Probiotic Microparticles with Plant-Based Walls: Production Processes and Uses

Description

/ 18 Probiotic microparticles with a plant-based wall: production processes and uses. Field of invention

[001] The present invention relates to probiotic microparticles obtained through the application of plant-based wall materials, more specifically yacon potato flour and green banana flour. The present invention also relates to the production processes of the microparticles and the use of plant-based wall materials for the microencapsulation of probiotics. The selection of the wall material used is of fundamental importance in microencapsulation, as it must protect the probiotic microorganism from adverse conditions during food processing and storage stages, not interfere with the sensory characteristics of the product, exhibit high stability and good solubility. Vegetable flours such as yacon potato flour and green banana flour are important alternatives for application as wall material for the microencapsulation of probiotics.The present invention is applied to the food sector, more specifically in the technology of functional products. Fundamentals of the invention

[002] In addition to providing nutrition, food can also influence the health and well-being of the consumer. In recent years, changes in eating habits have led consumers to seek healthier alternatives. This behavior has stimulated the food industry to develop products aimed at a public increasingly concerned with health and well-being. In this context, foods with functional appeal have emerged, which are those capable of providing beneficial health effects beyond basic nutrition. Petition 870250014841, dated 24 / 02 / 2025, page 10 / 36 / 18

[003] Among functional foods are foods fortified with probiotic microorganisms which, when consumed regularly and in sufficient quantities, are capable of promoting health benefits for the consumer, according to ARAYA, M., MORELLI, L., REID, G., SANDERS, ME, STANTON, C., PINEIRO, M., & BEN EMBAREK, P. in the “Guidelines for the evaluation of probiotics in food” published by the “Joint FAO / WHO Working Group Report on drafting guidelines for the evaluation of probiotics in food” in 2002. However, the vast majority of products containing probiotics are dairy-based, such as fermented milk, yogurt, dairy desserts, cheeses, and ice cream. However, the probiotic food market demands other non-dairy-based products in order to meet the needs of consumers who are lactose intolerant, allergic to milk proteins, or vegetarians.

[004] During the development of probiotic foods, steps such as formulation, processing, and storage of products must favor the survival of microorganisms. Similarly, technologies and the food matrix must protect probiotic cells against external stress factors, maintaining the viability of the cultures. Furthermore, once the food is consumed, the steps and effects generated during passage through the gastrointestinal system, such as the acidic pH of the stomach, the presence of bile salts, and digestive enzymes, can also affect the viability of microorganisms.

[005] One alternative to minimize damage to probiotics during processing and incorporation into different products is the microencapsulation technique, a strategy widely known for its ability to improve the physical protection and stability of probiotics in food. Among existing techniques, spray drying stands out due to the high flexibility of the process, low cost of microparticle production, and the possibility of large-scale production compared to other techniques. The principle of the technique involves spraying a solution containing Petition 870250014841, dated 24 / 02 / 2025, page 11 / 36 3 / 18 The wall material (or encapsulant) and the probiotics are placed in parallel with a stream of heated air (> 100 °C) inside the drying chamber to form the microparticle powder. The hot air causes the water in the solution to evaporate rapidly, forming the microparticle powder that encapsulates and protects the probiotics.

[006] The selection of the wall material used is of fundamental importance in microencapsulation, as it must protect the probiotic microorganism, not interfere with the sensory characteristics of the matrix, exhibit high stability and good solubility. Furthermore, the wall material must be safe for consumption when added to food, must be cost-effective, and highly available for application by the food industry. Microencapsulation using the spray drying technique allows working with different wall materials, thus increasing the possibilities for the development of new efficient microparticles.

[007] Among the most commonly used wall materials in the spray drying microencapsulation technique, water-soluble polymers such as modified starch, whey protein, maltodextrin, gum arabic, and their combinations stand out. These wall materials, despite helping to maintain the stability of probiotics, have some limitations. For example, when incorporating probiotics into products intended for lactose-free or gluten-free diets, some materials are unsuitable due to the risk of contamination.

[008] In recent years, there has been a growing search for new wall materials derived from natural sources or from the reuse of by-products from the food industry, which are readily available, safe and low-cost, thus enabling the use of these microparticles in different products developed by the industry.

