Bead-type microparticles comprising r-phycoerythrin pigment

Abstract

The present application relates to bead-type microparticles comprising a wall material formed by a mixture of biomaterials, sodium alginate and natural resin (shellac) that allows the pigment R-phycoerythrin (R-PE) to be contained in the microparticle so as to stabilise same in gastric juices, for the treatment of colon cancer and melanoma.

Description

[0001]PEARL-TYPE MICROPARTICLE COMPRISING R-PHYCOERYTHRIN PIGMENT DESCRIPTIVE SPECIFICATION FIELD OF THE INVENTION The present application relates to pearl-type microparticles comprising a wall material composed of a mixture of biomaterials, sodium alginate and natural resin (Shellac), which allow the retention of the R-phycoerythrin pigment (R-PE) inside for its stabilization against gastric juices and which are useful for treating colon cancer and melanoma. STATE OF THE ART Colorectal cancer is the cancer that begins in the large intestine (colon) or in the rectum (final part of the colon). Other types of cancer can also affect the colon and these include lymphoma, carcinoid tumors, melanoma and sarcomas. Colon cancer is a disease of extraordinary importance, both for its frequency and for the mortality rate that it entails, however, if detected early, the cure rates reach 90%.Treatment for colorectal cancer depends on the size, location, and spread of the cancer. The most common treatments include surgery to remove the cancer, chemotherapy, and radiation therapy. As researchers learn more about the cell changes that cause colon or rectal cancer, new types of drugs have been developed to specifically target these changes. These targeted therapy drugs work differently than those commonly used in chemotherapy. Sometimes, these drugs work when chemotherapy drugs are ineffective and often have different side effects. They can be used alongside chemotherapy or alone if chemotherapy is no longer effective. Based on the above, there is a great need to find more effective treatments to combat and / or prevent this type of cancer.This need can be addressed through a treatment using the bioactive R-phycoerythrin (R-PE), which must also be protected from gastric juices by a bioprotection method, which ensures its stability. In accordance with the above, the present application proposes pearl-type microparticles comprising a mixture of biomaterials, sodium alginate and natural resin (Shellac), which allow the retention of the R-phycoerythrin (R-PE) pigment inside, achieving its stabilization against gastric juices useful for treating cancer, specifically colon cancer and melanoma. R-phycoerythrin (also known as PE or R-PE) is an intensely bright phycobiliprotein isolated from red algae that exhibits a red-orange fluorescence with high quantum yields. In practical applications, the sensitivity of R-PE conjugates is typically 5 to 10 times higher than the sensitivity of the corresponding fluorescein conjugates.Like all phycobiliproteins, it is composed of a protein moiety that covalently binds to chromophores called phycobilins. Within the phycoerythrin family, the best-known phycobilins are phycoerythrobilin, a typical acceptor chromophore for phycoerythrin. Phycoerythrobilin is a linear tetrapyrrole molecule found in cyanobacteria, red algae, and cryptomonads. Along with other bilins such as phycocyanobilin, it serves as a light-harvesting pigment in the light-harvesting photosynthetic structures of cyanobacteria called phycobilisomes. Phycoerythrins are composed of monomers (αβ), usually organized in a disc-shaped trimer (αβ)3 or hexamer (αβ)6 (the latter being the functional unit of antennal rods). These typical complexes also contain a third type of subunit, the γ chain.Phycoerythrins, except for phycoerythrin 545 (PE545), are composed of (αβ) monomers assembled into disc-shaped (αβ)6 hexamers or (αβ)3 trimers with 32 or 3 symmetry and a central enveloping channel. In phycobilisomes (PBS), each trimer or hexamer contains at least one linker protein located in the central channel. B-phycoerythrin (B-PE) and R-phycoerythrin (R-PE) from red algae, in addition to the α and β chains, have a third γ subunit that contributes to both the linker function and light harvesting, because it contains chromophores. Specifically, R-phycoerythrin is predominantly produced by red algae. The protein is made up of at least three different subunits and varies depending on the algal species that produces it. The most common R-PE subunit structure is (αβ)6γ.The α subunit has two phycoerythrobilins (PEBs), the β subunit has 2 or 3 PEBs and one phycourobilin (PUB), while the different gamma subunits have 3 PEBs and 2 PUBs (γ1) or 1 or 2 PEBs and 1 PUB (γ2). The molecular weight of R-PE is 250,000 Daltons. Phycoerythrin R-phycoerythrin is useful in the laboratory as a fluorescence-based indicator for detecting the presence of cyanobacteria and for labeling antibodies, most commonly for flow cytometry. It is also used in microarray assays, ELISAs, and other applications requiring high sensitivity but not photostability. Its use is limited in immunofluorescence microscopy due to its rapid photobleaching characteristics. Phycobiliproteins, on the other hand, have wide applications in the pharmaceutical industry, but their instability under light or heat would greatly limit their application. Therefore, one