Curcumin-embedded red bean protein-corn starch composite condensation gel as well as preparation method and application thereof

Abstract

The invention discloses curcumin-embedded red bean protein-corn starch composite condensation gel as well as a preparation method and application thereof, and belongs to the technical field of functional food processing. In order to solve the technical problems that curcumin is relatively low in stability and bioavailability, and condensation gel only formed by protein cannot meet the application requirement of being used as a curcumin carrier, the invention provides a preparation method of curcumin-embedded red bean protein-corn starch composite condensation gel. Sequentially carrying out mixing co-heating and ultrahigh pressure treatment on red bean protein subjected to pH shift and ultrasonic treatment and corn starch with a specific straight / branched ratio, introducing curcumin into a protein-starch composite system, and carrying out high-pressure microjet homogenization treatment to finally form the composite condensation gel under the acid induction condition. The prepared composite condensation gel has high gel strength and physical and chemical stability, can provide better protection for sensitive bioactive compounds such as curcumin, realizes controlled release of the bioactive compounds and improves the bioaccessibility of the bioactive compounds.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of functional food processing, and particularly relates to a curcumin-embedded red bean protein-corn starch composite cold gel and a preparation method and application thereof. BACKGROUND

[0002] Natural polyphenols such as curcumin and other bioactive compounds or nutrients in food have various physiological functions such as antioxidant, antitumor and hypolipidemic effects, and are used as health-promoting factors in the functional food and pharmaceutical industries. However, due to their sensitivity to light, heat and other processing environments, as well as to the gastrointestinal environment and digestive enzymes, their stability and bioavailability are low. Protein gels have good hydrophilicity, biocompatibility and mechanical properties. Compared with traditional thermal gels, protein cold gels are formed at low temperatures and are more suitable for the protection, controlled release and delivery of sensitive bioactive compounds or nutrients. At present, legumes are widely used in food processing as a good source of high-quality dietary protein. Red bean protein, as a byproduct of the red bean starch industry, is inexpensive, high in nutritional value, easily digestible and biocompatible, making it a good raw material for forming protein cold gels. However, cold gels formed only from protein have low mechanical strength and weak network structure, which cannot meet the application requirements as a food processing raw material or as a carrier for bioactive compounds.

[0003] Proteins and starches are the main natural polymer polymers in food systems, and are closely related to the taste, texture and structure of food. Mixing the two and heating can denature the protein and gelatinize the starch, enhancing their interactions (including main forces: hydrophobic interaction and hydrogen bonding, as well as ionic bonds, electrostatic interactions and van der Waals forces, etc.); after cooling, the addition of an acid coagulant (GDL) at room temperature can induce the formation of a cold gel. Therefore, protein-starch composite gels are controlled-release carriers that are expected to be used for the embedding, protection and delivery of bioactive compounds such as curcumin, and have important significance for the development of functional composite gel carriers, the precise design of gel-based health foods and the industrial application of gel-based health foods.

[0004] However, there are still some technical obstacles for using protein-starch composite gel to embed, protect and deliver bioactive compounds such as curcumin. For example, starch is mainly composed of two types of starch, slightly branched amylose and highly branched amylopectin. Different types of starch have different structures and properties, which will affect their gelatinization and gel properties, and then affect their interaction with proteins and regulate the gel properties of proteins. At present, there is no report on the structure and properties of composite acid-induced cold gel formed by red bean protein and corn starch with different straight / branch ratios (amylose and amylopectin ratio) and its use for curcumin protection and delivery. In addition, although in recent years, ultrasound, ultrahigh pressure, high pressure microjet homogenization as an effective physical modification method has been widely used in food deep processing and application basic research; these physical treatments can modify the structure and physicochemical properties of proteins and starch, promote the interaction between them, and then improve the structure and stability of protein-starch composite cold gel. However, the effect of these physical treatment methods on the composite gel formed by red bean protein and corn starch with different straight / branch ratios is not yet known. Therefore, it is urgent to solve the above technical problems and provide a protein-starch composite cold gel with high gel strength and physicochemical stability. SUMMARY

[0005] To solve the technical problems of low stability and bioavailability of curcumin, low mechanical strength and weak network structure of cold gel formed only by protein, and inability to meet the application requirements as a curcumin carrier, the present application provides a preparation method of red bean protein-corn starch composite cold gel embedding curcumin. The preparation method sequentially mixes and co-heats red bean protein treated by pH shift and ultrasonic assistance combined treatment with corn starch with a specific straight / branch ratio and then performs ultrahigh pressure treatment. Then, curcumin is introduced into the protein-starch composite system at room temperature, and finally a red bean protein-corn starch composite cold gel embedding curcumin is formed under acid-induced conditions by high pressure microjet homogenization treatment. The composite cold gel prepared by the present application has high gel strength and physicochemical stability, can provide better protection for sensitive bioactive compounds such as curcumin, realize the controlled release of bioactive compounds and improve their bioavailability.

[0006] To solve the above technical problems and achieve the corresponding technical effects, the present application provides the following technical solutions: The first object of the present application is to provide a preparation method of red bean protein-corn starch composite cold gel embedding curcumin, which comprises the following steps: 1) Mix red bean protein with deionized water to prepare a protein solution with a mass fraction of 4%, adjust the pH to 12.0, and then perform ultrasonic treatment at a power of 100-500 W for 20-30 min. Then, adjust the pH of the protein solution to 7.0 to obtain a protein dispersion; 2) adding corn starch with a mass fraction of 2% into the protein dispersion obtained in step 1), stirring by magnetic force, heating at 95℃ for 20 min, rapidly cooling to room temperature, and then treating by ultra-high pressure at a pressure of 600-800 MPa for 5-15 min to obtain a protein-starch composite dispersion; the mass ratio of amylose to amylopectin in the corn starch is 1:4-6; 3) adding the curcumin anhydrous ethanol solution into the protein-starch composite dispersion obtained in step 2) dropwise to make the final concentration of curcumin reach 0.4 mg / mL, stirring by magnetic force, and then treating by high-pressure microjet homogenization at a pressure of 200-300 MPa for 1-5 times to obtain a mixture; 4) performing rotary evaporation on the mixture obtained in step 3) to remove ethanol, adding deionized water to make the concentration of curcumin reach 0.4 mg / mL, removing the unbound curcumin by centrifugation, adding 0.4% of gluconic acid-delta-lactone into the supernatant, stirring uniformly, acidifying at room temperature for 4 h, and then storing at 4℃ for 12 h to obtain the curcumin-embedded red bean protein-corn starch composite cold gel.

