Method for improving hydrophobic active substance encapsulation and intestinal slow release through pH treatment of whey protein

Abstract

The invention discloses a method for improving hydrophobic active substance encapsulation and intestinal slow release through pH treatment of whey protein, and relates to the technical field of functional food processing. The natural food-borne substance whey protein is subjected to pH pretreatment, so that a hydrophobic inner cavity of the natural food-borne substance whey protein is better opened, then the natural food-borne substance whey protein and the isoorientin form a self-assembled compound under the driving of hydrogen bonds and hydrophobic action, and the isoorientin is wrapped in the hydrophobic inner cavity of the protein, so that the stability and bioavailability of the isoorientin in the digestion process are improved. Compared with a traditional chemical modification or artificial synthesis delivery technology, the natural concentrated whey protein is used as the carrier, and the isoorientin is non-covalently combined through self-assembly, so that chemical crosslinking is not needed, the safety is higher, and the carrier is natural in source and has higher efficiency, greenness and sustainability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of functional food processing, and in particular to a method for improving the encapsulation and intestinal sustained release of hydrophobic active substances by pH treatment of whey protein. BACKGROUND

[0002] Whey protein is a natural protein mixture extracted from milk whey, which retains the rich bioactive components in whey through filtration process. Its core components are mainly β-lactoglobulin and α-lactalbumin, supplemented with bovine serum albumin. These components not only provide essential amino acids for the human body, but also have the characteristics of easy digestion and absorption, and enhanced immunity. Compared with high-purity separated whey protein, whey protein retains more natural active substances, and also contains a small amount of lactose and milk fat, and the nutritional structure is closer to natural whey. Due to its high biological value and cost-effectiveness, it is widely used in sports nutrition supplements, infant formula foods and functional health products, and is an ideal protein source that takes into account basic nutrition and physiological activity.

[0003] Isoorientin, as a natural C-glucosyl flavone, exists in many medicinal and edible plants. It shows rich biological activity functions, including antioxidant, improvement of type II diabetes, anti-inflammatory and anticancer, etc. These activities highlight the important research and application value of isoorientin in functional foods. However, the chemical instability and low water solubility of isoorientin limit its incorporation into foods, which is mainly due to the abundance of phenolic hydroxyl groups of isoorientin. During the digestion process, after oral intake of isoorientin, it is extensively metabolized in the intestinal tract, resulting in a significant decrease in its bioavailability. Therefore, it is crucial to choose an appropriate strategy to prevent the rapid degradation of isoorientin in the gastrointestinal environment to effectively improve its bioavailability.

[0004] The hydrophobic cavity of whey protein (especially β-lactoglobulin) is the key structural basis for its encapsulation of hydrophobic active ingredients. By adjusting the protein conformation through pH treatment, when the pH is close to the isoelectric point of β-Lg, the net charge on the surface of the protein approaches zero, the intermolecular attraction increases, leading to the contraction of the hydrophobic cavity and possibly triggering aggregation; while under acidic or alkaline conditions, the protein carries a large amount of net charge and undergoes molecular stretching, which promotes the exposure of the hydrophobic region, thereby improving the encapsulation efficiency.

[0005] The self-assembled system of whey protein encapsulating isofraxidin has multiple significant advantages. As a natural carrier, whey protein is rich in components such as beta-lactoglobulin. By adjusting the pH, the hydrophobic pocket in the molecular structure can be stably combined with isofraxidin through hydrophobic interaction, hydrogen bond and other non-covalent interactions, realizing efficient encapsulation. The self-assembly process is mild and does not require complex chemical modification, and can preserve the natural activity of both. The complex formed exhibits excellent sustained-release performance in the gastrointestinal environment, reducing the degradation loss of isofraxidin in the early stage of digestion. At the same time, the self-assembled structure can protect isofraxidin from the external environment, and the active ingredient is slowly released after digestion, significantly improving the bioavailability, providing an efficient solution for functional ingredient delivery. SUMMARY

