pH-responsive nanoparticle carriers and their application in encapsulating phycocyanin for use in health food products

Abstract

This invention provides a pH-responsive nanoparticle carrier encapsulating phycocyanin. The nanoparticles can encapsulate phycocyanin, allowing it to safely pass through the acidic environment of the stomach and be released in large quantities in the alkaline environment of the intestine. This avoids the structural damage and inactivation of phycocyanin in the acidic environment of the stomach and protects phycocyanin from the effects of high temperature (heat) and light. The phycocyanin nanoparticles are non-cytotoxic and can eliminate and promote the growth of probiotics, have antibacterial properties, and thus increase the immune regulation of the intestine.

Description

[Technical Field]

[0001] The present invention relates to nanoparticles that can successfully pass through the stomach and are released in large quantities in the intestines, especially nanoparticles coated with phycocyanin. [Previous Technology]

[0002] Phycocyanin is a type of phycobiliprotein and the most abundant protein among phycobiliproteins. It can be used in food and is often used as a food ingredient or dye. Phycocyanin is sensitive to temperature, light, and pH, which causes many inconveniences in processing, especially in food applications.

[0003] Phycocyanin is easily degraded in an acidic environment. Therefore, when it is used as an oral food, its structure is destroyed after contact with the strong acid environment of the stomach, resulting in its inactivation and inability to exert its effects in the body. [Summary of the Invention]

[0004] In view of this, the inventors are keenly aware of the shortcomings and defects of the previous work and are eager to improve and innovate it. After years of research, they have finally developed a pH-responsive carrier to encapsulate phycocyanin, so that it can encapsulate phycocyanin under acidic conditions and release phycocyanin under neutral conditions to achieve the purpose of intestinal administration.

[0005] The purpose of this invention is to provide a nanocomposite particle carrier, which comprises: positively charged chitosan, a negatively charged first crosslinking agent, and an active ingredient. The negatively charged first crosslinking agent is tripolyphosphate or fucoidan. The nanocomposite particles have the property of resisting gastric acid decomposition and can only be decomposed in the intestine, which allows the active ingredient to safely pass through the destruction of gastric acid and smoothly reach the intestinal part.

[0006] In one embodiment of the present invention, the positively charged chitosan and the first crosslinking agent are in a ratio of 1:5 to 320.

[0007] In one embodiment of the present invention, the positively charged chitosan:tripolyphosphate ratio is 1:5~160.

[0008] In one embodiment of the present invention, the ratio of positively charged chitosan to fucoidan is 1:10~320.

[0009] In one embodiment of the present invention, the active ingredient is phycocyanin.

[0010] Another objective of the present invention is to provide a method for preparing nanocomposite particles coated with phycocyanin, the method comprising: (1) preparation of positively charged chitosan: (1-1) dissolving chitosan powder in 1% (v / v) 100ml acetic acid solution, (1-2) after the chitosan is completely dissolved, adjusting its pH value to 5.0~7.0, (1-3) dialyzing with double-distilled water; (2) preparation of a premixed solution of chitosan and phycocyanin: mixing the positively charged chitosan and phycocyanin; (3) preparation of nanoparticles coated with phycocyanin: (3-1) adding a negatively charged first crosslinking agent aqueous solution to the premixed solution of chitosan and phycocyanin, (3-2) performing ultrasonic oscillation on ice for 3 seconds, resting for 7 seconds, repeating the step 6 times to produce nanoparticles coated with phycocyanin.

[0011] Another object of the present invention is the use of nanocomposite particle carriers in the preparation of a composition that promotes intestinal health, wherein the composition is a pharmaceutical composition or a food composition, and the promotion of intestinal health refers to enhancing the growth of probiotics, enhancing intestinal immunity, restoring a healthy intestinal flora, or reducing intestinal pathogens.

[0012] In one embodiment of the present invention, the probiotic is selected from the group consisting of species of the genera *Streptococcus*, *Lactococcus*, *Atrophic bacteria*, *Gastroenterobacter*, *Carnivora*, *Enterococcus*, *Lactobacillus*, *Leuconostoc*, *Sacchariformis*, *Micrococcus*, *Tetracoccus*, *Paralysinus*, *Weissella*, *Bifidobacterium*, and *Pediococcus*.

[0013] In one embodiment of the present invention, the probiotic strain is selected from the group consisting of B. bifidum, B. longum, L. paracasei, L. acidophilus, L. reuteri, L. rhamnosus, L. salivarius and L. rhamnosus.

[0014] In one embodiment of the present invention, the intestinal pathogenic bacteria are selected from the group consisting of Salmonella enterica subsp. enterica, Listeria monocytogenes, Escherichia coli, Vibrio parahaemolyticus, Staphylococcus aureus, Shigella boydii, Shigella dysenteriae, Yersinia enterocolitica, and Proteus vulgaris.

