Preparation method of fucoxanthin-loaded pectin@β-cyclodextrin and its application in lipid-lowering foods

Abstract

The present invention discloses a method for preparing fucoxanthin-loaded pectin@β-cyclodextrin, comprising the following steps: (1) preparing a fucoxanthin stock solution; (2) preparing a PBS stock solution; and (3) preparing fucoxanthin-loaded pectin@β-cyclodextrin. The method for preparing fucoxanthin-loaded pectin@β-cyclodextrin of the present invention has simple process steps and strong operability. By loading fucoxanthin on pectin@β-cyclodextrin, the stability of fucoxanthin in vitro and in vivo is improved, so that fucoxanthin can better reach the target site and improve its bioavailability in the gastrointestinal tract. The present invention also provides an application of fucoxanthin-loaded pectin@β-cyclodextrin in lipid-lowering foods, which can provide ideas for the application and further development of fucoxanthin in foods.

Description

Technical Field

[0001] The present invention relates to the field of food technology, and in particular to a method for preparing fucoxanthin-loaded pectin@β-cyclodextrin and application thereof in lipid-lowering foods. Background Art

[0002] Obesity can lead to a variety of chronic diseases, among which hyperlipidemia can seriously impact people's health and quality of life. Currently, the primary treatment for hypercholesterolemia is statins, but these drugs are associated with significant side effects with long-term use. Consequently, the research and development of environmentally friendly foods with lipid-lowering properties has become increasingly popular in recent years. Pancreatic lipase is a key enzyme in dietary fat metabolism, hydrolyzing fat for further absorption. Lipid absorption in the human digestive tract relies on bile acids, which effectively increase the solubility of oils and promote their emulsification. Therefore, inhibiting pancreatic lipase and absorbing bile acids in the gastrointestinal tract can achieve lipid-lowering effects.

[0003] Fucoxanthin is a highly active natural pigment primarily extracted from marine algae. Similar to other carotenoids, fucoxanthin exhibits excellent biological activities, such as antioxidant and lipid-lowering properties. However, due to its easy degradation and poor stability in light and acidic environments, if orally administered as a functional factor, fucoxanthin's instability and oxidative degradation when exposed to various factors hinder its absorption and bioavailability in the body. Therefore, it is necessary to develop a suitable delivery system to improve its stability both in vitro and in vivo, allowing fucoxanthin to better reach its target site and enhance its bioavailability in the gastrointestinal tract. Summary of the Invention

[0004] The present invention aims to provide a method for preparing pectin@β-cyclodextrin loaded with fucoxanthin, which has simple process steps and strong operability. By loading fucoxanthin on pectin@β-cyclodextrin, the stability of fucoxanthin in vitro and in vivo is improved, so that fucoxanthin can better reach the target site and improve its bioavailability in the gastrointestinal tract.

[0005] The present invention also provides an application of fucoxanthin-loaded pectin@β-cyclodextrin in lipid-lowering foods, which can provide ideas for the application and further development of fucoxanthin in foods.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: A method for preparing pectin@β-cyclodextrin loaded with fucoxanthin of the present invention comprises the following steps:

[0007] (1) Preparation of fucoxanthin stock solution: Dissolve fucoxanthin (FC) in methanol to obtain fucoxanthin stock solution.

[0008] (2) Prepare PBS stock solution: Dissolve NaCl, KCl, KH2PO4, and Na2HPO4 in water, add water to make up the volume, and adjust the pH to 7.4 to obtain PBS stock solution.

[0009] (3) Preparation of pectin@β-cyclodextrin loaded with fucoxanthin: Pectin@β-cyclodextrin (PT@β-CD) and fucoxanthin stock solution were added to PBS stock solution, magnetically stirred, and vacuum dried. The resulting solid was pectin@β-cyclodextrin loaded with fucoxanthin (FC-PT@β-CD). The present invention improves the stability of fucoxanthin in vitro and in vivo by encapsulating fucoxanthin in pectin@β-cyclodextrin, allowing fucoxanthin to better reach the target site, thereby improving its bioavailability in the gastrointestinal tract.

