterilized vegetable oil solution comprising of 99.8% (v/v) vegetable oil and 0.2% (v/v) Tween 80 under stirring at 500 rpm. The water-in-oil emulsion solution was allowed to stir for up to 10 minutes. 40mL of 0.1M CaCl ₂ solution was quickly added to the water-in-oil emulsion solution. The solution was allowed to stir for another 15 minutes. After 15 minutes, the stirring was stopped, and the solution was allowed to settle for 30 mins at 4°C. The probiotic microspheres settled to the bottom and was harvested from the top oil layer. The probiotic microspheres were spun down at 350xg for 5 minutes and rinsed 3 times with a 0.9% saline solution. The probiotic microspheres were stored in a solution including 0.1% peptone and 0.9%) saline at pH 7 in a 4°C refrigerator. The probiotic microspheres ranged in size 1 to 200 microns with an average size of 100 microns. FIG. 3 depicts example probiotic microspheres 50 generated using method 3.

[0061] In method 4, a lactic acid bacteria were grown for 24 hours in 50mL of MRS Broth at 37°C. The overnight culture was spun down at 4000 rpm for 5 minutes and washed 3 times with a 0.9%) saline solution. The probiotic pellet was approximately lg. The lactic acid bacteria were resuspended in 2mL of 0.9% saline solution to create a probiotic slurry. To create a polymer matrix solution, 5mL of sterile distilled water was first heated to 60°C. Then 0.2g of low viscosity sodium alginate, lg of maltodextrin, lg of galactooligosacchride/beta-glucan, 0.1 g of VEGIDAY™ plant-based protein powder (organic pea protein, organic pumpkin protein, organic sacha inchi protein, organic chia seed, organic spirulina, organic chlorella, organic quinoa powder), and O. lg of nano calcium carbonate (15-40nm in diameter) were added. The polymer solution was then Q.S. to 8mL using sterile distilled water. The polymer solution was mixed via vortexing. The polymer solution was then cooled to room temperature. 2mL of the probiotic slurry was then added to the polymer solution for a final concentration of 20% (v/v). The probiotic-polymer slurry was then mixed until homogenous. The probiotic-polymer slurry had the composition of 10% (w/v) maltodextrin, 10% (w/v) galactooligosaccharide/beta-glucan, 2% (w/v) low viscosity alginate, 2% (v/v) probiotic slurry, 1% (w/v) VegiDay™ plant-based protein powder, and 1% (w/v) nano calcium carbonate. To form the microspheres, lOmL of the probiotic-polymer slurry was then slowly added to 50mL of a heat sterilized vegetable oil solution comprising of 99.8% (v/v) vegetable oil and 0.2% (v/v) Tween 80 under stirring at 500 rpm. The water-in-oil emulsion solution was allowed to stir for up to 10 minutes. 40mL of 0.1M CaCl ₂ solution was quickly added to the water-in-oil emulsion solution. The solution was allowed to stir for another 15 minutes. After 15 minutes, the stirring was stopped and the solution was allowed to settle for 30 mins at 4°C. The probiotic microspheres settled to the bottom and was harvested from the top oil layer. The probiotic microspheres were spun down at 350xg for 5 minutes and rinsed 3 times with a 0.9% saline solution. The probiotic microspheres were stored in a solution including 0.1% peptone and 0.9% saline at pH 7 in a 4°C refrigerator. The probiotic microspheres ranged in size 1 to 200 microns with an average size of 100 microns. FIG. 4 depicts example probiotic microspheres 50 generated using method 4.

G. FREEZE DRYING OF PROBIOTIC MICRO SPHERES INTO POWDER:

[0062] Probiotics microspheres from methods 1 through 4 could be turned into a dry powder form by freeze drying overnight using a lyophilization machine. The composition of methods 1 through 4 can provide a protective matrix surrounding the probiotic bacteria compared to non- encapsulated probiotic bacteria. Probiotic microspheres from methods 1 and 3 were chosen for lyophilization. The presence of nano calcium carbonate in methods 2 and 4 had the same effect on the probiotic cell count as methods 1 and 3, respectively. To freeze dry the probiotic microspheres, ImL of the wet probiotic microspheres were frozen in cryovials for 24hrs in an 80°C freezer. The frozen microspheres were then dried in a lyophilization machine (Labconco Freeze Dryer FreeZone 4.5) for 24 hrs. The probiotic microspheres could be stored at room temperature or at 4°C conditions.

