Capsule of powdered probiotic for hot beverages

Abstract

Disclosed is a capsule of probiotics which, in addition to the probiotics, contains pea protein, inulin, starch and maltodextrin. The capsule is stable at the temperature of hot beverages (90°C) and beverages at ambient temperature (30°C), is resistant to water crystal formation caused by freezing, at acid and base pH, and delivers the probiotic to the user's intestines effectively.

Description

[0001] PROBIOTIC POWDER ENCAPSULATION FOR HOT DRINKS

[0002] FIELD OF INVENTION

[0003] The present invention relates to an encapsulation that provides the probiotic with a protective barrier, allowing it to remain stable and viable at hot beverage temperatures (70 to 90 °C) and ambient beverage temperatures (25 to 40 °C), as well as in frozen non-dairy beverages (-10 to -20 °C). It is resistant to aqueous environments for at least three months, resistant to crystal formation caused by freezing, thermostable, stable at acidic pH and degradable at basic pH, bioavailable, and ensures effective delivery of the probiotic to the consumer's intestine. The encapsulation ingredients are safe for the food industry, considered GRAS (Generally Recognized As Safe), and safe for human consumption.

[0004] BACKGROUND OF THE INVENTION

[0005] International Publication No. WO2015019307A1, dated December 2, 2015, entitled "Microcapsules Containing Probiotics and Methods for Preparing Them," describes microcapsules for protecting probiotics and methods for preparing them. In some embodiments, the microcapsules are used to deliver probiotics, probiotic-prebiotic combinations, and / or synbiotics through the stomach of a mammal attached to its lower gastrointestinal tract. In some embodiments, the microcapsules comprise a biopolymer and a plant-based protein. In some embodiments, the microcapsules include alginate, iotacarrageenan, or deacylated gellan gum as the biopolymer. In some embodiments, the microcapsules include chickpea protein, pea protein, or soy protein as the plant-based protein.An emulsion containing the biopolymer, the plant-based protein, and the probiotic material to be encapsulated can be crosslinked to form the microcapsule.

[0006] Wherein, a microcapsule is provided in which: the prebiotic optionally comprises a non-digestible carbohydrate such as resistant starch or a non-starch polysaccharide such as hemicellulose, pectin or gum; an oligosaccharide such as a galactooligosaccharide, fructooligosaccharide or maltoligosaccharide; lactulose; or inulin; and / or the probiotic and the prebiotic provide a symbiotic.

[0007] On the other hand, the present invention describes the process for obtaining protective microcapsules of a probiotic containing at least Lactobacillus, as well as vegetable protein, starch, polysaccharides, or inulin. These components confer novel characteristics to the probiotic, such as resistance to aqueous environments for at least 3 months, resistance to crystal formation caused by freezing water, thermostable (70-90 °C), stable at acidic pH and degradable at basic pH, bioavailable, and ensuring effective delivery of the probiotic to the consumer's intestine.

[0008] European Publication EP4042881A1, dated 2022-08-17, entitled "Microcapsule, method of preparation thereof and application thereof," describes a microcapsule and a method of preparing it, specifically a multilayer microcapsule containing an active substance and a method of preparing it, the microcapsule having a single-layer or multi-layer encapsulation structure. It also describes a probiotic microcapsule and a method of preparing it, the probiotic microcapsule having a single-layer or multi-layer encapsulation structure. Finally, it describes a food product or a health product containing a probiotic microcapsule (comprising a dairy product, a fermented-style food product, a beverage, chocolate, sweets, a baked product, a fruit or vegetable juice product, etc., containing a probiotic microcapsule) and a method of preparing it.A probiotic microcapsule soft powder and a method of preparing the same, the probiotic microcapsule soft powder comprises coconut cream probiotic microcapsule soft powder and probiotic microcapsule gel ball soft powder.

[0009] Wherein the wall material is selected from one or a combination of more than: vegetable protein, such as soy protein, rice protein, wheat protein, corn protein, etc.; preferably corn protein, or animal protein, such as whey protein, casein, etc.; preferably whey protein concentrate (WPC), whey protein isolate (WPI) or whey protein peptide, especially preferably whey protein isolate (WPI);or fats, such as fats with a melting point of 40°C or higher, preferably with a melting point of 40-50°C, particularly preferably palm oil, medium-chain glycerides, hydrogenated fats (e.g., hydrogenated palm oil, hardened oil, hydrogenated soybean oil), lecithin, cocoa butter substitutes, palm oil monoglycerides, coconut oil, soybean oil, peanut oil, sunflower oil, or other materials, such as glycerin, oleic acid, sodium alginate, shellac, CMC-Na, gellan, xanthan gum, k-carrageenan, cellulose acetate phthalate, maltodextrin, starch, dextrin, sucrose, lactose, dextran, corn syrup, pectin, gum, arabic fatty acid esters, chitosan, acetylated mono- or diglycerides, gum konjac, carrageenan, wax or gelatin, etc.; preferably, the wall material is selected from whey protein or fat, particularly preferably WPI or MCT.;

