Preparation method of theaflavin-loaded pH response type composite hydrogel

Abstract

The invention discloses a preparation method of theaflavin-loaded pH response type composite hydrogel, and belongs to the technical field of carrier systems in food preparation technologies. According to the preparation method disclosed by the invention, the theaflavin is physically embedded by preparing the theaflavin solution and preparing the whey protein / sodium alginate solution, so that the theaflavin is prevented from being damaged by a high-acidity environment in the stomach, and the theaflavin is released after reaching the intestinal tract. The method is simple and safe to operate, low in raw material cost, safe and non-toxic, and can be applied to various fields.

Description

Technical Field

[0001] The invention relates to a method for preparing a pH-responsive composite hydrogel loaded with theaflavins, and belongs to the technical field of carrier systems in food preparation technology. Background Art

[0002] Theaflavins are the main polyphenolic components in fermented black tea. They are the main red pigment in black tea and are associated with a variety of health benefits, such as weight loss, hypoglycemic, anti-cancer, and anti-inflammatory. In addition, studies have found that theaflavins in black tea are considered to be a bioactive compound with multiple beneficial effects, but they show low stability, especially in alkaline solutions, and poor bioavailability, which severely limits their application in the food industry.

[0003] At present, there are few studies on solving or improving the low bioavailability of free theaflavins. With the continuous development of new drug delivery systems, a variety of drug delivery methods are used to embed bioactive compounds. Applying these technologies to theaflavins can not only improve their bioavailability, but also promote their application in food and other fields. In the existing technology of embedding theaflavins, theaflavins are released in gastric juice, the release rate in the intestine is low, and the sustained release effect is poor.

[0004] In recent years, the encapsulation of hydrogels, especially natural polymer hydrogels, has been found to be feasible, economical, and efficient in enhancing the stability and bioavailability of nutrients and achieving sustained and controlled release in the gastrointestinal tract. The hydrogel network can create an isolated environment that helps protect the embedded components from destructive stimuli (pH, heat, and oxygen), thereby improving stability. Moreover, hydrogels have a significant feature, namely the ability to swell, which enables them to increase the degradation of encapsulated bioactive compounds during gastric digestion. However, most hydrogel delivery systems are mainly made by chemical cross-linking, however, the safety of the chemical cross-linking method may not be fully guaranteed. Summary of the invention

[0005] [Technical issues]

[0006] The technical problem to be solved by the present invention is how to prepare a safe composite hydrogel loaded with theaflavins, further reduce the release of theaflavins in the stomach, and improve its release rate and sustained-release effect in the intestine.

[0007] [Technical solution]

[0008] In order to solve the above problems, the present invention provides a method for preparing a pH-responsive composite hydrogel loaded with theaflavins, which uses whey protein (WP) and sodium alginate (SA) as the wall materials of the hydrogel, and then adds theaflavins to a mixed solution of whey protein / sodium alginate, and finally drips it into a cross-linking agent solution (a mixed solution of carboxymethyl chitosan solution and calcium chloride solution) to carry out a cross-linking reaction to obtain composite hydrogel beads. The composite hydrogel prepared by the present invention prevents theaflavins from being destroyed by the high acidity environment in the stomach, so that the theaflavins can be released only when they reach the intestine. The method of the present invention is simple to operate, safe to operate, has low raw material cost, and is safe and non-toxic.

[0009] The present invention provides a method for preparing a pH-responsive composite hydrogel loaded with theaflavins, which specifically comprises the following steps:

[0010] (1) dissolving whey protein and sodium alginate in water respectively to obtain a whey protein solution and a sodium alginate solution;

[0011] (2) mixing the whey protein solution and the sodium alginate solution to obtain a whey protein / sodium alginate mixed solution;

[0012] (3) dissolving theaflavins in water to obtain a theaflavins solution;

[0013] (4) mixing the theaflavins solution with the whey protein / sodium alginate mixed solution to obtain the theaflavins-whey protein / sodium alginate mixed solution;

[0014] (5) dissolving carboxymethyl chitosan and calcium chloride in water respectively to obtain a carboxymethyl chitosan solution and a calcium chloride solution;

[0015] (6) mixing the carboxymethyl chitosan solution and the calcium chloride solution to obtain a cross-linking agent solution;

[0016] (7) dropping the theaflavins-whey protein / sodium alginate mixed solution into a crosslinking agent solution to carry out a crosslinking reaction, filtering, and washing to obtain a pH-responsive composite hydrogel loaded with theaflavins.

