A hydrogel bead loaded with astaxanthin and a preparation method thereof

Abstract

The present invention discloses a hydrogel bead loaded with astaxanthin and a preparation method thereof, belonging to the technical field of bioactive ingredient stabilization and colloidal delivery. The specific steps include: mixing the above-mentioned 20% inner oil phase containing ASTA and 80% intermediate aqueous phase together according to the mass ratio, and then obtaining an O1 / W emulsion loaded with ASTA through high-pressure homogenization; mixing the gelatin solution and the O1 / W emulsion evenly, and then adding genipin to the mixture of gelatin and the O1 / W emulsion, and mixing 80% outer oil phase and 20% O1 / W emulsion by stirring or high-speed shear mixing to prepare an O1 / W / O2 emulsion loaded with ASTA; then obtaining the hydrogel bead loaded with ASTA through centrifugation and washing. Encapsulating astaxanthin in the hydrogel bead to ensure its stable arrival at the digestion site; encapsulating astaxanthin and oil together in the hydrogel bead, and reaching the small intestine for digestion along with the oil. The oil digestion produces FFA, and FFA assists the formation of astaxanthin micelles by bile salt micelles, thereby promoting absorption.

Description

Technical Field

[0001] The present invention belongs to the field of bioactive ingredient stabilization and colloidal delivery, and particularly relates to a method for preparing astaxanthin-loaded hydrogel beads by an emulsion templating method. Background Art

[0002] Astaxanthin (ASTA) is a red carotenoid mainly derived from marine organisms (such as salmon, rainbow trout, shrimp, lobster, and Haematococcus pluvialis). As a natural active substance, ASTA has various effects such as antioxidant, anti-inflammatory, anti-cancer, anti-diabetic, heart protection, weight loss, and blood sugar regulation, which can promote human health. Previous studies have shown that the antioxidant activity of ASTA is 10–500 times that of well-known natural antioxidants (such as β-carotene and vitamin E). However, ASTA has low stability, is easily oxidized, decomposed by light, and fades in a sensitive environment, losing its physiological activity. Therefore, its application in the food industry is limited. Moreover, there are no specific receptor cells on the small intestinal epithelial cells, resulting in a very low absorption rate of less than 10%, especially due to the water-insolubility of carotenoids, the absorption rate is less than 1%. Therefore, how to improve the stability and bioacceptability of ASTA has become one of the important research contents in the current functional food field.

[0003] In recent years, more and more researchers have focused on using hybrid-filled biopolymer gel beads as carriers for active substances. The filled biopolymer gel beads are composed of biopolymers, and the oil droplets are loaded inside the macromolecules. The structure and composition of biopolymer gel beads can be controlled by selecting the components and manufacturing methods used to produce them, which allows people to adjust their functions according to specific applications. For example, the composition, size, shape, pore size, polarity, and intermolecular interactions of biopolymer gel beads can all be regulated. After the biopolymer gel beads are formed, their properties can be changed by coating biopolymers or changing the surface polarity, charge, stability, aggregation state, release characteristics, or digestibility of colloidal particles. The present invention selects a suitable method to prepare relatively small and well-defined hydrogel beads for loading and protecting ASTA and improving the bioacceptability of ASTA. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing astaxanthin-loaded hydrogel beads by an emulsion templating method, which solves the problems of low stability and absorption performance of astaxanthin during consumption due to the influence of the external environment.

[0005] To achieve the above purpose, a preparation method for preparing astaxanthin-loaded hydrogel beads according to the present invention specifically includes the following steps:

[0006] (1) Preparation of O1 / W emulsion loaded with ASTA:

[0007] (101) Dissolve 1% of Q-natural in PBS buffer solution with pH = 7 and mix evenly to obtain an intermediate aqueous phase;

[0008] (102) Dissolve ASTA in linseed oil and mix evenly to obtain an inner oil phase;

[0009] (103) Mix the above 20% inner oil phase and 80% intermediate aqueous phase together by mass ratio, and then obtain the O1 / W emulsion loaded with ASTA through high-pressure homogenization;

[0010] (2) Preparation of O1 / W / O2 emulsion:

[0011] (201) Mix the gelatin solution and the O1 / W emulsion evenly, and then add genipin to the mixture of gelatin and O1 / W emulsion to ensure that the final concentrations of gelatin, genipin, and linseed oil in the O1 / W emulsion are 5%, 0.1%, and 15% respectively;