[0006] The purpose of the present application is to use natural food sources to provide a method for improving the encapsulation efficiency and biological activity of hydrophobic active substances by treating whey protein with pH. The purpose is to open the hydrophobic cavity of the protein structure by adjusting the pH, and to form a whey protein-isofraxidin self-assembled complex with more isofraxidin under the driving of hydrogen bond and hydrophobic interaction, so as to significantly improve the bioavailability of isofraxidin.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] A method for improving the encapsulation efficiency and biological activity of hydrophobic active substances by treating whey protein with pH, the specific steps are as follows: (1) dissolve whey protein in deionized water to form a protein solution, and hydrate overnight at 4℃; (2) divide the protein solution into 5 groups, respectively adjust the pH to 2.0, 3.0, 4.0, 5.0 with 0.1M HCL, the control group (pH 6.5) remains in the natural state, continuously stirring at 25℃ for 30 minutes to make the protein fully unfold; (3) isofraxidin is dissolved in ethanol solution to prepare isofraxidin stock solution; (4) mix the pH pretreated whey protein solution with the isofraxidin stock solution under light-proof conditions; (5) adjust the pH of the complex to neutral to promote partial refolding of the protein to stabilize the complex; (6) determine the surface hydrophobicity and particle size of the pretreated protein solution obtained in step (2); (7) determine the encapsulation efficiency and loading capacity of the complex solution obtained in step (4); (8) determine the release rate of whey protein-isofraxidin complex and free isofraxidin by in vitro simulated gastrointestinal digestion.

[0009] Further, in step (1), the whey protein solution has a concentration of 20mg / mL, and the proportion of beta-lactoglobulin in the total protein is 71.3%.

[0010] Further, in step (2), the optimal pH for pretreating whey protein is 4.0.

[0011] Further, in step (3), the concentration of the isofraxidin is 3 mg / mL.

[0012] Further, in step (6), the surface hydrophobicity is determined by using an ANS fluorescence probe method, with an excitation wavelength of 370 nm and an emission wavelength of 380-600 nm.

[0013] Further, in step (7), the encapsulation efficiency and loading capacity are determined by using a centrifuge to centrifuge at 8000 x g for 25 minutes to separate free isofraxidin from the encapsulated complex, preparing an isofraxidin solution with a concentration gradient of 0-0.6 μg / mL using deionized water, determining the absorbance at 350 nm, and plotting a standard curve, and then determining the absorbance at 340 nm, and substituting the standard curve to calculate the free concentration and total concentration, with the formula as follows:

[0014]

[0015] wherein C 游离 is the free isofraxidin concentration, V 上清液 is the volume after centrifugation, C 总 is the total isofraxidin concentration, V 总 is the total volume of the encapsulation solution, and m WPC is the mass of the added whey protein.

[0016] Further, in step (8), in the simulated gastrointestinal digestion, the simulated gastric digestion is performed for 2 h, and after the gastric digestion is completed, the pH is adjusted to 7.0.

[0017] Further, in step (8), in the simulated gastrointestinal digestion, the simulated intestinal digestion is performed for 4 h, and then the enzyme is inactivated at 90°C.

[0018] Compared with the conventional technology, the present application has the following advantages:

[0019] 1. The conventional polyphenol delivery often relies on chemically modified carriers or artificial synthetic materials, which has the risk of toxic residue. The present application uses natural whey protein as a carrier, and non-covalently combines isofraxidin through self-assembly, without chemical cross-linking, which is safer, and the carrier is natural and biodegradable, which is suitable for food and nutrition fields.

[0020] 2. The conventional free isofraxidin is easily degraded in the gastrointestinal tract, and has low bioavailability. The present application expands the hydrophobic cavity of the protein through pH pretreatment, and encapsulates isofraxidin better under the action of hydrophobicity and hydrogen bonds, which is slowly released in the gastrointestinal environment, significantly prolongs the action time, and reduces the loss of active ingredients.