[0017] In one embodiment of the present invention, in step (1), the mass concentration of the whey protein solution is 5-20%.

[0018] In one embodiment of the present invention, in step (1), the mass concentration of the sodium alginate solution is 0.5-5%.

[0019] In one embodiment of the present invention, in step (2), the volume ratio of the whey protein solution to the sodium alginate solution is 1:1-10.

[0020] In one embodiment of the present invention, in step (3), the concentration of the theaflavin solution is 0.1-5 mg / mL.

[0021] In one embodiment of the present invention, in step (4), the volume ratio of the theaflavin solution to the whey protein / sodium alginate mixed solution is 1:1-5.

[0022] In one embodiment of the present invention, in step (5), the mass concentration of the carboxymethyl chitosan solution is 0.5-5%; the mass concentration of the calcium chloride solution is 1-10%.

[0023] In one embodiment of the present invention, in step (6), the volume ratio of the carboxymethyl chitosan solution to the calcium chloride solution is 1:1-10.

[0024] In one embodiment of the present invention, in step (7), the volume ratio of the theaflavin-whey protein / sodium alginate mixed solution to the cross-linking agent solution is 1:2-5.

[0025] In one embodiment of the present invention, in step (7), the temperature of the cross-linking reaction is 10-30° C., and the time of the cross-linking reaction is 0.5-6 h.

[0026] In one embodiment of the present invention, in step (7), a syringe is used to drop the theaflavin-whey protein / sodium alginate mixed solution into the cross-linking agent solution.

[0027] The invention provides a pH-responsive composite hydrogel loaded with theaflavins prepared by the method.

[0028] Application of the pH-responsive composite hydrogel loaded with theaflavins of the present invention in the field of food technology.

[0029] [Beneficial Effects]

[0030] 1. The hydrogel obtained by the present invention has a controllable targeted release effect, which prevents theaflavins from being damaged by the high acidity environment in the stomach, allowing them to be absorbed by the intestines, thereby exerting the beneficial properties of theaflavins;

[0031] 2. The present invention prepares theaflavins into a hydrogel to protect them, so that they can avoid the adverse conditions in the stomach after oral administration and reach the intestinal tract in the largest amount; the present invention uses edible materials (whey protein, sodium alginate) as the wall material of the hydrogel, which have the advantages of good biocompatibility, low toxicity, relatively low cost, and chemical and thermal stability;

[0032] 3. The hydrogel prepared by the present invention has obvious pH response characteristics. Its swelling rate is small when the pH is 2, and its swelling rate is large when the pH is 7.4, which shows that the present invention can effectively reduce the release of theaflavins in the stomach; the present invention adopts a combination of ion gel method and physical embedding method to solve the problem of low bioavailability of free theaflavins. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The swelling test results of the composite hydrogels prepared in Examples 1-5, (a) is pH 2.0, (b) is pH 7.4;

[0034] Figure 2 The swelling test results of the composite hydrogels prepared in Comparative Examples 1-5 are as follows: (a) is pH 2.0, and (b) is pH 7.4;

[0035] Figure 3 The results of in vitro simulated digestion of the composite hydrogel prepared in Example 2-5;

[0036] Figure 4 The results of in vitro simulated digestion of whey protein / sodium alginate hydrogel beads loaded with theaflavins prepared in Comparative Examples 2-5;

[0037] Figure 5 is the bioaccessibility of theaflavins in the composite hydrogel bead systems of Examples 1-5 and Comparative Examples 1-5;

[0038] Figure 6 The swelling ratios of the composite hydrogels of Example 5 and Comparative Example 5 at 60° C. (a) is pH 2.0, and (b) is pH 7.4;

