Preparation method of white kidney bean protein-propylene glycol alginate composite carrier steady-state emulsion for enhancing alpha-amylase inhibitory activity

Abstract

The invention discloses a preparation method of a white kidney bean protein-propylene glycol alginate composite carrier steady-state emulsion for enhancing alpha-amylase inhibitory activity, and belongs to the technical field of emulsion preparation. The method comprises the following steps: S1, mixing white kidney bean protein with water to prepare a protein solution; s2, mixing propylene glycol alginate with water to prepare a propylene glycol alginate solution, and then mixing the propylene glycol alginate solution with the protein solution prepared in the step S1 to prepare a water phase; s3, fat-soluble active substances are added into edible oil, and an oil phase is prepared; and S4, mixing the water phase and the oil phase, and carrying out high-speed dispersion and high-pressure homogenization to prepare the steady-state emulsion. The preparation method is simple and convenient to operate, and the prepared emulsion has good milk stability, storage stability, high active substance protection capacity and high alpha-amylase inhibitor activity.

Description

Technical Field

[0001] This invention belongs to the field of emulsion preparation technology, specifically relating to a method for preparing a stabilized emulsion using a white kidney bean protein-propylene glycol alginate composite carrier that enhances α-amylase inhibitory activity. Background Technology

[0002] With rapid economic development and improved living standards, the prevalence of metabolic diseases such as obesity and diabetes is becoming increasingly serious, making their prevention and control both socially and economically significant. While currently widely used chemical hypoglycemic drugs (such as acarbose and voglibose) are highly effective, they may cause side effects such as abnormal liver enzyme activity. In contrast, naturally derived α-amylase inhibitors (α-AIs) have attracted considerable attention due to their safety profile. Among them, α-AIs extracted from white kidney beans not only exhibit stronger inhibitory activity than other legumes but also demonstrate excellent biocompatibility, showing greater application potential in the field of obesity intervention.

[0003] α-AI in white kidney bean (WKB) is essentially a glycoprotein with a glycosyl content of 8.6%–15%. It specifically inhibits the activity of α-amylase in the saliva and intestines of humans and other animals, hindering the breakdown and digestion of carbohydrates such as starch, thus exhibiting physiological effects such as reducing food intake, controlling weight gain, and slowing fat accumulation. However, α-AI from a single legume source still faces significant bottlenecks in the development of functional foods. In vitro studies show that its inhibition efficiency is generally below 35%, and its activity retention rate in complex food systems is less than 60%. To address this issue, current research strategies focus on constructing complex systems of white kidney bean protein (WKBP) and multiple bioactive components (including proteins, polysaccharides, and polyphenols), but significant progress has not yet been achieved.

[0004] Furthermore, in existing patents, Chinese patent CN 111150068 A discloses a method for processing the wall material of probiotic microcapsules, which mentions the use of kidney bean protein and propylene glycol alginate (PGA). However, the use of PGA and WKBP in this patent is only briefly described, and their use and effects are not described in any embodiment. PGA and WKBP are also excluded from the preferred scope.

[0005] Chinese patent CN 119498414 A discloses a novel plant protein-polysaccharide composite nanoparticle stabilized powder oil and its preparation method, which uses PGA and other plant proteins for compounding, but does not conduct research on WKBP. Summary of the Invention

[0006] Technical issues

[0007] WKB contains α-AI, which has good α-amylase inhibitory activity and can be used for obesity intervention. However, using WKB or WKBP alone does not produce good results. Furthermore, the complex system composed of WKBP and multiple bioactive components still requires further investigation.

[0008] Technical solution

[0009] To address the aforementioned issues, this invention employs PGA as a functional carrier. This modified polysaccharide, prepared through esterification, possesses a main chain structure composed of specific uronic acid units, and the simultaneous presence of hydrophilic and hydrophobic groups in its molecule endows it with excellent amphiphilic properties. Through electrostatic interactions, hydrophobic interactions, and hydrogen bonding mechanisms, PGA can form a stable complex with WKBP, significantly improving the emulsification stability of the system. Using PGA and WKBP as the outer shell to encapsulate volatile, low-water-soluble, and poorly stable fat-soluble active substances (such as benzyl isothiocyanate (BITC)) not only enhances the α-amylase inhibition efficiency but also optimizes its bioavailability through a targeted delivery system of the active ingredient. This innovative technical approach of the present invention opens up new possibilities for the development of functional foods that regulate body fat.