[0015] The nanoparticles of the present invention can encapsulate phycocyanin to successfully pass through the stomach and are released in large quantities in the intestines, thus avoiding the destruction and inactivation of phycocyanin in the acidic environment of the stomach and protecting phycocyanin from the effects of high temperature (heat) and light. The phycocyanin nanoparticles are not cytotoxic and can clear and promote the growth of probiotics, have antibacterial properties, and thus increase the immune regulation of the intestines.

Implementation Method

[0033] The present invention is illustrated by the following embodiments, but the present invention is not limited to the following embodiments.

[0034] [Terminology Definitions] Many technical and scientific terms commonly used in the field of biotechnology are extensively used in this specification. In the following description, the following definitions are provided to provide a clear and consistent understanding of this specification and the scope of the claims, as well as the scope to which such terms are given. Other terms not specifically defined below have meanings that are commonly understood by those skilled in the art.

[0035] Unless otherwise specified, all materials used in this invention are commercially available and readily available.

[0036] The words "or," "and," and "and" used in this specification refer to "or / and" unless otherwise stated. Furthermore, the terms "comprising" and "including" are not restrictive open-ended conjunctions. The foregoing paragraphs are for systematic reference only and should not be construed as limiting the subject of the invention.

[0037] Unless otherwise specified, the "%" used in this specification refers to "weight percentage (wt%)"; the numerical range (e.g., 10%~11% of A) includes upper and lower limits unless otherwise specified (i.e., 10%≦A≦11%); if the numerical range does not define a lower limit (e.g., less than 0.2% of B, or less than 0.2% of B), it means that its lower limit may be 0 (i.e., 0%≦B≦0.2%); the proportional relationship of "weight percentage" of each component can also be replaced by the proportional relationship of "parts by weight".

[0038] All values ​​disclosed in this specification may have a standard technical measurement error (standard deviation) of ±10%. The term "about" is intended to indicate ±10%, ±5%, ±2.5%, or ±1% relative to a given value; that is, "about 20%" represents 20 ±2%, 20 ±1%, 20 ±0.5%, or 20 ±0.25%.

[0039] Example 1: Preparation steps of nanoparticles (a1) Preparation of positively charged chitosan: Dissolve 1 g of chitosan powder in 1% (v / v) 100 ml of acetic acid solution to prepare positively charged chitosan. After the chitosan is completely dissolved, adjust the pH to 6 with 1 M NaOH and filter through a 0.22 µm filter membrane. Dialyze the filtrate through a dialysis membrane bag in double-distilled water for 24 hours and store at 4°C for later use. (a2) Mix the positively charged chitosan with phycocyanin. (a3) ​​Add the negatively charged first crosslinking agent aqueous solution to the premixed solution of chitosan and phycocyanin. The negatively charged first crosslinking agent is tripolyphosphate (TPP) or fucoidan. Use a probe-type cell disruptor to perform ultrasonic oscillation on an ice bath (oscillation for 3 seconds, rest for 7 seconds). Repeat the oscillation step 6 times for a total of 1 minute to produce nanoparticles (aqua blue) coated with phycocyanin. After centrifuging at 13200 rpm, the supernatant was removed. The nanoparticles precipitated at the bottom were mixed with 1 ml of phosphate-buffered saline (PBS) solution to form a nanoparticle suspension.

[0040] Example 2: The optimal ratio of chitosan to tripolyphosphate to form nanoparticles

[0041] Nanoparticles were prepared using chitosan and tripolyphosphate at different concentrations in a 1:1 volume ratio. The hydration diameter (Dh) and dispersibility index (PDI) of the nanoparticles were measured using dynamic light scattering (DLS) to determine the optimal concentration for nanoparticle formation. Table 1 shows that when the chitosan concentration was 1 and 0.5 mg / ml, the hydration diameter of the nanoparticles decreased with decreasing tripolyphosphate concentration. When the chitosan concentration was 2 and 0.25 mg / ml, the minimum hydration diameter of the nanoparticles was 629.8 ± 4.9 nm and 455.33 ± 19.2 nm, respectively, when the tripolyphosphate concentration was 10 mg / ml. When the chitosan concentration is 1 mg / ml and 2 mg / ml, the prepared nanoparticles have a large interfacial potential and high stability. Therefore, chitosan at a concentration of approximately 0.25–2 mg / ml and tripolyphosphate at a concentration of approximately 10–40 mg / ml, or a chitosan to tripolyphosphate ratio of approximately 1:5–160, can form nanoparticles. The optimal conditions for the formation of nanoparticles from chitosan and tripolyphosphate are 1 mg / ml chitosan and 40 mg / ml tripolyphosphate. Nanoparticles formed from chitosan and TPP will be abbreviated as CTP (Chitosan, TPP, Phycocyanin); nanoparticles formed from chitosan and fucoidan will be abbreviated as CFP (Chitosan, Fucoidan, Phycocyanin).