[0039] Figure 7 The swelling ratios of the composite hydrogels of Example 5 and Comparative Example 5 at a humidity of RH 75%, (a) is pH 2.0, (b) is pH 7.4;

[0040] Figure 8 The swelling ratios of the composite hydrogels of Example 5 and Comparative Example 5 under strong light (4500 lx), (a) is pH 2.0, and (b) is pH 7.4. DETAILED DESCRIPTION

[0041] Swelling performance test:

[0042] The swelling properties of the freeze-dried gel were measured in solutions simulating the pH environment of the gastrointestinal tract. The freeze-dried gel was weighed and immersed in PBS media containing different pH values ​​and allowed to swell at 37°C. The excess water on the sample surface was removed with filter paper at specific time points and then weighed. The swelling ratio was calculated as follows: Swelling ratio (mg / mg) = (W s -W d ) / W d , where W s is the weight of the swollen sample, W d is the weight of the initial dry sample.

[0043] Theaflavins in vitro release test:

[0044] The in vitro release behavior of whey protein / sodium alginate hydrogel loaded with theaflavins was analyzed under simulated gastric fluid (pH 2.0) and simulated intestinal fluid (pH 7.4). 5-10 mg of freeze-dried hydrogel was incubated in a thermostatic oscillator at 37°C and 100 rpm in simulated gastric fluid for 2 h. Subsequently, the beads were transferred to simulated intestinal fluid and incubated for another 2 h. 0.2 mL of sample solution was extracted every half an hour for theaflavins content determination and replaced with an equal amount of fresh PBS to keep the system volume constant. The absorbance value of the test solution was measured at 380 nm using a UV spectrophotometer, and the content of theaflavins in the supernatant was calculated.

[0045] Assessment of theaflavin bioaccessibility:

[0046] The samples (10 mL of theaflavins and an equal amount of hydrogel beads loaded with theaflavins) were added to 10 mL of simulated gastrointestinal fluid. After simulated gastrointestinal digestion, the samples were immersed in an ice bath to terminate the digestion. The digestate was collected and centrifuged at 10,000 rpm for 30 min at 4°C. The resulting supernatant was then collected and the theaflavins content was determined.

[0047] Theaflavins Stability:

[0048] After drying at low temperature (37°C), the stability of the hydrogel beads was tested under high temperature, high humidity and strong light. At different time intervals, the samples were taken out and the swelling performance and drug content were determined. The freeze-dried hydrogel beads were immersed in simulated gastrointestinal fluids with pH values ​​of 2.0 and 7.4, respectively, and the swelling test was carried out at 37°C. After 3h, the swollen hydrogel beads were taken out, the surface moisture was absorbed with filter paper, and then weighed to calculate the swelling ratio (mg / mg). The samples before and after treatment were placed in PBS buffer (pH7.4) to investigate the retention rate of theaflavins after 3h release at 37°C.

[0049] Example 1

[0050] A method for preparing a pH-responsive composite hydrogel loaded with theaflavins comprises the following steps:

[0051] (1) dissolving whey protein powder in deionized water to prepare a whey protein solution, then stirring at room temperature for 2 hours, and then at 4° C. overnight to ensure that the protein is fully hydrated, to obtain a whey protein solution with a mass concentration of 10%;

[0052] (2) At the same time, a sodium alginate solution with a mass concentration of 1% was prepared in deionized water at 80° C. using magnetic stirring, and the sodium alginate solution and the whey protein solution of step (1) were stirred at a volume ratio of 1:1 on a magnetic stirrer at 500 rpm for 30 min to prepare a whey protein / sodium alginate mixed solution, and then the solution was cooled to room temperature to ensure complete dissolution, and allowed to stand for 30 min until the bubbles disappeared;

[0053] (3) dissolving theaflavins in water to obtain a theaflavins solution with a concentration of 2 mg / mL;

[0054] (4) mixing the theaflavins solution with the whey protein / sodium alginate mixed solution to obtain a theaflavins-whey protein / sodium alginate mixed solution, wherein the volume ratio of theaflavins to the whey protein / sodium alginate mixed solution is 1:3;