H. PROBIOTIC CELL COUNT OF PROBIOTIC MICROSPHERES BEFORE AND

AFTER FREEZE DRYING:

[0063] The composition can include a protective polymer matrix composition that can protect probiotics during the freeze drying. To determine the extent at which the polymer matrix of the present disclosure could protect probiotic cells, free non-encapsulated probiotic cells and wet probiotic microspheres from methods 1 and 3 were subjected to testing cell count before and after freeze drying. Before freeze drying, 500uL of wet probiotic microspheres from methods 1 and 3 were each suspended in 4.5mL of 0.1M Sodium Citrate Buffer (pH 6.0) and shaken for 10 mins. The 0.1M Sodium Citrate buffer was used to dissolve the protective polymer matrix in order to release the probiotic cells. 500uL of free non-encapsulated probiotic cells were also subjected to the same conditions as the probiotic microspheres to ensure consistency. A dilution series was prepared using the dissolved probiotic microspheres (or free non-encapsulated probiotic cells) in sterile Buffered Saline Gelatin water (0.9% sodium chloride, 0.1% Gelatin). The dilutions were spotted onto MRS agar plates and incubated for 48 hours at 37°C before colonies were counted.

[0064] After the probiotic microspheres were freeze dried into a powder, the samples were Q.S. to the original ImL volume using 0.1M Sodium Citrate Buffer (pH 6.0) and shaken for 10 mins. Free non-encapsulated probiotic slurry in 0.9% saline solution were also frozen for 24hrs at 80°C and dried using the lyophilization machine for 24 hrs. The free probiotic freeze dried powders were also Q.S. to the original ImL volume using 0.1M Sodium Citrate Buffer (pH 6.0) and shaken for 10 mins. 500uL of the dissolved probiotic microspheres (or free cells) were suspended in 4.5mL of BSG and mixed. A dilution series was prepared in sterile Buffered Saline Gelatin water. The dilutions were spotted onto MRS agar plates and incubated for 48 hours at 37°C before colonies were counted. [0065] The probiotic microspheres from method 1 had greater cell viability than the free non- encapsulated probiotic cells. The addition of the VEGIDAY™ proteins in method 3 resulted in greater cell viability and less cell death than method 1. Both method 1 and 3 had greater cell viability than non-encapsulated probiotic cells. FIG. 5 depicts the cell count in Logio CFU/mL before and after freeze drying of free probiotic cells and probiotic microspheres from methods 1 and 3. Percent reduction of cell count was calculated according to the following formula:

Percent reduction

= 100

Before Freeze Drying CFU /mL) — After Freeze Drying CFU /mL)

Before Freeze Drying CFU /mL)

[0066] The probiotic microspheres from method 3 had a 59.18% reduction in cell count (CFU/mL) while probiotic microspheres from method had a 79.13%) reduction in cell count (CFU/mL) and non-encapsulated (free cells) probiotic cells had a 99.04%> reduction in cell count (CFU/mL). FIG. 6 depicts the percent reduction of the cell counts after freeze drying of free probiotic cells and probiotic microspheres from methods 1 and 3.

[0067] Without wishing to be bound by theory, it is believed that the addition of VegiDay™ proteins created a superior protective matrix surrounding the probiotic cells during freeze drying. This is believed to be due to the presence of sugars, lipids, amino acids, and vitamins in the protein powder mix stabilizing the probiotic cells. Literature indicates that the presence of proteins such as whey protein enhances probiotic survival while freeze drying due to its lipid and sugar content. The addition of VEGIDAY™ proteins may further protect the probiotic cells during freeze drying. The non-encapsulated probiotic cells suffered the highest percent reduction because the cells were not protected from large ice crystals and the cell membranes were not stabilized during freeze drying.