[009] Thus, in this patent application, the present invention seeks to meet the demand for new wall materials from natural sources that are low cost and highly available, and at the same time Petition 870250014841, dated 24 / 02 / 2025, page 12 / 36 / 18 efficient methods for promoting the protection of sensitive compounds such as probiotic microorganisms, using yacon potato and green banana flours. Furthermore, the present invention aims to offer an efficient alternative for incorporating probiotics into different food matrices while maintaining the viability of the probiotic and preserving the characteristics of the product.

[0010] Soy protein was used for spray-drying microencapsulation of curcumin nanocomplexes by CHEN, FP, LIU, LL, & TANG, CH in the article "Spray-drying microencapsulation of curcumin nanocomplexes with soy protein isolate: Encapsulation, water dispersion, bioaccessibility and bioactivities of curcumin" published in the journal Food Hydrocolloids, volume 105, p. 1-10, in 2020; carrot fiber was also used by NISHAD, Jyoti; MIR, Shabir Ahmad; WALIA, Kanika in the article "Optimization of spray drying technology for sugarcane juice using natural and synthetic encapsulating agents" published in the journal Sugar tech, v. 21, p. 749-755 in the year 2019. Orange juice by-products have also been used as wall material by Kaderides, K., & Goula, AM in the article "Encapsulation of pomegranate peel extract with a new carrier material from orange juice by-products," published in the Journal of Food Engineering, volume 253, p. 1-13 in the year 2019.Although many studies exist on new wall materials for microencapsulation, none address the use of vegetable flours, such as yacon potato flour and green banana flour, as wall materials for the microencapsulation of probiotics by spray drying, as proposed by the present invention. In addition to their ability to form microparticles, yacon potato and green banana flours also showed good encapsulation efficiency, resistance to different thermal conditions, and pH variations. Brief description of the drawings