[0042] Table 1. Analysis of the properties of nanoparticles composed of chitosan and tripolyphosphate (TPP) at different concentrations.

[0043] Example 3: The optimal ratio of chitosan and fucoidan to form nanoparticles

[0044] Nanoparticles were prepared using chitosan and fucoidan (FUC) at different concentrations in a 1:1 volume ratio. The hydration diameter (Dh) and dispersibility index (PDI) of the nanoparticles were measured using DLS to determine the optimal concentration for nanoparticle formation. As shown in Table 2, when the chitosan concentration was 1 mg / ml and 2 mg / ml, the hydration diameter decreased with increasing fucoidan concentration. However, when the chitosan concentration was 0.5 mg / ml and 0.25 mg / ml, the hydration diameter of the nanoparticles increased with increasing fucoidan concentration. When the chitosan concentration is 1 mg / ml and 2 mg / ml, the prepared nanoparticles have a large interface potential, indicating high stability. Therefore, chitosan with a concentration of about 0.25~2 mg / ml and fucoidan with a concentration of about 20~80 mg / ml, or a chitosan to fucoidan ratio of about 1:10~320, can form nanoparticles. The optimal conditions for the formation of nanoparticles from chitosan and fucoidan are 1 mg / ml chitosan and 80 mg / ml fucoidan.

[0045] Table 2. Analysis of the properties of nanoparticles composed of chitosan and fucoidan (FUC) at different concentrations.

[0046] Example 4: Confirmation of the formation of chitosan nanoparticles (CFP and CTP) coated with phycocyanin

[0047] The chemical properties of the nanoparticles before and after preparation were analyzed using Fourier transform infrared spectroscopy (FT-IR) to explore the formation of chitosan nanoparticles and their coating of phycocyanin. Figure 1 shows the infrared spectra of chitosan and fucoidan-coated phycocyanin nanoparticles (CFP), chitosan and tripolyphosphate-coated phycocyanin nanoparticles (CFP), fucoidan (FUC), TPP, chitosan (CS), and phycocyanin (PC). The spectra of CFP and CTP both contain signals of ionic cross-linking reactants and phycocyanin, confirming the obtained chitosan nanoparticles coated with phycocyanin.

[0048] Example 5: Morphological observation of chitosan nanoparticles (CFP and CTP) coated with phycocyanin

[0049] The morphology and size of the chitosan nanoparticles coated with phycocyanin were observed using a scanning electron microscope (SEM) as shown in Figure 2. The SEM magnification was 200x (Figure 2a) and 4000x (Figure 2c); the SEM magnification was 200x (Figure 2b) and 4000x (Figure 2d). The nanoparticles were irregular in shape, but at 4000x magnification, their layered coating structure was visible.

[0050] Example 6: Calculation of the ratio and physical properties of phycocyanin coated with chitosan nanoparticles (CTP and CFP)

[0051] After completing the coating of phycocyanin with chitosan nanoparticles, the nanoparticles and supernatant were separated by centrifugation (13200 rpm, 20 min). The supernatant contained uncoated phycocyanin. The absorbance intensity at 615 nm of the supernatant was measured using a UV / Vis spectrophotometer and compared with a pre-prepared calibration curve to determine the concentration of uncoated phycocyanin in the supernatant. The coating rate of phycocyanin in the nanoparticles was obtained by subtracting the concentration after coating from the concentration before coating and dividing by the concentration before coating.

[0052] The formula for calculating the coating rate is: Coating rate = [(content before coating - content without coating) / content before coating] × 100%.

[0053] The coating ratio (EE (%)), hydration diameter, dispersibility index, and interfacial potential of phycocyanin nanoparticles using the optimal concentrations of chitosan (C), tripolyphosphate (T), and fucoidan (F) are shown in Table 3. The table shows that the nanoparticles synthesized from chitosan and tripolyphosphate achieved a coating rate of 86% for phycocyanin, while the nanoparticles synthesized from chitosan and fucoidan achieved a coating rate of 88% for phycocyanin.