[0010] The first objective of this invention is to provide a stabilized emulsion using WKBP and PGA as composite carriers, wherein the stabilized emulsion contains 0.9–0.95 wt% WKBP, 0.1–1 wt% PGA, 7.5–8.5 wt% edible oil, 89–91 wt% water, and 0.04–0.06 wt% fat-soluble active substances.

[0011] Furthermore, the stabilized emulsion contains 0.9–0.95 wt% WKBP, 0.3–0.8 wt% PGA, 7.5–8.5 wt% edible oil, 89–91 wt% water, and 0.04–0.06 wt% fat-soluble active substances.

[0012] Furthermore, the stabilized emulsion is composed of 0.9–0.95 wt% WKBP, 0.3–0.8 wt% PGA, 7.5–8.5 wt% edible oil, 89–91 wt% water and 0.04–0.06 wt% fat-soluble active substances.

[0013] Furthermore, fat-soluble active substances include one or more of BITC, proanthocyanidins, carotenoids, phytosterols, and polyunsaturated fatty acids.

[0014] Furthermore, the carotenoids include one or more of α-carotene, β-carotene, lutein, lycopene, and astaxanthin.

[0015] Furthermore, the phytosterols include one or more of β-sitosterol, stigmasterol, and campesterol.

[0016] Furthermore, the polyunsaturated fatty acids include one or more of linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).

[0017] Furthermore, edible oils include one or more of the following: corn oil, peanut oil, sunflower oil, soybean oil, and rapeseed oil.

[0018] The second objective of this invention is to provide a method for preparing a stabilized emulsion using WKBP and PGA as a composite carrier, comprising the following steps:

[0019] S1. Protein solution preparation: Mix WKBP with water to prepare a protein solution;

[0020] S2, Aqueous phase preparation: PGA is mixed with water to prepare a PGA solution, and then the PGA solution is mixed with the protein solution prepared in step S1 to prepare an aqueous phase;

[0021] S3. Oil phase preparation: The fat-soluble active substance is added to edible oil to prepare the oil phase;

[0022] S4. Mixing: The aqueous phase prepared in step S2 is mixed with the oil phase prepared in step S3, dispersed at high speed, and then homogenized under high pressure to obtain a stabilized emulsion.

[0023] In one embodiment of the present invention, in step S1, the mass concentration of WKBP in the protein solution is 1% to 10%.

[0024] In one embodiment of the present invention, in step S1, the pH of the protein solution is adjusted to 5.5 to 6.5.

[0025] In one embodiment of the present invention, in step S2, the mass concentration of PGA in the PGA solution is 1% to 10%.

[0026] In one embodiment of the present invention, water may be added during the preparation of the aqueous phase in step S2.

[0027] In one embodiment of the present invention, in step S2, the final concentration of PGA in the aqueous phase is 1% to 2%.

[0028] In one embodiment of the present invention, in step S2, the final concentration of WKBP in the aqueous phase is 0.2% to 1%.

[0029] In one embodiment of the present invention, in step S3, the fat-soluble active substances include one or more of BITC, proanthocyanidins, carotenoids, phytosterols, and polyunsaturated fatty acids.

[0030] In one embodiment of the present invention, the carotenoids include one or more of α-carotene, β-carotene, lutein, lycopene, and astaxanthin.

[0031] In one embodiment of the present invention, the phytosterols include one or more of β-sitosterol, stigmasterol, and campesterol.

[0032] As one embodiment of the present invention, the polyunsaturated fatty acids include one or more of linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA).

[0033] In one embodiment of the present invention, in step S3, the edible oil includes one or more of corn oil, peanut oil, sunflower seed oil, soybean oil, and rapeseed oil.

[0034] In one embodiment of the present invention, in step S3, the mass concentration of the fat-soluble active substance in the oil phase is 1 to 10 mg / mL.

[0035] As one embodiment of the present invention, in S1 to S3, the preparation process can be one or more of the following operations used to dissolve the solute in the solvent: stirring, heating, and sonication.

[0036] In one embodiment of the present invention, in step S1, the preparation involves stirring followed by ultrasonication; the stirring time is 0.5 to 2 hours, the ultrasonic power is 240 to 720 W, and the time is 10 to 20 minutes.