[0011] The figures below illustrate the present invention in a non-limiting manner: Petition 870250014841, dated 24 / 02 / 2025, page 13 / 36 5 / 18 Figure 1: Presents the thermal resistance graph of probiotics, comparing free and microencapsulated probiotics in different concentrations of wall materials, using yacon potato flour. The probiotics were subjected to thermal conditions of rapid pasteurization (72 °C / 15 s) and slow pasteurization (63 °C / 30 min) to verify the survival rate (%). Means followed by the same letter in the same thermal process do not differ by Tukey's test at 5% probability.Free cell composed of 100% L rhamnosus GG pellet (Black); A (Red): microparticle containing 16.67% maltodextrin, 3.33% gelatin and 5% L rhamnosus GG pellet; B (Green): microparticle containing 12.5% ​​maltodextrin, 2.5% gelatin, 5% yacon potato flour and 5% L rhamnosus GG pellet; C (Yellow): microparticle containing 11.11% maltodextrin, 2.22% gelatin and 6.67% yacon potato flour and 5% L rhamnosus GG pellet; D (Blue): microparticle containing 10% maltodextrin, 2% gelatin and 8% yacon potato flour and 5% L rhamnosus GG pellet. - Figure 2: Presents the resistance graph of microparticles containing L. rhamnosus GG when subjected to different pH conditions, being basic (7.4) and acidic (2.0), in order to verify the survival rate (%) of the probiotics. Means followed by the same letters at the same pH do not differ by Tukey's test at 5% probability. Free cell composed of 100% L. rhamnosus GG pellet (Black); A (Red): microparticle containing 16.67% maltodextrin, 3.33% gelatin and 5% L. rhamnosus GG pellet; B (Green): microparticle containing 12.5% ​​maltodextrin, 2.5% gelatin, 5% yacon potato flour and 5% L. rhamnosus GG pellet; C (Yellow): microparticle containing 11.11% maltodextrin, 2.22% gelatin, 6.67% yacon potato flour and 5% L rhamnosus GG pellet; D (Blue): microparticle containing 10% maltodextrin, 2% gelatin, 8% yacon potato flour and 5% L rhamnosus GG pellet. Petition 870250014841, dated 24 / 02 / 2025, p. 14 / 36 / 18 - Figure 3: Shows micrographs of microparticles containing L. rhamnosus GG (C) encapsulated with yacon potato flour, obtained by spray drying. The images demonstrate that the microparticles have a spherical shape, without cracks or pores, indicating efficient and stable encapsulation. Figure 4: Presents the survival graph of the probiotic L. rhamnosus GG present in the selected microparticle with the highest resistance (11.11% maltodextrin, 2.22% gelatin, 6.67% yacon potato flour and 5% L. rhamnosus GG pellet - C) under different storage temperatures (-18, 8 and 25 °C) over 120 days of storage. Different uppercase letters indicate a significant difference (p<0.05) between samples at the same time. Different lowercase letters indicate a significant difference (p<0.05) over time for each sample. Figure 5: Presents the survival rate graph of L. rhamnosus GG under cell-free and microencapsulated conditions in the microparticle developed under simulated in vitro gastrointestinal conditions (gastric phase - stomach, enteric phase I - small intestine and enteric phase II - large intestine). Cell-free (Gray); Microparticle (Blue): containing 11.11% maltodextrin, 2.22% gelatin, 6.67% yacon potato flour and 5% L. rhamnosus GG pellet. Different uppercase letters indicate a significant difference (p<0.05) between samples in the same phase. Different lowercase letters indicate a significant difference (p<0.05) for the same sample across phases. - Figure 6: Presents the viability graph and survival rate of the free and microencapsulated probiotic when added to a food matrix such as pitaya jelly under different storage conditions (8 and 25 °C) for 60 days. M8 (Dark Pink): 100 g of jelly; 10 g of microparticles containing L. rhamnosus GG stored at 8°C; M25 (Light Pink): 100 g of jelly; 10 g of microparticles containing L. rhamnosus GG stored at 25 °C; Petition 870250014841, dated 24 / 02 / 2025, page 15 / 36 / 18 F8 (Dark Purple): 100 g of jelly; 1 g of L. rhamnosus GG cell-free stored at 8 °C; F25 (Light Purple): 100 g of jelly; 1 g of L. rhamnosus GG cell-free stored at 25 °C. ND: not determined. Different uppercase letters indicate a significant difference (p<0.05) between samples in the same phase. Different lowercase letters indicate a significant difference (p<0.05) for the same sample across phases. - Figure 7: Presents the thermal resistance graph of the free and microencapsulated probiotic in different concentrations of wall materials using green banana flour when subjected to different thermal conditions, such as rapid pasteurization (72 °C / 15 s) and slow pasteurization (63 °C / 30 min), in order to verify the survival rate (%) of the probiotics. Means followed by the same letter in the same heat treatment do not differ by Tukey's test at 5% probability. Free cell composed of 100% L. rhamnosus GG pellet (Black); E (Red): microparticle containing 16.67% maltodextrin, 3.33% gelatin and 5% L. rhamnosus GG pellet; F (Green): microparticle containing 12.5% ​​maltodextrin, 2.5% gelatin, 5% green banana flour and 5% L. rhamnosus GG pellet; G (Yellow): microparticle containing 15% maltodextrin, 1% gelatin, 4% green banana flour and 5% L pellet.rhamnosus GG; H (Blue): microparticle containing 15% maltodextrin, 2% gelatin and 3% green banana and 5% L. rhamnosus GG pellet. Figure 8: Presents the resistance graph of the free and microencapsulated probiotic using green banana flour when subjected to different pH conditions, such as basic pH (7.4) and acidic pH (2.0) in order to verify the survival rate (%) of the probiotics. Means followed by the same letter at the same rupture pH do not differ by Tukey's test at 5% probability. Free cell composed of 100% L. rhamnosus pellet GG (Black); E (Red): microparticle containing 16.67% maltodextrin, 3.33% gelatin and 5% L. rhamnosus pellet. Petition 870250014841, dated 24 / 02 / 2025, page 16 / 36 / 18 rhamnosus GG; F (Green): microparticle containing 12.5% ​​maltodextrin, 2.5% gelatin, 5% green banana flour and 5% L. rhamnosus GG pellet; G (Yellow): microparticle containing 15% maltodextrin, 1% gelatin and 4% green banana flour and 5% L. rhamnosus GG pellet; H (Blue): microparticle containing 15% maltodextrin, 2% gelatin and 3% green banana and 5% L. rhamnosus GG pellet. - Figure 9: Presents micrographs of microparticles using green banana flour containing L. rhamnosus GG obtained by spray drying (F) and demonstrates that the microparticles obtained have a spherical shape with no cracks or pores. Figure 10: Shows the survival graph of the probiotic L. rhamnosus GG present in the selected microparticle with the highest resistance (12.5% ​​maltodextrin, 2.5% gelatin, 5% green banana flour and 5% L. rhamnosus GG pellet (F)) under different storage temperatures (-18, 8 and 25 °C) over 120 days of storage. Different uppercase letters indicate a significant difference (p<0.05) between samples at the same time. Different lowercase letters indicate a significant difference (p<0.05) over time for each sample. Figure 11: Presents the survival rate (%) graph of L. rhamnosus GG under cell-free and microencapsulated conditions in a microparticle developed with green banana flour (F) under simulated in vitro gastrointestinal conditions (gastric phase - stomach, enteric phase I - small intestine and enteric phase II - large intestine). Cell-free (Blue); Microparticle (Yellow): containing 12.5% ​​maltodextrin, 2.5% gelatin, 5% green banana flour and 5% L. rhamnosus GG pellet. Different uppercase letters indicate a significant difference (p<0.05) between samples in the same phase. Different lowercase letters indicate a significant difference (p<0.05) for the same sample across phases. Petition 870250014841, dated 24 / 02 / 2025, page 17 / 36 9 / 18 Figure 12: Presents the viability graph of free and microencapsulated L. rhamnosus GG added to mass-produced banana candy. CL (Brown): mass-produced banana candy containing free L. rhamnosus GG; M (Yellow): mass-produced banana candy containing microencapsulated L. rhamnosus GG. Initial: before simulation; FG: gastric phase; FE-I: enteric phase I; FE-II: enteric phase II; Different uppercase letters indicate a significant difference (p<0.05) between samples in the same phase. Different lowercase letters indicate a significant difference (p<0.05) for the same sample across phases. Description of the invention