[0054] Table 3. Property analysis of phycocyanin-coated nanoparticles (CTP and CFP)

[0055] Example 7: Calculation of the ratio of phycocyanin released by chitosan nanoparticles (CFP and CTP) coated with phycocyanin under various pH conditions

[0056] In the release experiment, nanoparticles coated with phycocyanin were added to buffer solutions with different pH values ​​to form nanoparticle suspensions. Samples were collected at various time intervals (0 minutes, 1, 2, 3, 4, 6, 8, 16, 20, 22, and 24 hours). The supernatant was collected, and the phycocyanin content was measured using a UV / Vis spectrophotometer. The result was compared with the phycocyanin content used to determine the rate of phycocyanin release from the nanoparticles. Figure 3 shows the rate of phycocyanin release from chitosan nanoparticles (Figure 3a: CFP, Figure 3b: CTP) in buffer solutions with pH 2, 3, 4, 5, 6, and 7. The release rate of the nanoparticles was relatively low in buffer solutions with pH < 6, and there was no significant and rapid release during the process. After 4 hours, the curve flattened out, and the release rate was only below 40% by the 24th hour. In buffer solutions with pH > 5, the release rate is relatively high, and there is a significant and intense release from the first to the fourth hour. By the fourth hour, the release rates at pH 6 and pH 7 have reached as high as 31% and 38%, respectively, and the release continues for the next 20 hours. By the 24th hour, the release rates are 62% and 72%, respectively.

[0057] To simulate the pH value and corresponding time of the oral carrier passing through the gastrointestinal environment and circulating in the body after absorption, chitosan nanoparticles (CFP and CTP) coated with phycocyanin were placed in a simulated gastric acid environment of pH 2 for 2 hours, followed by a simulated intestinal fluid environment of pH 6.8 for 4 hours, and then a simulated human blood environment of pH 7.4 for 14 hours. The amount of phycocyanin released at each time period was calculated to simulate the release versus time relationship in the body. As shown in Figure 4, there was no large release of phycocyanin in the simulated gastric environment (pH 2) for two hours, while the release increased significantly in the simulated intestinal (pH 6.8) and blood (pH 7.4) environments. This indicates that most of the phycocyanin coated with chitosan nanoparticles can successfully pass through the stomach and be released in large quantities in the intestine and body.

[0058] Example 8: Thermal stability, light stability and swelling tests of chitosan nanoparticles (CFP and CTP) coated with phycocyanin

[0059] In the thermal stability test, the suspension of phycocyanin-coated nanoparticles was placed in a constant temperature environment (pH=7) at 27°C, 37°C, 47°C, and 57°C. At specific time points, the corresponding microcentrifuge tubes were removed, and the nanoparticles and supernatant were separated by centrifugation. The concentration of phycocyanin in the supernatant was measured using a UV / Vis spectrophotometer. Figure 5 shows that the release rate of chitosan nanoparticles coated with phycocyanin (Figure 5a shows CFP, Figure 5b shows CTP) did not change significantly with temperature differences. Therefore, the stability of the chitosan nanoparticles is not affected by high temperature (heat). In the photostability test, suspensions of chitosan nanoparticles (CFP and CTP) coated with phycocyanin were placed in a constant-temperature environment (pH=7) under both light-protected and light-protected conditions. Small amounts of the nanoparticle suspension were taken out at specific time intervals, and the nanoparticles and supernatant were separated by centrifugation. The concentration of phycocyanin in the supernatant was measured using a UV / Vis spectrophotometer. Figure 6 shows that the release rate of the nanoparticles (Figure 6a: CFP, Figure 6b: CTP) did not differ significantly between light-protected and light-protected environments. This indicates that the stability of the chitosan nanoparticles is not affected by light. To understand the relationship between the electrostatic attraction of the chitosan nanoparticles and the pH of the solution, chitosan nanoparticles (CFP and CTP) were suspended in PBS buffer solutions with different pH values, and the particle size change was measured after 4 hours. As shown in Figure 7, at time 0 (Figure 7a: CFP, Figure 7c: CTP), the chitosan nanoparticles at pH values ​​of 2, 3, 4, and 5 had particle sizes between approximately 200-500 nm, while those at pH values ​​of 6 and 7 had particle sizes between approximately 500-700 nm. After 4 hours (Figure 7b: CFP, Figure 7d: CTP), the particle sizes of nanoparticles at pH values ​​of 2, 3, and 4 remained within the 300-600 nm range, while those at pH value 6 increased to 700-1100 nm, and those at pH value 7 increased to 700-2000 nm, mainly concentrated between 900-1500 nm. This observation indicates that a neutral environment affects the electrostatic attraction of chitosan nanoparticles, thus affecting their particle size. This also suggests that pH adjustment can control the release behavior of chitosan nanoparticles.