[0007] In an embodiment of the present application, the ultrasonic treatment in step 1) is performed at a power of 300 W for 25 min.

[0008] In an embodiment of the present application, the mass ratio of amylose to amylopectin in the corn starch in step 2) is 1:5.

[0009] In an embodiment of the present application, the ultra-high pressure treatment in step 2) is performed at a pressure of 700 MPa for 10 min.

[0010] In an embodiment of the present application, the high-pressure microjet homogenization treatment in step 3) is performed at a pressure of 250 MPa for 3 times.

[0011] In an embodiment of the present application, the time of magnetic stirring in steps 1) and 2) is 30 min, and the time of magnetic stirring in step 3) is 2 h.

[0012] In an embodiment of the present application, the concentration of curcumin in the curcumin anhydrous ethanol solution in step 3) is 10 mg / mL.

[0013] In an embodiment of the present application, the temperature of rotary evaporation in step 4) is 35℃, and the centrifugation is performed at 5000 g for 10 min.

[0014] A second object of the present application is to provide the curcumin-embedded red bean protein-corn starch composite cold gel prepared by the preparation method.

[0015] A third object of the present application is to provide the use of the curcumin-embedded red bean protein-corn starch composite cold gel described above in the preparation of a food, health product, cosmetic or pharmaceutical product containing curcumin.

[0016] The beneficial effects of the present application are: In the present application, the red bean protein is subjected to pH shift and ultrasonic-assisted combined treatment, which greatly promotes the solubility and dispersion of the protein, and at the same time modifies the structure of the protein, which is beneficial to the subsequent interaction with corn starch; the specific straight / branched ratio of corn starch is mixed with red bean protein for co-heating, so that the protein is denatured and the starch is gelatinized, inducing the protein and starch to be fully crosslinked, and at the same time, the ultrahigh pressure treatment is further used to promote the protein-starch interaction, forming a more stable non-covalent complex; then curcumin is introduced into the protein-starch composite system at room temperature, and the system is dispersed more uniformly through high-pressure microjet homogenization treatment, promoting the combination and crosslinking of curcumin with protein and starch, and finally forming a curcumin-embedded red bean protein-corn starch composite cold gel under the condition of acid induction. The method has the advantages of low raw material cost, high product added value, green safety, etc., mainly through the synergistic regulation of three physical modification methods of ultrasonic, ultrahigh pressure and high-pressure microjet homogenization and the straight / branched ratio of corn starch to regulate the structure and physicochemical properties of the red bean protein-corn starch composite, finally improving the gel strength, water holding capacity, rheological properties, physical stability of the composite gel, and the embedding, stabilization, controlled release and delivery of curcumin, and at the same time, the composite gel is endowed with antioxidant activity, which can be applied to the development and production of multifunctional composite gel carriers and health foods. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The gel strength and water holding capacity determination results of the curcumin-embedded red bean protein-corn starch composite cold gel prepared by the examples and comparative examples provided in the present application are shown in the following figures: Figure 2 The microstructure of the curcumin-embedded red bean protein-corn starch composite cold gel prepared by the examples and comparative examples provided in the present application is shown in the following figure: Figure 3 The dynamic rheological property determination results of the curcumin-embedded red bean protein-corn starch composite cold gel prepared by the examples and comparative examples provided in the present application are shown in the following figures: Figure 4 The creep-recovery curve of the curcumin-embedded red bean protein-corn starch composite cold gel prepared by the examples and comparative examples provided in the present application is shown in the following figure: Figure 5 The intermolecular interaction, X-ray diffraction pattern, Fourier transform infrared spectrum and endogenous fluorescence spectrum detection results of the curcumin-embedded red bean protein-corn starch composite cold gel prepared by the examples and comparative examples provided in the present application are shown in the following figures:Figure 5 A in FIG. 6 is a molecular interaction detection result graph, Figure 5 B in FIG. 7 is an X-ray diffraction pattern detection result graph, Figure 5 C in FIG. 8 is a Fourier transform infrared spectrum detection result graph, Figure 5 D in FIG. 9 is an endogenous fluorescence spectrum detection result graph; Figure 6 FIG. 10 is a curcumin embedding rate detection result graph of the curcumin-embedded red bean protein-corn starch composite cold gel prepared by each embodiment and the comparative example provided by the present application; Figure 7 FIG. 11 is an in vitro antioxidant activity detection result graph of the curcumin-embedded red bean protein-corn starch composite cold gel prepared by each embodiment and the comparative example provided by the present application; Figure 8 FIG. 12 is a chemical stability detection result graph of curcumin in the curcumin-embedded red bean protein-corn starch composite cold gel prepared by each embodiment and the comparative example provided by the present application; wherein, Figure 8 A in FIG. 13 is a light stability detection result graph, Figure 8 B in FIG. 14 is a thermal stability detection result graph at 45℃, Figure 8 C in FIG. 15 is a thermal stability detection result graph at 75℃, Figure 8 D in FIG. 16 is a thermal stability detection result graph at 95℃; Figure 9 FIG. 17 is a release rate and bioavailability detection result graph of curcumin in the curcumin-embedded red bean protein-corn starch composite cold gel prepared by each embodiment and the comparative example provided by the present application; wherein, Figure 9 A in FIG. 18 is a curcumin release rate detection result graph, SGF represents a gastric digestion process, and SIF represents an intestinal digestion process, Figure 9 B in FIG. 19 is a curcumin bioavailability detection result graph. DETAILED DESCRIPTION

[0018] For the purposes of the present invention, the technical solutions and advantages are more clearly apparent, the present invention will be further described in detail below with specific embodiments and the accompanying drawings. It should be noted that the following examples are only applicable to explain the present invention, but not to limit the scope of the present invention. The following examples are only a part of the embodiments of the present invention, not all. Those skilled in the art can refer to the content herein, and appropriately improve the process parameters to achieve the purpose of the present invention. In particular, it should be pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present invention. The method and application of the present invention have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention, to realize and apply the present invention. In the art, other technicians will not make creative efforts, and the examples they obtain are protected by the present invention.