[0021] 3. The equipment used in the preparation of traditional complexes is complex or the reaction conditions are harsh; the self-assembly process of the present application has mild conditions, simple operation, low energy consumption, easy industrialization and low production cost. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a process flow diagram of the present application;

[0023] Figure 2 is the hydrophobic cavity of the β-barrel structure of β-lactoglobulin;

[0024] Figure 3 is the surface hydrophobicity of whey protein under different pH conditions (2, 3, 4, 5) pretreatment and non-pretreatment (6.5);

[0025] Figure 4 is the average particle size of whey protein under different pH conditions (2, 3, 4, 5) pretreatment and non-pretreatment (6.5);

[0026] Figure 5 is the encapsulation efficiency and loading capacity of isovanillin of whey protein under different pH conditions (2, 3, 4, 5) pretreatment and non-pretreatment (6.5);

[0027] Figure 6 is the in vitro simulated gastrointestinal digestion of isovanillin release amount. DETAILED DESCRIPTION

[0028] The specific embodiments are further described below in conjunction with the accompanying drawings.

[0029] Example 1

[0030] (1) Add whey protein powder to water, stir well with a magnetic stirrer to make a 20 mg / mL whey protein solution, and hydrate overnight at 4°C;

[0031] (2) Add 0.1M HCL solution to the protein solution prepared in step (1) to adjust the pH to 2;

[0032] (3) Dissolve isovanillin in ethanol solution to make it fully dissolved to obtain an isovanillin preparation solution;

[0033] (4) Mix the isovanillin preparation solution prepared in step (3) with the pH pretreated whey protein solution in step (2) to form a complex, so that the concentration of isovanillin is 1.0 mg / mL, and then adjust the pH to neutral;

[0034] (5) Mix the complex obtained in step (4) with simulated gastric juice 1:1, simulate gastric digestion for 2h, and adjust the pH to 7.0 after gastric digestion;

[0035] (6) Mix the remaining gastric juice obtained in step (5) with simulated intestinal juice at a ratio of 1:1, simulate intestinal digestion for 4 hours, and place the simulated digestion at 90°C for 10 minutes to inactivate enzymes.

[0036] (7) The surface hydrophobicity of whey protein in the pH pretreated protein solution obtained in step (2) was measured to be 161.9±3.86, indicating that after pH pretreatment, some hydrophobic groups of β-lactoglobulin were exposed, and the hydrophobicity was higher than that of the untreated group.

[0037] (8) The average particle size of the protein solution after pH pretreatment obtained in step (2) was measured to be 397.47±2.08nm, indicating that acidity may cause protein denaturation and aggregation, and the average particle size of the protein increases.

[0038] (9) The encapsulation efficiency and loading of isopropionol in the complex obtained in step (4) were determined. The encapsulation efficiency was 62.25±0.26% and the loading was 7.55±0.22. Compared with the complex without pH treatment, the encapsulation efficiency increased by 17.91% and the loading increased by 1.68%.

[0039] (10) Measure the amount of isochoric acid released from the intestinal digestive fluid obtained in step (6), such as... Figure 5 As shown, the release of isoharmonin in the intestinal digestive fluid was 74.31±0.25%, which was 8.85% lower than that of free isoharmonin. This indicates that after pH pretreatment, whey protein better encapsulates isoharmonin in the hydrophobic cavity, which can effectively alleviate the release of isoharmonin in the intestine and improve the intestinal stability of isoharmonin.

[0040] Example 2

[0041] (1) Add whey protein powder to water and stir thoroughly with a magnetic stirrer to dissolve it completely, prepare a whey protein solution of 20 mg / mL, and hydrate it overnight at 4°C.

[0042] (2) Add 0.1M HCl solution to the protein solution prepared in step (1) to adjust the pH to 3;

[0043] (3) Dissolve isopropionol in an ethanol solution to obtain isopropionol preparation solution;

[0044] (4) The isoharmone preparation solution prepared in step (3) is thoroughly mixed with the pH pretreated whey protein solution in step (2) to form a complex, so that the concentration of isoharmone is 1.0 mg / mL, and then the pH is adjusted to neutral.

[0045] (5) Mix the complex obtained in step (4) with simulated gastric juice at a ratio of 1:1, simulate gastric digestion for 2 hours, and adjust the pH to 7.0 after gastric digestion is completed;

[0046] (6) Mix the remaining gastric juice obtained in step (5) with simulated intestinal juice at a ratio of 1:1, simulate intestinal digestion for 4 hours, and place the simulated digestion at 90°C for 10 minutes to inactivate enzymes.