[0012] (202) Mix 10% of the oil-soluble surfactant (PGPR) and 90% of corn oil by mass ratio to obtain an outer oil phase. Mix 80% of the outer oil phase and 20% of the O1 / W emulsion by mass ratio through stirring or high-speed shearing to prepare the O1 / W / O2 emulsion loaded with ASTA;

[0013] (3) Preparation of hydrogel beads:

[0014] Place the above three O1 / W / O2 emulsions at room temperature of 20 °C, gel and crosslink in the dark for 24 h, then centrifuge to separate the hydrogel beads, remove the outer oil phase, wash to remove the residual outer oil phase, and finally remove the excess n-hexane by vacuum filtration to obtain semi-solid particles filled with hydrogel particles. Collect the hydrogel beads, wash with ultrapure water and PBS to remove the excess ions on the surface to obtain the hydrogel beads loaded with ASTA.

[0015] Specifically, in step (103), after mixing 20% inner oil phase and 80% intermediate aqueous phase, mix them with a high-speed stirrer at 20000 rpm for 2 min, and then circulate three times under the pressure of 13000 psi through microfluidics, and naturally cool to room temperature to obtain the O1 / W emulsion loaded with ASTA.

[0016] Specifically, the specific mixing method in step (202) is:

[0017] Method a: Rapidly mix 20% of the O1 / W emulsion and 80% of the outer oil phase, and stir with a spiral stirrer for 2 min;

[0018] Method b: Slowly add 20% of the O1 / W emulsion to 80% of the external oil phase, and stir with a high-speed stirrer at 20,000 rpm while adding. After all is added, stir for another 2 min;

[0019] Method c: Use a magnetic stirrer to drop 20% of the O1 / W emulsion into 80% of the external oil phase and stir at room temperature for 2 min.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) Encapsulating astaxanthin in hydrogel beads effectively solves the problems of its easy oxidation in the external environment and easy decomposition when exposed to light. At the same time, it also solves the damage to its structure by the digestive tract environment before the small intestine, ensuring its stable delivery to the digestion site. More importantly, it can ensure its slow release in the small intestine;

[0022] (2) Mixing astaxanthin with oil and encapsulating them together in hydrogel beads, and reaching the small intestine for digestion along with the oil. The oil digestion produces FFA, and FFA assists bile salt micelles to form astaxanthin micelles, thereby promoting absorption;

[0023] (3) By simulating the digestive environments of the stomach and small intestine, calculating the ratio of astaxanthin in the end product to the initially loaded astaxanthin to measure the bioavailability (loading efficiency of hydrogel beads) of astaxanthin, the results show that the loading has basically no effect on the structure of astaxanthin;

[0024] (4) The hydrogel beads loaded with astaxanthin can be dispersed in an aqueous solution containing a hydrophilic emulsifier (Q-natural), greatly reducing the aggregation degree of the hydrogel beads, and thus promoting the water dispersibility of the hydrogel beads;

[0025] (5) Linseed oil is an edible oil rich in ω-3 fatty acids, and corn oil, as a kind of long-chain fatty acid, can effectively improve the retention rate of loaded astaxanthin. Description of the Drawings

[0026] Figure 1 It is a flowchart of the preparation method of the hydrogel beads loaded with astaxanthin ASTA involved in Example 1.

[0027] Figure 2 It is the microscopic images of the O1 / W emulsions (a, c, and e) loaded with ASTA and the hydrogel beads loaded with ASTA prepared in Example 1 (b, d, and f), from top to bottom are Method a, Method b, and Method c.

[0028] Figure 3 It is a photo of the dispersion state of the hydrogel beads loaded with ASTA prepared in Example 1 in an aqueous solution (containing 1% Q-natural), from left to right are Method a, Method b, and Method c.

[0029] Figure 4 Microscopic images of the ASTA-loaded hydrogel beads prepared in Example 1 in an aqueous solution (containing 1% Q-natural), from top to bottom are Method a, Method b, and Method c.

[0030] Figure 5 CLSM images of the ASTA-loaded hydrogel beads prepared in Example 1 in an aqueous solution (containing 1% Q-natural), from left to right are Method a, Method b, and Method c.

[0031] Figure 6 Process diagram of the pH stat detecting the release of FFA from the hydrogel beads digested by the in vitro model.