[0019] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and instruments used are conventional materials, reagents and instruments in the art unless otherwise specified, and can be obtained by commercial channels by those skilled in the art.

[0020] The detection method involved in the present invention is specifically as follows: 1) Gel strength determination A texture analyzer equipped with a P / 0.5 probe was used to determine the gel strength of the gel sample: the gel was equilibrated at room temperature for 30 min, and penetrated the gel at a constant speed of 1 mm / s, with a compression ratio of 50% and a trigger force of 5.0 g. The gel strength was defined as the maximum force used during penetration.

[0021] 2) Water holding capacity (WHC) determination The gel was formed in a centrifuge tube, and the water not wrapped by the gel in the centrifuge tube was discharged. The total mass of water in the gel was the difference between the mass of water added before gel formation and the mass of water not combined after gel formation. After centrifugation at 10000 g for 20 min, the water was discharged, and the water remaining on the surface of the sample and the wall of the centrifuge tube was absorbed with dry filter paper. The mass difference between the centrifuge tube containing the gel before and after centrifugation was the mass of water released from the gel. The WHC of the gel was calculated as follows:

[0022] In the formula: M t is the total mass of water in the gel, M r is the mass of water released from the gel after centrifugation.

[0023] 3) Scanning electron microscope (SEM) determination Microstructure of the gel sample was observed by SEM: the freeze-dried gel sample was fixed by double-sided tape, then sputter-coated with gold, and observed and photographed under the condition of an accelerating voltage of 10 kV and a magnification of x500.

[0024] 4) Dynamic rheological property determination The gel sample was placed on the test table of the rheometer, a circular flat plate with a diameter of 40 mm was used, the flat plate distance was 1 mm, the excess gel exposed to the outer edge of the parallel plate was wiped off, paraffin oil was used for sealing to prevent moisture evaporation, then the heat preservation cover was added for preparation of determination, the strain was set to 0.05%, the temperature was set to 25°C, the sample was scanned at a frequency of 0.01-10 Hz, and the changes of storage modulus (G') and loss modulus (G'') were recorded in the process.

[0025] 5) Creep-recovery property determination The gel sample was placed on the test table of the rheometer, the parallel plate distance was 1 mm, the gel was applied with a constant stress of 8 Pa at 25°C, and the creep behavior was evaluated. In the creep stage, the creep response of the gel was measured for 300 s, and in the recovery stage, the recovery response of the gel after removal of the stress was measured for 300 s.

[0026] 6) Intermolecular interaction determination a solution: 2 g of the gel sample was mixed with 10 mL of 0.05 mol / L NaCl solution; b solution: 2 g of the gel sample was mixed with 10 mL of 0.6 mol / L NaCl solution; c solution: 2 g of the gel sample was mixed with 10 mL of a mixed solution (0.6 mol / L NaCl solution + 1.5 mol / L urea solution); d solution: 2 g of the gel sample was mixed with 10 mL of a mixed solution (0.6 mol / L NaCl solution + 8 mol / L urea solution); e solution: 2 g of the gel sample was mixed with 10 mL of a mixed solution (0.6 mol / L NaCl solution + 8 mol / L urea solution + 0.05 mol / L β-mercaptoethanol).

[0027] The above 5 kinds of solutions were placed at 4°C for 60 min, and the protein mass concentration in the supernatant was determined after centrifugal treatment (10000 r / min, 15 min, 4°C). Among them: the ionic bond is the difference between the protein mass concentration in the b solution and the protein mass concentration in the a solution; the hydrogen bond is the difference between the protein mass concentration in the c solution and the protein mass concentration in the b solution; the hydrophobic interaction is the difference between the protein mass concentration in the d solution and the protein mass concentration in the c solution; the disulfide bond is the difference between the protein mass concentration in the e solution and the protein mass concentration in the d solution.

[0028] 7) X-ray diffraction (XRD) determination The gel samples were placed on the XRD instrument for analysis, the scanning range 2 theta was 5°-60°, the scanning rate was 6° / min, the step width was 0.02°, the voltage was 40 kV, and the current was 30 mA.

[0029] 8) Fourier transform infrared spectroscopy (FTIR) determination The 2 mg lyophilized gel was mixed with 200 mg anhydrous potassium bromide (KBr), ground and pressed into a wafer for FTIR analysis. The FTIR spectrum was recorded in the wave number scanning range of 400-4000 cm -1 , with a resolution of 4 cm -1 -1 and a cumulative scanning number of 64.

[0030] 9) Endogenous fluorescence spectrum After the gel was freeze-dried, it was dispersed in a phosphate buffer (0.01 M, pH 7.0) to make a dispersion of 2 mg / mL. The excitation wavelength was 280 nm, the slit width was 5 nm, the scanning rate was 60 nm / min, and the fluorescence emission spectrum was recorded in the wavelength range of 300-450 nm.

[0031] 10) Curcumin entrapment efficiency (EE) determination 2 g of lyophilized gel powder was dispersed in 10 mL of anhydrous ethanol, and the curcumin was extracted by ultrasonic-assisted ethanol method, then the supernatant was collected by centrifugation, and the absorbance of curcumin in the supernatant at 426 nm was measured by spectrophotometer. The content of curcumin entrapped in the gel was quantitatively determined according to the absorbance-concentration standard curve. The EE of curcumin was calculated as follows:

[0032] In the formula: A e is the content of curcumin entrapped in the gel, A t is the total amount of curcumin added to the gel.

[0033] 11) In vitro antioxidant activity determination The DPPH free radical scavenging capacity was used to evaluate the in vitro antioxidant activity of the gel samples: 1.0 mL of 0.5 mg / mL sample was mixed with 2.0 mL of 0.2 mM DPPH-ethanol solution, and the reaction was carried out in the dark for 30 min. The absorbance was measured at 517 nm after the reaction. The DPPH distilled water solution was used as a blank. The DPPH free radical scavenging capacity was calculated as follows:

[0034] In the formula: A b is the absorbance of the blank, A s is the absorbance of the sample.