[0037] In one embodiment of the present invention, in step S2, the preparation is carried out by heating and stirring; the heating temperature is 30-40°C and the time is 0.5-2 hours.

[0038] In one embodiment of the present invention, in step S3, the preparation is carried out by stirring; the stirring time is 0.5 to 2 hours.

[0039] In one embodiment of the present invention, in step S4, the volume ratio of the oil phase to the water phase is 1:4 to 1:15.

[0040] In one embodiment of the present invention, in step S4, the volume ratio of the oil phase to the water phase is specifically 1:10.

[0041] In one embodiment of the present invention, in step S4, the parameters for high-speed dispersion are 8000–12000 r / min and the time is 2–4 min.

[0042] In one embodiment of the present invention, in step S4, the parameters of high-pressure homogenization are 5000-10000 psi and the number of times is 6-8.

[0043] The present invention provides a stabilized emulsion prepared based on the above method.

[0044] The application of the stabilized emulsion provided by this invention in the food industry.

[0045] The application of the stabilized emulsion provided by this invention in the field of health products.

[0046] Furthermore, the aforementioned health supplement is one that helps control body fat.

[0047] The beneficial effects of this invention are:

[0048] (1) The composite carrier of WKBP and PGA prepared in this invention can not only significantly improve the stability of the emulsion, but also change the tertiary conformation of WKBP through interaction with PGA, thereby improving the ability to inhibit α-amylase (compared to WKBP), and has a long-term regulatory effect on carbohydrate hydrolysis.

[0049] (2) The emulsion interface of the present invention has high stability. Figures 5-11 When the PGA concentration was 0.6%, the oil-water interface angle approached the critical value of -90°, and the prepared oil-in-water emulsion was the most stable. Furthermore, after measuring the average particle size, zeta potential, appearance, and oil droplet distribution of the WKBP and PGA composite emulsion stored at 4°C for 14 days, it was found that the composite system did not undergo significant stratification during storage. Figures 14-18 The zeta potential changed relatively little throughout the storage process. Figure 13 This indicates that the emulsion has good storage stability; finally, when the PGA concentration is 0.6%, the BITC retention rate and α-AI activity are the highest (p<0.05). Figure 24 , Figure 25 This indicates that the composite emulsion possesses high protection capability against bioactive substances (BITC) and strong α-AI activity. In summary, the emulsion of this invention exhibits multiple superior effects and is a product with broad market application prospects. Attached Figure Description

[0050] Figure 1 These are the ultraviolet spectra of Comparative Examples 1-2 and Examples 1-5 in this invention;

[0051] Figure 2 These are the fluorescence spectra of Comparative Examples 1-2 and Examples 1-5 in this invention;

[0052] Figure 3These are the X-ray diffraction patterns of Comparative Examples 1-2 and Examples 1-5 in this invention;

[0053] Figure 4 These are the Fourier transform infrared spectra of Comparative Examples 1-2 and Examples 1-5 in this invention;

[0054] Figure 5 This is a diagram showing the measurement of the contact angle in Comparative Example 1 of this invention;

[0055] Figure 6 This is a diagram showing the measurement of the contact angle in Comparative Example 2 of this invention;

[0056] Figure 7 This is a diagram showing the measurement of the contact angle in Example 1 of this invention;

[0057] Figure 8 This is a diagram showing the measurement of the contact angle in Example 2 of this invention;

[0058] Figure 9 This is a diagram showing the contact angle measurement in Example 3 of this invention;

[0059] Figure 10 This is a diagram showing the measurement of the contact angle in Example 4 of this invention;

[0060] Figure 11 This is a diagram showing the contact angle measurement in Example 5 of this invention;

[0061] Figure 12 This is a particle size change graph of Comparative Examples 3-4 and Examples 6-10 of the present invention after being stored at 4°C for 1, 4, 7, 10 and 14 days (different lowercase letters represent the significant differences between different series in the same number of days, and different uppercase letters represent the significant differences between the same series in different numbers of days (p<0.05)).

[0062] Figure 13 This is a potential change graph of Comparative Examples 3-4 and Examples 6-10 of the present invention stored at 4°C for 1, 4, 7, 10 and 14 days (different lowercase letters represent the significance of differences between different series in the same number of days, and different uppercase letters represent the significance of differences between the same series in different numbers of days (p<0.05)).