[0047] (7) The surface hydrophobicity of whey protein in the pH pretreated protein solution obtained in step (2) was measured to be 140.97±2.32, indicating that after pH pretreatment of the protein, some hydrophobic groups of β-lactoglobulin were exposed, and the hydrophobicity was higher than that of the untreated group.

[0048] (8) The average particle size of the protein solution after pH pretreatment obtained in step (2) was measured to be 275±7.56nm, indicating that acidic pH treatment caused the internal hydrophobic cavities of some proteins to expand, thereby increasing their average particle size.

[0049] (9) The encapsulation efficiency and loading of isopropionate obtained in step (4) were determined. The encapsulation efficiency was 69.99±0.91% and the loading was 7.66±0.12%. Compared with the untreated complex, the encapsulation efficiency increased by 25.65% and the loading increased by 1.79%.

[0050] (10) Measure the amount of isochoric acid released from the intestinal digestive fluid obtained in step (6), such as... Figure 5 As shown, the release of isoharmonin in the intestinal digestive fluid was 69.18±1.31%, which was 13.98% lower than that of free isoharmonin. This indicates that after pH pretreatment, whey protein better encapsulates isoharmonin in the hydrophobic cavity, which can effectively alleviate the release of isoharmonin in the intestine and improve the intestinal stability of isoharmonin.

[0051] Example 3

[0052] (1) Add whey protein powder to water and stir thoroughly with a magnetic stirrer to dissolve it completely, prepare a whey protein solution of 20 mg / mL, and hydrate it overnight at 4°C.

[0053] (2) Add 0.1M HCl solution to the protein solution prepared in step (1) to adjust the pH to 4;

[0054] (3) Dissolve isopropionol in an ethanol solution to obtain isopropionol preparation solution;

[0055] (4) The isoharmone preparation solution prepared in step (3) is thoroughly mixed with the pH pretreated whey protein solution in step (2) to form a complex, so that the concentration of isoharmone is 1.0 mg / mL, and then the pH is adjusted to neutral.

[0056] (5) Mix the complex obtained in step (4) with simulated gastric juice at a ratio of 1:1, simulate gastric digestion for 2 hours, and adjust the pH to 7.0 after gastric digestion is completed;

[0057] (6) Mix the remaining gastric juice obtained in step (5) with simulated intestinal juice at a ratio of 1:1, simulate intestinal digestion for 4 hours, and place the simulated digestion at 90°C for 10 minutes to inactivate enzymes.

[0058] (7) The surface hydrophobicity of whey protein in the pH pretreated protein solution obtained in step (2) was measured to be 162.6±4.72, indicating that after pH pretreatment, most of the hydrophobic groups of β-lactoglobulin were exposed, and the hydrophobicity was higher than that of the untreated group, which facilitated the binding of more hydrophobic compounds.

[0059] (8) The average particle size of the protein solution after pH pretreatment obtained in step (2) was measured to be 195.87±0.7nm, indicating that the pretreatment at pH 4 caused the internal hydrophobic cavity of the protein to expand, thereby increasing its average particle size.

[0060] (9) The encapsulation efficiency and loading of isopropionate in the complex obtained in step (4) were determined. The encapsulation efficiency was 72.28±0.52% and the loading was 8.08±0.13%. Compared with the complex without pH treatment, the encapsulation efficiency and loading were significantly improved, by 27.94% and 2.21%, respectively. This indicates that under pH 4 treatment, the protein exposed more hydrophobic sites to bind with isopropionate.

[0061] (10) Measure the amount of isochoric acid released from the intestinal digestive fluid obtained in step (6), such as... Figure 5 As shown, the release of isoharmonin in the intestinal digestive fluid was 66.85±0.60%, which was 16.31% lower than that of free isoharmonin. This indicates that after pH pretreatment, whey protein better encapsulates isoharmonin in the hydrophobic cavity, which can effectively alleviate the release of isoharmonin in the intestine and improve the intestinal stability of isoharmonin.

[0062] Example 4

[0063] (1) Add whey protein powder to water and stir thoroughly with a magnetic stirrer to dissolve it completely, prepare a whey protein solution of 20 mg / mL, and hydrate it overnight at 4°C.