[0010] On the other hand, the present invention describes the process for obtaining protective microcapsules of a probiotic containing at least Lactobacillus, as well as vegetable protein, starch, polysaccharides, or inulin. These components confer novel characteristics to the probiotic, such as resistance to aqueous environments for at least 3 months, resistance to crystal formation caused by freezing water, thermostable (70-90 °C), stable at acidic pH and degradable at basic pH, bioavailable, and ensuring effective delivery of the probiotic to the consumer's intestine.

[0011] Mexican Patent Application MX / a / 2023 / 005340, which describes an invention to develop a scalable and affordable encapsulation that allows probiotics to survive high-temperature baking processes, minimizing the over-addition necessary to achieve the CFU required to make the corresponding declaration.

[0012] On the other hand, the present invention describes the process for obtaining protective microcapsules of a probiotic containing at least Lactobacillus, as well as vegetable protein, starch, polysaccharides, or inulin, and a probiotic powder encapsulation formulation for beverages comprising a powdered ingredient that allows probiotics to be incorporated into a hot or frozen beverage.

[0013] These characteristics give the probiotic novel features such as being resistant to aqueous environments for at least 3 months, resistant to the generation of crystals produced by freezing water, thermostable (70-90 °C), stable at acidic pH and degrading at basic pH, bioavailable and providing effective delivery of the probiotic to the consumer's intestine.

[0014] BRIEF DESCRIPTION OF THE INVENTION

[0015] The object of the invention is a probiotic encapsulation formulation, with functional ingredients, as well as its obtaining process, useful for consumer beverages comprising probiotics in both hot beverages (70 to 90 °C), room temperature beverages (25 to 40 °C) and frozen non-dairy beverages (-10 to -20 °C).

[0016] This ingredient is characterized by its resistance to aqueous environments for at least 3 months, resistance to crystal formation caused by freezing water, and its thermostable nature. It also remains stable at acidic pH levels and degrades at basic pH levels, which enhances the bioavailability and effective delivery of probiotics to the consumer's gut. Synergistic effects were found among these ingredients, resulting in greater prebiotic stability and more effective delivery to the gut. Furthermore, the wall materials are safe for use in the food industry, are considered GRAS (Generally Recognized As Safe), and are safe for human consumption.

[0017] The probiotic powder encapsulation for beverages comprises a microcapsule in which: the prebiotic optionally comprises a non-digestible carbohydrate such as resistant starch or a non-starch polysaccharide such as hemicellulose, pectin, or gum; an oligosaccharide such as a galactooligosaccharide, fructooligosaccharide, or maltoligosaccharide; lactulose; or inulin; and / or the probiotic and prebiotic provide a symbiotic, crosslinked with an oligosaccharide such as lactulose, inulin, dextrin, or maltodextrin.

[0018] Hemicelluloses are heteropolysaccharides (polysaccharides composed of more than one type of monomer), formed by a heterogeneous group of polysaccharides, which in turn are formed by two types of monosaccharides linked by O(1-4) bonds (primarily xylose, arabinose, galactose, mannose, glucose, and glucuronic acid), forming a branched linear chain. Among these monosaccharides, glucose, galactose, and fructose are the most prominent.

[0019] It forms part of the walls of plant cells, covering the surface of cellulose fibers and allowing pectin to bind.

[0020] Galactooligosaccharides (GOS) are oligosaccharides composed of a terminal glucose unit and two or more galactose units. These substances are considered prebiotics because they promote the growth of beneficial gut bacteria.

[0021] Pectin is a natural product present in the cell wall of all higher plants and is used by the food, cosmetic and pharmaceutical industries for its gelling, thickening and stabilizing properties.

[0022] Inulin is a non-digestible carbohydrate found in many vegetables, fruits, and grains. Currently, it is industrially extracted from chicory root (Cichorium intybus) and widely used as an ingredient in functional foods.

[0023] A symbiotic is a pharmaceutical or food preparation that contains one or more species of probiotics and prebiotic ingredients.

[0024] A fructooligosaccharide is a linear oligosaccharide composed of 10 to 20 fructose monomers linked by (1→2) bonds, and may contain an initial glucose molecule. A typical example of a fructooligosaccharide is 1-kestose.

[0025] Fructooligosaccharides, sometimes also called oligofructose or oligofructans, or abbreviated FOS, are commonly used as sugar substitutes. These polysaccharides exhibit a sweetening power that, for the same weight, varies between 30 and 50 percent of the sweetening power of common sugar in commercial syrup preparations. They frequently appear in a multitude of natural products and experienced a commercial boom in the 1980s in response to consumer demand for healthier, lower-calorie food products.