[0032] Figure 7 Final FFA released from the hydrogel beads digested by the pH stat detected by the in vitro model at different pH values.

[0033] Figure 8 Bioacceptance rate of ASTA after digestion of the hydrogel beads. Detailed implementation mode

[0034] The present invention will be further described below through specific implementation modes.

[0035] Example 1

[0036] A method for preparing ASTA-loaded hydrogel beads by an emulsion templating method involved in this example specifically includes the following steps:

[0037] (1) Preparation of the O1 / W emulsion loaded with ASTA:

[0038] (101) Dissolve 1% of Q-natural in PBS buffer solution with pH = 7, stir for 2 h, and place it in a refrigerator at 4 °C overnight to ensure complete dissolution, obtaining the intermediate aqueous phase; the Q-natural (Ingredion Incorporated, Q-NATURALE 200Dry-17955923) is a natural food-grade surfactant isolated from the bark of Quillaja saponaria Molina;

[0039] (102) Dissolve ASTA in linseed oil, heat to 50 °C and stir for 30 min until completely dissolved, obtaining the internal oil phase;

[0040] (103) Mix the above 20% internal oil phase and 80% intermediate aqueous phase together according to the mass ratio, then mix them with a high-speed stirrer at 20000 rpm for 2 min, and then circulate three times under a pressure of 13000 psi through microfluidics, and naturally cool to room temperature to obtain the O1 / W emulsion loaded with ASTA, and store it at 4 °C for later use.

[0041] (2) Preparation of O1 / W / O2 emulsion:

[0042] (201) Prepare a gelatin solution with an appropriate concentration, stir overnight, mix the completely dissolved gelatin with the O1 / W emulsion and stir, then add genipin to the mixture of gelatin and O1 / W emulsion, ensuring that the final concentrations in the O1 / W emulsion are 5% gelatin, 0.1% genipin, and 15% linseed oil;

[0043] (202) Mix 10% of the oil-soluble surfactant (PGPR) with 90% of corn oil by mass to obtain the outer oil phase. Mix 80% of the outer oil phase with 20% of the O1 / W emulsion by mass to prepare the O1 / W / O2 emulsion loaded with ASTA. The specific mixing method is as follows:

[0044] Method a: Rapidly mix 20% of the O1 / W emulsion with 80% of the outer oil phase and stir with a spiral stirrer for 2 min;

[0045] Method b: Slowly add 20% of the O1 / W emulsion to 80% of the outer oil phase and stir with a high-speed stirrer at 20000 rpm, stirring while adding. After adding all, stir for another 2 min;

[0046] Method c: Use a magnetic stirrer to drop 20% of the O1 / W emulsion into 80% of the outer oil phase and stir at room temperature for 2 min.

[0047] (3) Preparation of hydrogel beads:

[0048] Place the above three kinds of O1 / W / O2 emulsions at 20 °C room temperature, gel and crosslink in the dark for 24 h, then centrifuge to separate the hydrogel beads, remove the outer oil phase, add an equal volume of n-hexane, stir and disperse, and centrifuge. Repeat this three times to remove the residual outer oil phase. Finally, remove the excess n-hexane by vacuum filtration to obtain semi-solid particles filled with hydrogel particles. Collect the hydrogel beads, wash them with ultrapure water and PBS to remove the excess ions on the surface, and obtain the hydrogel beads loaded with ASTA, which are stored at 4 °C.

[0049] 1. Characteristics of hydrogel beads

[0050] According to Figure 3 it can be understood that the hydrogel beads can be well dispersed in pure water containing an emulsifier (1% Q-natural) ( Figure 3)。The dispersion phases of the ASTA-loaded hydrogel beads prepared by the three methods were all turbid and light yellow, indicating that the hydrogel beads could be well dispersed. This was because Q-natural was a molecular emulsifier, which could more effectively adsorb on the hydrogel beads to cover the non-polar parts of the surface, thus greatly reducing the aggregation degree of the hydrogel beads and further promoting the aqueous dispersibility of the hydrogel beads. By observing the microscopic images of the hydrogel beads dispersed in 1% Q-natural aqueous solution ( Figure 4 ), it could be seen that the re-dispersed hydrogel bead particles were intact and had a more uniform shape. The beads were stable discrete particles without adhesion or aggregation. However, the hydrogel beads prepared by the high-speed stirrer were small in size and there was also partial aggregation. The CLSM images showed that the oil droplets (stained red) were completely encapsulated in the hydrogel beads (stained green) ( Figure 5 ), and the oil phase could be evenly distributed throughout the O1 / W system, indicating that a delivery system loaded with ASTA could be prepared.