[0064] (2) The protein solution prepared in step (1) is added dropwise with 0.1M HCL solution to adjust the pH to 5;

[0065] (3) The isohumulones are placed in an ethanol solution to fully dissolve, obtaining an isohumulone preparation solution;

[0066] (4) The isohumulone solution prepared in step (3) is fully mixed with the pH pretreated whey protein solution in step (2) to form a complex, with the concentration of isohumulone being 1.0 mg / mL, and then the pH is adjusted to neutral;

[0067] (5) The complex obtained in step (4) is mixed with simulated gastric juice 1:1, simulated gastric digestion is performed for 2h, and after the gastric digestion is completed, the pH is adjusted to 7.0;

[0068] (6) The remaining gastric juice obtained in step (5) is mixed with simulated intestinal juice 1:1, simulated intestinal digestion is performed for 4h, and the simulated digestion is placed at 90°C high temperature for 10min to inactivate the enzyme;

[0069] (7) The pH pretreated protein solution obtained in step (2) is measured, and the surface hydrophobicity of whey protein is 82.77±2.17, indicating that after the pH pretreatment of the protein, the partial hydrophobic group of β-lactoglobulin is exposed, and the hydrophobicity is slightly higher than that of the untreated group;

[0070] (8) The pH pretreated protein solution obtained in step (2) is measured, and the average particle size of the protein after pH pretreatment is 149.63±1.29nm, and part of the hydrophobic cavity is opened, compared with the control group, the particle size is slightly increased;

[0071] (9) The complex obtained in step (4) is measured for the encapsulation efficiency and loading capacity of isohumulone, the encapsulation efficiency is 58.31±0.79%, and the loading capacity is 4.63±0.30%, compared with the complex without pH treatment, the encapsulation efficiency is increased by 13.97, and the loading capacity is slightly decreased;

[0072] (10) The intestinal digestion solution obtained in step (6) is measured for the release amount of isohumulone, as shown in Figure 5 , the release amount of isohumulone in the intestinal digestion solution is 71.92±0.34%, which is reduced by 11.24% compared with free isohumulone, indicating that the pH pretreated whey protein can better wrap isohumulone in the hydrophobic cavity, which can effectively alleviate the release of isohumulone in the intestinal tract, and improve the intestinal stability of isohumulone.

[0073] Example 5

[0074] (1) Whey protein powder is added to water, fully stirred with a magnetic stirrer to fully dissolve, and a 20mg / mL whey protein solution is prepared, and is placed in 4°C hydration overnight;

[0075] (2) Maintain the original pH of the protein solution prepared in step (1);

[0076] (3) Dissolve isopropionol in an ethanol solution to obtain isopropionol preparation solution;

[0077] (4) The isoharmone solution prepared in step (3) is thoroughly mixed with the pH pretreated whey protein solution in step (2) to form a complex, so that the concentration of isoharmone is 1.0 mg / mL, and then the pH is adjusted to neutral.

[0078] (5) Mix the complex obtained in step (4) with simulated gastric juice at a ratio of 1:1, simulate gastric digestion for 2 hours, and adjust the pH to 7.0 after gastric digestion is completed;

[0079] (6) Mix the remaining gastric juice obtained in step (5) with simulated intestinal juice at a ratio of 1:1, simulate intestinal digestion for 4 hours, and place the simulated digestion at 90°C for 10 minutes to inactivate enzymes.

[0080] (7) The surface hydrophobicity of whey protein in the pretreated protein solution obtained in step (2) was measured to be 65.08±3.43.

[0081] (8) The average particle size of the untreated protein solution obtained in step (2) was measured to be 98.06±2.38 nm.

[0082] (9) The encapsulation efficiency and loading of isopropionate in the complex obtained in step (4) were determined. The encapsulation efficiency was 44.34±1.34% and the loading was 5.87±0.24%.

[0083] (10) Measure the amount of isochoric acid released from the intestinal digestive fluid obtained in step (6), such as... Figure 5 As shown, the release of isoharmonin in the intestinal digestive fluid was 71.22±0.73%, which was 11.94% lower than that of free isoharmonin.