[0026] Maltodextrin is a mixture of glucose polymers that result from the hydrolysis of starch. It is typically sold as a white powder composed of several glucose oligomers, each containing 5 to 10 units.

[0027] Dextrins are a group of low molecular weight oligosaccharides produced by the hydrolysis of starch. They have the same general formula as polysaccharides but a shorter chain length.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a diagram of a spray drying system. The main components of a spray drying system are a container holding the solution or suspension to be spray dried. Inside the drying chamber are the atomizing nozzle where the solution is sprayed. Very close to this nozzle is a hot air outlet that dries the small droplets formed from the solution. Finally, the dried product is obtained at the end of the drying chamber, generally with a particle diameter similar to the opening of the atomizing nozzle.

[0030] Figure 2 shows the microencapsulation production process diagram. This process consists of weighing each of the ingredients according to the formulation optimized for this invention. The appropriate proportion of water is then added, and the mixture is homogenized. Once the ingredients are thoroughly homogenized, the probiotic to be encapsulated is added, and the suspension is kept under constant agitation. This mixture is then injected into the spray drying equipment to obtain a dry, whitish powder. This product is stored at 4 o C.

[0031] Figure 3 shows a procedure diagram for the hot beverage test. The functional ingredient is poured into the hot beverage, which is then stirred. Aliquots of the beverage are taken at the following times: 0 seconds, 15 seconds, 30 seconds, 60 seconds, 5 minutes, 10 minutes, 20 minutes, and 30 minutes. These aliquots are then used to assess the viability of the probiotic by plate count at different dilutions.

[0032] Figure 4 shows how the hot beverage experiment was conducted. Distilled water was used and heated to 90 °C. The probiotic was then added, and aliquots were taken at regular intervals. The temperature was constantly monitored.

[0033] Figure 5 shows the probiotic content during the hot beverage test. It can be observed that, with the formulation proposed for the present invention (different proportions of the ingredients within the ranges described in the invention), in all cases the viability of the probiotic was greater than 1x10 7 Colony Forming Units per mL of beverage (CFU / mL)

[0034] Figure 6 shows the detail of the in-vitro digestion experiment.

[0035] Figure 7 shows the viscosity comparison. On the left is a 200 µL drop of commercial BC30 coffee, in the middle is a 200 µL drop of coffee with milk from the same establishment (without probiotics), and on the right is a 200 µL drop of black coffee with the Boosting technology proposed in the present invention as a functional ingredient.

[0036] Figure 8 shows a summary comparison of Boosting and commercial BC30 coffee. It can be seen that the functional ingredient results in a coffee with a viscosity similar to water and a translucent appearance. In contrast, the commercial BC30 coffee produces a viscous, dense, and opaque coffee.

[0037] Figure 9 shows the viability of the probiotic in the proposed functional ingredient in an aqueous solution stored at approximately 35 °C. It also shows the viability of the probiotic in the functional ingredient in an aqueous solution that was stored at approximately 35 °C and then frozen at -10 °C and subsequently thawed to measure its viability after thawing. These are compared with the non-encapsulated probiotic.

[0038] Figure 10 shows the viability of the probiotic in the proposed functional ingredient in an aqueous solution stored at -20°C. It also shows the viability of the probiotic in the functional ingredient that was stored at -20°C but was thawed and refrozen. These are compared with the unencapsulated probiotic.

[0039] DETAILED DESCRIPTION OF THE INVENTION

[0040] Although probiotics have traditionally been associated with dairy products, their presence is expanding into other food categories, such as juices, cereals, and bars. However, their sensitivity to heat poses significant challenges during thermal processes, especially in the manufacture and storage of baked goods like bread. The survival and successful growth of probiotics within the host organism are essential for their beneficial effects, requiring ideal conditions in terms of pH, oxygen presence, temperature, and humidity.

[0041] The encapsulation of nutraceuticals, including probiotics, is emerging as an effective strategy to ensure the stability of these ingredients under specific conditions. This technology addresses challenges by capturing the functional ingredient as its core and coating it with an inert or protective layer that enhances its stability during passage through the gastrointestinal tract. In the context of probiotics used in baked goods, preserving their viability during baking and stabilizing them for delivery to the human gut are imperative to ensure their health benefits.

[0042] Encapsulation is positioned as an essential alternative for the implementation of probiotics in food, ensuring their effective arrival at the site of action, protecting them from adverse environmental conditions such as heat, light, air and humidity.

[0043] Despite the diversity of encapsulation methods, such as emulsion, liposome formation, electrospinning and spray drying, the latter stands out for being an industrially scalable, fast, low-cost operating technique that does not require solvents, which defines it as a sustainable option in the industrial encapsulation process.