[0012] The present invention relates to probiotic microparticles obtained through the use of plant-based wall materials, to the processes for producing the microparticles and to the use of plant-based wall materials for the microencapsulation of probiotics.

[0013] The plant-based wall materials used in the present invention refer to yacon potato flour and green banana flour.

[0014] The production process for yacon potato flour-based microparticles comprises the following steps:

[0015] The microparticle is prepared using 10 to 16.67% maltodextrin, 2 to 3.33% gelatin, and 5 to 8% yacon potato flour. These materials are dissolved in distilled water and homogenized in a magnetic stirrer for 1 hour. Then, the resulting encapsulating solutions, containing maltodextrin, gelatin, and yacon potato flour, are filtered and autoclaved at 121 °C for 15 minutes. After cooling to between 45 and 50 °C, the cell suspension of the microorganism of interest is added to the encapsulating solution at a concentration of 5% relative to the amount of wall material (w / w) and homogenized for 10 minutes at 25 °C. To obtain the microparticles, the encapsulating solutions containing the probiotic are dried in a spray dryer. Petition 870250014841, dated 24 / 02 / 2025, page 18 / 36 10 / 18 using an inlet temperature of 130 °C and an outlet temperature of 73 °C, with a feed flow rate of 0.25 L / h and a drying air flow rate of 3.00 m³ / min. After obtaining the powdered microparticles, they were stored under refrigeration at 8 °C.

[0016] The production process for microparticles made from green banana flour comprises the following steps:

[0017] To prepare the microparticle based on green banana flour, 12.5 to 15% maltodextrin, 1 to 2.5% gelatin, and 3 to 5% green banana flour are used. These materials are dissolved in distilled water and homogenized for 1 hour at 25 °C. The resulting encapsulating solutions, containing maltodextrin, gelatin, and green banana flour, are filtered and autoclaved at 121 °C for 15 minutes. After cooling to between 45 and 50 °C, the cell suspension of the microorganism of interest is added to the encapsulation solution at a concentration of 5% relative to the amount of cell wall material and homogenized for 10 minutes at 25 °C. For the production of microparticles, the encapsulating solutions containing probiotics are dried in a spray dryer using an inlet temperature of around 130 °C and an outlet temperature of 73 °C, with a feed flow rate of 0.25 L / h-1 and a drying air flow rate of 3.00 m3 / min-1.After extraction, the powdered microparticles were stored under refrigeration at 8 °C.