Claims

1. A method for improving the encapsulation of hydrophobic active substances and intestinal sustained release by pH treatment of whey protein, the specific steps are as follows: (1) Dissolve whey protein in deionized water to form a protein solution, and place it in a 4℃ condition for hydration overnight; (2) Divide the protein solution into 5 groups, respectively adjust the pH to 2.0, 3.0, 4.0, 5.0 with 0.1M HCL, the control group (pH 6.5) remains in a natural state, continuously stir at 65℃ for 30 minutes to fully unfold the protein; (3) Use ethanol solution to dissolve the hydrophobic active substance isorhamnetin to prepare an isorhamnetin stock solution; (4) Mix the whey protein solution pretreated by pH with the isorhamnetin stock solution under light-proof conditions. (5) The complex was allowed to refold at 25℃ by adjusting its pH to neutral to stabilize the complex; (6) The pretreated protein solution obtained in step (2) was subjected to surface hydrophobicity determination and particle size determination; (7) The complex solution obtained in step (5) was subjected to encapsulation efficiency and loading capacity determination; (8) The whey protein-isoflavone complex and free isoflavone were subjected to in vitro simulated gastrointestinal digestion to determine their release rates.

2. The method of claim 1, wherein the pH-treated whey protein enhances the encapsulation efficiency of the hydrophobic active substance and the intestinal sustained release, and the pH-treated whey protein is a protein having a pH of 4.5 to 7.

0. The whey protein solution had a concentration of 20 mg / mL, and the proportion of beta-lactoglobulin in the total protein was 71.3%.

3. The method of claim 1, wherein the pH-treated whey protein enhances the encapsulation efficiency of the hydrophobic active substance and the intestinal sustained release, and the pH-treated whey protein is a protein having a pH of 4.5 to 7.

0. The optimal pH for pretreating the whey protein was 4.

0.

4. The method of claim 1, wherein the pH-treated whey protein enhances the encapsulation efficiency of the hydrophobic active substance and the intestinal sustained release, and the pH-treated whey protein is a protein having a pH of 4.5 to 7.

0. The concentration of the isoflavone was 3 mg / mL.

5. The method of claim 1, wherein the pH-treated whey protein enhances the encapsulation efficiency of the hydrophobic active substance and the intestinal sustained release, and the pH-treated whey protein is a protein having a pH of 4.5 to 7.

0. The surface hydrophobicity was determined by ANS fluorescence probe method, with an excitation wavelength of 370 nm and an emission wavelength of 380-600 nm.

6. The method of claim 1, wherein the pH-treated whey protein enhances the encapsulation efficiency of the hydrophobic active substance and the intestinal sustained release, and the pH-treated whey protein is a protein having a pH of 4.5 to 7.

0. The encapsulation efficiency and loading capacity were determined as follows: the free isoflavone in the encapsulation system was separated from the encapsulation complex by centrifugation at 8000 x g for 25 minutes, an isoflavone solution with a concentration gradient of 0-0.6 μg / mL was prepared using deionized water, the absorbance at 350 nm was determined, and a standard curve was plotted; then the absorbance at 340 nm was determined, and the free concentration and total concentration were calculated by substituting the standard curve, with the calculation formula being as follows: Encapsulation efficiency Loadings wherein Cfree is the free isoflavone concentration, Vsupernatant is the volume after centrifugation, Ctotal is the total isoflavone concentration, Vtotal is the total volume of the encapsulation solution, and mWPC is the mass of the added concentrated whey protein.

7. The method of claim 1, wherein the pH-treated whey protein is a pH-treated whey protein isolate. In the simulated gastrointestinal digestion, the simulated gastric digestion was performed for 2 h, and the pH was adjusted to 7.0 after the gastric digestion was completed.

8. The method of claim 1, wherein the pH-treated whey protein enhances the encapsulation efficiency of the hydrophobic active substance and the intestinal sustained release, and the pH-treated whey protein is a protein having a pH of 4.5 to 7.

0. In the simulated gastrointestinal digestion, the simulated intestinal digestion was performed for 4 h, and then the enzymes were inactivated at 90℃.

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