[0063] Figure 14 These are actual images of Comparative Examples 3-4 and Examples 6-10 of this invention stored at 4°C for 1 day;

[0064] Figure 15 These are actual images of Comparative Examples 3-4 and Examples 6-10 of this invention stored at 4°C for 4 days;

[0065] Figure 16 These are actual images of Comparative Examples 3-4 and Examples 6-10 of this invention stored at 4°C for 7 days;

[0066] Figure 17 These are actual images of Comparative Examples 3-4 and Examples 6-10 of this invention stored at 4°C for 10 days;

[0067] Figure 18 These are actual images of Comparative Examples 3-4 and Examples 6-10 of this invention stored at 4°C for 14 days;

[0068] Figure 19 These are fluorescence microscope images of Comparative Examples 3-4 and Examples 6-10 of this invention stored at 4°C for 1 day;

[0069] Figure 20 These are fluorescence microscope images of Comparative Examples 3-4 and Examples 6-10 of this invention stored at 4°C for 4 days;

[0070] Figure 21 These are fluorescence microscope images of Comparative Examples 3-4 and Examples 6-10 of this invention stored at 4°C for 7 days;

[0071] Figure 22 These are fluorescence microscope images of Comparative Examples 3-4 and Examples 6-10 of this invention stored at 4°C for 10 days;

[0072] Figure 23 These are fluorescence microscope images of Comparative Examples 3-4 and Examples 6-10 of this invention stored at 4°C for 14 days;

[0073] Figure 24 This is a graph showing the encapsulation rate of BITC stored at 4°C for 1 day, 7 days and 14 days in Comparative Examples 3-4 and Examples 6-10 of this invention (different uppercase letters represent significant differences between different series at the same number of days, and different lowercase letters represent significant differences between the same series at different numbers of days (p<0.05)).

[0074] Figure 25 These are α-AI activity assay graphs for Comparative Examples 3-4 and Examples 6-10 of this invention (different lowercase letters indicate significant differences between groups (p<0.05)). Detailed Implementation

[0075] Source of raw materials

[0076] White kidney bean protein was purchased from Yunnan Tianbaohua Biological Resources Development Co., Ltd.; other materials and reagents used in the following examples are commercially available unless otherwise specified.

[0077] Comparative Example 1

[0078] S1. Prepare a 1% WKBP solution and stir it with a magnetic stirrer at room temperature until dissolved.

[0079] S2. Adjust pH to 6, then perform ultrasonic treatment with an ultrasonic power of 480W for 15 minutes.

[0080] Comparative Example 2

[0081] S1. Prepare a 1% PGA solution and heat it in a water bath at 37°C with thorough stirring until completely dissolved.

[0082] Comparative Example 3

[0083] S1. Protein solution preparation: Prepare a 1% WKBP solution, stir with a magnetic stirrer at room temperature for 1 hour, adjust the pH to 6, and then perform ultrasonic treatment with an ultrasonic power of 480W for 15 minutes.

[0084] S2. Oil phase preparation: Dissolve BITC in corn oil to prepare a BITC-corn oil solution with a mass concentration of 5 mg / mL, which is the oil phase.

[0085] S3. Mix the oil phase prepared in step S2 with the WKBP solution prepared in step S1 at a volume ratio of 1:10, and disperse at 10000r / min for 2min.

[0086] S4. The emulsion dispersed at high speed in S3 was homogenized at 7252psi for 6 times to obtain the control group emulsion of WKBP alone.

[0087] Comparative Example 4

[0088] S1. Preparation of protein solution: Prepare a 1% PGA solution and heat it in a water bath at 37°C with thorough stirring until completely dissolved;

[0089] S2. Oil phase preparation: Dissolve BITC in corn oil to prepare a BITC-corn oil solution with a mass concentration of 5 mg / mL, which is the oil phase.

[0090] S3. Mix the oil phase prepared in step S2 with the WKBP solution prepared in step S1 at a volume ratio of 1:10, and disperse at 10000r / min for 2min.

[0091] S4. The emulsion dispersed at high speed in S3 was homogenized at 7252psi for 6 times to obtain the control group emulsion with pure PGA.

[0092] Example 1

[0093] S1. Preparation of protein solution: Prepare a 2% WKBP solution and stir it thoroughly with a magnetic stirrer at room temperature for 1 hour. After adjusting the pH to 6, perform ultrasonic treatment with an ultrasonic power of 480W for 15 minutes.