[0018] For the production of microparticles based on yacon potato flour and green banana flour, different microorganisms can be used, including those of the genus Lactobacillus, such as Lacticaseibacillus rhamnosus GG and Lactiplantibacillus plantarum. Examples of embodiments of the invention

[0019] For culturing the microorganism, a capsule of L. rhamnosus GG ATCC53103 (Culturelle®) was activated in 10 mL of Man Rogosa and Sharp (MRS) broth and incubated at 37 °C for 24 hours. The contents were then transferred Petition 870250014841, dated 24 / 02 / 2025, p. 19 / 36 / 18 for a flask containing 500 mL of Man Rogosa and Sharp (MRS) broth and incubated again at 37 °C for 24 hours. The cell pellet was resuspended twice in sterile saline solution (0.85%) and centrifuged at 5,000 rpm for 10 minutes. The resulting pellet was stored under refrigeration until use. Subsequently, the broth was centrifuged at 5,000 rpm for 10 min at 8°C.

[0020] To prepare yacon potato flour, the potatoes were immersed in a solution containing 100 mg / L of active chlorine for 10 minutes. Subsequently, they were rinsed in a solution of 10 mg / L of active chlorine, and then peeled and cut into thin slices that were immersed in a 2% citric acid solution for 5 minutes to prevent browning. The slices were dried for 11 hours at 65 °C to achieve a water content of less than 10%, but subsequently, the dried slices were ground in a mill, and the flour was packaged in polypropylene bags and sealed with a vacuum sealer, then stored in a cool, ventilated place until use.

[0021] To obtain the microparticles using yacon potato flour, the process described in the invention description was used.

[0022] Four microparticles were produced using yacon potato flour, with microparticle containing 16.67% maltodextrin, 3.33% gelatin and 5% L. rhamnosus GG pellet (A); microparticle containing 12.5% ​​maltodextrin, 2.5% gelatin, 5% yacon potato flour and 5% L. rhamnosus GG pellet (B); microparticle containing 11.11% maltodextrin, 2.22% gelatin and 6.67% yacon potato flour and 5% L. rhamnosus GG (C) and microparticle containing 10% maltodextrin, 2% gelatin and 8% yacon potato flour and 5% L. rhamnosus GG pellet (D).

[0023] Microparticles obtained using yacon potato flour as wall material were evaluated for encapsulation efficiency, probiotic resistance to heat, and pH variations. The most resistant microparticle was selected according to the data described in Table 1. Petition 870250014841, dated 24 / 02 / 2025, page 20 / 36 / 18 Table 1: Viability of L. rhamnosus GG before and after spray drying and encapsulation efficiency (%). Microparticle Before drying (log CFU.g-1) After drying (log CFU.g-1) EE (%) A 10.02 ± 0.08ab 6.56 ± 0.06c 65.47 ± 0.33c B 10.22 ± 0.56ab 7.29 ± 0.12b 71.44 ± 2.85b C 10.62 ± 0.03a 7.42 ± 0.03b 69.92 ± 0.52b D 9.55 ± 0.04b 7.79 ± 0.05a 81.61 ± 0.62a EE: encapsulation efficiency. *Means followed by the same letter in the column do not differ by Tukey's test at 5% probability.

[0024] Among the treatments evaluated, the microparticle containing 11.11% maltodextrin, 2.22% gelatin and 6.67% yacon potato flour (C) showed an encapsulation efficiency of 69.92% as shown in Table 1.

[0025] The developed microparticles also showed high thermal resistance (> 64%) of the probiotic (Table 2 and Figure 1) and survival of the same in acidic pH (> 76%) as described in Table 3 and Figure 2. Table 2: Viability of free and microencapsulated L. rhamnosus GG (log CFU.g1) before and after different heat treatments. Initial Treatment 63 °C / 30 min 72 °C / 15 s CL 12.09 ± 0.21a 5.69 ± 0.08a 5.45 ± 0.67b A 6.56 ± 0.06d 3.00 ± 0.25c 3.84 ± 0.07d B 7.29 ± 0.12c 3.46 ± 0.18c 4.43 ± 0.01c C 7.42 ± 0.03c 4.81 ± 0.06b 5.83 ± 0.13a D 7.79 ± 0.05b 4.50 ± 0.04b 5.38 ± 0.10b *Means followed by the same letter in the column (L. rhamnosus GG count - log CFU.g- 1) do not differ by Tukey's test at 5% probability. Petition 870250014841, dated 24 / 02 / 2025, page 21 / 36 / 18 Table 3: Viability of free and microencapsulated L. rhamnosus GG (log CFU.g) under different pH conditions. Initial Treatment pH 7.4 pH 2.0 CL 12.02 ± 0.24a 10.43 ± 1.79a 5.45 ± 0.67a A 6.56 ± 0.06d 6.78 ± 0.40b 3.84 ± 0.07b B 7.29 ± 0.12c 7.39 ± 0.38b 4.43 ± 0.01b C 7.42 ± 0.03c 7.69 ± 0.32b 5.83 ± 0.13a D 7.79 ± 0.05b 7.80 ± 0.30b 5.38 ± 0.10a * Means followed by the same letter in the column (L. rhamnosus GG count - log CFU.g-1) do not differ by Tukey's test at 5% probability.