[0035] 12) Curcumin chemical stability determination Fresh gels were placed in glass bottles and evaluated for light stability under 254 nm UV light at 15 W power and 25 °C, and for thermal stability by placing them in the dark at 45 °C, 75 °C and 95 °C for 6 h. Samples were collected at intervals and curcumin was extracted by ultrasonic-assisted ethanol method. The content of curcumin was determined according to the standard curve. The retention rate of curcumin was expressed as the ratio of curcumin content in the treated sample to the initial curcumin content in the sample before treatment.

[0036] 13) Curcumin release rate determination during simulated digestion A 1 g gel sample was immersed in 100 mL simulated gastric fluid (SGF, containing 150 mM NaCl), pH adjusted to 2.0 with 1 M HCl, 3 mg / mL pepsin was added, and the sample was subjected to simulated gastric digestion at 37 °C for 2 h with a shaking rate of 100 rpm; subsequently, the gastric digest was mixed with an equal volume of simulated intestinal fluid (SIF, containing 150 mM NaCl, 8 mM KCl, 10 mM CaCl2and 5 mg / mL cholate), pH adjusted to 7.0 with 1 M NaOH, 1 mg / mL trypsin was added, and the sample was subjected to simulated intestinal digestion at the same temperature and shaking rate for 4 h, and the digestion reaction was terminated by placing the sample in an ice bath for 30 min. At specific time intervals, 2 mL of the gastro-intestinal digest was mixed with 6 mL of absolute ethanol, and curcumin was extracted by ultrasonic-assisted ethanol method. The content of curcumin released in the digest was determined according to the standard curve. The formula for calculating the release rate of curcumin is as follows:

[0037] wherein: A r is the content of curcumin released in the digest, A e is the content of curcumin embedded in the gel.

[0038] 14) Curcumin bioaccessibility determination After the end of the simulated gastro-intestinal digestion, the supernatant was considered to be the part of the mixed micelles in which curcumin was solubilized, by centrifugation at 10,000 g for 30 min at 4 °C. 2 mL of the supernatant was mixed with 6 mL of absolute ethanol, and curcumin was extracted by ultrasonic-assisted ethanol method. The content of curcumin in the mixed micelles was determined according to the standard curve. The formula for calculating the bioaccessibility of curcumin in the gel after digestion is as follows:

[0039] wherein: C m is the content of curcumin in the micelles, C e is the content of curcumin embedded in the gel.

[0040] Example 1 1. Preparation of protein dispersion: The red bean protein was mixed with deionized water to prepare a protein dispersion with a mass fraction of 4%. The pH of the dispersion was adjusted to 12.0 using 1 M NaOH, and the mixture was stirred magnetically for 30 min. The protein dispersion was then treated with ultrasound at an ultrasonic power of 300 W for 25 min. The pH of the protein dispersion was then adjusted to 7.0.

[0041] 2. Preparation of protein-starch composite dispersion: The protein dispersion obtained in step 1 was added with straight-chain corn starch and branched-chain corn starch. The total amount of corn starch added was 2%, and the mass ratio of straight-chain corn starch to branched-chain corn starch was 1:5. The obtained protein-starch mixed dispersion was stirred magnetically for 30 min, heated at 95°C while stirring for 20 min, quickly cooled to room temperature, and then treated with ultra-high pressure at a pressure of 700 MPa for 10 min to obtain a protein-starch composite dispersion.

[0042] 3. Addition of curcumin and homogenization: Curcumin crystals were dissolved in anhydrous ethanol to prepare a curcumin solution with a concentration of 10 mg / mL. The curcumin solution was then added dropwise to the protein-starch composite dispersion obtained in step 2 to achieve a final concentration of 0.4 mg / mL. The mixture was stirred magnetically at room temperature for 2 h, and then treated with high-pressure microjet homogenization at a homogenization pressure of 250 MPa for 3 times.

[0043] 4. Preparation of red bean protein-corn starch composite cold gel embedded with curcumin: The mixture obtained in step 3 was subjected to rotary evaporation at 35°C to remove ethanol, and then deionized water was added to achieve a curcumin concentration of 0.4 mg / mL. The mixture was then centrifuged at 5000 g for 10 min to remove unbound curcumin. Glucose acid-δ-lactone was added to the supernatant at a concentration of 0.4%, and the mixture was stirred uniformly. The mixture was then acidified at room temperature for 4 h, and then stored at 4°C for 12 h to obtain a red bean protein-corn starch composite acid-induced cold gel embedded with curcumin.

[0044] Example 2 1. Preparation of protein dispersion: The red bean protein was mixed with deionized water to prepare a protein dispersion with a mass fraction of 4%. The pH of the dispersion was adjusted to 12.0 using 1 M NaOH, and the mixture was stirred magnetically for 30 min. The protein dispersion was then treated with ultrasound at an ultrasonic power of 200 W for 30 min. The pH of the protein dispersion was then adjusted to 7.0.

[0045] 2. Preparation of protein-starch composite dispersion: To the protein dispersion obtained in step 1, linear corn starch and branched corn starch were added, the total amount of corn starch added was 2%, and the mass ratio of linear corn starch to branched corn starch was 1:6. The obtained protein-starch mixed dispersion was magnetically stirred for 30 min, heated at 95°C while stirring for 20 min, rapidly cooled to room temperature, and then subjected to ultra-high pressure treatment at a pressure of 800 MPa for 5 min to obtain a protein-starch composite dispersion.

[0046] 3. Addition of curcumin and homogenization: Curcumin crystals were dissolved in anhydrous ethanol to prepare a curcumin solution of 10 mg / mL, which was then added dropwise to the protein-starch composite dispersion obtained in step 2 to achieve a final curcumin concentration of 0.4 mg / mL. The mixture was magnetically stirred at room temperature for 2 h, and then subjected to high-pressure microfluidization homogenization at a pressure of 300 MPa for 2 times.