[0044] Spray drying is a widely used technology in the food industry for producing powdered ingredients such as instant coffee and infant formula. The advantages of spray drying include its robustness, scalability, and high yields. Figure 1 presents a detailed diagram of the spray drying process, highlighting the key components of specialized equipment for this technique.

[0045] First, the container holding the solution to be spray-dried is highlighted. This fluid, crucial for the method's success, is carefully atomized in the drying chamber using a specialized nozzle. Strategically positioned near the solution, the atomizing nozzle plays a central role in transforming it into tiny droplets.

[0046] In the next stage, a stream of hot air is directed toward the tiny droplets formed by the atomized solution. The main purpose of this hot air stream is to remove moisture from the droplets, bringing them to a dry state. The proximity of the hot air outlet to the atomizing nozzle ensures efficient and rapid drying.

[0047] Finally, in the last section of the drying chamber, the dry product is obtained. It is important to note that the diameter of the resulting particles is typically similar to the opening of the spray nozzle. This precise control of particle size is essential for obtaining a homogeneous, high-quality final product.

[0048] This diagram provides a detailed view of the spray drying process, highlighting the importance of each component in obtaining dry products in an efficient and controlled manner.

[0049] The main objective was to develop a powdered ingredient that would allow probiotics to be incorporated into a hot beverage, minimizing the amount (1 to 4 g) needed to achieve the required CFUs for the corresponding claim. The wall materials provide the probiotic with a protective barrier, giving it thermostability and stability in acidic pH environments. These materials are safe for human consumption and are used in the food industry.

[0050] Traditionally, a large quantity of probiotics must be added to food because they cannot survive processing temperatures above 37°C, and many also cannot withstand the acidic environment of the stomach. Technology now allows probiotics to survive temperatures above 100°C and acidic pH conditions, enabling a greater number to reach the consumer's intestine and exert their beneficial effects.

[0051] The following stages were considered for carrying out the process:

[0052] 1) Development of a Functional Ingredient. Generate an optimized formulation that allows for stable encapsulation at the temperature of a hot beverage, and that withstands being in an aqueous environment while maintaining its thermostability properties. The aim is to obtain more than 1x10 6UFC per milliliter, with the least amount of ingredient incorporation.

[0053] 2) Hot beverage test generation. Evaluate the behavior of the formulation in a hot beverage from 70°C to 90°C until it cools to room temperature (30°C).

[0054] 3) In vitro digestion evaluation of the functional ingredient developed in a hot beverage. Evaluate the quantity of viable microorganisms by performing an in vitro digestion assay to determine the number of viable microorganisms that would potentially reach the human intestine.

[0055] 4) Comparison with commercial technology. Since there is a commercial coffee that is labeled as having added probiotics, it was compared with respect to the proposed invention, in terms of CFU per milliliter, and the appearance of the final product (coffee).

[0056] 5) Evaluation of stability and viability in aqueous solution at room temperature (approximately 35 °C) for at least 3 months.

[0057] 6) Evaluation of stability and viability in frozen aqueous solution at approximately -20 °C for at least 3 months.

[0058] Figure 2 illustrates the encapsulation methodology, where the probiotic microorganism is located in the center, and the shell is composed primarily of protein, a prebiotic polymer, and a crosslinking agent. This methodology and formulation ensure a functional ingredient that allows the addition of a viable probiotic and a prebiotic to hot beverages.

[0059] Figure 2 shows the Microencapsulation Production Process Diagram. It consists of the following steps:

[0060] A) The ingredients are weighed according to the optimized formulation: To encapsulate 1 probiotic of a size between 0.5 qm and 3 qm

[0061] • 20 to 40% Inulin (prebiotic)

[0062] • 2 to 7% High amylose starch (Hylon) (prebiotic)

[0063] • 50 to 70% Pea protein (globular protein)

[0064] • 2 to 8% Maltodextrin (cross-linker)

[0065] B) Potable water is added to the coating ingredients. The amount of water depends on the spray dryer used and the amount of dissolved solids it can tolerate. In the case of the spray dryer used, the solids are equivalent to 9 to 14% of the solution.

[0066] C) Stir the ingredients in the water until a homogeneous solution is formed

[0067] D) The probiotics are added to the solution while stirring to maintain a homogeneous mixture. In this case, 2x10 were added. 12 CFU / g of Bacillus clausi (small, spore-forming probiotic about the size of half a millimeter) or 2x10 12CFU / g of Lactobacillus (large, non-spore-forming probiotic, larger than a pea)

[0068] E) This solution, which is under constant agitation, feeds the spray drying equipment under the conditions shown in the diagram (inlet temperature of 120 °C, outlet temperature of 60 °C, at a pressure of 0.17 MPa).