[0010] Preferably, in step (1), the mass concentration of the fucoxanthin stock solution is 0.5 mg / mL.

[0011] Preferably, in step (2), 4 g NaCl, 100 mg KCl, 120 mg KH2PO4, and 0.72 g Na2HPO4 are dissolved in water, and water is added to make the volume to 500 mL.

[0012] Preferably, in step (3), the pectin@β-cyclodextrin is prepared by the following method: adding pectin (PT) to an aqueous HCl solution, dissolving until there are no lumps, adding β-cyclodextrin (β-CD) and stirring evenly, heating and aging in a water bath until the solution is transparent, adding an appropriate amount of glutaraldehyde dropwise, heating and shaking in a water bath to cross-link until it turns yellow, precipitating with anhydrous ethanol solution, centrifuging, and drying the precipitate to obtain pectin@β-cyclodextrin. In the present invention, glutaraldehyde is used as a cross-linking agent, and the loading and encapsulation effect is good.

[0013] Preferably, 1 g of pectin is added to 120 mL of a 1 mol / L aqueous solution of HCl; 8 g of β-cyclodextrin is added; the solution is heated and aged at 85°C in a water bath until the solution becomes transparent; 3 mL of a 50% mass concentration glutaraldehyde solution is added dropwise; the solution is heated in a water bath at 60°C and shaken at 130 rpm for cross-linking until it turns yellow; the solution is centrifuged at 8000 rpm; and the precipitate is dried at 30°C.

[0014] Preferably, in step (3), the amount of pectin@β-cyclodextrin added is 10 mg, the mass ratio of fucoxanthin to pectin@β-cyclodextrin is 1:8, and the amount of PBS stock solution added is 10 mL. The mass ratio of fucoxanthin to pectin@β-cyclodextrin is a key factor in the present invention. The applicant has found through experiments that the mass ratio of fucoxanthin to pectin@β-cyclodextrin directly affects the entrapment efficiency of fucoxanthin. The inventors have found through experiments that a mass ratio of fucoxanthin to pectin@β-cyclodextrin of 1:8 can achieve the best entrapment efficiency and good encapsulation effect.

[0015] A fucoxanthin-loaded pectin@β-cyclodextrin formulation is used in lipid-lowering foods. The fucoxanthin-loaded pectin@β-cyclodextrin formulation prepared by the present invention has an adsorption effect on bile salts and an inhibitory effect on pancreatic lipase, and can be added as a functional ingredient in health products, foods, and the like.

[0016] Therefore, the present invention has the following beneficial effects:

[0017] (1) A method for preparing pectin@β-cyclodextrin loaded with fucoxanthin is provided. The process steps are simple and the operability is strong. By loading fucoxanthin on pectin@β-cyclodextrin, the stability of fucoxanthin in vitro and in vivo is improved, so that fucoxanthin can better reach the target site and improve its bioavailability in the gastrointestinal tract;

[0018] (2) This paper provides an application of fucoxanthin-loaded pectin@β-cyclodextrin in lipid-lowering foods, which can provide ideas for the application and further development of fucoxanthin in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the particle size distribution diagram of fucoxanthin-loaded pectin@β-cyclodextrin in Example 1.

[0020] Figure 2 This is an electron micrograph of the fucoxanthin-loaded pectin@β-cyclodextrin in Example 1.

[0021] Figure 3 This is the thermogravimetric analysis chart of fucoxanthin-loaded pectin@β-cyclodextrin and pectin@β-cyclodextrin in Example 1.

[0022] Figure 4 This is the infrared spectrum of the fucoxanthin-loaded pectin@β-cyclodextrin in Example 1.

[0023] Figure 5 This is a standard curve diagram of the concentration of FC-PT@β-CD solution and sodium taurocholate in Example 1.

[0024] Figure 6 This is a standard curve diagram of the FC-PT@β-CD solution concentration and sodium glycocholate in Example 1.

[0025] Figure 7 This is a curve diagram showing the change in FC-PT@β-CD solution concentration and pancreatic lipase inhibition rate in Example 1.