[0047] 4. Preparation of red bean protein-corn starch composite cold gel embedded with curcumin: The mixture obtained in step 3 was subjected to rotary evaporation at 35°C to remove ethanol, and then deionized water was added to achieve a curcumin concentration of 0.4 mg / mL. The mixture was then centrifuged at 5000 g for 10 min to remove unbound curcumin. Glucaric acid-δ-lactone was added to the supernatant at a concentration of 0.4%, and the mixture was stirred uniformly and then acidified at room temperature for 4 h. The mixture was then stored at 4°C for 12 h to obtain red bean protein-corn starch composite acid-induced cold gel embedded with curcumin.

[0048] Example 3: 1. Preparation of protein dispersion: Red bean protein was mixed with deionized water to prepare a protein dispersion with a mass fraction of 4%. The pH of the dispersion was adjusted to 12.0 using 1 M NaOH, and the mixture was magnetically stirred for 30 min. The mixture was then subjected to ultrasonic treatment at an ultrasonic power of 400 W for 20 min. The pH of the protein dispersion was then adjusted to 7.0.

[0049] 2. Preparation of protein-starch composite dispersion: To the protein dispersion obtained in step 1, linear corn starch and branched corn starch were added, the total amount of corn starch added was 2%, and the mass ratio of linear corn starch to branched corn starch was 1:4. The obtained protein-starch mixed dispersion was magnetically stirred for 30 min, heated at 95°C while stirring for 20 min, rapidly cooled to room temperature, and then subjected to ultra-high pressure treatment at a pressure of 600 MPa for 15 min to obtain a protein-starch composite dispersion.

[0050] 3. Addition of curcumin and homogenization: Curcumin crystals were dissolved in anhydrous ethanol to prepare a curcumin solution of 10 mg / mL, which was then added dropwise to the protein-starch complex dispersion obtained in step 2 to achieve a final curcumin concentration of 0.4 mg / mL. The mixture was magnetically stirred at room temperature for 2 h, and then subjected to high-pressure microfluidization homogenization at a homogenization pressure of 200 MPa for 4 times.

[0051] 4. Preparation of curcumin-embedded red bean protein-corn starch complex cold gel: The mixture obtained in step 3 was subjected to rotary evaporation at 35°C to remove ethanol, and then supplemented with deionized water to achieve a curcumin concentration of 0.4 mg / mL. The mixture was then centrifuged at 5000 g for 10 min to remove unbound curcumin. 0.4% of gluconic acid-delta-lactone was added to the supernatant, which was stirred uniformly and then acidified at room temperature for 4 h. The mixture was then refrigerated at 4°C for 12 h to obtain curcumin-embedded red bean protein-corn starch complex acid-induced cold gel.

[0052] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that step 1 omits the ultrasonic treatment process, step 2 omits the ultra-high pressure treatment process and the corn starch composition is different, and step 3 omits the high-pressure microfluidization homogenization process. The specific preparation method is as follows: 1. Preparation of protein dispersion: Red bean protein was mixed with deionized water to prepare a protein dispersion with a mass fraction of 4%. The pH of the dispersion was adjusted to 12.0 using 1 M NaOH, and the mixture was magnetically stirred for 30 min. The pH of the protein dispersion was then adjusted to 7.0.

[0053] 2. Preparation of protein-starch complex dispersion: 2% of ordinary corn starch (mass ratio of amylose to amylopectin of 1:3.76) was added to the protein dispersion obtained in step 1. The obtained protein-starch mixed dispersion was magnetically stirred for 30 min, heated at 95°C for 20 min while stirring, and then rapidly cooled to room temperature to obtain a protein-starch complex dispersion.

[0054] 3. Addition of curcumin and homogenization: Curcumin crystals were dissolved in anhydrous ethanol to prepare a curcumin solution of 10 mg / mL, which was then added dropwise to the protein-starch complex dispersion obtained in step 2 to achieve a final curcumin concentration of 0.4 mg / mL. The mixture was magnetically stirred at room temperature for 2 h.

[0055] 4. Preparation of curcumin-embedded red bean protein-corn starch complex cold gel: The mixture obtained in step 3 was placed in a rotary evaporator at 35°C to remove ethanol, and then deionized water was added to reach a curcumin concentration of 0.4 mg / mL. The mixture was then centrifuged at 5000 g for 10 min to remove unbound curcumin. Glucono-delta-lactone was added to the supernatant at a concentration of 0.4%, and the mixture was stirred uniformly. After acidification at room temperature for 4 h, the mixture was placed in a refrigerator at 4°C for 12 h to obtain a red bean protein-corn starch composite acid-induced cold gel embedding curcumin.

[0056] Comparative Example 2: Comparative Example 1 and Example 1 differ only in that the step 2 ultra-high pressure treatment process is omitted and the corn starch composition is different, and the step 3 high-pressure micro-jet homogenization process is omitted. The specific preparation method is as follows: 1. Preparation of protein dispersion: Red bean protein was mixed with deionized water to prepare a protein dispersion with a mass fraction of 4%. The pH of the dispersion was adjusted to 12.0 using 1 M NaOH, and the mixture was stirred magnetically for 30 min. The protein dispersion was then subjected to ultrasonic treatment at an ultrasonic power of 300 W for 25 min. The pH of the protein dispersion was then adjusted to 7.0.

[0057] 2. Preparation of protein-starch composite dispersion: To the protein dispersion obtained in step 1, 2% of ordinary corn starch (mass ratio of amylose to amylopectin 1:3.76) was added. The obtained protein-starch mixed dispersion was stirred magnetically for 30 min, and then heated at 95°C while stirring for 20 min. After rapid cooling to room temperature, a protein-starch composite dispersion was obtained.

[0058] 3. Addition of curcumin and homogenization: Curcumin crystals were dissolved in anhydrous ethanol to prepare a curcumin solution with a concentration of 10 mg / mL. The curcumin solution was then added dropwise to the protein-starch composite dispersion obtained in step 2 to achieve a final curcumin concentration of 0.4 mg / mL. The mixture was stirred magnetically at room temperature for 2 h.

[0059] 4. Preparation of red bean protein-corn starch composite cold gel embedding curcumin: The mixture obtained in step 3 was placed in a rotary evaporator at 35°C to remove ethanol, and then deionized water was added to reach a curcumin concentration of 0.4 mg / mL. The mixture was then centrifuged at 5000 g for 10 min to remove unbound curcumin. Glucono-delta-lactone was added to the supernatant at a concentration of 0.4%, and the mixture was stirred uniformly. After acidification at room temperature for 4 h, the mixture was placed in a refrigerator at 4°C for 12 h to obtain a red bean protein-corn starch composite acid-induced cold gel embedding curcumin.