[0055] (5) dissolving carboxymethyl chitosan and calcium chloride in water to obtain a carboxymethyl chitosan solution and a calcium chloride solution, with mass concentrations of 3.5% and 2%, respectively;

[0056] (6) mixing the carboxymethyl chitosan solution and the calcium chloride solution in a mixing volume ratio of 1:1 to obtain a cross-linking agent solution;

[0057] (7) Theaflavin-lactalbumin / sodium alginate mixed solution was dripped into a crosslinker solution containing carboxymethyl chitosan and calcium chloride solution using a syringe for crosslinking. The volume ratio of theaflavin-lactalbumin / sodium alginate mixed solution to the crosslinker solution was 1:4. The crosslinking reaction temperature was 25 °C, and the crosslinking reaction time was 0.5 h. The mixture was then filtered and rinsed with deionized water to remove the crosslinker containing carboxymethyl chitosan and calcium chloride solution. A portion of the hydrogel was placed in a culture dish, the moisture on the surface of the gel was removed, and then freeze-dried for 48 h and stored at -20 °C, while the rest was stored in water at 4 °C in the dark until further analysis.

[0058] Example 2

[0059] The preparation method is the same as that of Example 1, except that in step (2), the volume ratio of the whey protein solution to the sodium alginate solution is 1:2.

[0060] Example 3

[0061] The preparation method is the same as that of Example 1, except that in step (2), the volume ratio of the whey protein solution to the sodium alginate solution is 1:4.

[0062] Example 4

[0063] The preparation method is the same as that of Example 1, except that in step (2), the volume ratio of the whey protein solution to the sodium alginate solution is 1:5.

[0064] Example 5

[0065] The preparation method is the same as that of Example 1, except that in step (2), the volume ratio of the whey protein solution to the sodium alginate solution is 1:10.

[0066] Comparative Example 1

[0067] The preparation method is the same as that of Example 1, except that in step (6), the carboxymethyl chitosan solution is omitted and the cross-linking agent solution is a calcium chloride solution with a mass concentration of 2%.

[0068] Comparative Example 2

[0069] The preparation method is consistent with that of Example 2, except that in step (6), the carboxymethyl chitosan solution is omitted and the cross-linking agent solution is a calcium chloride solution with a mass concentration of 2%.

[0070] Comparative Example 3

[0071] The preparation method is the same as that of Example 3, except that in step (6), the carboxymethyl chitosan solution is omitted and the cross-linking agent solution is a calcium chloride solution with a mass concentration of 2%.

[0072] Comparative Example 4

[0073] The preparation method is the same as that of Example 4, except that in step (6), the carboxymethyl chitosan solution is omitted and the cross-linking agent solution is a calcium chloride solution with a mass concentration of 2%.

[0074] Comparative Example 5

[0075] The preparation method is consistent with that of Example 5, except that in step (6), the carboxymethyl chitosan solution is omitted and the cross-linking agent solution is a calcium chloride solution with a mass concentration of 2%.

[0076] The hydrogels prepared in Examples 1-5 were tested for swelling properties. Figure 1 It is known that A1-A5 correspond to the volume ratios of whey protein solution and sodium alginate solution of 1:1, 1:2, 1:4, 1:5 and 1:10 respectively, and the hydrogel obtained by the present invention has obvious pH response characteristics, that is, the swelling ratio in the simulated gastric fluid with a pH of 2 is smaller than the swelling ratio in the simulated intestinal fluid with a pH of 7.4. Figure 1 b shows that as the sodium alginate content increases, the swelling ratio of each hydrogel bead gradually increases. It can be seen that the present invention can effectively reduce the release of theaflavins in simulated gastric fluid.