way to address this instability is through microencapsulation.In the state of the art, some documents related to the aforementioned have been found, among them: The scientific article Preparation and Characterization of Phycobiliprotein Mcirocapsule which discloses that phycobiliprotein is a type of bioactive material that is widely used in the fields of food and medicine. This document refers to the microencapsulation of phycobiliprotein using a chitosan-sodium alginate encapsulation method. However, this article refers to a complex or mixture of phycobiliproteins, while the invention proposed in the present application proposes pearl-type microparticles comprising a wall material composed of a mixture of sodium alginate and shellac biomaterials inside which the bioactive pigment R-phycoerythrin (R-PE) is retained, which is extracted and concentrated to a cut-off size of 30KDa, achieving a purity of 1.57.Unlike this document, the invention proposed in the present application does not refer to phycobiliproteins, which are a family of biliproteins made up of phycoerythrin, phycocyanin, and allophycocyanin. It should also be noted that this article does not propose the use of microparticles comprising the bioactive R-PE retained inside to treat colon cancer and melanoma. On the other hand, microencapsulation of active ingredients is also known in the state of the art, such as, for example, in document US2021308279 (A1), which discloses hydrogel nanoparticles retaining a conjugated charge of salvianolic acid-gelatin useful for the oral administration of salvianolic acid, pharmaceutical compositions comprising them, and methods of use and preparation thereof.Salvianolic acids are the main bioactive components of the traditional Chinese medicinal herb Salvia miltiorrhiza and are known to exhibit several therapeutic properties, such as antioxidant, anticancer, anti-inflammatory, and cardioprotective properties. In contrast, the pearl-like microparticle proposed in the present application, without a defined core, comprises an internal and external structure provided by the mixture of sodium alginate and shellac throughout the pearl, and the pigment R-phycoerythrin, useful for treating colon cancer and melanoma, is immobilized inside.It should be noted that the pearl-type microparticles proposed in the present application have been designed by means of the shrinking phenomenon based on the ionic interactions between anionic salts (sodium alginate and shellac) and cationic salts (calcium chloride), which, at certain process operations, allows the formation of a new biomaterial architecture with high chemical affinity with phycoerythrin (R-PE). SUMMARY DESCRIPTION OF THE INVENTION The present application addresses the technical problem of providing bioprotection of phycobiliproteins, specifically the bioactive R-phycoerythrin (R-PE), with the aim of making them useful for treating colon cancer and melanoma.In order to solve the technical problem posed, the present application proposes a pearl-type microparticle comprising a wall material composed of a mixture of biomaterials, sodium alginate and natural resin (Shellac) that allows the retention of the pigment R-phycoerythrin (R-PE) and that, in addition, allows to solve the technical problem related to providing bioprotection of the bioactive R-phycoerythrin against gastric juices, bioprotection that provides stability to said pigment. The present invention also provides the process that provides bioprotection of phycobiliproteins, specifically R-phycoerythrin (R-PE). DESCRIPTION OF THE FIGURES Figure 1 shows the process steps and working conditions for the formation of pearl-type microparticles comprising aglinate / shellac loaded with R-PE and in vitro gastrointestinal evaluation. Figure 2 shows a flow diagram of the simulated gastrointestinal system (SIMUGIT).Figure 3 shows a confocal microscopy of 1) the design spot #1 (97.5% EE) and 2) the presence of R-phycoerythrin detected by fluorescence. Figure 4 shows a morphology of the particles (beads) by Scanning Electron Microscopy (SEM) for a) design spot #1, b) design spot #4 and c) 500X magnification for the surface of the beads. Figure 5 shows the bioavailability (%) of R-phycoerythrin extract and encapsulates during in vitro gastrointestinal simulation at the human intestinal level. Lines with asterisks indicate significant differences (p<0.05) between the concentrated extract and encapsulates. Figure 6 shows a Nuclear Magnetic Resonance (NMR) spectra of 1H NMR (DMSO- d6+D2O) of the digested enzyme fraction (A) and the intestinal absorption (permeate) (B).DESCRIPTION OF THE INVENTION The present patent application relates to pearl-type microparticles comprising a wall material composed of a mixture of the biomaterials sodium alginate and natural resin (Shellac) at a ratio of 75 / 25 (%V / V), which by chemical affinity manage to mix with the pigment R-phycoerythrin (R-PE). The presence of calcium ion provided by the calcium chloride salt allows the occurrence of ionic gelation, generating a chemical reaction between L-guluronic acid and D-mannuronic acid of sodium alginate together with the jaularic acid of shellac (Shellac), which results in a structural rearrangement, creating a new polymeric matrix. The process conditions that allow this new conformation of pearls are: flow of 