[0060] Comparative Example 3: The difference between Comparative Example 1 and Example 1 is only that the ultrasonic treatment process is deleted in Step 1, the corn starch composition is different in Step 2, and the high-pressure micro-jet homogenization process is deleted in Step 3. The specific preparation method is as follows: 1. Preparation of protein dispersion liquid: The red bean protein was mixed with deionized water to prepare a protein dispersion liquid with a mass fraction of 4%. The pH of the dispersion liquid was adjusted to 12.0 with 1 M NaOH, and the dispersion liquid was magnetically stirred for 30 min. Then the pH of the protein dispersion liquid was adjusted to 7.0.

[0061] 2. Preparation of protein-starch composite dispersion liquid: 2% of ordinary corn starch (the mass ratio of amylose to amylopectin is 1:3.76) was added to the protein dispersion liquid obtained in Step 1, and the obtained protein-starch mixed dispersion liquid was magnetically stirred for 30 min. The mixture was heated at 95°C while stirring for 20 min, and then rapidly cooled to room temperature. The mixture was subjected to ultrahigh pressure treatment at a pressure of 700 MPa for 10 min to obtain a protein-starch composite dispersion liquid.

[0062] 3. Addition of curcumin and homogenization: Curcumin crystals were dissolved in anhydrous ethanol to prepare a curcumin solution with a concentration of 10 mg / mL. The curcumin solution was then added dropwise to the protein-starch composite dispersion liquid obtained in Step 2 to obtain a final curcumin concentration of 0.4 mg / mL. The mixture was magnetically stirred at room temperature for 2 h.

[0063] 4. Preparation of red bean protein-corn starch composite cold gel embedded with curcumin: The mixture obtained in Step 3 was subjected to rotary evaporation at 35°C to remove ethanol, and deionized water was added to the mixture to obtain a curcumin concentration of 0.4 mg / mL. The mixture was then centrifuged at 5000 g for 10 min to remove unbound curcumin. 0.4% of gluconic acid-delta-lactone was added to the supernatant, and the mixture was stirred uniformly. The mixture was then acidified at room temperature for 4 h, and then stored at 4°C for 12 h to obtain a red bean protein-corn starch composite acid-induced cold gel embedded with curcumin.

[0064] Comparative Example 4: The difference between Comparative Example 1 and Example 1 is only that the ultrasonic treatment process is deleted in Step 1, the ultrahigh pressure treatment process is deleted in Step 2, and the corn starch composition is different. The specific preparation method is as follows: 1. Preparation of protein dispersion liquid: The red bean protein was mixed with deionized water to prepare a protein dispersion liquid with a mass fraction of 4%. The pH of the dispersion liquid was adjusted to 12.0 with 1 M NaOH, and the dispersion liquid was magnetically stirred for 30 min. Then the pH of the protein dispersion liquid was adjusted to 7.0.

[0065] 2. Preparation of protein-starch complex dispersion: To the protein dispersion obtained in step 1, 2% of normal corn starch (mass ratio of amylose to amylopectin 1:3.76) was added, and the obtained protein-starch mixed dispersion was magnetically stirred for 30 min, heated at 95°C while stirring for 20 min, and then rapidly cooled to room temperature to obtain a protein-starch complex dispersion.

[0066] 3. Addition of curcumin and homogenization: Curcumin crystals were dissolved in anhydrous ethanol to prepare a curcumin solution of 10 mg / mL, which was then added dropwise to the protein-starch complex dispersion obtained in step 2 to achieve a final curcumin concentration of 0.4 mg / mL. The mixture was magnetically stirred at room temperature for 2 h, and then subjected to high-pressure microfluidization homogenization under a homogenization pressure of 250 MPa for 3 times.

[0067] 4. Preparation of red bean protein-corn starch complex cold gel embedding curcumin: The mixture obtained in step 3 was subjected to rotary evaporation at 35°C to remove ethanol, and then supplemented with deionized water to achieve a curcumin concentration of 0.4 mg / mL. The mixture was then centrifuged at 5000 g for 10 min to remove unbound curcumin. 0.4% of gluconic acid-δ-lactone was added to the supernatant, which was stirred uniformly and then acidified at room temperature for 4 h. The mixture was then stored at 4°C for 12 h to obtain a red bean protein-corn starch complex acid-induced cold gel embedding curcumin.

[0068] The gel strength, water holding capacity (WHC), microstructure, dynamic rheological properties, creep-recovery properties, intermolecular interactions, X-ray diffraction pattern (XRD), Fourier transform infrared spectrum (FTIR), intrinsic fluorescence spectrum, curcumin embedding efficiency (EE), in vitro antioxidant activity, curcumin chemical stability, curcumin release rate during simulated digestion, and curcumin bioaccessibility of the red bean protein-corn starch complex cold gel embedding curcumin prepared in each example and comparative example were detected to optimize the preparation process and parameters of the red bean protein-corn starch complex cold gel embedding curcumin.

[0069] 1. Results of gel strength and water holding capacity determination: The complex gel prepared in Comparative Example 1 had relatively low gel strength and water holding capacity, and the gel strength and water holding capacity of the complex gels prepared in Comparative Examples 2-4 were slightly increased but still relatively low. The gel strength and water holding capacity of the complex gels prepared in the three examples were significantly increased, and Example 1 had the highest gel strength and water holding capacity (P<0.05). Figure 1). This shows that the preparation process of the curcumin-embedded red bean protein-corn starch composite cold gel provided by the present application can obtain a composite gel with higher mechanical properties and stability.

[0070] 2. Microstructure observation results: Compared with the four comparative examples, the microstructure of the composite gel prepared in the three examples is more compact and has better continuity, resulting in the formation of a composite gel structure with higher strength and stability, especially the gel network prepared in example 1 has greater cross-linking degree and smaller porosity. Figure 2 Therefore, the preparation process of the curcumin-embedded red bean protein-corn starch composite cold gel provided by the present application can obtain a composite gel with a compact microstructure.