[0077] like Figure 3 As shown, the release rate of theaflavins in the hydrogel prepared in Example 2-5 in the simulated gastric fluid environment with a pH of 2 is less than 15%; while in the simulated intestinal fluid environment with a pH of 7.4, as the hydrogel gradually disintegrates, the cumulative release rate of theaflavins in the simulated intestinal fluid after 4 hours of release can reach more than 75%. Figure 4The release rate of theaflavins embedded in the hydrogel prepared in comparative examples 2-5 (A2-A5 correspond to the volume ratios of whey protein solution and sodium alginate solution of 1:2, 1:4, 1:5 and 1:10, respectively) in simulated gastric fluid is high, and it is completely released only within 2 hours of simulated intestinal fluid as the hydrogel gradually disintegrates. This shows that the hydrogel prepared by the present invention has the potential to achieve targeted release and has a sustained release effect.

[0078] like Figure 5 As shown, compared with free theaflavins, the bioaccessibility of theaflavins in the composite hydrogel bead systems of Comparative Examples 1-5 and Examples 1-5 was improved. The bioaccessibility of free theaflavins was 13.80%. The bioaccessibility of theaflavins in both hydrogel systems in SIF was greatly improved. It is worth noting that the bioaccessibility of Comparative Examples 1-5 was about 26.61-36.11%, which was significantly higher than the composite hydrogel beads of Example 1 (24.28-26.07%). The results show that the composite hydrogel beads of Examples 1-5 of the present invention, as a delivery system, can transport theaflavins to the small intestine through the highly acidic gastric environment, thereby improving the bioaccessibility of theaflavins. However, compared with the composite hydrogel beads of Comparative Examples 1-5, their ability to improve bioaccessibility is lower. This may be due to the controlled release of the composite hydrogel beads of Examples 1-5, which provide a more stable and longer-lasting theaflavins concentration under digestive conditions. The "shell-core" structure formed in the hydrogel beads stabilizes theaflavins during digestion, preventing them from degradation in the mouth and stomach and delaying their release. This controlled release mechanism improves the sustained delivery capability of the hydrogel.

[0079] The effect of high temperature (60°C) on the composite hydrogel delivery system of Example 5 and Comparative Example 5 over time was studied. The morphology, color, dispersion and roundness of the beads did not change significantly. Samples were taken on the 0th, 5th and 10th days to determine their content. The results are shown in Figure 5. Figure 6 As shown, for the two hydrogel delivery systems, the swelling rate after high temperature treatment at different time periods increased compared with that before treatment. This may be due to the evaporation of the water in the hydrogel beads after long-term treatment at high temperature, resulting in a smaller mass and a relatively increased swelling rate. However, for the composite hydrogel beads of Example 5 at pH 7.4, the swelling rate decreased slightly during this period. As can be seen from Table 1, compared with Comparative Example 5, high temperature has little effect on the content of theaflavins in Example 5, and the range of variation of the sample data within 10 days is less than 10%. Theaflavins are loaded into the hydrogel delivery system through hydrogen bonding, which enhances the thermal stability of theaflavins to a certain extent. The above results indicate that the samples should be stored at a lower temperature.

[0080] Table 1 Theaflavin content in the composite hydrogels of Example 5 and Comparative Example 5 at 60°C (n=3)

[0081]

[0082] The effect of high humidity (RH 75%) on the composite hydrogel delivery system of Example 5 and Comparative Example 5 was studied based on changes over time. Samples were taken on days 0, 5, and 10 to determine their content. There was almost no significant difference in color, indicating that more moisture was absorbed. As shown in Table 2, the percentage change range of the composite hydrogel beads of Example 5 was less than 8% on the 5th day, but the percentage changes of the composite hydrogel beads of Comparative Example 5 and Example 5 on the 10th day were approximately 20% and 14%, respectively. This indicates that high humidity is not suitable for storing these two hydrogel beads. This may be because the wall material used to prepare the hydrogel beads is water-soluble. In a humid environment, the hydrogel beads absorb moisture, destroy or dissolve the structure of the wall material, resulting in gaps or cracks, and further affecting the content. Therefore, due to the problem of moisture absorption and reduced content of the hydrogel bead samples under high humidity conditions, the hydrogel bead samples loaded with theaflavins should be stored under dry conditions.