[0026] After selection, the microparticle was characterized and evaluated in relation to the viability of the probiotic for 120 days under different storage conditions (-18, 8 and 25 °C), survival under in vitro gastrointestinal conditions and its behavior in pitaya jelly.

[0027] Figure 3 shows Scanning Electron Microscopy (SEM) images, which is a type of microscope that uses electron beams to create three-dimensional images of solid objects such as the microparticles developed in the study. In this image, it is possible to observe numerous rounded microparticles (Formulation containing 11.11% maltodextrin, 2.22% gelatin, 6.67% yacon potato flour and 5% L. rhamnosus GG pellet).

[0028] During storage for 120 days, the probiotic remained viable in the microparticle with counts > 6 log CFU.g-1 under freezing (Figure 4) and withstood simulated in vitro gastrointestinal conditions with approximately 5.64 log CFU.g-1 of viable cells (Table 4 and Figure 5). Table 4: Viability of free and microencapsulated L. rhamnosus GG (log CFU.g1) during in vitro gastrointestinal simulation. Petition 870250014841, dated 24 / 02 / 2025, page 22 / 36 14 / 18 Phases Free Cell Initial Microparticle 10.41 ±0.29Aa 6.16 ± 0.19Ba Gastric Phase 3.21 ± 0.11Bb 5.25 ± 0.15Ab Enteric Phase 1 3.48 ± 0.05Bb 5.53 ± 0.06Ab Enteric Phase II 3.88 ± 0.71Bb 5.64 ± 0.27Ab *Different uppercase letters indicate a significant difference (p<0.05) between samples in the same phase. Different lowercase letters indicate a significant difference (p<0.05) for the same sample across phases.

[0029] The microparticle provided high survival (> 70%) of the probiotic after 60 days of storage at 8 °C in pitaya jelly (Table 5 and Figure 6), causing small variations in the pH and viscosity of the product. This indicates that the microparticle composed of maltodextrin, yacon potato flour and gelatin is efficient in protecting and maintaining the viability of the probiotic during the processing and storage of the jelly, being a new alternative to be used in spray drying microencapsulation technology and the development of potentially symbiotic foods. Table 5: Survival rate (%) of free and microencapsulated L. rhamnosus GG (log CFU.g-1) added to pitaya jelly during 60 days of storage at 8 °C and 25 °C. Treatments 15 days 30 days 45 days 60 days M8 90.48 ± 3.73b 88.29 ± 2.55a 78.25 ± 1.41a 77.83 ± 3.47a M25 81.14 ± 4.27b 79.13 ± 2.64b 70.26 ± 3.49b ND F8 100.62 ± 1.65a 89.53 ± 2.58a 73.03 ± 1.55ab 63.94 ± 2.65b F25 61.85 ± 1.09c 23.33 ± 1.94c 0.00 0.00 *M8: 100 g of jelly; 10 g of microparticles containing L. rhamnosus GG, stored at 8°C; M25: 100 g of jelly; 10 g of microparticles containing Petition 870250014841, dated 24 / 02 / 2025, page 23 / 36 / 18 L. rhamnosus GG stored at 25 °C; F8: 100 g of jelly; 1 g of cell-free L. rhamnosus GG stored at 8 °C; F25: 100 g of jelly; 1 g of cell-free L. rhamnosus GG stored at 25 °C. ND: not determined. Different uppercase letters indicate a significant difference (p<0.05) between samples in the same phase. Different lowercase letters indicate a significant difference (p<0.05) for the same sample across phases.