[0094] S2. Preparation of aqueous phase: Prepare a 2% PGA solution and heat it in a water bath at 37°C until it is completely dissolved. Mix the WKBP solution prepared in step S1, the PGA solution prepared in step S2, and deionized water in a certain proportion, heat them in a water bath at 37°C, and stir for 1 hour to prepare a solution with a final WKBP concentration of 1 wt% and a final PGA concentration of 0.2 wt%, which is the aqueous phase.

[0095] Example 2

[0096] The difference from Example 1 is that the final concentration of PGA in step S2 is 0.4 wt%.

[0097] Example 3

[0098] The difference from Example 1 is that the final concentration of PGA in step S2 is 0.6 wt%.

[0099] Example 4

[0100] The difference compared to Example 1 is that the final concentration of PGA in step S2 is 0.8 wt%.

[0101] Example 5

[0102] The difference from Example 1 is that the final concentration of PGA in step S2 is 1 wt%.

[0103] Example 6

[0104] S1. Preparation of protein solution: Prepare a 2% WKBP solution and stir it thoroughly with a magnetic stirrer at room temperature for 1 hour until it is completely dissolved. Then adjust the pH to 6 and perform ultrasonic treatment at a power of 480W for 15 minutes.

[0105] S2. Preparation of aqueous phase: Prepare a 2% PGA solution and heat it in a water bath at 37°C with thorough stirring until completely dissolved; mix the WKBP solution prepared in step S1, the PGA solution prepared in step S2, and deionized water in a certain proportion, heat and stir in a water bath at 37°C for 1 hour to prepare a solution with a final WKBP concentration of 1 wt% and a final PGA concentration of 0.2 wt%, which is the aqueous phase;

[0106] S3. Oil phase preparation: Dissolve BITC in corn oil to prepare a BITC-corn oil solution with a mass concentration of 5 mg / mL, which is the oil phase.

[0107] S4. Mix the oil phase prepared in step S3 with the aqueous phase prepared in step S2 at a volume ratio of 1:10, and disperse at 10000 r / min for 2 min.

[0108] S5. The emulsion dispersed at high speed in S3 is then homogenized at 7252psi for 6 times to obtain an emulsion with WKBP and PGA as composite carriers.

[0109] Example 7

[0110] The difference from Example 6 is that the final concentration of PGA in step S2 is 0.4 wt%.

[0111] Example 8

[0112] The difference compared to Example 6 is that the final concentration of PGA in step S2 is 0.6 wt%.

[0113] Example 9

[0114] The difference from Example 6 is that the final concentration of PGA in step S2 is 0.8 wt%.

[0115] Example 10

[0116] The difference from Example 6 is that the final concentration of PGA in step S2 is 1 wt%.

[0117] Test methods

[0118] Ultraviolet fluorescence spectrum Comparative Examples 1-2 and Examples 1-5 were diluted 2-fold. Ultraviolet (UV) spectroscopy analysis was performed using a UV spectrometer, with all samples scanned in the wavelength range of 200-700 nm.

[0119] Fluorescence spectroscopy determination Comparative Examples 1-2 and Examples 1-5 were diluted 2-fold. The fluorescence spectra of the samples were measured using a fluorescence spectrometer with a scanning range of 300-500 nm, an excitation wavelength of 280 nm, and a scan rate of 50 nm / min.

[0120] Infrared spectroscopy measurement Fourier transform infrared spectroscopy was used to measure the interactions of Comparative Examples 1-2 and Examples 1-5. 10 mg of sample was mixed with dry potassium bromide powder at a 100-fold ratio and ground to prepare thin films. Wavenumbers were measured in the range of 4000–400 cm⁻¹. -1 The scan.

[0121] X-ray diffraction measurement Comparative Examples 1-2 and Examples 1-5 lyophilized powder samples were prepared into circular sample sheets with a thickness of 1-2 mm in a tableting chamber. The diffraction angle was 10-50° at a voltage of 40 kV and a current of 40 mA. The scanning speed was set to 10° / min.