[0060] Example 9: Degradation test of phycocyanin

[0061] Edible proteins and peptides are rapidly degraded by enzymes and gastric acid in the stomach, resulting in their inability to be completely delivered to the intestines for absorption. This experiment tested the stability of 40 µg / ml phycocyanin in a simulated gastric acid solution (84 mM HCl, 70 mM NaHCO3 and 0.2 mg pepsin, pH 1.2), and the degree of degradation of phycocyanin was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE results in Figure 8 show characteristic blocks of phycocyanin in the 13-18 kDa range. Phycocyanin was mixed with a simulated gastric acid solution, and a quenching solution (300 mM NaHCO3, pH 11) was added at a specific time to terminate the reaction. The reaction was prepared by adding phycocyanin after adding the quenching solution to the gastric acid solution at time t = 0 min. Figure 8 shows that phycocyanin was rapidly degraded after 5 minutes of reaction and completely degraded after 30 minutes, indicating that phycocyanin will be completely degraded in the stomach within 30 minutes and is destroyed before being delivered to the intestines, thus failing to exert its effect.

[0062] The thermal degradation test of phycocyanin involved placing 40 µg / ml phycocyanin in a constant temperature environment at 27°C, 37°C, 47°C, and 57°C for 1, 4, 8, 16, 24, 48, and 72 hours. The degree of degradation of phycocyanin at different temperatures and times was then analyzed by SDS-PAGE. The content of undegraded phycocyanin is shown in Figure 9. Figure 9 shows that after 72 hours at 27°C and 37°C, phycocyanin showed no significant degradation. After 16 hours at 47°C, it had degraded to approximately 68%, and after 72 hours, it had degraded to 1.5%, indicating almost complete degradation. After being placed at 57°C for 1 hour, the phycocyanin content was reduced to 48.3%, and after 4 hours it was reduced to 13.5%. After 8 hours, only 7.2% of the phycocyanin remained, indicating that it was almost completely degraded. Therefore, the denaturation temperature of phycocyanin is approximately 47°C.

[0063] Photodegradation test of phycocyanin: 40 µg / ml phycocyanin was placed in a dark room and under light for 1, 4, 8, 16, 20, 24, 36, 48, and 96 hours. The degradation degree of phycocyanin under dark and light conditions over time was analyzed by SDS-PAGE. The content of undegraded phycocyanin is shown in Figure 10. Figure 10 shows that after 96 hours in the dark environment, phycocyanin did not show significant degradation, maintaining 95% of its initial concentration. Under light conditions, after 16 hours, it degraded to approximately 62.5% of its initial concentration, and after 96 hours, it had degraded to 4.3% of its initial concentration. This indicates that phycocyanin is susceptible to photodegradation, and the degree of photodegradation is related to the duration of light exposure.

[0064] Example 10: Free radical scavenging ability test of phycocyanin-chitosan nanoparticles (CFP and CTP)

[0065] The free radical scavenging ability of a drug in the body can be correlated with its antioxidant capacity. The DPPH colorimetric method is a common antioxidant analysis for determining free radical scavenging ability.

[0066] Figure 11 shows the relationship between the concentration of uncoated phycocyanin (PC) and chitosan nanoparticles coated with phycocyanin (CFP and CTP) at different concentrations and their free radical scavenging capacity, with ascorbic acid as the control group. The results show that phycocyanin has the greatest free radical scavenging capacity at concentrations of 10-40 µg / ml, and phycocyanin coated with chitosan still retains a considerable free radical scavenging capacity. Taking a concentration of 20 µg / ml as an example, compared with ascorbic acid, uncoated phycocyanin (PC) and chitosan nanoparticles coated with phycocyanin (CFP and CTP) both retain more than 80% of their free radical scavenging capacity.

[0067] Figure 12 shows the relationship between the concentration and free radical scavenging ability of phycocyanin (Figure 12a) and chitosan nanoparticles coated with phycocyanin (Figure 12b for CFP and Figure 12c for CTP) after 48 hours of light protection and light exposure. It can be seen that the free radical scavenging ability of phycocyanin decreased significantly after photodegradation, while the free radical scavenging ability of CFP and CTP was not affected by light exposure. This confirms that coating phycocyanin with a carrier can block light exposure and retain its free radical scavenging ability.

[0068] Figure 13 shows the relationship between the concentration and free radical scavenging capacity of phycocyanin (Figure 13a) and chitosan nanoparticles coated with phycocyanin (Figure 13b: CFP, Figure 13c: CTP) after being placed in environments of 27°C, 37°C, 47°C, and 57°C for 72 hours. It can be seen that the free radical scavenging capacity of phycocyanin did not decrease significantly after being placed in environments of 27°C and 37°C for 72 hours, but decreased significantly after being placed in environments of 47°C and 57°C for 72 hours. At 57°C, the free radical scavenging capacity of phycocyanin was less than 50%. The free radical scavenging capacity of the chitosan nanoparticles coated with phycocyanin (CFP and CTP) was not affected by temperature, indicating that the chitosan coating can protect phycocyanin from thermal degradation and maintain its antioxidant activity.