I. PROBIOTIC MICROSPHERE CELL COUNT IN SIMULATED GASTRIC

CONDITIONS:

[0068] The probiotic microspheres of the present disclosure can also protect probiotics in gastric conditions to enable active probiotics to be released in the small intestine. The mean gastric emptying time is 2 hours. To determine the extent at which the microspheres can protect probiotic cells, probiotic microspheres from methods 1 through 4 and free non-encapsulated probiotic cells were subjected to 2 hours of simulated gastric conditions. 500uL of probiotic microspheres or free probiotic cells were added to 4.5mL of simulated gastric juice (0.5% saline, 0.1% pepsin (250UI), pH 1.5) for 2 hours at 37°C. After 2 hours, 500uL of the gastric samples were added to 4.5mL of 0.1M Sodium Citrate Buffer (pH 6.0) and shaken for 10 mins to dissolve the protective matrix. Free probiotic cells were treated the same. A dilution series was prepared in sterile Buffered Saline Gelatin water. The dilutions were spotted onto MRS agar plates and incubated for 48 hours at 37°C before colonies were counted.

[0069] The probiotic microspheres from methods 1 through 4 had greater cell viability than the free non-encapsulated probiotic cells. FIG. 7 depicts the cell count (Logio CFU/mL) before and after 2 hours of simulated gastric conditions. FIG. 8 depicts the percent reduction in cell count (CFU/mL) after 2 hours of simulated gastric conditions. Percent reduction of cell count was calculated according to the following formula:

Percent reduction

= 100

Before Gastric Buffer CFU /mL) — After Gastric Buffer CFU /mL)

Before Gastric Buffer CFU /mL)

[0070] Non-encapsulated probiotic cells had a 99.999997% reduction in cell count (CFU/mL), method 1 had a 56.01%) reduction in cell count (CFU/mL), method 2 had a 65.71%) reduction in cell count (CFU/mL), method 3 had a 57.69%> reduction in cell count (CFU/mL), and method 4 had a 63.88%> reduction in cell count (CFU/mL) after 2 hours in simulated gastric conditions.

[0071] Without wishing to be bound by theory, it is believed that the microencapsulation compositions in methods 1 to 4 were able to form a protective matrix surrounding the probiotic cells. The sodium alginate formed a protective hydrogel matrix around the probiotic cells and the dissolution of CaC0 ₃ into Ca ²⁺ , as described earlier, strengthened the hydrogel matrix. Method 2 and 4 with nano calcium carbonate had a higher reduction in cell counts. This is likely due to the particle sizes of the nano calcium carbonate that may interfere with the cross-linking of the sodium alginate hydrogel. The particles can be seen in FIGS. 2 and 4. J. PROBIOTIC MICROSPHERE CELL COUNT IN SIMULATED GASTRIC

CONDITIONS WITH AMPICILLIN ANTIBIOTIC:

[0072] The probiotic microspheres of the present disclosure can survive in gastric conditions with ampicillin antibiotic to allow active probiotics to be released in the small intestine. Millions of antibiotics are prescribed each year in the United States for various infections. The presence of antibiotics in the person or animals' system can negate and inactive probiotics. The mean gastric emptying time is 2 hours and antibiotics such as ampicillin are absorbed throughout the gastrointestinal tract. Probiotic microspheres from methods 1 and 3 and free non-encapsulated probiotic cells were subjected to 2 hours of simulated gastric conditions with ampicillin antibiotics. To determine the extent at which the microspheres can protect probiotic cells, probiotic microspheres from methods 1 and 3 and free non-encapsulated probiotic cells were subjected to 2 hours of simulated gastric conditions with ampicillin antibiotic. 500uL of probiotic microspheres or free probiotic cells were added to 4.5mL of simulated gastric juice with ampicillin (0.5% saline, 0.1% pepsin (250UI), pH 1.5, 100μg/mL Ampicillin) for 2 hours at 37°C. After 1 hour and 2 hours, 500uL of the gastric samples were added to 4.5mL of 0.1M Sodium Citrate Buffer (pH 6.0) and shaken for 10 mins to dissolve the protective matrix. Free probiotic cells were treated the same. A dilution series was prepared in sterile Buffered Saline Gelatin water. The dilutions were spotted onto MRS agar plates and incubated for 48 hours at 37°C before colonies were counted.