[0122] Determination of contact angle of composite systemThe freeze-dried powder samples from Comparative Examples 1-2 and Examples 1-5 were prepared into circular sample sheets with a thickness of 1-2 mm in a tableting tank. These sample sheets were then immersed in corn oil for a thorough oil bath. After this process, they were placed on a glass slide, and ultrapure water was slowly added dropwise to the oil-bathed sample using the seated drop method. The angle between the droplet and the plane was measured and quantified. The specific analytical basis is as follows: a contact angle of 90° represents the critical value for amphiphilicity. Above this critical value, the oil-water interface tends to be hydrophobic, and below it, it tends to be hydrophilic. The oil-in-water emulsion prepared at the critical value is the most stable.

[0123] Observation of the appearance and structure of the emulsion Appropriate amounts of the emulsions prepared in Examples 6-10 and the emulsions in Comparative Examples 3-4 (stored at 4°C for 1 day, 4 days, 7 days, 10 days, and 14 days, respectively) were taken and their appearance and structure were observed. The specific analysis was based on the fact that the appearance of the emulsions did not change significantly, indicating strong system stability.

[0124] Emulsion Particle Size and Potential Measurement A suitable amount of the emulsions prepared in Examples 6-10 were diluted 200 times with the emulsions in Comparative Examples 3-4 (stored at 4°C for 1 day, 4 days, 7 days, 10 days, and 14 days, respectively). The average particle size and zeta potential were then measured using a nanoparticle size analyzer. Specific analysis and theoretical basis include:

[0125] (1) The smaller the average particle size of the emulsion, the more stable the system.

[0126] (2) The larger the absolute value of the emulsion potential, the more stable the system.

[0127] Observation of oil droplet distribution in emulsion: Appropriate amounts of the emulsions prepared in Examples 6-10 and Comparative Examples 3-4 were taken (stored at 4°C for 1, 4, 7, 10, and 14 days, respectively). WKBP and PGA were labeled with fluorescein isothiocyanate as wall materials, and corn oil was labeled with Nile Red as core material. The dye and emulsion samples were mixed evenly at a ratio of 1:25. 5 μL of the stained sample was placed on a glass slide, and images of each sample were obtained using a fluorescence microscope. The specific analytical basis was that the more uniform the oil droplet distribution in the fluorescence microscope image, the more stable the system.

[0128] BITC retention rate determination Take appropriate amounts of the product emulsions prepared in Examples 6-10 and the emulsions of Comparative Examples 3-4 (stored at 4℃ for 1 day, 7 days, and 14 days), and extract BITC from the emulsions using hexane and methanol by centrifugation (5000 rpm, 15 min). Finally, analyze the BITC content using high performance liquid chromatography. Based on the positive correlation between the concentration of BITC in the solution and the peak content, calculate the retention rate of BITC in the emulsion: Retention rate (%) = Peak area of ​​BITC in the emulsion / Peak area of ​​BITC in the standard.

[0129] α-AI activity assayWeigh 11g of iodine and 22g of potassium iodide, dissolve the iodine completely in a small amount of water, and bring the volume to 500mL. Then, take 2mL of the above solution, add 20g of potassium iodide, dissolve in water, and bring the volume to 500mL to prepare a dilute iodine solution, which is then stored in a brown bottle.

[0130] Prepare a 2 mg / mL soluble starch solution (prepared fresh before use) and a 0.15 mg / mL porcine pancreatic amylase standard solution. Mix 5 mL of the porcine pancreatic amylase solution (α-amylase solution) with 5 mL of each of Examples 6-10 and Comparative Examples 3-4, and preheat in a water bath at 37 ± 0.2 °C for 8 min. Add each solution sequentially to the test tubes according to the table below. Incubate the sample tubes in a 37 °C water bath for 5 min, and terminate the reaction by adding 5 mL of 0.1 mol / L dilute hydrochloric acid. Take 1 mL of each of the above solutions, add it to 10 mL of dilute iodine solution, shake well, and measure the absorbance (A) at 660 nm. Record the data.

[0131] Table 1

[0132]

[0133]

[0134]

[0135] U – amylase inhibitor activity (U / g);

[0136] M1 – Sample amount (g) of white kidney bean extract;

[0137] B – The slope in the regression equation of the starch standard curve;

[0138] A1 – Absorbance of amylase reference standard;

[0139] A2 – Absorbance of white kidney bean extract;

[0140] 342.3 — Molar mass of maltose (g / mol);

[0141] T — Reaction time (min);

[0142] F – Conversion coefficient of the sample, 1000;

[0143] 10 6 — The unit conversion factor between moles and micromoles.