[0069] F) Once the entire solution has passed through the equipment, a dry white powder is obtained. This is the encapsulated prebiotic and protein coating, which contains the viable microorganism at its core. This white powder is weighed and stored in bags to prevent it from becoming damp.

[0070] G) The powder is stored, preferably at 4 °C and taking care that it does not get wet.

[0071] The results obtained are:

[0072] STAGE 1: Development of Functional Ingredient.

[0073] Objective: To develop a formulation that allows for temperature-stable encapsulation of a hot beverage and that can withstand exposure to an aqueous environment while maintaining its thermostability properties. The aim is to obtain a ratio greater than 1:1 O 6 UFC per milliliter, with the least amount of ingredient incorporation.

[0074] The aim was to generate from two initial formulations, to create, through spray drying, an encapsulation that would remain stable in water and that would confer thermostability properties to a probiotic.

[0075] Bacillus clausii was chosen as the strain to be encapsulated. Its properties, in addition to maintaining and restoring the microbiota of the digestive system and supporting the immune system, have been reported to maintain intestinal permeability, and therefore improve nutrient absorption. It also reduces the sensation of bloating, flatulence, diarrhea or constipation and relieves gastrointestinal pain and inflammation (Muhammad et al., 2018).

[0076] The results of the two initial formulations tested are listed below. a) GHOST formulation:

[0077] • 2 to 75% innulin

[0078] • 50 to 70% Pea protein

[0079] • 2 to 8% Maltodextrin

[0080] • 10 units (4x10 12 UFC / g) b) G-HYLON formulation:

[0081] • 2 to 75% Hylon (high amylose starch)

[0082] • 50 to 70% Pea protein

[0083] • 2 to 8% Maltodextrin

[0084] • 10 units (4x10 12 UFC / g)

[0085] The following conclusions were drawn from these formulations:

[0086] The first formulation is thermostable with 100% efficiency; however, a loss of probiotics (33%) is observed. This is because the protective layer of the probiotic dissolves rapidly in water, and therefore a percentage of these probiotics are affected by the water temperature.

[0087] The second formulation also exhibited thermostability, as it successfully protected a portion of the probiotic (65%). However, approximately 35% of the probiotic was lost due to temperature. Since this percentage loss was higher than that of the first formulation, it was concluded that the first formulation was more effective. This conclusion was verified through beverage monitoring and is explained in stage 2.

[0088] Thanks to these two initial formulations, the formulation was optimized by testing secondary formulations: one with a higher proportion of Hylon (high amylose starch) and the other with a higher proportion of inulin. The optimized formulation was as follows: c) BOOSTING formulation:

[0089] • 30% Inulin 5% Hylon

[0090] 60% Pea Protein

[0091] • 5% Maltodextrin

[0092] • 5 units (2x10 12 UFC / g)

[0093] With the optimized BOOSTING formulation, the initial amount of probiotics was reduced by half, and there were significantly fewer losses in the solution simulating the hot beverage at 90°C (11%), demonstrating that it is thermostable and stable in hot aqueous solution that cools to room temperature.

[0094] In this stage, the prototype of the functional ingredient was obtained, which, when added 1g to the beverage, is capable of providing around 6.90x10 8 UFC / mL of probiotics and therefore be labelled as such.

[0095] STAGE 2: Hot beverage test generation

[0096] The objective of this stage was to generate a test in a hot beverage. To evaluate the behavior of the formulation in a hot beverage from 90 °C until it cools to room temperature (30 °C).

[0097] In this stage, the time a beverage stays hot was evaluated; this was determined by measuring the temperature at specific times, as shown in the diagram in Figure 3.

[0098] Figure 3. Procedure diagram for the hot beverage test:

[0099] A) Distilled water was brought to a temperature of 90 °C. It was kept under constant stirring and with a thermometer throughout the experiment.

[0100] B) Once 90 °C was reached, the heat source was turned off and 1 g of the developed ingredient was added. The temperature was measured and 1 ml aliquots were taken at the following time intervals: 0 seconds, 15 seconds, 30 seconds, 60 seconds (1 minute), 5 minutes, 10 minutes, 20 minutes, and 30 minutes.

[0101] C) Each aliquot was plateped in duplicate at three different dilutions to determine the number of viable Colony Forming Units per milliliter at each time interval.

[0102] The laboratory test was observed as shown in Figure 4. It was conducted in a fume hood to prevent environmental contamination. This ensures that the microorganism cultivated in the aliquot at each time point is indeed the probiotic incorporated into the beverage through the ingredient formulated in this project.