[0069] Figure 14 shows the relationship between the concentration and free radical scavenging capacity of phycocyanin (Figure 14a) and chitosan nanoparticles coated with phycocyanin (Figure 14b: CFP, Figure 14c: CTP) after treatment with neutral buffer solution and acidic simulated gastric acid solution for one hour. Figure 14 shows that the free radical scavenging capacity of phycocyanin decreases significantly after degradation by gastric acid. However, the free radical scavenging capacity of chitosan nanoparticles coated with phycocyanin (CFP and CTP) is comparable in both acidic and neutral environments, indicating that their free radical scavenging capacity is not affected by gastric acid. This shows that chitosan coating can protect phycocyanin from acid degradation and maintain its antioxidant activity.

[0070] Example 11: Analysis of the effect of phycocyanin-chitosan nanoparticles (CTP and CFP) on the growth of probiotics

[0071] Undegraded proteins can be used as a fermentation source for probiotics after entering the intestines. Therefore, in this experiment, 40 µg / ml of phycocyanin, phycocyanin degraded by simulated gastric acid solution (Digested PC), and phycocyanin-coated chitosan nanoparticles (CTP and CFP) were added to the probiotics B. bifidum and L. paracasei. The probiotics without added probiotics served as the control group. The results were compared with MRS medium and MRS medium with added PC. The absorbance at 600 nm was used to represent the growth of probiotics. The growth curves of B. bifidum and L. paracasei were analyzed, as shown in Figure 15. It can be seen that when phycocyanin was added to the medium, the OD600 increased significantly in the first 24 hours. This stage may be the proliferation phase. Compared with the control group, the proliferation phase of the phycocyanin-added group increased more and faster. However, after adding phycocyanin degraded by gastric acid to the medium, the OD600 was significantly lower than that of the control group. There were no significant changes at any time point, indicating that the degraded phycocyanin could not serve as a fermentation source in the intestine. The increase in CTP and CFP during the proliferation phase was relatively slow, possibly because the phycocyanin had not yet been released and could not be fermented by probiotics. By hour 60, the control group had entered the death phase, with OD600 gradually decreasing, but the group with added phycocyanin did not show a significant death phase. This shows that phycocyanin can increase the stationary phase of probiotics, thus increasing their survival rate. The CTP and CFP groups reached their maximum bacterial count at approximately 48 and 60 hours, respectively, possibly because the phycocyanin had been released in large quantities and stably, providing a continuous and sufficient fermentation source for probiotics. Therefore, it can be concluded that phycocyanin can act as a prebiotic to improve the survival rate of probiotics.

[0072] Example 12: Antibacterial activity test of phycocyanin-chitosan nanoparticles (CTP and CFP)

[0073] The fermentation broth of probiotics is rich in growth metabolites, and its components include many antibacterial substances such as organic acids. This experiment aims to detect whether adding phycocyanin and phycocyanin-encapsulated chitosan nanoparticles (CTP and CFP) to the probiotic culture medium as fermentation components affects the growth and metabolism of probiotics, thereby giving their products a higher ability to resist exogenous pathogens. This experiment used eight strains of probiotics (B. bifidum, B. longum, L. paracasei, L. acidophilus, L. reuteri, L. rhamnosus, L. salivarius, L. rhamnosus) as test subjects. After culturing them for 50 hours, cell-free supernatant was used as an antibacterial agent to test the inhibition zone against Escherichia coli and compared with the antibiotic ampicillin. The control group was the MRS culture medium group without additional additives. Table 4 shows that the inhibition zone of cell-free supernatant of probiotics against Escherichia coli was about 5-6 mm. The inhibition zone of cell-free supernatant of probiotics with added phycocyanin, CTP, and CFP increased significantly, and it had a significant effect on all eight strains of probiotics. This shows that the addition of phycocyanin to the culture medium affected the metabolism of probiotics, thereby enhancing their antibacterial effect against Escherichia coli, which also means enhancing the immune regulation of the intestine.

[0074] Table 4. Inhibition zone test of cell-free supernatant containing probiotics with added phycocyanin (PC) and phycocyanin-encapsulated chitosan nanoparticles (CTP and CFP) against Escherichia coli.