[0030] To prepare green banana flour, the bananas were washed with soap and water. Subsequently, the bananas were sanitized in a solution containing 100 mg / L of active chlorine for 10 minutes and rinsed in a solution of 10 mg / L of active chlorine. After sanitization, the product was peeled and cut into thin slices, about 3 to 4 mm thick, to facilitate the drying process. The material was placed in a forced-air oven at 65 to 70 °C for 6 hours to dehydrate the fruit. Finally, the material was ground / crushed and sieved through a sieve with a 0.208 mm mesh opening, resulting in green banana flour. The flour was packaged in polypropylene bags and sealed with a vacuum sealer, then stored in a cool, ventilated place until use.

[0031] The process described in the invention description was used to produce microparticles using green banana flour.

[0032] Four microparticles were produced using green banana flour, with the following microparticles: one containing 16.67% maltodextrin, 3.33% gelatin, and 5% L. rhamnosus GG pellet (E); one containing 12.5% ​​maltodextrin, 2.5% gelatin, 5% green banana flour, and 5% L. rhamnosus GG pellet (F); one containing 15% maltodextrin, 1% gelatin, 4% green banana flour, and 5% L. rhamnosus GG pellet (G); and one containing 15% maltodextrin, 2% gelatin, 3% green banana flour, and 5% L. rhamnosus GG pellet (H).

[0033] The microparticles produced were evaluated in relation to encapsulation efficiency, thermal resistance of the probiotic and pH variations. Petition 870250014841, dated 24 / 02 / 2025, page 24 / 36 / 18 Subsequently, the microparticle that provided the greatest resistance to the probiotic was characterized and evaluated for its viability over 120 days under different storage conditions (-18, 8, and 25 °C), survival under simulated in vitro gastrointestinal conditions, and probiotic viability when added to banana jam. Among the treatments evaluated, the microparticle containing 12.5% ​​maltodextrin, 2.5% gelatin, and 5% green banana flour (F) showed an encapsulation efficiency of > 80%, as described in Table 6. Table 6. Lacticaseibacillus rhamnosus GG count and encapsulation efficiency (%). Microparticle Before drying (log CFU.g-1) After drying (log CFU.g-1) EE (%) E 8.44 ± 0.21a 7.62 ± 0.25b 90.29 ± 1.61b F 8.86 ± 0.07a 7.17 ± 0.05b 80.94 ± 0.27c G 9.21 ± 0.09a 8.53 ± 0.28a 92.61 ± 3.16a H 8.85 ± 0.05a 7.70 ± 0.05b 87.06 ± 0.77b EE: encapsulation efficiency. *Means followed by the same letter in the column do not differ by Tukey's test at 5% probability.

[0034] Furthermore, the developed microparticles showed high thermal resistance of the probiotic (> 77%) in different heat treatments (Table 7 and Figure 7) and survival in acidic pH (> 74%) as shown in Table 8 and Figure 8. Table 7: Viability of free and microencapsulated L. rhamnosus GG (log CFU.g1) before and after different heat treatments. Initial Treatment 72 °C / 15 s 63 °C / 30 min CL 12.95 ± 0.56a5.02 ± 0.40b4.84 ± 0.24b Petition 870250014841, dated 24 / 02 / 2025, page 25 / 36 / 18 E 7.62 ± 0.25c 4.50 ± 0.25b 4.24 ± 0.13b F 7.17 ± 0.05c 5.50 ± 0.11a 5.73 ± 0.18a G 8.53 ± 0.28b 5.96 ± 0.41a 6.01 ± 0.20a H 7.70 ± 0.05c 5.88 ± 0.33a 5.35 ± 0.18ab *Means followed by the same letter in the column (L. rhamnosus GG count - log CFU.g- 1) do not differ by Tukey's test at 5% probability. Table 8: Viability of free and microencapsulated L. rhamnosus GG (log CFU.g1) under different pH conditions. Initial Treatment pH 2.0 pH 7.4 CL 12.95 ± 0.56a 3.82 ± 0.66d 10.25 ± 0.28a E 7.62 ± 0.25c 4.67 ± 0.31c 6.72 ± 0.54d F 7.17 ± 0.05d 5.35 ± 0.44ab 7.76±0.69b G 8.53±0.28b 5.21±0.33b 8.00±0.06b H 7.70±0.05c 5.52±0.15a 7.35±0.43c *Means followed by the same letter in the column (L. rhamnosus GG count - log CFU.g-1) do not differ by Tukey's test at 5% probability.