[0144] Results Analysis

[0145] This invention constructs emulsions simultaneously encapsulating BITC using WKBP and PGA as carriers and different proportions of WKBP-PGA composite carriers. The study found that:

[0146] Except for Comparative Example 2, all samples showed a peak around 260 nm. Figure 1 The WKBP, containing tryptophan and tyrosine, exhibited a significant increase in maximum absorption peak intensity without any red or blue shift. This phenomenon indicates that the complexation of WKBP with PGA leads to the transfer of amino acid residues in the WKBP molecule to a hydrophobic environment. Compared to Comparative Example 1, the addition of PGA resulted in a slight blue shift in the position of the maximum absorption peak at different PGA concentrations. Figure 2 Furthermore, the fluorescence of both WKBP and PGA showed enhanced fluorescence, exhibiting a dose-response relationship, indicating an interaction between WKBP and PGA and a change in their tertiary conformation. This may be because the formation of the WKBP-PGA complex system causes the fluorophore to shift from a polar solvent-exposed state to a nonpolar hydrophobic region, creating a more hydrophobic microenvironment within the complex system. This result corresponds to the UV results. Figure 1 ).

[0147] Figure 3 The X-ray diffraction patterns for Comparative Examples 1-2 and Examples 1-5 show no sharp peaks, indicating the presence of an amorphous structure in WKBP, PGA, and the WKBP-PGA complex. All samples elute at approximately 22°, representing the presence of a β-plate-like structure. Furthermore, the peak value significantly decreases after WKBP combines with PGA, indicating that the addition of PGA alters the tertiary conformation of WKBP, causing rearrangement of larger aggregates and forming a more stable structure. After the addition of PGA, Examples 1-5 show peaks at 1742 cm⁻¹. -1 PGA characteristic peaks appear ( Figure 4 This indicates that WKBP interacts with PGA. The WKBP-PGA complex system is located in the amide A band (3100–3500 cm⁻¹). -1 The characteristic peaks of the unsaturated carbon (CH stretching) exhibited varying degrees of vibrational shift. These changes are due to the formation of hydrogen bonds through condensation between the functional groups of WKBP and PGA. The WKBP-PGA complex system shows a significant vibrational shift in the amide I band (1600–1700 cm⁻¹). -1 ) and amide II band (1400-1500cm) -1 The characteristic peak of WKBP shifts to different degrees with increasing PGA concentration, indicating that there are electrostatic and hydrophobic interactions between WKBP and PGA.

[0148] Contact angle ( Figures 5-11The results showed that, compared with Comparative Examples 1-2, the contact angles of Examples 1-5 all increased, exhibiting a trend of first increasing and then decreasing with increasing PGA concentration. This is attributed to the exposure of hydrophobic amino acids in WKBP due to the binding of proteins with low concentrations of polysaccharides. This, in turn, increases the affinity of the particles for oil, enhancing their amphiphilicity and thus increasing the contact angle. When excessive polysaccharides bind to proteins, the hydrophobic interaction between them is enhanced, resulting in an overall hydrophobic state and a decrease in the contact angle. These results indicate that adding a specific proportion of PGA can alter the oil-water interface adsorption capacity of the system and improve the stability of the WKBP-PGA complex.

[0149] Depend on Figure 12 It can be seen that after 14 days of storage, compared with the freshly prepared compound emulsion, the average particle size of Comparative Examples 3-4 and Examples 6, 7, 9, and 10 was significantly increased (p<0.05). This indicates that with the extension of storage time, the probability of collision between droplets increases, leading to droplet aggregation and an increase in average particle size. In contrast, Comparative Example 8 showed a smaller particle size and no significant change during 14 days of storage, and the potential also showed no significant difference. Figure 13 (p<0.05) indicates that the emulsion corresponding to Example 8 is the most stable.

[0150] Figures 14-18 The image shows the appearance and structure of the WKBP-PGA composite emulsion stored at 4°C for 14 days. The results show that Comparative Example 3 exhibited significant stratification on the 4th day of storage, and Comparative Example 4 showed stratification on the 7th day of storage, indicating poor storage stability in both groups of emulsions. In stark contrast, Examples 6-10 remained stable throughout these 14 days, without stratification, flocculation, or precipitation, demonstrating excellent physical stability.