[0051] 2. Release of free fatty acid FFA during the digestion of hydrogel beads

[0052] Astaxanthin is lipophilic, insoluble in water and soluble in organic solvents. Free astaxanthin is extremely unstable and easily oxidized. In the present invention, astaxanthin is loaded in hydrogel beads to make full use of its lipophilic characteristics while protecting it and improving its stability in the environment. The oil phase of the hydrogel beads is lipid, which is mainly digested in the small intestine. By monitoring the release degree and rate of free fatty acid FFA in the small intestine, the encapsulation effect of the hydrogel beads on the loaded ASTA is evaluated. The automatic acid-base titration method (pH-stat) is used to monitor lipid digestion and free fatty acid FFA release under simulated small intestine conditions. Figure 6FFA release curves of hydrogel beads in SIF. Hydrogel beads loaded with ASTA prepared by three methods had low digestibility during the entire small intestine phase. They were rapidly digested in the first 20 min due to the presence of free oil droplets on the surface of the hydrogel beads. In the subsequent longer period, the FFA release was slower because the oil droplets were inside the hydrogel beads. It reached a relatively stable level until 50 min. These results clearly showed that the loading of hydrogel beads could slow down the rate of lipid digestion. However, there were obvious differences in the FFA release rates among hydrogel beads prepared by different methods. This might be because under simulated GIT conditions, the sizes of the hydrogel bead spheres were different. After being digested by pepsin, the larger hydrogel beads disintegrated more irregularly and were more likely to release oil droplets in the small intestine phase, and lipase molecules were more likely to contact the oil droplets. During lipid digestion, the rate and extent of lipid digestion significantly depended on the oil droplet loading rate. The larger the original hydrogel bead diameter, the more oil droplets were loaded, and the FFA release amount was about 45–55% within 2 h. The hydrogel beads prepared by high-speed stirring had a release amount of only 40% in the first 2 h, which might also be attributed to the fact that after the large hydrogel beads were crosslinked with genipin, the crosslinking density was not as high as that of the smaller diameter hydrogel beads, and the ATSA molecules were more likely to escape. There were several reasons to explain the lower digestibility of oil droplets in the hydrogel beads. First, lipid digestion relied on the adsorption of lipase on the surface of the oil droplets so that it could contact the triacylglycerol molecules. In the case of free oil droplets, lipase could easily and unhinderedly migrate to the surface of the oil droplets. On the contrary, since the oil droplets were loaded inside the hydrogel beads, lipase had to penetrate the hydrogel matrix. Second, if the long-chain FFA generated during digestion accumulated at the oil-water interface, lipid digestion would be hindered. These FFA were usually removed by forming mixed micelles with bile salts and phospholipids, or forming insoluble salts with calcium ions. The removal of FFA on the surface of the oil droplets by mixed micelles or calcium salts might be inhibited in the presence of hydrogel beads. This might be due to the inhibition of the molecular diffusion process by the hydrogel particles. In summary, partial release was due to the disintegration of the hydrogel beads. Therefore, we could control the release rate of gastric juice and intestinal juice and the disintegration kinetics of intestinal juice by changing the size of the hydrogel beads. Using the internationally recognized lipid digestion method, the free fatty acid release rate of the hydrogel beads at the end of the small intestine was only 41–57%, lower than the in vitro digestion method of nanoemulsion widely used earlier (>90%). This phenomenon might occur because when using a low calcium level, some FFA did not ionize at neutral pH and could not be titrated. To verify this hypothesis, we used an additional back-titration step to detect the total amount of FFA released at pH = 9. In this case, the final FFA release amount was much higher, about 100–187%( Figure 7)。Assuming that each triglyceride molecule releases two free fatty acids, the final FFA value is far higher than the expected value of 100%. Several other studies have also reported that this effect of having a free fatty acid value of >100% at the end of lipid digestion may be due to the alkaline hydrolysis of triacylglycerol molecules at high pH, resulting in more than two FFAs per triacylglycerol.