20mL / min, 15g / L of CaCl. 2and at a collection distance of 5 cm. The process for obtaining a pearl-type microparticle according to the present application comprises the following steps: a) mixing sodium alginate powder with water to obtain a sodium alginate solution; b) combining the mixture from step a) with a shellac solution to obtain a wall material; c) adjusting the pH of the solution obtained in step b); d) incorporating an R-phycoerythrin (R-PE) pigment into the solution from step d); e) extruding the mixture obtained in step e); and f) pouring the extruded mixture from step e) into a gelling bath containing CaCl2.The mixture of sodium alginate and shellac biomaterials (75 / 25% v / v) proposed in the present application, in the presence of the bioactive R-PE at a ratio of 80 / 20% v / v, allows the creation of a new solid biomaterial architecture with gel characteristics by means of an optimized ionic gelling type process under operating conditions of 20 mL / min; 5 g / L of CaCl. 2and a distance of 5 cm, providing pearl-like microparticles capable of tolerating intestinal barriers and achieving a controlled release of the bioactive component. This is verified by evaluating human gastric simulation where permeate currents, equivalent to the absorption of encapsulated R-PE, present a bioavailability 2.5 times significantly greater than that of non-encapsulated R-PE. In turn, NMR results indicate the presence of R-PE and its methylamino acids and oligosaccharides at a concentration between 0.5-2.5 and 3.8-6.8 ppm.Furthermore, the R-phycoerythrin used corresponds to a 100% natural pigment, without additives like E-120, and it has nutritional properties as it is a protein-based pigment, antioxidants, and anticarcinogenic against human colon carcinogen-type cells. This is due to its ability to delay or prevent the oxidation of other molecules and is a strong pigment, with a degree of purity suitable for the food industry. Furthermore, since it is an algae (plant organism), it does not pose any problems for vegans to consume. It is worth noting that the biomaterial mixture will protect and retain the phycoerythrin pigment (R-PE), providing human gastrointestinal barriers and achieving a therapeutic effect at the intestinal level. To date, only information exists on the formulation of alginate and shellac for the protection of compounds of hydrophilic and lipophilic origin.However, to date, there is no information on the protection (encapsulation formulation) of protein-based pigment compounds with an anticariogenic effect using the technique proposed in this application. APPLICATION EXAMPLES EXAMPLES OF PREFERRED EMBODIMENTS The following are application examples of this patent application. These examples are provided for illustrative purposes only to provide a better understanding of the proposed invention, but in no case should they be considered to limit the scope of the protection requested. Additionally, specifications of different technical characteristics described in the examples may be combined with each other, or with other previously described technical characteristics, without this limiting the scope of the protection requested.EXAMPLE 1 Wall material formulation A sodium alginate solution (20 g / L) is prepared by mixing alginate powder with distilled water under stirring at 700 rpm using a magnetic stirrer. The suspension is kept under gentle stirring (1 h) until complete hydration and is left to stand for 60 min to cause deaeration. Subsequently, the wall material is prepared by mixing an alginate solution (20 g / L) and a shellac solution (250 g / L) at a ratio of 75 / 25 (%v / v) with constant stirring for 30 min at 500 rpm using a magnetic stirrer. Then, the pH of the resulting solution is adjusted to pH 7.5 with a 1 M NaOH solution. EXAMPLE 2 Formulation of pearl-type microparticles by external ionic gelation Figure 1 illustrates the process for preparing the alginate / shellac pearl-type microparticles with the bioactive component inside.Bioactive / wall material ratios (80 / 20 to 20 / 80 % v / v) are prepared by mixing the bioactive (R-PE) with the previously prepared wall material under continuous stirring (200 rpm for 15 min). The bioactive / wall material ratios are extruded by a controlled flow system pump (SyringePump, Inc.) through a 0.3 mm nozzle and poured into 100 ml of a gelling bath containing CaCl. 2(5-15 g / l) to produce the bead-like microparticles containing the bioactive component inside. The gelling bath is prepared by dissolving CaCl2 in an ethanol / water mixture (1:1 v / v). The flow rate (30-90 ml / h) and the distance (5-15 cm) from the syringe tips to the surface of the gelling bath are evaluated. The formed bead-like microparticles are left to stand in the gelation bath for 30 min to ensure crosslinking. EXAMPLE 3 Experimental design for aqueous encapsulation To optimize the encapsulation of R-PE with high EE (encapsulation efficiency %), a Taguchi experimental design is applied. A L4 matrix(2 3) with three independent variables and two working levels (L1 and L2), applying the “bigger is better” criterion: fed flow solutions of Bioactive (R-PE) / wall material (Variable Flow; L1: 20 mL / h; L2: 90 mL / h), distance to the gelling bath surface (variable Distance; L1: 5 cm; L2: 15 cm) and concentration of calcium chloride solution (variable