[0026] Figure 8 This is a graph showing the relationship between the FC-PT@β-CD concentration and the inhibition of pancreatic lipase in Example 1.

[0027] Figure 9 This is a double reciprocal curve diagram of the effect of FC-PT@β-CD on pancreatic lipase in Example 1.

[0028] Figure 10 This is a graph showing the results of the loading rate determination.

[0029] Figure 11 This is a graph showing the results of an in vitro release test in simulated gastrointestinal fluid. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0031] Example 1

[0032] (1) Preparation of fucoxanthin stock solution: Dissolve fucoxanthin in methanol to prepare a fucoxanthin stock solution with a mass concentration of 0.5 mg / mL;

[0033] (2) Prepare PBS stock solution: Dissolve 4 g NaCl, 100 mg KCl, 120 mg KH2PO4, and 0.72 g Na2HPO4 in water, add water to make up to 500 mL, and adjust the pH to 7.4 to obtain PBS stock solution.

[0034] (3) Preparation of pectin@β-cyclodextrin loaded with fucoxanthin: 10 mg of pectin@β-cyclodextrin and fucoxanthin stock solution were added to 10 mL of PBS stock solution. The fucoxanthin stock solution was added at a mass ratio of fucoxanthin to pectin@β-cyclodextrin of 1:8. After magnetic stirring, the solution was dried in vacuum. The resulting solid was pectin@β-cyclodextrin loaded with fucoxanthin (FC-PT@β-CD). Pectin@β-cyclodextrin was prepared by the following method: 1 g of pectin was added to 120 mL of 1 mol / L HCl aqueous solution, dissolved until there were no lumps, 8 g of β-cyclodextrin was added and stirred evenly, heated in a water bath at 85°C for aging until the solution was transparent, 3 mL of 50% glutaraldehyde solution was added dropwise, heated in a water bath at 60°C for oscillation at 130 rpm for cross-linking until it turned yellow, and then precipitated with 4 times the volume of anhydrous ethanol solution. The solution was centrifuged and dried at 30°C to obtain pectin@β-cyclodextrin.

[0035] 1. Particle size and morphology determination

[0036] The particle size and morphology of the obtained fucoxanthin-loaded pectin@β-cyclodextrin were measured. The particle size and morphology determination method was as follows: dilute 10 times with deionized water, perform three independent measurements before 2 minutes of equilibrium at 25°C, and record all data. The morphology of FC-PT@β-CD will be observed by scanning electron microscopy. In order to obtain higher image quality, a gold layer is sprayed on the FC-PT@β-CD. The obtained particle size distribution and electron microscopy images are shown in Figure 2. Figure 1 、 Figure 2 shown.

[0037] from Figure 1 It can be seen that the particle size is normally distributed, and the particle size range of FC-PT@β-CD is between 100-400nm; Figure 2 It can be seen that the obtained fucoxanthin-loaded pectin@β-cyclodextrin is spherical, which well encapsulates fucoxanthin (FC) and is consistent with the particle size data.

[0038] 2. Thermogravimetric Analysis

[0039] The obtained fucoxanthin-loaded pectin@β-cyclodextrin (FC-PT@β-CD) and pectin@β-cyclodextrin (PT@β-CD) were subjected to thermogravimetric analysis. The thermogravimetric analysis method is as follows: PT@β-CD and FC-PT@β-CD were subjected to thermogravimetric analysis using a thermogravimetric analyzer; PT@β-CD and FC-PT@β-CD were weighed 5 mg each and heated from 50°C to 550°C at a heating gradient of 10°C, under a constant nitrogen flow rate (100 mL / min). The thermogravimetric analysis results are shown in Figure 2. Figure 3 shown.

[0040] from Figure 3 It can be seen that in the temperature range of 50℃-150℃, the initial weight loss of the two samples (about 4-9%) can be attributed to the evaporation of adsorbed water and organic reagents, among which PT@β-CD showed a higher weight loss in this initial stage, indicating that there was more residual solvent in the pores of PT@β-CD; in contrast, during the same heating period, the initial weight loss of FC-PT@β-CD was relatively slow; the weight of PT@β-CD decreased significantly at 184℃; and after fucoxanthin was loaded into PT@β-CD, the temperature of FC-PT@β-CD's obvious weight loss was 259.4℃. The delayed thermal degradation and mass loss of FC-PT@β-CD were observed, which may be due to the fact that fucoxanthin was loaded into PT@β-CD through hydrogen bonds, van der Waals forces, etc., and the thermal stability of fucoxanthin in FC-PT@β-CD was improved.