[0035] Figure 9 shows the images obtained through Scanning Electron Microscopy (SEM), which is a type of microscope that uses electron beams to create three-dimensional images of solid objects from the microparticle developed using green banana flour containing the probiotic. It is possible to verify that the microparticles obtained presented a rounded shape and different sizes, characteristic of the microencapsulation technique used.

[0036] Furthermore, L. rhamnosus GG remained viable with counts of 7.24 log CFU.g-1 when stored at 8 °C and 7.97 log CFU.g-1 when stored at -18 °C as described in Figure 10. L. rhamnosus GG withstood the simulation. Petition 870250014841, dated 24 / 02 / 2025, page 26 / 36 / 18 gastrointestinal in vitro with approximately 5.53 log CFU.g-1 of viable cells (Figure 11).

[0037] When the microparticles were added to the banana dessert, microencapsulated L. rhamnosus GG showed greater viability compared to the free probiotic stored at 25 °C (Figure 12). Thus, the microparticle developed using maltodextrin, green banana flour and gelatin can be considered a good and efficient prebiotic carrier matrix to protect probiotic microorganisms in the development of potentially symbiotic foods. Petition 870250014841, dated 24 / 02 / 2025, page 27 / 36

Claims

1 / 2 CLAIMS 1. Microparticle characterized by comprising 10 to 16.67% maltodextrin, 2 to 3.33% gelatin, 5 to 8% yacon potato flour and 5% probiotic microorganisms in relation to the amount of wall material (m / m).

2. Production process of the microparticle, described in claim 1, characterized by comprising the following steps: - Dissolving 10 to 16.67% maltodextrin, 2 to 3.33% gelatin and 5 to 8% yacon potato flour in distilled water; - Homogenizing for 1 hour; - Filtering the resulting encapsulating solutions; - Subjecting the filtered solutions to heat treatment; - Cooling between 45 and 50 °C; - Adding the suspension of probiotic microorganism cells at a concentration of 5% relative to the amount of wall material (m / m); - Homogenizing for 10 minutes at 25 °C; - Drying the resulting solution; - Storing the resulting microparticles under refrigeration at 8 °C.

3. Microparticle characterized by comprising 12.5 to 15% maltodextrin, 1 to 2.5% gelatin, 3 to 5% green banana flour and 5% probiotic microorganisms in relation to the amount of wall material (m / m).

4. Production process of the microparticle, described in claim 3, characterized by comprising the following steps: Petition 870250014841, dated 24 / 02 / 2025, page 28 / 36 2 / 2 - Dissolve 12.5 to 15% maltodextrin, 1 to 2.5% gelatin, 3 to 5% green banana flour in distilled water; - Homogenize for 1 hour at 25 °C; - Filter the resulting encapsulating solutions; - Subject the filtered solutions to heat treatment; - Cool to between 45 and 50 °C; - Add the suspension of probiotic microorganism cells at a concentration of 5% relative to the amount of wall material (m / m); - Homogenize for 10 minutes at 25 °C; - Dry the resulting solutions; - Store the obtained microparticles under refrigeration at 8 °C.

5. Process, according to claims 2 and 4, characterized by the possibility of using different microorganisms, including those of the genus Lactobacillus, selected from the group comprising Lacticaseibacillus rhamnosus GG or Lactiplantibacillus plantarum.

6. Process, according to claims 2 and 4, characterized in that the heat treatment can be carried out by autoclaving at 121 °C for 15 minutes.

7. Process, according to claims 2 and 4, characterized in that the drying of the obtained solution can be done in a Spray Dryer, using an inlet temperature of 130 °C and an outlet temperature of 73 °C, with a feed flow rate of 0.25 L / h-1 and a drying air flow rate of 3.00 m3 / min'1.

8. Use of yacon potato flour, described in claim 1, characterized by being for microencapsulation of probiotics.

9. Use of green banana flour, described in claim 3, characterized by being for microencapsulation of probiotics. Petition 870250014841, dated 24 / 02 / 2025, pp. 29 / 36

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