[0103] According to the tests performed, it was determined that 30 minutes was sufficient for the beverage to return to room temperature (35 to 30 °C). The results of this monitoring are shown in the following table. Time Temperature (°C)

[0104] The 90s

[0105] 15s 87

[0106] 30s 85

[0107] 60s 80

[0108] 5m 71

[0109] 10m 67

[0110] 20m 49

[0111] 30m 35

[0112] It was determined that the 1g addition amount is similar to other additives commonly added to hot beverages, such as sweeteners or cream substitutes. Furthermore, up to 4g could be added, based on the amount typically used to increase the probiotic content per beverage, if the intention were to increase the amount of probiotics per drink.

[0113] The results of the three formulations in the hot beverage test were performed in triplicate and the results are shown below.

[0114] Time (s) GHOST G-Hylon Boosting (CFU / mL)

[0115] (CFU / mL) (CFU / mL)

[0116] It is reaffirmed that the amount of probiotics contained in the hot beverage, by adding only 1g of the functional ingredient, is significantly higher than 1x10 6 CFU / mL which is required to label the product as containing probiotics, according to current Mexican Standards.

[0117] From the GHOST formulation, it is concluded that it is stable at high temperatures, since there were no variations in the initial concentration throughout the experiment, although the loss of viability of the probiotic was 33%.

[0118] From the G-Hylon formulation, it is concluded that it is less stable at a temperature of 90°C, as abrupt variations of the probiotic are observed, which apparently increase when the beverage is cooled, and there is a loss of viability of 35% of the initial probiotic.

[0119] The Boosting formulation was found to be stable at high temperatures, as it maintained the probiotic concentration throughout the experiment. It was also confirmed that the highest concentration of stable probiotics was achieved with the Boosting formulation. This was because it started with half the probiotic concentration and because the probiotic viability loss was approximately 11%.

[0120] STAGE 3: In vitro digestion evaluation of the developed ingredient in a hot beverage

[0121] The aim was to evaluate the number of viable microorganisms by performing an in vitro digestion assay to determine the number of viable microorganisms that would potentially reach the human intestine.

[0122] The samples were digested in triplicate. One gram of each sample was subjected to:

[0123] • ORAL PHASE: Digestion with porcine amylase at pH 4 for 20 minutes

[0124] • GASTRIC PHASE: Digestion with porcine pepsin at pH 2 for 120 minutes

[0125] • INTESTINAL PHASE: Digestion with porcine pancreatin at pH 9.1 for 120 minutes

[0126] This experiment was performed with the details contained in Figure 6.

[0127] After digestion, the cell viability of the samples was measured, obtaining the following results shown in the following table.

[0128] In vitro digestion CFU / ml count

[0129] Initial 1.51X10 8

[0130] Oral phase 3.80X10 7

[0131] Gastric phase 2.02X10 7

[0132] Intestinal phase 1.09X10 7

[0133] The results show that the concentration obtained in the beverage is sufficient to qualify it as an ingredient in probiotic drinks. Regarding digestion, the microorganism withstood in-vitro digestion (Figure 6), and the number of microorganisms reaching the simulated intestinal phase exceeded 1:1. 6 CFU per milliliter as stipulated in Mexican regulations. STAGE 4: Comparison with commercial technology

[0134] There is a commercial coffee fortified with probiotics (https: / / 7-eleven.com.mx / cafe-select / sabe-bien-te-sientes-mejor / ), but we don't know the initial probiotic content. However, since it's labeled as probiotic, it's assumed to contain at least 1 x 10⁻⁶ CFU / mL. 6UFC / ml of coffee.

[0135] The coffee was analyzed to verify its probiotic content and compare it with the technologies developed. The results are shown in the following table.

[0136] Technology Counting CFU / ml

[0137] GHOST 3.38xl0 8

[0138] G-Hylon 1.16xl0 9

[0139] Boosting 2.23X10 8

[0140] BC Commercial Coffee 30 1.99xl0 6

[0141] As can be seen using the same counting method, the three developed technologies were superior in the quantity of probiotics by two orders of magnitude. This means that while the quantity of probiotics in commercial BC30 coffee was 1,000,000 CFU / ml, in the developed technologies we are at the order of 200,000,000 CFU / ml. This is achieved by adding, in the case of Boosting, only 1g of the encapsulated product to 250 ml of coffee.

[0142] Another important characteristic worth mentioning is the effect of adding technologies to coffee on its sensory properties. It was observed that commercial BC30 coffee has a significantly more viscous and opaque consistency compared to the control commercial coffee (without probiotics). In fact, due to this milky consistency, it would not be possible to add it to black coffee. In the case of Boosting, due to its wall material composition, adding only 1g of the encapsulated product allows it to be added to black coffee without affecting its sensory properties. See Figure 7.

[0143] As shown in Figure 7, commercial BC30 coffee has a significantly higher viscosity than the coffee with milk from the same establishment, as the coffee droplet does not deform. Its consistency is heavier and denser, possibly due to the high concentration of probiotic added, which increases the final product's density (overdosing), and also because this technology uses starch as a diluent excipient, not as an encapsulant.