[0041] 3. Infrared spectroscopy analysis

[0042] In order to further prove whether FC was successfully loaded on PT@β-CD, the obtained fucoxanthin-loaded pectin@β-cyclodextrin (FC-PT@β-CD) and fucoxanthin (FC) were subjected to infrared spectroscopy analysis. The specific method was as follows: 2 mg of sample was accurately weighed and mixed with 200 mg of dried spectral KBr powder, with KBr as the blank control. The wavenumber measurement range was 4000-400 cm -1 , with a resolution of 4 cm -1 The infrared spectrum obtained is as follows Figure 4 shown.

[0043] from Figure 4It can be seen that at 3400cm -1 The peak at 1930 cm is -OH. -1 There is an absorption peak, which is the allyl bond, which is considered to be the characteristic functional group of fucoxanthin. −1 The peaks at 1714 and 1630 cm show that there are asymmetric and symmetric stretching vibrations of methylene and methyl groups. -1 The peak at 1665cm is C=O stretching. −1 The peak at 958 cm− 1 The peak at 1930cm is the external torsion vibration of the CH bond in the double bond, which is the characteristic peak of trans-substituted ethylene. After loading fucoxanthin, the related absorption peak of fucoxanthin also appeared in FC-PT@β-CD. In comparison, after loading fucoxanthin, the absorption peak at 1930cm -1 Weaker or even disappeared, which may be due to the attachment of fucoxanthin to the inner core by hydrogen bonds and was not detected in the spectrometer.

[0044] 4. Detection of the binding ability of FC-PT@β-CD with sodium taurocholate and sodium glycocholate

[0045] Glycocholic acid and taurocholic acid are important bile acids in the human body. They bind to lipids in the digestive tract to form complexes, increasing their solubility and emulsification, thereby promoting their digestion and absorption. Furthermore, bile acids aid in the absorption of cholesterol, meeting the body's cholesterol needs.

[0046] FC-PT@β-CD solutions of different concentrations (1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, and 5.0 mg / mL) were prepared and dissolved in a pH 7.0 NaHPO4 buffer. The same volume of 1.0 mg / mL sodium taurocholate or sodium glycocholate was then added. After incubation at 37°C for 2 hours, the supernatant was mixed with 60% sulfuric acid solution, incubated in a 70°C water bath for 30 minutes, and then on ice for 5 minutes. The absorbance at 387 nm was measured using a microplate reader. The sodium taurocholate standard curve was established as y = 2.0523x + 0.0952, R 2 = 0.9989 (e.g. Figure 5 As shown), the standard curve of sodium glycocholate is y = 3.9688x + 0.1283, R 2 = 0.9922 (e.g. Figure 6 shown).

[0047] from Figure 5 、 Figure 6It can be seen that when the concentration of FC-PT@β-CD is between 1-3 μg / mL, the adsorption of glycocholic acid increases with increasing concentration. At 4-5 μg / mL, the adsorption of sodium glycocholate by FC-PT@β-CD / C tends to be flat with increasing concentration, with an adsorption rate of 25.25±0.82 (%). The adsorption of sodium taurocholate tends to be flat at 4-5 μg / mL, with an adsorption rate of 56.21±1.04 (%). This shows that FC-PT@β-CD has a good ability to adsorb bile acid.