[0073] The probiotic microspheres from methods 1 and 3 had negligible cell death compared to the free non-encapsulated probiotic cells. After 1 hour, the non-encapsulated probiotic cells were completely inactivated. FIG. 9 depicts the cell count (Logio CFU/mL) before and after 1 and 2 hours of simulated gastric conditions with ampicillin antibiotic.

[0074] Without wishing to be bound by theory, it is believed that the microencapsulation compositions in methods 1 and 3 were able to form a protective matrix surrounding the probiotic cells. The sodium alginate formed a protective hydrogel matrix around the probiotic cells and the dissolution of CaC0 ₃ into Ca ²⁺ , as described earlier, strengthened the hydrogel matrix. Thus, the probiotic cells were not able to diffuse into the harsh stomach buffer and the CaC0 ₃ was able to buffer and act as an antacid within the microsphere. K. PROBIOTIC MICROSPHERE CELL COUNT IN SIMULATED GASTRIC AND

INTESTINAL CONDITIONS:

[0075] The probiotic microspheres of the present disclosure can protect probiotics in gastric and intestinal conditions. The mean gastric emptying time is 2 hours and the mean small intestinal emptying time is 4 hours. To determine the extent the microspheres can protect probiotic cells, probiotic microspheres from method 1 and free non-encapsulated probiotic cells were subjected to 2 hours of simulated gastric conditions and subsequently 2 hours of small intestine conditions. 500uL of probiotic microspheres or free probiotic cells were added to 4.5mL of simulated gastricjuice (0.5% saline, 0.1% pepsin (250UI), pH 1.5) for 2 hours at 37°C. After 2 hours, 500uL of the gastric samples were added to 4.5mL of 0.1M Sodium Citrate Buffer (pH 6.0) and shaken for 10 mins to dissolve the protective matrix. Free probiotic cells were treated the same. A dilution series was prepared in sterile Buffered Saline Gelatin water. The dilutions were spotted onto MRS agar plates and incubated for 48 hours at 37°C before colonies were counted.

[0076] The remaining samples in simulated gastric buffer were spun down at 4000 rpm for 5 mins. 4.5mL of the gastricjuice were removed from the samples. 4.5mL of small intestine buffer (0.5%) saline, 0.1%> pancreatin, 4.5% bile salts, pH 8) was added to each sample. After 2 hours, 500uL of the small intestine samples were added to 4.5mL of 0.1M Sodium Citrate Buffer (pH 6.0) and shaken for 10 mins to dissolve the protective matrix. Free probiotic cells were treated the same. A dilution series was prepared in sterile Buffered Saline Gelatin water. The dilutions were spotted onto MRS agar plates and incubated for 48 hours at 37°C before colonies were counted.

[0077] The probiotic microspheres from method 1 had greater cell count compared to the free non-encapsulated probiotic cells. After 2 hours in gastric buffer, the non-encapsulated probiotic cells were a Log of 1.27 compared to a Log of 7.46 in method 1. After 2 hours in small intestine buffer, the non-encapsulated probiotic cells were completely inactivated and the method 1 had a cell count of Log of 5.09. FIG. 10 depicts the cell count before and after 1 and 2 hours of simulated gastric conditions with ampicillin antibiotic. [0078] Without wishing to be bound by theory, it is believed that the microencapsulation compositions in method 1 were able to form a protective matrix surrounding the probiotic cells. The sodium alginate formed a protective hydrogel matrix around the probiotic cells and the dissolution of CaC0 ₃ into Ca ²⁺ , as described earlier, strengthened the hydrogel matrix in the stomach acid. In the alkaline conditions of the small intestine the protective matrix swelled and started to erode, releasing the probiotic cells.