[0144] Furthermore, this same image shows that the Boosting technology can be added to black coffee without affecting its color (translucent coffee) or consistency (viscosity similar to water). This is because the amount of encapsulated material added to the coffee is very small (1g per 250ml), and also because its wall material is not only starch, but also contains a proportion of protein, inulin, and hylon, as described in STAGE 1.

[0145] Finally, its behavior was compared in an in-vitro digestion simulation to approximate how it would behave in the human digestive system. This was developed as described in STAGE 3. The results are described in the following table:

[0146] Results of in-vitro digestion tests on the functional ingredient Boosting and commercial BC30 coffee

[0147] In vitro digestion boosting (CFU / ml) Commercial BC3o coffee

[0148] Initial 1.51X10 8 1.83X10 6

[0149] Oral phase 3.80X10 7 1.62X10 5

[0150] Gastric phase 2.02X10 7 1.75X10 4

[0151] Intestinal phase 1.09X10 7 1.00X10 4

[0152] According to the results obtained, Boosting loses one order of magnitude during digestion; however, the BC30 commercial coffee technology loses two orders of magnitude during the CFU / ml process. This may be because the organism in the BC30 commercial coffee technology is not encapsulated.

[0153] The following table presents a comparison of both technologies.

[0154] Comparison summary of Boosting and BC30 Commercial Coffee

[0155] STAGE 5: Comparison of stability in aqueous solution at room temperature for at least 3 months.

[0156] The objective of this stage was to evaluate the stability and viability of the probiotic in a beverage stored at room temperature for an extended period (approximately 3 months). The evaluation was performed as follows: 1 gram of the Boosting encapsulation was dissolved in an aqueous solution at approximately 35 °C and stored under ambient light and temperature conditions. Each week, a 1-milliliter aliquot was taken and used to assess the viability of the beverage by plate count at 3 different dilutions with 2 replicates. The ability of the Boosting encapsulation to withstand freezing and thawing of the beverage and to remain viable at room temperature was also evaluated.This test was performed using 250 mL of aqueous solution with 1 gram of booster added. The solution was stored at approximately 35 °C and then frozen at -10 °C for one day. After thawing, viability was measured. These two conditions (room temperature only and room temperature plus freezing) were compared to the unencapsulated probiotic. The unencapsulated probiotic was diluted in the same proportion in 250 mL of aqueous solution and maintained at approximately 35 °C. A 1 mL aliquot was taken weekly to perform viability counts per plate at three dilutions with two replicates.

[0157] As shown in Figure 9, the Boosting formulation maintained probiotic viability above 100,000,000 Colony Forming Units per milliliter for more than 6 weeks. This was true both at room temperature and under room temperature conditions followed by freezing. This is achieved because the encapsulation keeps the probiotic isolated from the beverage, thus keeping its cellular metabolism dormant and preventing activation by the temperature, thereby maintaining its viability for a longer period. As can be seen, the unencapsulated probiotic did not survive more than 4 weeks under these temperature conditions. This is because, in the aqueous solution at room temperature (35 °C), the bacteria's metabolism becomes active, and without available food, the probiotic loses viability over time.

[0158] STAGE 6: Comparison of stability in aqueous solution kept frozen for at least 3 months.

[0159] The objective of this stage was to evaluate the stability and viability of the probiotic in a beverage stored at freezing temperatures for an extended period (approximately 3 months). The evaluation was performed as follows: 1 gram of the Boosting encapsulation was dissolved in an aqueous solution at approximately 35 °C, then frozen and maintained under frozen conditions (approximately -20 °C). Each week, a 1-milliliter aliquot of the added aqueous solution was thawed and used to assess the viability of the beverage by plate count at 3 different dilutions and with 2 replicates.

[0160] The ability of the Boosting encapsulation to withstand freezing and thawing of the beverage and maintain its freezing temperature was also evaluated. This test was performed on 250 milliliters of aqueous solution containing 1 gram of Boosting, which was stored at approximately -20 °C. Each sample was thawed, and a 1-milliliter aliquot was taken for counting. It was then refrozen and held until the following week, when it was thawed again for another 1-milliliter aliquot to be taken for plate counting. The counts were performed in the same way as under the other conditions (plate counting at Y1).

[0161] 3 different dilutions and with 2 replicates).

[0162] As a control for both experiments, the results were compared with the unencapsulated probiotic, which was also diluted in 250 milliliters of aqueous solution and kept frozen at -20°C. This solution was thawed, an aliquot was taken for plate viability counting, and then refrozen until the following week, when it was thawed again for another plate count. The counts were performed in the same way as in the other conditions (plate count at 3 different dilutions and with 2 replicates).