[0075] Example 12: Cellular uptake and toxicity test of phycocyanin-chitosan nanoparticles (CTP and CFP)

[0076] Cellular uptake assays are commonly used in the fields of nutrient uptake and drug delivery to assess drug accessibility. This study evaluates the efficacy of phycocyanin as a health food by assessing the time-dependent uptake of phycocyanin (PC) and phycocyanin-coated chitosan nanoparticles (CTP and CFP) by human colon adenocarcinoma cells Caco-2. Caco-2 cells were seeded in 24-well plates (1 × 10⁵ cells / well) and cultured at 37°C in a 5% CO₂ incubator for 24 hours. After cell attachment, phycocyanin-coated nanoparticles were added. After 2, 4, 5, and 6 hours of cell uptake, the old culture medium was removed, and residual waste liquid was washed with PBS. Cells were collected using a cell scraper, and the intracellular phycocyanin content was measured using a fluorescence spectrometer (excitation light 620 nm, emission light 650 nm). Figure 16 shows that at hour 2, PC uptake was higher than CTP and CFP uptake. After hour 4, CTP and CFP uptake exceeded PC uptake, and after hour 5, uptake did not increase significantly, indicating that adding CTP and CFP to Caco-2 cells resulted in optimal uptake after 5 hours. The cytotoxicity of Caco-2 intestinal cells was analyzed using the MTT assay to evaluate the biocompatibility of phycocyanin, CTP, and CFP.

[0077] Figure 17 shows the cytotoxicity analysis of human colon adenocarcinoma cells Caco-2 using the MTT assay to evaluate the biocompatibility of phycocyanin (PC) and phycocyanin-encapsulated chitosan nanoparticles (CTP and CFP). At concentrations of phycocyanin, CTP, and CFP below 40 µg / ml, the survival rate of Caco-2 intestinal cells did not show a significant decreasing trend, indicating that phycocyanin, CTP, and CFP at this concentration were not significantly toxic to the cells.

[0078] In summary, the nanoparticles of the present invention can successfully pass through the stomach and are released in large quantities in the intestines. The nanoparticles can encapsulate phycocyanin, preventing the structure of phycocyanin from being easily destroyed and inactivated in the acidic environment of the stomach, and protecting phycocyanin from the effects of high temperature (heat) and light. The phycocyanin nanoparticles are not cytotoxic, can clear and promote the growth of probiotics, have antibacterial properties, and thus increase the immune regulation of the intestines.

[0079] The foregoing detailed description is a specific illustration of feasible embodiments of the present invention. However, these embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the spirit of the present invention should be included within the patent scope of this application. The aforementioned multiple effects fully meet the statutory patent requirements of novelty and inventiveness. Therefore, this application is filed in accordance with the law, and we respectfully request your office to approve this invention patent application to encourage invention. [Simplified Explanation of the Diagram]

[0016] Figure 1 shows the infrared spectra of chitosan and fucoidan-coated phycocyanin nanoparticles (CFP), chitosan and tripolyphosphate-coated phycocyanin nanoparticles (CTP), fucoidan (FUC), tripolyphosphate (TPP), chitosan (CS), and phycocyanin (PC) analyzed by a Fourier transform infrared spectrometer (FT-IR).

[0017] Figure 2 shows images of chitosan nanoparticles observed using a scanning electron microscope (SEM). Figure 2a shows CFP images observed and photographed at 200x magnification; Figure 2b shows CTP images observed and photographed at 200x magnification; Figure 2c shows CFP images observed and photographed at 400x magnification; Figure 2d shows CTP images observed and photographed at 4000x magnification.

[0018] Figure 3 shows the rate at which chitosan nanoparticles (Figure 3a is CFP, Figure 3b is CTP) release phycocyanin in buffer solutions with pH 2, 3, 4, 5, 6, and 7.

[0019] Figure 4 shows the release rate of chitosan nanoparticles (CFP and CTP) coated with phycocyanin over time at pH 2, pH 6.8 and pH 7.4.

[0020] Figure 5 shows the relationship between the phycocyanin release rate and time of chitosan nanoparticles coated with phycocyanin (Figure 5a is CFP, Figure 5b is CTP) at different temperatures.

[0021] Figure 6 shows the relationship between the rate of phycocyanin release and time of chitosan nanoparticles coated with phycocyanin (Figure 6a is CFP, Figure 6b is CTP) under dark and light conditions.

[0022] Figure 7 shows the particle size distribution of chitosan nanoparticles coated with phycocyanin at different pH values. Figure 7a shows CFP placed in PBS buffer solution at different pH values ​​for 0 hours; Figure 7b shows CFP placed in PBS buffer solution at different pH values ​​for 4 hours; Figure 7c shows CTP placed in PBS buffer solution at different pH values ​​for 0 hours; Figure 7d shows CTP placed in PBS buffer solution at different pH values ​​for 4 hours.

[0023] Figure 8 is a gel electrophoresis image of sodium dodecyl sulfate polyacrylamide. Pepsin, unreacted phycocyanin (PC), and phycocyanin were reacted in a simulated gastric acid solution for 0 to 180 minutes.

[0024] Figure 9 is a quantitative analysis diagram of the degradation degree of phycocyanin at different temperatures over time.