CaCl 2 ; L1:5 and L2:15 g / L). An optimized theoretical equation (OTE) is determined considering the average of the highest impact response, identifying the most important variables and working levels. Equation 1 where V is the variable, T is the average of the experimental runs responded, L corresponds to the working levels. EXAMPLE 4 Determination of the encapsulation efficiency (EE) of the R-PE cargo The concentration of phycoerythrin (R-PE) is determined spectrophotometrically, using the Bennett and Bogorad equation, (1973) as follows: Equation 2 Equation 3 Equation 4 The encapsulation efficiency of the microcapsule was calculated following the method of Ge et al., (2009) with some modifications, measuring the concentration of uncoated phycoerythrin and the concentration of phycocyanin added at the beginning of the microencapsulation process as follows: Equation 5 EXAMPLE 5 Sphericity factor (SF) of bead-like microparticles The size and shape of the bead-like microparticles are determined using Image software (National Institutes of Health, USA). A digital camera coupled to a binocular microscope is used to capture the images of the bead-like microparticles. SF is used to indicate the roundness of the beads, as described by Chan (2011), where a value equal to zero indicates a perfect sphere and higher values ​​indicate a higher degree of shape distortion. SF is calculated according to the following equation: Equation 6 where D maxis the maximum diameter passing through the centroid of the cord (mm) and D per is the diameter perpendicular to D max passing through the centroid of the bead (mm). All samples were analyzed in triplicate to determine the mean and standard error. The size and shape of the bead-like microparticles were determined based on the measurement of 50 beads per sample. Table 1. Encapsulation efficiency of R-phycoerythrin, sphericity factor, particle size using orthogonal L4 matrix (2 3 – Orthogonal matrix 2 levels and 3 factors). Orthogonal Matrix Point L4 (2 3 ) Characterization of the Efficiency of Pearl Encapsulation Design Flow Distance CaCl 2 Factor Size %EE (mL / h) (cms) (gr L -1 ) sphericity (mm) (SF) 1 20 (L 1 ) 5 (L 1 ) 5 (L 1 ) 0.06 ± 0.02 1.2 ± 0.2 97.5 ± 0.7 2 20 (L 1 ) 10 (L 2 ) 15 (L 2 ) 0.10 ± 0.03 1.6 ± 0.1 89 ± 1.41 3 90 (L 2 ) 5 (L1 ) 15 (L 2 ) 0.09 ± 0.05 1.1 ± 0.2 93.5 ± 0.70 4 90 (L 2 ) 10 (L 2 ) 5 (L 1) 0.07 ± 0.01 1.1 ± 0.1 95.5 ± 0.1 EXAMPLE 6 Gastrointestinal tract simulating membrane bioreactor (GITSMB): SimuGIT The dynamic gastrointestinal simulator system (Figure 2) used (SimuGIT) was developed by the TEP025 Research Group of the Department of Chemical Engineering, University of Granada and Malaga (Rivas-Montoya et al., 2016). The GITSMB dynamic in vitro membrane bioreactor (SimuGIT) simulating the gastrointestinal tract consists of a continuous stirred tank reactor (CSTR) connected in series to a continuous plug flow tubular reactor (PFTR) equipped with a single-channel tubular ceramic microfiltration membrane. The CSTR used to simulate gastric digestion in the stomach is a benchtop fermenter supplied by Braun Biotech International (model Biostat B).It consists of a conventional autoclavable stirred tank glass vessel equipped with an impeller stirrer (180 W, Rushton model) and a proportional-integral-derivative (PID) control system for temperature, level, foam, dissolved oxygen and pH. The control system unit includes an RS-422 interface that allows the CSTR to be controlled by a computer. The CSTR has an external jacket to maintain constant temperature, so the temperature in the vessel is measured by a digital Pt-100 sensor and is accurately controlled (Tsetpoint ± 0.1. °C) by the PID circuit connected to a thermostatic laboratory bath. The CSTR system also has sampling ports and reagent addition via several peristaltic pumps (Eyela, model MP-3). The pH inside the CSTR is measured by a pH electrode (Hamilton, model Easyferm Plus K8) immersed in the vessel and adjusted by a proprietary pH control system based on data acquisition modules, which acts on two different peristaltic pumps that dose acid and basic solutions (HCl or NaHCO3). The digested solution exiting the CSTR is continuously conducted to the PFR.To achieve this, the PID control system acts on the delivery and return peristaltic pumps (two of the four peristaltic pumps integrated into the benchtop fermentation system, as described above). By varying the flow rates of these pumps, the pressure inside the hydraulic circuits, as well as the product filtration rate, can be regulated. In addition to this proprietary pressure control mechanism, the operating pressure can be precisely adjusted (set point P ± 10 mmHg) using a spring-loaded pressure regulating valve (model SS-R4512MM-SP, Swagelok) and monitored by a digital pressure gauge (Endress + Hauser, model Ceraphant PTC31). The continuous PFR connected in series to the CSTR is used to simulate intestinal conditions.It consists of a cylindrical stainless steel tube (provided by Prozesstechnik GmbH, Basel, Switzerland) equipped with a single-channel ceramic microfiltration (MF) membrane in a tubular configuration provided by Atech Innovations GmbH. The MF membrane used for the experiments is an inorganic surface-active α-Al membrane. 