[0048] 5. Investigating the inhibitory effect of FC-PT@β-CD on pancreatic lipase

[0049] A 5 mmol / L sodium acetate solution was prepared. 4-Nitrophenyl laurate was added to the prepared sodium acetate solution to prepare a 0.8 mg / mL solution. The solution was heated in an 85°C water bath to dissolve and cooled to room temperature for later use. Pancreatic lipase was prepared to a 5 mg / mL solution. Different concentrations of FC-PT@β-CD (0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 8 mg / mL, 12 mg / mL, 16 mg / mL, and 20 mg / mL) were prepared. 80 μL of 4-Nitrophenyl laurate, 60 μL of PBS, 20 μL of FC-PT@β-CD of different concentrations, and 60 μL of pancreatic lipase solution were added to each well of the ELISA plate. The reaction was incubated at 37°C for 30 minutes, and the absorbance was measured at 405 nm. The relationship between different concentrations of FC-PT@β-CD and the pancreatic lipase inhibition rate is shown in the figure. Figure 7 shown.

[0050] The substrate concentration of 4-nitrophenyl laurate was kept constant at 0.8 mmol / mL and the preparation was as shown above. The IC50 was calculated and pancreatic lipase was prepared into solutions of different concentrations (2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL). The reaction system was followed by incubation at 37°C for 10 min. The absorbance was measured at a wavelength of 405 nm and an inhibition type diagram was plotted (see Figure 2). Figure 8 shown).

[0051] The concentration of pancreatic lipase remained unchanged (10 mg / mL), and the IC50 of FC-PT@β-CD remained unchanged. The concentration of the substrate 4-nitrophenyl laurate was changed to 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, and 0.8 mg / mL. The reaction was carried out according to the above system at 37°C for 10 min. After the reaction, the absorbance was measured at 405 nm and a double reciprocal curve (Lineweaver-Burk) was plotted, as shown in Figure 2. Figure 9As shown, the inhibition type was determined: FC-PT@β-CD: y = 11.26981x + 11.48636, Con: y = 4.95718x + 15.65496.

[0052] from Figure 7 It can be seen that the inhibition rate of FC-PT@β-CD on pancreatic lipase increases with the increase of FC-PT@β-CD concentration at different concentrations and finally tends to be stable, indicating that FC-PT@β-CD has a certain inhibitory effect on pancreatic lipase. Through software analysis, the IC50 of FC-PT@β-CD on pancreatic lipase is 28.45 mg·mL -1 .

[0053] from Figure 8 、 Figure 9 It can be seen that when the double reciprocal plot is performed using 1 / [S] and 1 / V, the two straight lines of the substrate intersect at the origin, and the slope decreases after the addition of FC-PT@β-CD. After adding different concentrations of FC-PT@β-CD, the two straight lines intersect in the first quadrant, indicating that the type of inhibition of FC-PT@β-CD and pancreatic lipase is mixed.

[0054] Comparative Example 1

[0055] Comparative Example 1 is different from Example 1 in that the fucoxanthin stock solution is added at a mass ratio of fucoxanthin to pectin@β-cyclodextrin of 1:6, and the rest is exactly the same as Example 1.

[0056] Comparative Example 2

[0057] Comparative Example 2 is different from Example 1 in that the fucoxanthin stock solution is added at a mass ratio of fucoxanthin to pectin@β-cyclodextrin of 1:10, and the rest is exactly the same as Example 2.

[0058] The fucoxanthin-loaded pectin@β-cyclodextrin prepared in Example 1, Comparative Example 1 and Comparative Example 2 were subjected to entrapment efficiency determination and in vitro release test in simulated gastrointestinal fluid.

[0059] The encapsulation efficiency was determined by washing with methanol to remove excess fucoxanthin in the solution; measuring different concentrations of fucoxanthin at a wavelength of 500 nm using a microplate reader, and plotting a fucoxanthin standard curve based on the absorbance; and calculating the encapsulation efficiency (EE, %) of FC-PT@β-CD using the following formula: EE% = (amount of encapsulated FC) / total amount of FC × 100%.

[0060] The specific method of in vitro release test in simulated gastrointestinal fluid is:

[0061] Prepare simulated gastric fluid: Mix 3.2 g / L pepsin solution and 2.0 g / L NaCl solution, then adjust the pH of the mixture to 1.2 with hydrochloric acid. To simulate gastric release, precisely weigh 50 mg of sample and mix with simulated gastric fluid (SGF), shake well, and incubate in a 37°C shaking incubator for 1 hour.