[0151] Fluorescence microscopy revealed that during storage at 4°C for 14 days ( Figures 19-23 In Comparative Examples 3-4, a large amount of oil droplet aggregation was observed. Compared to Comparative Examples 3-4, the distribution of emulsified oil droplets in Examples 7-9 was more uniform. Conversely, oil droplet aggregation occurred in Examples 6 and 10. After 14 days of storage, the microstructure size of all compound emulsions increased compared to freshly prepared emulsions. This result is consistent with the trend of emulsion particle size change. Figure 12 ).

[0152] Depend on Figure 24It can be seen that the BITC encapsulation rates of Examples 8 to 10 on the first day of storage were 89.27±1.24%, 85.16±2.35%, and 82±2.27%, respectively, which were significantly higher than those of other compound concentrations (p<0.05). After 14 days of storage, the BITC encapsulation rate in the compound emulsion with a PGA concentration of 0.6% (Example 8) was the highest (66.34±1.10%), indicating that the BITC encapsulation effect of Example 8 was the best after 14 days of storage.

[0153] Compared with Comparative Example 3, the α-AI activity of the compound systems in Examples 6-10 was enhanced. Figure 25 The reason for this is that the interaction between WKBP and PGA alters the tertiary structure of WKBP, leading to the exposure of more fragments within WKBP that inhibit α-amylase. As the concentration of PGA increases, α-AI activity gradually increases, reaching a peak at a concentration of 0.6%, and then decreasing. This may be because after PGA binds to WKBP, excess polysaccharides in the solution adhere to the protein surface or remain free in the solution, preventing the inhibitory protein fragments from fully contacting the amylase, thus reducing the inhibition rate.

[0154] The results above indicate that, under the condition of 1% WKBP and 0.6% PGA compounding, the composite emulsion has good storage stability, high protection ability of active substances (BITC) and strong α-AI activity.

[0155] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A stabilized emulsion, characterized in that, The stabilized emulsion contains 0.9–0.95 wt% white kidney bean protein, 0.1–1 wt% propylene glycol alginate, 7.5–8.5 wt% edible oil, 89–91 wt% water, and 0.04–0.06 wt% fat-soluble active substances.

2. The stabilized emulsion according to claim 1, characterized in that, The stabilized emulsion contains 0.9–0.95 wt% white kidney bean protein, 0.3–0.8 wt% propylene glycol alginate, 7.5–8.5 wt% edible oil, 89–91 wt% water, and 0.04–0.06 wt% fat-soluble active substances.

3. The stabilized emulsion according to claim 1 or 2, characterized in that, Fat-soluble active substances include one or more of benzyl isothiocyanate, proanthocyanidins, carotenoids, phytosterols, and polyunsaturated fatty acids.

4. The method for preparing the stabilized emulsion according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Preparation of protein solution: Mix white kidney bean protein with water to prepare a protein solution; S2, Aqueous phase preparation: Propylene glycol alginate is mixed with water to prepare a propylene glycol alginate solution. Then, the propylene glycol alginate solution is mixed with the protein solution prepared in step S1 to prepare an aqueous phase. S3. Oil phase preparation: The fat-soluble active substance is added to edible oil to prepare the oil phase; S4. Mixing: The aqueous phase prepared in step S2 is mixed with the oil phase prepared in step S3, dispersed at high speed, and then homogenized under high pressure to obtain a stabilized emulsion.

5. The preparation method according to claim 4, characterized in that, In step S1, the mass concentration of white kidney bean protein in the protein solution is 1% to 10%.

6. The preparation method according to claim 4, characterized in that, In S2, the mass concentration of propylene glycol alginate in the propylene glycol alginate solution is 1% to 10%, the final concentration of propylene glycol alginate in the aqueous phase is 1% to 2%, and the final concentration of white kidney bean protein in the aqueous phase is 0.2% to 1%.

7. The preparation method according to claim 4, characterized in that, In S3, the mass concentration of the fat-soluble active substance in the oil phase is 1–10 mg / mL.

8. The preparation method according to claim 4, characterized in that, In S4, the volume ratio of the oil phase to the water phase is 1:4 to 1:

15.

9. The preparation method according to claim 4, characterized in that, In S4, the parameters for high-speed dispersion are 8000–12000 r / min and the time is 2–4 min, while the parameters for high-pressure homogenization are 5000–10000 psi and the number of times is 6–8.

10. The application of the stabilized emulsion according to any one of claims 1 to 3 in the food or health product fields.

Images