[0025] Figure 10 shows the quantitative analysis of the degradation degree of phycocyanin over time under light or dark conditions.

[0026] Figure 11 shows the relationship between the concentrations of ascorbic acid, phycocyanin (PC), and chitosan nanoparticles (CTP and CFP) encapsulating phycocyanin and their free radical scavenging capabilities.

[0027] Figure 12 shows the relationship between concentration and free radical scavenging ability after 48 hours of light and light-proof environment. Figure 12a shows uncoated phycocyanin, Figure 12b shows phycocyanin-coated chitosan nanoparticles (CFP), and Figure 12c shows phycocyanin-coated chitosan nanoparticles (CTP).

[0028] Figure 13 shows the relationship between concentration and free radical scavenging ability at different temperatures. Figure 13a shows uncoated phycocyanin, Figure 13b shows phycocyanin-coated chitosan nanoparticles (CFP), and Figure 13c shows phycocyanin-coated chitosan nanoparticles (CTP).

[0029] Figure 14 is a graph showing the relationship between the concentration and free radical scavenging ability after treatment with simulated gastric acid solution. Figure 14a shows uncoated phycocyanin, Figure 14b shows phycocyanin-coated chitosan nanoparticles (CFP), and Figure 14c shows phycocyanin-coated chitosan nanoparticles (CTP).

[0030] Figure 15 shows the absorbance values ​​(OD600) of the growth curves of probiotics (Figure 15a is L. paracasei, Figure 15b is B. bifidum). Phycocyanin (PC), phycocyanin-encapsulated chitosan nanoparticles (CTP and CFP), and phycocyanin degraded by simulated gastric acid solution (Digestion PC) were added to the probiotics. The control group represents the group with only probiotics and no added probiotics. MRS represents the sample without probiotics and only MRS medium. MRS+PC represents the MRS medium without probiotics and PC added.

[0031] Figure 16 shows the relationship between the uptake of phycocyanin (PC) and phycocyanin-encapsulated chitosan nanoparticles (CTP and CFP) by human intestinal Caco-2 cells and time.

[0032] Figure 17 shows the cell viability of human intestinal Caco-2 cells cultured with the addition of phycocyanin (PC) and phycocyanin-encapsulated chitosan nanoparticles (CTP and CFP).

Claims

1. A nanocomposite particle carrier comprising: positively charged chitosan, a negatively charged first cross-linking agent, and an active ingredient, wherein the negatively charged first cross-linking agent is tripolyphosphate or fucoidan, and the active ingredient is phycocyanin, wherein: (1) When the negatively charged first crosslinking agent is tripolyphosphate, the concentration of the positively charged chitosan is 1 mg / ml and the concentration of the tripolyphosphate is 40 mg / ml; (2) When the negatively charged first crosslinking agent is fucoidan, the concentration of the positively charged chitosan is 1 mg / ml and the concentration of the fucoidan is 80 mg / ml; The nano-composite particle carrier has the characteristic of resisting gastric acid decomposition and being decomposed only in the intestine, which allows the active ingredient to safely pass through the destruction of gastric acid and smoothly reach the intestine to exert its effect. The nano-composite particle carrier has good biocompatibility with intestinal cells, has antioxidant activity, and can improve the stability of the phycocyanin.

2. The nanocomposite particle carrier as described in claim 1, the preparation method of the carrier includes: (1) preparation of positively charged chitosan: (1-1) dissolving chitosan powder in 1% (v / v) 100ml acetic acid solution, (1-2) after the chitosan is completely dissolved, adjusting its pH value to 5.0~7.0, (1-3) dialyzing with double-distilled water; (2) preparation of a premixed solution of chitosan and phycocyanin: mixing the positively charged chitosan and phycocyanin; (3) preparation of nanoparticles coated with phycocyanin: (3-1) adding a negatively charged first crosslinking agent aqueous solution to the premixed solution of chitosan and phycocyanin, the negatively charged first crosslinking agent being tripolyphosphate (TPP) or fucoidan, (3-2) performing ultrasonic oscillation on ice for 3 seconds, resting for 7 seconds, repeating the step 6 times to produce nanoparticles coated with phycocyanin.

3. Use of a nanocomposite particle carrier as claimed in claim 1 or 2 for preparing a composition that promotes gut health, wherein the composition is a pharmaceutical composition or a food composition, and the promotion of gut health refers to enhancing the growth of probiotics, enhancing gut immunity, restoring a healthy gut microbiota, or reducing gut pathogens.

4. As claimed in claim 3, the probiotic strain is selected from the group consisting of B. bifidum, B. longum, L. paracasei, L. acidophilus, L. reuteri, L. rhamnosus, L. salivarius and L. rhamnosus.

5. As requested in item 3, the enteric pathogen is Escherichia coli.

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