L. A SCALABLE METHOD FOR PRODUCING MICROENCAPSULATED

PROBIOTIC S/ACTIVE COMPONENTS IN POWDER FORM USING AN EFFICIENT AND CONTINUOUS CLOSED SYSTEM:

[0079] The methods described herein can provide an efficient and continuous closed system scalable to commercially produce microspheres in powder form. These methods can incorporate the in-situ cross-linking technique with a spray dryer system as described earlier. The spray dryer system can be the last step in producing microspheres in powder form. The components in this method may be at the bench scale, pilot scale, or commercial scale. Premature gelation of the active components such as probiotics, microencapsulation formulation with the alginate biopolymer, and the weakly acidic solution of glucono-delta-lactone will occur if all three of the components are added and allowed to mix and incubate in an extended period of time before converting the mixture into a microsphere powder. The components can be separated to prevent premature gelation. In some implementations, the reactors and pipes are kept at about 4°C.

[0080] FIG. 11 illustrates an example system 100 for producing the microspheres described herein. The system 100 can include tank 1, tank 2, and tank 3. The tanks can also be referred to as reactors. The tanks can be connected using pipes or tubing. Active components, such as probiotics, can be stored in either reactors 1 or 2. The microencapsulation formulation (e.g., the microencapsulation formulation described in relation to methods 1 to 4) can be stored in either reactors 1 or 2. For example, a first tank can include the active components and a second tank can include the microencapsulation formulation. The weak acid glucono-delta-lactone can be stored in reactor 3. Reactors 1 and 2 can be kept at 4°C. Reactor 3 may be kept at room temperature or 4°C. The system 100 can include a spray dryer system 14. Valves 16, 17, 18, and 19 can be used to control passage of the solutions and mixtures from the reactors to the spray dryer system 14. When the values are opened, pumps 4, 5, 6, and 7 can pump the solutions and mixtures form the reactors to the tubing between the reactors and towards the spray dryer system 14. The pumps can control the rate at which the solutions from each reactor enter the mixers 12 and 13 and the spray dryer system 14. The system 100 can include a plurality of pressure gauges 8, 9, 10, and 11 that can measure the pressure within the pipes or tubing. The system 100 can include a controller that can control the pumps (e.g., their activation and rate of flow) and monitor the pressures measured by the pressure gauges. The mixers 12 and 13 may be static mixers or motorized mixers. The mixers are used to mix and homogenize solutions from the reactors. The arrows depicting the mixers indicate the direction or flow of the solutions from each reactor. Each of the reactors are connected to each other via pipes or tubing. The pipes or tubing can include insulation 15 to maintain the temperature within the tubing or pipes. The components of the system 100 can be manufactured from seriahzable materials that can be such that the system 100 can be sterilized between uses.

[0081] In some implementations, the active components in reactors 1 or 2 may be in a liquid or a powder form. The active components may be pharmaceuticals, nutraceuticals, biologies, microorganisms, bacteria based products, probiotics, or any combination thereof. The active components may be premixed and added into reactors 1 and 2 or be mixed in reactors 1 or 2. The microencapsulation formulation in reactors 1 or 2 may be the same compositions from methods 1 to 4 as described herein. In some implementations, the microencapsulation formulation can include any biopolymer and additives at any combination and concentration thereof. The microencapsulation formulation may be premixed and added into reactors 1 or 2 or mixed in reactors 1 or 2. The acid in reactor 3 may be glucono-delta-lactone, acetic acid, lactic acid, tartaric acid, gluconic acid, malic acid, citric acid, or any combinations thereof and at any concentration. The acid may be premixed and added to reactor 3 or be mixed in reactor 3. The reactors 1, 2, and 3 may be tanks, holding tanks, bioreactors, fermentation reactors, mammalian cell reactors, or any type of reactors and any combination thereof.

[0082] The valves 16, 17, 18, and 19 may be one-way, two-way, or three-way hydraulic valves, pneumatic valves, manual valves, solenoid valves, motor valves, and or any combinations thereof. The pumps 4,5, 6, and 7 may be direct lift pumps, displacement pumps, gravity pumps, and or any combinations thereof. The pipe or tubing insulation 15 may be mineral wool, glass wool, flexible elastomeric foams, rigid foam, polyethylene, cellular glass, aerogel, and or any combinations thereof. The spray dryer system 14 can include a drying chamber, hot air supply system, atomizing device, powder/fines recovery system, and or fines return system, and or powder after-treatment system.