2 EITHER 3with an average pore diameter of 0.2 µm and a molecular weight cut-off in the range of 1.2 kDa (model 20N). The selected membrane dimensions are 1,000 mm in length, 6 mm in duct diameter and 2 ± 0.5 mm in thickness. This type of MF membrane also ensures several advantageous characteristics, such as high resistance to temperature (suitable for steam sterilization at 121°C) and pressure (up to 10 bar) during cleaning protocols, pH stability (0 to 14), the possibility of backpulsing, very high abrasion resistance (against aggressive chemical reagents), optimal permeability recovery, as well as high selectivity and performance, and a long service life. The PFR is integrated with the CSTR by means of a drive and return system made of chemically resistant polyethylene tubing.Finally, a precision electronic mass balance with USB connectivity (Sartorius, model Quintix 5102, accuracy equal to 1 mg) is coupled to an automatic sampling and data logging system to record permeate values ​​(bioaccessible fraction that crossed the membrane) over time. Conditions of the membrane bioreactor simulating the gastrointestinal tract GITSMB - Gastric Phase At the beginning of the test, the temperature of the bath that keeps the reactor jacket warm is set at 37.5 ± 1 °C throughout the process, and the reactor stirring speed is set at 50 rpm to simulate the peristaltic movements of the stomach. Next, 800 mL of the simulated liquid gastric solution (GSF) and 14 mL of the simulated liquid salivary solution (SSF) are prepared according to the concentrations by Brodkorb et al., (2019). Once prepared, the GSF is introduced into the reactor and heated to a temperature of 37.5°C.At this point, the pH drops to 3, as an empty stomach is simulated before adding the food with the microcapsules. At the same time, 20 g of mineral water are weighed, and 1 g of phycoerythrin is added. Since two different samples were chosen (phycoerythrin-encapsulated and non-encapsulated), two separate tests were carried out. The SSF is then mixed with the food preparation and microcapsules; 1 mL of alpha-amylase solution (75 U / mL) and 0.1 mL of CaCl are added. 2 *2H 2Or 0.3 M concentration and 4.9 mL of distilled water (final ratio of salivary fluid to feed preparation of 1:1). The mixture is stirred for 2 min and the pH is adjusted to 7 (when necessary). Once the GSF is heated, the feed mixture with the SSF is added to the reactor tank, stirred for a few seconds and an initial sample is taken. Then, a solution of 10 mL of pepsin (2000 U) and 50 mg of phospholipid (Lipoid p45) is added, and the pH is adjusted to 3 by controlled dosing of 6 M HCl. From this moment on, a sample is taken every 5 min for the next half hour. - Duodenal phase When the gastric phase ends with the last sample, the pH of the GSF is raised to 6.5 by dosing NaHCO 31 M at a rate of 2.05 ml / min and a sample is taken after adjustment. This pH simulates the action of pancreatic juices on the food being digested. Then, intestinal enzymes are added: 10 mL of pancreatic lipase solution (2,000 U / mL), bile salts (for a final concentration of 5 mM), 10 mL of pancreatin solution (10%), and 1 mL of trypsin solution (50 mg / mL). Digestion in the duodenum takes about 10 min after enzyme addition, and a sample is taken after this digestion period. The approximate total duration of this phase (including pH increase, enzyme addition, and digestion) is approximately 30 min. - Intestinal absorption phase To simulate intestinal absorption, fluids are pumped from the bioreactor to flow through the modular filtration system described by Brodkorb et al. (2019). For these tests, a membrane size of 0.2 µm was chosen, based on previously performed tests (Abad et al., 2019; Gonzalez et al., 2018). The system overpressure limit was set at 50 mmHg, so diffusion of phycoerythrin through the membrane was due solely to passive transport. It is important to remember that the ionization characteristics of a compound can have a profound effect on the rate of its passive diffusion transfer because only non-ionized species are capable of passive diffusion through the membrane. Once the circuit is primed and fluids begin to flow into the bioreactor, the intestinal absorption phase, which lasts 180 min, is considered to have begun. Samples are taken at 30, 60, 90, 120, 150, and 180 min. Two samples are taken each time, one of the fluid filtered through the membrane (permeate) and another of the retained fluid, which forms part of the colonic residue (retentate). In summary, each simulation test lasts approximately 4 h: gastric phase (30 min), duodenal phase (30 min) and intestinal absorption phase (180 min).Each test is performed in duplicate. Throughout the test, the system program generates a data file in which all events and processes that occurred during the simulation are recorded. This file is then processed in the computer to obtain information on all variables, variables that are related to the phases of digestion. That is, this refers to the monitoring of variables such as feed rate, pH, temperature, time and pressure of the reactor that simulates the conditions in the stomach EXAMPLE 7 Sample Analysis All samples collected during the test are refrigerated and, subsequently, the concentration of phycoerythrin present in the fluids of each sample is measured by spectrophotometric analysis according to Bennet & Bogoard, (1976), polyacrylamide gel electrophoresis (SDS-Page), polyacrylamide gel electrophoresis (SDS-Page) nuclear magnetic resonance (NMR).The concepts of release or bioaccessibility and bioavailability are used to measure the amount of phycoerythrin released along the digestive tract that is present in the chyme and the amount absorbed by the membrane, respectively. The release corresponds to the fraction of pigment released by the microparticles in the gastrointestinal tract during the gastric and duodenal phases, and is calculated using Equation (7), adapted from Rivas-Montoya et al., (2016). When the release and precipitation processes occurred simultaneously, the same equation is used to calculate the bioaccessible percentage as the fraction released and not precipitated in the chyme. For its part, bioavailability corresponds to the fraction of pigment absorbed by diffusion through the membrane during the intestinal absorption phase, and is calculated using Equation (8), adapted from Ariza et al., (2018). Equation 7 Equation 8 where IAS means intestinal absorption samples.In both equations, the amounts of phycoerythrin present in the chyme and in the bead-like microparticles are expressed in milligrams of R-PE per total biomass (mg g. -1). Figure 5 shows the bioavailability of encapsulated and non-encapsulated R-PE at the end of intestinal digestion. Figure 5 comparing R-PE samples, it is observed that encapsulated R-PE presents the highest percentages (6.0%) of bioavailability compared to non-encapsulated R-PE (2.41%) at 180 min of intestinal digestion. At the beginning of the intestinal absorption phase (permeate samples), it is observed that small percentages of non-encapsulated and encapsulated R-PE of the total initial content were available and passed through the membrane. It is observed that during each digestion time for all samples the R-PE content increases showing similar contents, but at the end of intestinal digestion, a significant difference is detected at 180 min reached for non-encapsulated and encapsulated R-PE 0.123 and 0.31 mg of R-PE g −1of biomass, respectively. EXAMPLE 8 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Encapsulated and non-encapsulated phycoerythrin hydrolysates were prepared for SDS-PAGE using the protocol developed by Wang et al. (2006), which combines TCA / acetone and methanol washes and a phenol step for protein extraction. SDS-PAGE is performed according to the procedure of Laemmli (1970). In this essay, the SDS-PAGE profile of the different digestion stages of the samples is described, where multiple protein bands ranging from 20 to 100 kDa can be observed, except in the mouth and stomach. More intense bands eluted in the duodenum and intestine around 50, 20–37 kDa, suggesting that some bands correspond to phycoerythrin. Thus, it is possible to understand that the matrix core of the phycoerythrin encapsulation has good resistance to oral and stomach levels because no signal is reported.However, the signals between the concentrated and permeate phases in the intestine are similar in the intense bands at different reaction times (30 and 180 min). EXAMPLE 9 Nuclear Magnetic Resonance-NMR Methodology A 400 MHz Plus Avance II NMR spectrometer (Bruker, Switzerland) is used for nuclear magnetic resonance analyses. 1H NMR spectra are recorded at 400 MHz at room temperature (21 °C). Samples (15–30 mg) are dissolved in deuterated dimethyl sulfoxide (DMSO-d. 6 , 0.75 ml), with residual solvent peaks of ^= 2.50 (DMSO) and ^= 3.42 (H2O) ppm for 1 H. In some samples D is added 2O (3 drops). NMR results indicate the presence of R-PE and its methylamino acids and oligosaccharides between 0.5 and 2.5 and 3.8–6.8 ppm, respectively. EXAMPLE 10 Cell cultures The following five cell lines are chosen to perform the cytotoxicity analysis: human fibroblasts (1064sk, CIC-UGA, ES), human leukemia cell line (U-937, ATCC, USA), human malignant melanoma (G-361, ATCC, USA), and colon cancer cell line (HCT-116, ATCC, USA) stored in liquid nitrogen in the cell culture unit of the Central Research Support Services (SCAI) of the University of Malaga (UMA). To maintain cells, 1064sk, G-361 and HCT-116 are pre-cultured using Dulbecco's modified Eagle's medium (DMEM) (Capricorn Scientific, ref. DMEM-HPSTA) supplemented with 10% fetal bovine serum (Biowest, ref. S1810-500)), 100x 1% penicillin-streptomycin solution (Capricorn Scientific, ref.PS-B), and 0.5% amphotericin B (Biowest ref. L0009-100). U-937 cells are cultured in RPMI-1640 medium (BioWhittaker, ref. BE12-167F) supplemented with 10% fetal bovine serum (Biowest ref. S1810-500), 1% penicillin-streptomycin solution 100x and 0.5% amphotericin B. In the case of suspension cells (U-937), they are harvested at 70-75% confluence and centrifuged at 1500 rpm for 5 min at room temperature. Once centrifuged, the corresponding assay or cell subculture is performed. The cells are maintained at subconfluence in a controlled atmosphere incubator with 5% CO. 2 at 37 °C. Finally, the R-PE loaded into the bead-like microparticles can be used as a potential delivery system for the aqueous extract and give a therapeutic response according to the IC 50 values ​​(144 μg mL −1) found for R-PE to treat colon cancer. EXAMPLE 11 Analytical Method (MTT Assay) For the cancer and healthy cell viability assay, U-937, HTC-116, G-361, and 1064sk cells are incubated in R-PE encapsulate