[0071] 3. Dynamic rheological property determination results: The G' of all the composite gel samples prepared in each example and comparative example is higher than the G'', indicating the formation of an elastic gel structure; all the G' increases with the increase of the frequency, showing frequency dependence, which indicates that all the composite gels are physical gels, and in addition to covalent disulfide bonds, there may be more non-covalent interactions for stabilizing the gel structure; the composite gel prepared in comparative example 1 has relatively low G' and G'', the G' and G'' of the composite gel samples prepared in comparative examples 2-4 increase slightly, but are still relatively low; the G' and G'' of the composite gels prepared in the three examples increase significantly, and example 1 has the highest G' and G''. Figure 3 This shows that the composite gel prepared by the preparation process provided in the examples of the present application exhibits more prominent solid elastic behavior, and the gel network is strengthened and the viscoelasticity is increased.

[0072] 4. Creep-recovery property determination results: All the composite gel samples prepared in each example and comparative example exhibit a typical creep-recovery curve with viscoelasticity characteristics, indicating the degree of deformation of the gel system during the external force process. The composite gel prepared in comparative example 1 has the largest deformation, the deformation of the composite gel samples prepared in comparative examples 2-4 decreases slightly, but is still relatively large; the deformation of the composite gels prepared in the three examples decreases sharply, and the deformation degree of example 1 is the lowest. Figure 4 This shows that the preparation process of the curcumin-embedded red bean protein-corn starch composite cold gel provided by the examples of the present application promotes the formation of a more compact and stronger composite gel network structure, and under the condition of applying a constant stress in the creep process, the high-strength gel has higher anti-deformation ability.

[0073] 5. Intermolecular interaction, X-ray diffraction pattern, Fourier transform infrared spectrum and endogenous fluorescence spectrum detection results: Figure 5The results of intermolecular interaction, X-ray diffraction pattern, Fourier transform infrared spectrum and endogenous fluorescence spectrum of the composite gels prepared for each example and comparative example were detected. As can be seen from A in Figure 5 , the order of intermolecular interaction from large to small in all composite gels is: hydrophobic interaction > disulfide bond > hydrogen bond > ionic bond, wherein the content of hydrophobic interaction and disulfide bond is much higher than that of ionic bond and hydrogen bond, which dominates in the gel, and the change degree of ionic bond and hydrogen bond is small, which does not have a significant impact on the gel. Compared with the composite gel prepared in comparative example 1, the hydrophobic interaction of the composite gel samples prepared in comparative examples 2-4 increases slightly, while the hydrophobic interaction of the composite gels prepared in three examples increases significantly, and the hydrophobic interaction of the composite gel prepared in example 1 is the highest. The hydrophobic interaction plays an important role in the gel network, and the hydrophobic aggregation of the protein can promote the formation of a dense gel network structure. In addition, the disulfide bond of all composite gel samples does not change significantly. The results show that the improvement of the composite gel prepared in the example is mainly due to the enhancement of the hydrophobic interaction in the gel system.

[0074] As can be seen from B in Figure 5 , all composite gel samples have a wide diffraction peak near 20.2°, which indicates that they are amorphous polymers. The diffraction peak shape and position of the composite gels prepared in three examples are similar to those of the composite gels prepared in four comparative examples, but the composite gels prepared in examples have higher diffraction peak intensity, indicating that the protein and starch form a more compact complex. Example 1 has the largest diffraction peak intensity, the protein and starch are more tightly combined, and a more stable complex structure is formed, which further improves the gel network. As can be seen from C in Figure 5 , all composite gels have a wide absorption peak at 3200-3600 cm -1 , which is caused by the stretching vibration of hydroxyl group, and the change of hydroxyl vibration of different samples is small, indicating that the hydrogen bonding between the protein and starch is small. The composite gel has new absorption peaks at 933 cm -1 , 1026 cm -1 and 1153 cm -1 , which is related to the introduction of glycosidic bond in starch. In addition, the intensity and shape of the amide I band absorption peak of the composite gel change to different degrees, indicating that the structure of the composite gel is stable through hydrophobic interaction. As can be seen from D in Figure 5 , compared with the composite gels prepared in four comparative examples, the fluorescence intensity of the composite gels prepared in three examples is significantly reduced, which means that the protein and starch are combined through hydrophobic interaction, and the endogenous fluorescence of the protein is quenched. The fluorescence intensity of example 1 is the lowest, indicating that the protein and starch have stronger hydrophobic interaction, forming a stronger and more compact non-covalent gel structure.

[0075] 6. Results of the encapsulation efficiency and antioxidant activity determination of curcumin: Depend on Figure 6 It can be seen that the composite gel prepared in Comparative Example 1 has a low encapsulation rate, while the encapsulation rates of the composite gel samples prepared in Comparative Examples 2-4 have increased. The encapsulation rates of the composite gels prepared in the three examples have further increased, with the composite gel sample prepared in Example 1 exhibiting the highest encapsulation rate. This is related to the higher mechanical properties, intermolecular interactions, structural stability, and denser microstructure of the composite gel. Furthermore, the images of the composite gels also show that the composite gels prepared in the comparative examples have more liquid expulsion, while the sample samples in the examples have almost no liquid expulsion. This also promotes the encapsulation of curcumin in the composite gel. The trend of the DPPH radical scavenging ability of the composite gels is consistent with the curcumin encapsulation rate. The DPPH radical scavenging ability of the composite gels prepared in all examples is significantly higher than that of all comparative examples, especially the composite gel sample prepared in Example 1, which has the highest DPPH radical scavenging ability and exhibits the strongest antioxidant activity. Figure 7 This is mainly due to the high curcumin encapsulation rate of the composite gel prepared in Example 1.