[0163] As shown in Figure 10, the Boosting formulation maintained probiotic viability above 10,000,000 Colony Forming Units per milliliter for more than 6 weeks. This was achieved both under frozen conditions and under conditions of freezing followed by thawing for counting and subsequent refreezing. This sustained viability is due to the encapsulation's ability to protect the probiotic's cell membrane from damage caused by the formation of water crystals.As can be seen, the unencapsulated probiotic does not survive more than 3 weeks under these temperature conditions. This is because the aqueous solution, when frozen, forms water crystals that decrease the probiotic's viability by damaging the integrity of the cell membrane. When thawed, the crystals melt, but the probiotic's cell membrane is unable to regenerate and loses viability, therefore it is unable to grow even under optimal plate culture conditions.

[0164] Optimal formulations were obtained for microencapsulating Bacillus clausii to be incorporated into hot beverages at average temperatures of 90°C.

[0165] The three formulations presented in this project are capable of ensuring a quantity greater than 1x10 6 UFC / mL in a beverage.

[0166] The boosting formulation was tested as the one that manages to maintain the thermostability properties in aqueous solution, with a lower amount of initial probiotics.

[0167] The optimal dose for incorporation into the beverage is 1-4 g; this range reliably ensures a number significantly higher than 1x10 6 UFC / mL that regulates Mexican laws for declaring probiotics.

[0168] Comparing Boosting technology to commercial coffee, it is observed that with a lower dosage, it is possible to achieve 2 orders of magnitude more CFU / mL, without affecting the sensory properties of the final product.

[0169] Current commercial technology has significant changes in its sensory properties due, possibly, to overdosing of the ingredient.

[0170] In in vitro digestion tests, the BC30 commercial coffee technology loses significantly more CFU during the process (2 orders of magnitude). This may be because the organism in the BC30 commercial coffee technology is not encapsulated.

[0171] Boosting technology is able to maintain the viability of the probiotic long-term (more than 3 months) when added to an aqueous solution and kept under ambient light and temperature conditions (approximately 35 °C). This characteristic was not observed in the unencapsulated probiotic, which loses its viability in approximately 4 weeks.

[0172] Finally, Boosting technology maintains the long-term viability of the probiotic by adding it to an aqueous solution that is subsequently frozen and stored at approximately -20°C. Boosting technology also allows the probiotic to remain viable despite prolonged freezing and thawing cycles. It was observed that unencapsulated probiotics lose their long-term viability under freezing conditions because water crystals rupture the cell membrane, preventing regeneration.

Claims

CLAIMS 1. A probiotic powder encapsulation for beverages characterized in that it comprises: 1 to 4g of probiotic necessary to achieve the CFU required for its realization, 20 to 40% Inulin, 2 to 7% high amylose starch (Hylon), 50 to 70% pea protein, 2 to 8% maltodextrin; wherein the size of the encapsulation is between 0.5 qm to 3 qm.

2. The probiotic powder encapsulation for beverages according to claim 1, wherein its wall material is comprised of 1 or more prebiotics, 1 crosslinker and 1 globular protein, with an optimal formulation, • 20 to 40% Inulin (prebiotic) • 2 to 7% High amylose starch (Hylon) (prebiotic) • 50 to 70% Pea protein (globular protein) • 2 to 8% Maltodextrin (cross-linker) 3. The probiotic powder encapsulation for beverages according to claim 2, wherein a microcapsule is comprised in which: the prebiotic optionally comprises a non-digestible carbohydrate such as resistant starch or a non-starch polysaccharide such as hemicellulose, pectin or gum; an oligosaccharide such as a galactooligosaccharide, fructoligosaccharide or maltoligosaccharide; lactulose; or inulin; and / or the probiotic and the prebiotic provide a symbiotic, crosslinked with an oligosaccharide such as lactulose, inulin, dextrin or maltodextrin.

4. The probiotic powder encapsulation for beverages according to claim 3, wherein the wall materials are selected from one or a combination of more than: globular protein, whether vegetable or animal, such as soy protein, rice protein, wheat protein, corn protein, whey protein, casein, etc.; preferably pea protein.

5. The probiotic powder encapsulation for beverages according to claim 4, wherein it is able to withstand being added to hot beverages (70 to 90 °C) and maintain 89% of the initial viability.

6. The probiotic powder encapsulation for beverages according to claim 5, wherein it is also able to withstand being added to a beverage and remain viable for more than 3 months at room temperature (approximately 35 °C) 7. The encapsulation of probiotic powder for beverages in accordance with the claim 6, wherein it is also able to resist the formation of water crystals caused by the freezing of the beverage and remain viable for more than 3 months under freezing conditions (-20 °C) 8. The probiotic powder encapsulation for beverages according to claim 7, wherein it is also able to resist the repeated formation and degradation of water crystals caused by freezing and thawing of the beverage and remain viable for more than 3 months.

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