[0062] Prepare simulated intestinal fluid: Mix 2.0 g / L trypsin, 8.8 g / L sodium chloride, 6.8 g / L potassium dihydrogen phosphate, and 12.0 g / L bile salt solution to prepare simulated intestinal fluid. Add 18 mL of simulated intestinal fluid (SIF) to the sample digested with the aforementioned gastric fluid and shake well. Test the digested fluid every 30 min, allowing the entire simulated small intestinal digestion to proceed for 2 h. All samples collected during the simulated digestion process were centrifuged at 4°C (10,000 × g) for 10 min before testing.

[0063] The results of the loading rate determination are as follows Figure 10 The results of in vitro release test in simulated gastrointestinal fluid are shown in Figure 11 shown.

[0064] from Figure 10 It can be seen that the entrapment rate of the pectin@β-cyclodextrin loaded with fucoxanthin prepared in Comparative Examples 1 and 2 is low, and the fucoxanthin is not effectively encapsulated, resulting in raw material loss. Figure 11 It can be seen that the cumulative release rate of fucoxanthin from the fucoxanthin-loaded pectin@β-cyclodextrin prepared in Comparative Examples 1 and 2 is low, which may indicate that a large amount of fucoxanthin reacts and the encapsulation effect is poor, resulting in a low in vitro release rate.

[0065] The embodiment described above is only a preferred solution of the present invention and does not limit the present invention in any form. Other variations and modifications are possible without exceeding the technical solution described in the claims.

Claims

1. A method for preparing fucoxanthin-loaded pectin@β-cyclodextrin, characterized in that: The following steps are involved: (1) Preparation of fucoxanthin stock solution: dissolve fucoxanthin in methanol to obtain a fucoxanthin stock solution with a fucoxanthin concentration of 0.5 mg / mL; (2) Prepare PBS stock solution: Dissolve NaCl, KCl, KH2PO4, and Na2HPO4 in water, add water to make up the volume, and adjust the pH to 7.4 to obtain PBS stock solution. (3) Preparation of pectin@β-cyclodextrin loaded with fucoxanthin: pectin@β-cyclodextrin and fucoxanthin stock solution were added to PBS stock solution, magnetically stirred, and vacuum dried. The obtained solid was pectin@β-cyclodextrin loaded with fucoxanthin. The amount of pectin@β-cyclodextrin added was 10 mg. The fucoxanthin stock solution was added at a mass ratio of fucoxanthin to pectin@β-cyclodextrin of 1:

8. The amount of PBS stock solution added was 10 mL. The pectin@β-cyclodextrin was prepared by the following method: 1 g pectin was added to 120 mL of 1 mol / L HCl aqueous solution, dissolved until no lumps were left, 8 g β-cyclodextrin was added and stirred evenly, heated in a water bath for aging until the solution was transparent, 3 mL of 50% mass concentration glutaraldehyde solution was added dropwise, heated in a water bath for oscillation and cross-linking until it turned yellow, precipitated with anhydrous ethanol solution, centrifuged, and the precipitate was dried to obtain pectin@β-cyclodextrin.

2. The method for preparing fucoxanthin-loaded pectin@β-cyclodextrin according to claim 1, wherein: In step (2), 4 g NaCl, 100 mg KCl, 120 mg KH2PO4, and 0.72 g Na2HPO4 were dissolved in water, and water was added to make the volume to 500 mL.

3. The method for preparing fucoxanthin-loaded pectin@β-cyclodextrin according to claim 1, wherein: In step (3), the solution is heated and aged in a water bath at 85°C until the solution becomes transparent; 3 mL of a 50% mass concentration glutaraldehyde solution is added dropwise; the solution is heated in a water bath at 60°C and shaken at 130 rpm for cross-linking until the solution turns yellow; the solution is centrifuged at 8000 rpm; and the precipitate is dried at 30°C.

4. Use of the fucoxanthin-loaded pectin@β-cyclodextrin prepared by the fucoxanthin-loaded pectin@β-cyclodextrin preparation method according to claim 1 in preparing a food having an auxiliary lipid-lowering effect.

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