[0083] Table 2 Theaflavin content in the composite hydrogels of Example 5 and Comparative Example 5 at RH 75% (n=3)

[0084]

[0085] The changes of Example 5 and Comparative Example 5 hydrogel beads loaded with theaflavins over time under strong light (4500lx) were studied. After being treated with strong light for different time periods, the morphology, color, dispersibility and roundness of the hydrogel beads did not change significantly. The results are shown in Table 3. It can be seen that the percentage change range of the composite hydrogel beads of Example 5 was less than 6% on the 5th day. However, on the 10th day, strong light had a greater effect on the content of the hydrogel beads of Comparative Example 5 and Example 5 loaded with theaflavins, resulting in a decrease in the theaflavins content in the hydrogel beads, which showed an overall downward trend, indicating that the samples should be stored away from light.

[0086] Table 3 Theaflavin content in the composite hydrogels of Example 5 and Comparative Example 5 under strong light (n=3)

[0087]

[0088] The embodiments provided above are not intended to limit the scope of the present invention, and the steps described are not intended to limit the execution order thereof. Those skilled in the art may make obvious improvements to the present invention in combination with existing common knowledge, which also fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a pH-responsive composite hydrogel loaded with theaflavins, characterized in that: The steps include: (1) dissolving whey protein and sodium alginate in water respectively to obtain a whey protein solution and a sodium alginate solution, and mixing the whey protein and sodium alginate solution to obtain a whey protein / sodium alginate mixed solution; (2) dissolving theaflavins in water to obtain a theaflavins solution; (3) mixing the theaflavins solution with the whey protein / sodium alginate mixed solution to obtain the theaflavins-whey protein / sodium alginate mixed solution; (4) dissolving carboxymethyl chitosan and calcium chloride in water respectively to obtain a carboxymethyl chitosan solution and a calcium chloride solution, and mixing the solution to obtain a cross-linking agent solution; (5) dropping the theaflavins-whey protein / sodium alginate mixed solution into a crosslinking agent solution to carry out a crosslinking reaction, filtering, and washing to obtain a pH-responsive composite hydrogel loaded with theaflavins.

2. The method for preparing a pH-responsive composite hydrogel loaded with theaflavins according to claim 1, characterized in that: In step (1), the mass concentration of the whey protein solution is 5-20%; the mass concentration of the sodium alginate solution is 0.5-5%.

3. The method for preparing a pH-responsive composite hydrogel loaded with theaflavins according to claim 1, characterized in that: In step (1), the volume ratio of the whey protein solution to the sodium alginate solution is 1:1-10.

4. The method for preparing a pH-responsive composite hydrogel loaded with theaflavins according to claim 1, characterized in that: In step (2), the concentration of theaflavin solution is 0.1-5 mg / mL.

5. The method for preparing a pH-responsive composite hydrogel loaded with theaflavins according to claim 1, characterized in that: In step (3), the volume ratio of the theaflavin solution to the whey protein / sodium alginate mixed solution is 1:1-5.

6. The method for preparing a pH-responsive composite hydrogel loaded with theaflavins according to claim 1, characterized in that: In step (4), the mass concentration of the carboxymethyl chitosan solution is 0.5-5%; the mass concentration of the calcium chloride solution is 1-10%.

7. The method for preparing a pH-responsive composite hydrogel loaded with theaflavins according to claim 1, characterized in that: In step (4), the volume ratio of the carboxymethyl chitosan solution to the calcium chloride solution is 1:1-10.

8. The method for preparing a pH-responsive composite hydrogel loaded with theaflavins according to claim 1, characterized in that: In step (5), the volume ratio of the theaflavin-whey protein / sodium alginate mixed solution to the cross-linking agent solution is 1:2-5; the temperature of the cross-linking reaction is 10-30° C., and the time of the cross-linking reaction is 0.5-6 h.

9. The pH-responsive composite hydrogel loaded with theaflavins prepared by the method according to any one of claims 1 to 8.

10. Use of the pH-responsive composite hydrogel loaded with theaflavins according to claim 9 in the field of food technology.

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