[0083] FIG. 12 illustrates a block diagram of an example method 1200 to form powdered, probiotic microspheres with the system 100 illustrated in FIG. 11. The method 1200 can include mixing a polymer solution (step 1202). The method 1200 can include mixing a probiotic slurry (step 1204). The method can include forming a gel (step 1206). The method can include spray drying the gel (step 1208).

[0084] The method 1200 can include mixing a polymer solution (step 1202). The polymer solution can be any of the polymer solutions described herein. The polymer solution can be any of the polymer solutions described above in relation to methods 1-4. For example, to create a polymer matrix solution, 5mL of sterile distilled water was first heated to 60°C. Then 0.2g of low viscosity sodium alginate, lg of maltodextrin, lg of galactooligosacchride/beta-glucan, and O. lg of calcium carbonate were added. The polymer solution was then Q.S. to 8mL using sterile distilled water. Also referring to FIG. 11, among others, the polymer solution can be stored in a first tank 1. The polymer solution was mixed by vortexing or by a mixing mechanism within the tank 1. The tank can maintain the polymer solution at about room temperature.

[0085] The method 1200 can include mixing a probiotic slurry (step 1204). The probiotic slurry can be any of the probiotic slurries described herein. The probiotic slurry can be any of the probiotic slurries described above in relation to methods 1-4. For example, a lactic acid bacteria can be grown for 24 hours in 50mL of MRS Broth at 37°C. The overnight culture can be spun down at 4000 rpm for 5 minutes and washed 3 times with a 0.9% saline solution. The probiotic pellet was approximately lg. The lactic acid bacteria can be resuspended in 2mL of 0.9% saline solution to create a probiotic slurry. Also referring to FIG. 11, among others, the probiotic slurry can be stored in a second tank 2. In some implementations, a saline solution can be stored in the second tank 2. Probiotic pellets (formed, for example, using the above technique) can be added to the tank 2 and saline solution. Mixers in the tank 2 can resuspend the probiotics within the saline solution.

[0086] The method 1200 can include forming a gel (step 1206). Also referring to FIG. 11, among others, the system's controller can release the polymer solution and the probiotic slurry from their respective tanks. The controller can also release acid from a third tank 3. The acid can include glucono-delta-lactone, acetic acid, lactic acid, tartaric acid, gluconic acid, malic acid, citric acid, or any combinations thereof and at any concentration. Mixers within the pipping connecting the first, second, and third tanks can mix the acid, polymer solution, and the probiotic slurry, which can form a gel. The method 1200 can include spray drying the gel (step 1208). The gel that is formed during step 1206 can be fed into a spray dryer which can atomize and dry the gel to form a powered.

M. FOOD GRADE DISSOLVABLE FILMS FOR PACKAGING POWDER

MICROSPHERES

[0087] In some implementations, the powder microspheres (e.g., the probiotic microspheres) can be stored or otherwise incorporated into dissolvable films. The dissolvable films can be easily incorporated into liquids such as water, juice, milk, soda, shakes, infant formula, or any type of beverage for oral administration. The dissolvable films can include polymers and polysaccharides. The following Table 2 details example food grade dissolvable film

compositions:

Materials Comp Comp Comp Comp Comp Comp Comp Comp osition osition osition osition osition osition osition osition 1 2 3 4 5 6 7 8

Kelcogel Low acyl 0.2 0.2 - - 0.2 0.15 0.15 0.15

Gellan Gum(g)

Hydroxypropylmeth - - 0.2 0.2 . . . . yl cellulose (10,000

Mn, 6 cP at 20

degrees Celsius) (g) MarCoat 125N 0.04 0.04

Shellac 25%

concentration (mL)

2% Nanocellulose 0.05 0.5 fibers solution (mL)

Sterile distilled water 9.8 9.76 9.8 9.76 9.8 9.85 9.8 9.35

(mL)

[0088] Compositions 1 through 10 were made by adding the appropriate amount of materials to the corresponding volume of distilled water. The mixtures were then heated to 75°C and kept constant at this temperature. The mixtures were then mixed by magnetic stirring (400-500 rpm) for 30 minutes until the solution is homogenized. The polymer solution was solvent casted onto plastic molds and dried in a 40°C oven for 24 hours to make films.