[0076] 7. Results of the chemical stability test of curcumin: Due to its unique chemical structure, curcumin is extremely unstable under light and high temperature conditions and is prone to oxidative degradation, which limits its application in food. Figure 8 The chemical stability of curcumin in the composite gels prepared in each embodiment and comparative example is shown, under light exposure and storage at 45°C, 75°C, and 95°C. Figure 8 As shown in Figure A, the curcumin retention rate in all composite gels decreased rapidly within 1 hour of light exposure, and continued to decline within 1-6 hours of light exposure. Compared with the composite gels prepared in the four comparative examples (52%-61%), the composite gels prepared in the three examples showed higher curcumin retention rates after 6 hours of light exposure, with the composite gel prepared in Example 1 exhibiting the highest curcumin retention rate (65.51%), demonstrating the highest photostability. During storage at 45℃, 75℃, and 95℃, the curcumin retention rate in the composite gels showed a similar trend to that under light-exposed storage (…). Figure 8 B in Figure 8 C and Figure 8(D in the example). Compared with the composite gels prepared in the four comparative examples, the composite gels prepared in the three examples showed higher curcumin retention rates after 6 h of heat treatment, with the retention rate decreasing as the heat treatment temperature increased. After 6 h of storage at 45°C, 75°C, and 95°C, the composite gel prepared in Example 1 showed the highest curcumin retention rates, at 59.22%, 41.26%, and 34.82%, respectively, representing increases of 23.92%, 23.02%, and 21.96% compared to Comparative Example 1. The results indicate that the stronger and denser gel network structure of the composite gel prepared in Example 1 provides a better physical barrier and protection for curcumin, resisting partial chemical degradation of curcumin caused by external environmental stresses such as light and high temperature, thus maintaining good chemical stability of the curcumin embedded in the composite gel during light and high temperature storage.

[0077] 8. Results of curcumin release rate and bioavailability assays: When the food matrix is ​​digested, the embedded bioactive compounds are released. The rate and location of release will affect the bioavailability of the bioactive compounds, and thus affect the human body's absorption and bioutilization of the bioactive compounds. Figure 9 The results of curcumin release rate and bioavailability assays for the composite gels prepared in each embodiment and comparative example during simulated gastrointestinal digestion are presented. Figure 9 As shown in Figure A, during simulated gastric digestion, the composite gel prepared in the examples exhibited a lower curcumin release rate than the composite gel prepared in the comparative example, indicating that the composite gel prepared in the examples has stronger resistance to gastric digestion. During simulated intestinal digestion, the composite gel prepared in the examples exhibited a higher curcumin release rate than the composite gel prepared in the comparative example, demonstrating a promotion of curcumin release, especially the composite gel prepared in Example 1, which achieved the highest curcumin release rate during simulated intestinal digestion. The results indicate that the method provided in Example 1 promotes the targeted release of curcumin during intestinal digestion, exhibiting a better controlled-release effect. This alteration in the digestive behavior of the composite gel and its controlled-release effect on curcumin improves the bioavailability of curcumin, resulting in the composite gel prepared in Example 1 having the highest curcumin bioavailability. Figure 9 (B in the original text), which may be related to its improved chemical stability of curcumin. Higher biological accessibility leads to higher bioavailability, indicating that the body has a higher absorption efficiency of nutrients or active ingredients.

[0078] In conclusion, the red bean protein-corn starch composite gel prepared by the preparation method has high gel strength, water retention and physical stability, the rheological properties are improved, the polyphenol sensitive bioactive compounds such as curcumin can be effectively embedded, the antioxidant activity of the composite gel and the chemical stability of curcumin are improved, the controlled release of curcumin is realized and the bioavailability of curcumin is improved, and the method can be applied to the development of functional composite gel products.

[0079] Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore, the protection scope of the present application should be defined by the claims.

Claims

1. A method for preparing a red bean protein-corn starch composite cryogel with encapsulated curcumin, characterized in that, Includes the following steps: 1) Mix red bean protein with deionized water to prepare a protein solution with a mass fraction of 4%. Adjust the pH to 12.

0. After stirring with a magnetic force, sonicate at a power of 100-500 W for 20-30 minutes. Then adjust the pH of the protein solution to 7.0 to obtain a protein dispersion. 2) Add 2% corn starch by mass to the protein dispersion obtained in step 1), stir magnetically, heat at 95°C while stirring for 20 min, rapidly cool to room temperature, and then treat under ultra-high pressure at 600-800 MPa for 5-15 min to obtain a protein-starch composite dispersion; the mass ratio of amylose to amylopectin in the corn starch is 1:4-6. 3) Add anhydrous ethanol solution of curcumin dropwise to the protein-starch composite dispersion obtained in step 2) to make the final concentration of curcumin reach 0.4 mg / mL. After magnetic stirring, perform high-pressure micro-jet homogenization treatment at a pressure of 200-300 MPa and a number of homogenization times of 1-5 times to obtain a mixture. 4) The mixture obtained in step 3) was subjected to rotary evaporation to remove ethanol, and deionized water was added to make the curcumin concentration 0.4 mg / mL. Unbound curcumin was removed by centrifugation, and 0.4% gluconate-δ-lactone was added to the supernatant. After stirring evenly, the mixture was acidified at room temperature for 4 h, and then refrigerated at 4℃ for 12 h to obtain the curcumin-encapsulated red bean protein-corn starch composite cold gel.

2. The preparation method according to claim 1, characterized in that, Step 1) The ultrasonic treatment is performed at a power of 300 W for 25 minutes.

3. The preparation method according to claim 1, characterized in that, Step 2) The mass ratio of amylose to amylopectin in the corn starch is 1:

5.

4. The preparation method according to claim 1, characterized in that, Step 2) The ultra-high pressure treatment is performed at a pressure of 700 MPa for 10 min.

5. The preparation method according to claim 1, characterized in that, Step 3) The high-pressure microjet homogenization process involves homogenizing three times at a pressure of 250 MPa.

6. The preparation method according to claim 1, characterized in that, The magnetic stirring time in steps 1) and 2) is 30 min, and the magnetic stirring time in step 3) is 2 h.

7. The preparation method according to claim 1, characterized in that, Step 3) The concentration of curcumin in the anhydrous ethanol solution of curcumin is 10 mg / mL.

8. The preparation method according to claim 1, characterized in that, Step 4) The rotary evaporation temperature is 35°C, and the centrifugation is performed at 5000 g for 10 min.

9. The curcumin-encapsulated red bean protein-corn starch composite cold gel prepared by any of the preparation methods described in claims 1-8.

10. The use of the curcumin-encapsulated red bean protein-corn starch composite cryogel of claim 9 in the preparation of food, health products, cosmetics or pharmaceuticals containing curcumin.

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