[0053] If astaxanthin is simply transported to the small intestine, there are no specific receptor cells on the small intestinal epithelial cells, resulting in a very low absorption rate, less than 10%. Therefore, in the present invention, astaxanthin is dissolved in oil, and the digestion of the oil produces FFA. The FFA assists in the formation of astaxanthin micelles by bile salt micelles, thereby promoting absorption.

[0054] From Figure 8 it is known that the bioavailability of ASTA in all hydrogel beads is about 65.5% (the absorbance of ASTA is measured at 474 nm using a UV-visible spectrophotometer. The bioavailability of ASTA is calculated by the following formula: bioavailability = concentration of ASTA in micelles / concentration of ASTA in the original sample). This is because after most of the ASTA passes through the digestive tract, the soluble part is mixed with the digested micelles, resulting in a relatively high bioavailability. ASTA is loaded in this relatively stable hydrogel bead delivery system, and the degradation amount is relatively low.

Claims

1. A preparation method of a shrimp astaxanthin-loaded hydrogel bead, characterized in that, Specifically, it includes the following steps: (1) Preparation of the O1 / W emulsion loaded with astaxanthin, where the astaxanthin is dissolved in the oil phase; (2) Preparation of the O1 / W / O2 emulsion: (201) Mix the gelatin solution and the O1 / W emulsion loaded with astaxanthin evenly, and then add genipin to the mixture of gelatin and the O1 / W emulsion loaded with astaxanthin; (202) According to the mass ratio, mix 80% of the external oil phase and 20% of the O1 / W emulsion treated in step (201) by stirring or high-speed shearing to prepare the O1 / W / O2 emulsion loaded with astaxanthin; (3) Preparation of hydrogel beads: Place the above-mentioned O1 / W / O2 emulsion loaded with astaxanthin at room temperature of 20 °C, and gel and crosslink it in the dark for 24 h. Then, centrifuge to separate the hydrogel beads, remove the external oil phase, wash to remove the residual external oil phase. Finally, obtain the semi-solid particles filled with hydrogel particles by vacuum filtration, wash, and obtain the hydrogel beads loaded with astaxanthin.

2. The preparation method of the shrimp astaxanthin-loaded hydrogel beads according to claim 1, wherein Step (1) is specifically as follows: (101) Dissolve 1% of Q-natural in the PBS buffer solution with pH = 7, and mix evenly to obtain the intermediate aqueous phase. Q-natural is a natural food-grade surfactant isolated from the bark of Quillaja saponaria Molina; (102) Dissolve astaxanthin in linseed oil and mix evenly to obtain the internal oil phase; (103) According to the mass ratio, mix the above-mentioned 20% internal oil phase and 80% intermediate aqueous phase together, and then obtain the O1 / W emulsion loaded with astaxanthin by high-pressure homogenization.

3. The preparation method of the shrimp astaxanthin-loaded hydrogel beads according to claim 2, characterized in that, The final concentrations of gelatin, genipin, and linseed oil in the O1 / W emulsion treated in step (201) are 5%, 0.1%, and 15% respectively.

4. The preparation method of the shrimp astaxanthin-loaded hydrogel beads according to claim 1, wherein, In step (202), according to the mass ratio, mix 10% of the oil-soluble surfactant and 90% of corn oil to obtain the external oil phase.

5. The preparation method of the shrimp astaxanthin-loaded hydrogel beads according to claim 2, characterized in that, In step (103), after mixing the 20% internal oil phase and 80% intermediate aqueous phase, mix them with a high-speed stirrer at 20000 rpm for 2 min, then circulate three times under the pressure of 13000 psi through microfluidics, and naturally cool to room temperature to obtain the O1 / W emulsion loaded with astaxanthin.

6. The preparation method of the shrimp astaxanthin-loaded hydrogel beads according to claim 1, wherein The specific mixing method in step (202) is as follows: Method a: Quickly mix 20% of the O1 / W emulsion and 80% of the external oil phase, and stir with a helical stirrer for 2 min; Method b: Slowly add 20% of the O1 / W emulsion to 80% of the external oil phase, and use a high-speed stirrer to stir at 20000 rpm, stirring while adding. After all are added, stir for another 2 min; Method c: Use a magnetic stirrer to drop 20% of the O1 / W emulsion into 80% of the external oil phase, and stir at room temperature for 2 min.

7. Hydrogel beads loaded with astaxanthin prepared by the preparation method according to any one of claims 1-6.

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