[0089] In some implementations, the films are formed into capsules or pouches (which can also be referred to as pods. FIG. 13 illustrates a front and a side view of an example capsule 200. The capsule 200 can include a first dissolvable film 202(1) that is coupled with a second dissolvable film 202(2). The first dissolvable film 202(1) can be coupled with the second dissolvable film 202(2) toward the edge 204 of each of the respective dissolvable films 202. The dissolvable films 202 can be sealed together using heat or food grade adhesives during the manufacturing process. The powder microspheres are filled into the volume 206 formed between the dissolvable films 202. The volume 206 can be spherical or globular in shape.

[0090] FIG. 14 illustrates an example pod 250. The pod 250 can include a first dissolvable film 202(1) and a second dissolvable film 202(2). The dissolvable films 202 can be sealed together by using heat or food grade adhesives during the manufacturing process only near the edges of the dissolvable films 202 to form a volume 206 within the pod 250. In some implementations, initially three of the edges of the two dissolvable films 202 can be sealed together to form the volume 206. The powder microspheres can be loaded into the volume 206 and the remaining, fourth set of edges can be sealed together to form the completed pod 250.

[0091] Having now described some illustrative implementations, it is apparent that the foregoing is illustrative and not limiting, having been presented by way of example. In particular, although many of the examples presented herein involve specific combinations of method acts or system elements, those acts and those elements may be combined in other ways to accomplish the same objectives. Acts, elements and features discussed in connection with one

implementation are not intended to be excluded from a similar role in other implementations or implementations.

[0092] The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including" "comprising" "having" "containing" "involving" "characterized by" "characterized in that" and variations thereof herein, is meant to encompass the items listed thereafter, equivalents thereof, and additional items, as well as alternate implementations consisting of the items listed thereafter exclusively. In one

implementation, the systems and methods described herein consist of one, each combination of more than one, or all of the described elements, acts, or components.

[0093] As used herein, the term "about" and "substantially" will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, "about" will mean up to plus or minus 10% of the particular term.

[0094] Any references to implementations or elements or acts of the systems and methods herein referred to in the singular may also embrace implementations including a plurality of these elements, and any references in plural to any implementation or element or act herein may also embrace implementations including only a single element. References in the singular or plural form are not intended to limit the presently disclosed systems or methods, their components, acts, or elements to single or plural configurations. References to any act or element being based on any information, act or element may include implementations where the act or element is based at least in part on any information, act, or element.

[0095] Any implementation disclosed herein may be combined with any other implementation or embodiment, and references to "an implementation," "some implementations," "one implementation" or the like are not necessarily mutually exclusive and are intended to indicate that a particular feature, structure, or characteristic described in connection with the implementation may be included in at least one implementation or embodiment. Such terms as used herein are not necessarily all referring to the same implementation. Any implementation may be combined with any other implementation, inclusively or exclusively, in any manner consistent with the aspects and implementations disclosed herein.

[0096] The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0097] References to "or" may be construed as inclusive so that any terms described using "or" may indicate any of a single, more than one, and all of the described terms. For example, a reference to "at least one of 'A' and 'Β'" can include only Ά', only 'Β', as well as both 'A' and 'Β' . Such references used in conjunction with "comprising" or other open terminology can include additional items.

[0098] Where technical features in the drawings, detailed description or any claim are followed by reference signs, the reference signs have been included to increase the intelligibility of the drawings, detailed description, and claims. Accordingly, neither the reference signs nor their absence has any limiting effect on the scope of any claim elements.

[0099] The systems and methods described herein may be embodied in other specific forms without departing from the characteristics thereof. The foregoing implementations are illustrative rather than limiting of the described systems and methods. Scope of the systems and methods described herein is thus indicated by the appended claims, rather than the foregoing description, and changes that come within the meaning and range of equivalency of the claims are embraced therein.