concentrations of 5 to 4.76 × 10 −6 mg mL −1 in serial dilutions (1:1) of the alga S. skottsbergii. The experiment is performed individually with each cell line in a 96-well microplate for 72 h (37 °C, 5% CO2 in a humidified atmosphere). The proliferation of these cell lines is estimated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Abdala-Díaz et al. 2019). Briefly, a volume of 10 μL of the MTT solution (5 mg mL −1in phosphate-buffered saline). The plates are incubated at 37 °C for 4 h. The yellow tetrazolium salt of MTT is reduced by metabolically active mitochondrial dehydrogenases of viable cells to form insoluble purple formazan crystals. The formazan is dissolved by the addition of acid-isopropanol (150 μL of 0.04 N HCl-2 propanol) and measured spectrophotometrically at 550 nm (Micro Plate Reader 2001, Whittaker Bioproducts, USA). Relative cell viability is expressed as the mean percentage of viable cells compared to untreated cells. Four samples for each concentration tested are included in each experiment. Measurements were carried out in independent triplicate experiments. Table 2.Cytotoxicity indices (IC50) and selectivity index (SI) for encapsulated and non-encapsulated R-PE on different human cell lines: 1064 sK (human gingival fibroblasts cell line), HCT-116 (human colon cancer), G-361 (human melanoma), U-937 (human leukemia). Samples Active cell lines Cytotoxicity index Selectivity index Antiproliferative IC. 50 (µg mL- (SI) and health 1 ). Encapsulated 1064sK 3516 ±15.3 - HCT-116 1076 ± 8.46 3.27 U-937 1251 ± 4.83 0.86 G-361 4132 ± 10.86 0.26 Non-Encapsulated 1064sK 4438 ± 10.3 - (R-PE extract). HCT-116 144.5 ± 3.96 30.71 U-937 2702 ±12.6 1.59 G-361 2788 ±11.5 1.64 Finally, R-PE loaded into the bead-like microparticles can be used as a potential delivery system for the aqueous extract and give a therapeutic response according to the IC 50 values ​​(144 μg mL −1) found for R-PE to treat colon cancer. Example 12 Mathematical model of phycoerythrin release For this purpose, a mathematical model was proposed to describe the results of phycoerythrin release tests. The proposed model corresponds to a second-order differential equation, which is presented as follows: Equation 9 In Equation 9, x(t) describes the time in which the pigment is released from the bead-like microparticles into the aqueous solution from the beginning at time t = 0 to the end of the experiment. In contrast, y(t) represents the amount of phycoerythrin released measured in percentage units. The initial conditions for x(t), y(t), and their derivative terms were all zero. The Laplace transform is used to obtain a transfer function Equation 10 between the input signal x(t) and the output signal y(t) using Equation 9, where s represents the complex frequency. Equation 10 Where: The second-order transfer function exhibits a two-step first-order response, with the first step occurring at t = 0 and the second at td. From this equation, several parameters of the release response can be calculated, such as the maximum release (MR) of the first (S1) and second (S2) stages, the time to reach the maximum value or stabilization time (ST), and the time delay (td) between each phase, calculated as follows: STS1 = 5 / STS2 = 5 / Time: td The parameters a1, a2, c1, c2, and td are fitted to data showing phycoerythrin release at three different pH values: 1, 3, and 5. Parameter fitting is performed using an evolutionary algorithm. The evolutionary algorithm considers five parameters, with the minimization of the normalized mean square error between the data and the model as the evaluation function, following the algorithm presented by others. Table 3. Parameters of the second-order model for phycoerythrin release pH=1 0.125 0.995 0.075 0.243 101.573 15.944 pH=3 0.038 0.297 0.092 0.258 73.452 28.948 pH=5 0.130 0.239 0.273 0.266 77.107 24.870 Table 4. Pigment release response. Release Time Lag Time Lag Time Lag Time Lag Time Maximum 1 Maximum 2 Stabilization Stabilization 1 (min) 2 (min) pH=1 1.657 5.758 66.490 20.617 101.573 pH=3 0.416 1.565 54.571 19.357 73.452 pH=5 0.476 1.371 18.315 18.765 77.107 Conclusions: The application of this model provides information on the process conditions for preparing R-PE bead-like microparticles with high EE (encapsulation efficiency) and their evolution during in vitro gastrointestinal digestion. The process variables for the encapsulation of R-PE into the bead-like microparticles are obtained using a feed flow rate of 20 mL h −1 , 5 cm distance and 5 g L −1 of CaCl 2. Encapsulation under optimal conditions shows 97.5%, providing a good match with the OTE predictive values ​​(OPTIMIZED THEORETICAL EQUATION-OTE) (97%). The bead-like microparticles have a hemispherical shape and are free of pores with a size of 1.2 mm. In vitro digestion shows that the alginate / shellac bead-like microparticles have a 2.5-fold higher bioavailability than the non-encapsulated R-PE extract in intestinal stage digestion at a concentration of 0.31 mg of R-PE. Finally, the R-PE loaded into the bead-like microparticles can be used as a potential delivery system for the aqueous extract and give a therapeutic response according to the IC values. 50 (144 μg mL −1 ) found for R-PE to treat colon cancer. These R-PE bead-like microparticles could be incorporated into a food matrix for nutraceutical applications.

Claims

CLAIMS 1. A pearl-type microparticle CHARACTERIZED in that it comprises a wall material composed of a mixture of biomaterials that includes sodium alginate and natural resin (Shellac) wherein a bioactive component, pigment R-phycoerythrin (R-PE), is retained inside said wall material.

2. The pearl-type microparticle of claim 1, CHARACTERIZED in that it comprises that the sodium alginate and shellac are in a proportion of 75 / 25% v / v in the mixture of biomaterials.

3. The pearl-type microparticle of claim 1, CHARACTERIZED in that it comprises that the mixture of biomaterials of sodium alginate and shellac is in a proportion of 80 / 20% v / v with respect to the bioactive component R-PE. 4.Process for obtaining a pearl-type microparticle, comprising: a) mixing sodium alginate powder with water to obtain a sodium alginate solution; b) combining the mixture from step a) with a shellac solution to obtain a wall material; c) adjusting the pH of the solution obtained in step b); d) incorporating an R-phycoerythrin (R-PE) pigment into the solution from step d); e) extruding the mixture obtained in step e); and f) pouring the extruded mixture from step e) into a gelling bath containing CaCl2.

5. The process according to claim 4, CHARACTERIZED in that it comprises mixing sodium alginate powder with water by stirring until the sodium alginate is hydrated, and subsequently allowing said mixture to stand.

6. The process according to claim 4, CHARACTERIZED in that it comprises obtaining a wall material by mixing the mixture of biomaterials. which includes sodium alginate and natural resin (Shellac) and a shellac solution at a ratio of 75 / 25 (% v / v) with constant stirring.

7. The process according to claims 4 to 6, CHARACTERIZED in that it comprises adjusting the pH of the resulting solution to pH 7.5 with a NaOH solution.

8. The process according to claims 4 to 7, CHARACTERIZED in that incorporating an R-phycoerythrin (R-PE) pigment comprises mixing said pigment (R-PE) with the wall material by means of continuous stirring.

9. The process according to claims 4 to 8, CHARACTERIZED in that it comprises mixing said pigment (R-PE) with the wall material at ratios between 80 / 20 and 20 / 80 % v / v. 10.The method according to claims 4 to 9, CHARACTERIZED in that it comprises extruding the mixture comprising said pigment (R-PE) and the wall material by means of a controlled flow system pump to obtain the pearl-type microparticles.

11. The method according to claim 4, CHARACTERIZED in that it comprises pouring the pearl-type microparticles into a gelling bath containing CaCl2 and further comprising preparing said gelling bath by dissolving CaCl2 in an ethanol / water mixture at a ratio of 1:1 v / v.

12. The method according to claims 4 to 11, CHARACTERIZED in that it further comprises allowing the pearl-type microparticles to rest in said gelling bath to promote crosslinking of said microparticles.

13. Use of a pearl-type microparticle CHARACTERIZED in that it is used to prepare a medicament useful for treating colon cancer and melanoma.

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