Water-in-oil-in-water dual emulsion loaded with dihydroquercetin and epigallocatechin gallate and application

Abstract

The invention belongs to the technical field of food processing, particularly relates to an oil-in-water-in-oil double emulsion loaded with dihydroquercetin and epigallocatechin gallate, and further relates to application of the emulsion as a fresh-keeping material of dried products such as black-skin termitomyces albuminosus and other edible mushrooms, Chinese yam and strawberries. The preparation method of the double emulsion comprises the following steps: preparing an inner water phase W1, preparing a W1 / O primary emulsion, preparing an outer water phase W2, and preparing the double emulsion W1 / O / W2 double emulsion. According to the invention, a stable W1 / O / W2 double-emulsion system is constructed to realize loading of hydrophilic and lipophilic functional substances, so that the problem that hydrophilic EGCG and lipophilic DHQ cannot be directly combined and applied due to large solubility difference is solved; therefore, the stability and bioavailability of the dual emulsion loading the two components in different environments are effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and specifically relates to a water-in-oil-in-water double emulsion loaded with dihydroquercetin and epigallocatechin gallate, and also relates to the application of the emulsion as a fresh-keeping material for edible fungi such as black-skinned chicken mushroom and dried products such as strawberries. Background Art

[0002] Dihydroquercetin (DHQ), also known as taxifolin, is an important dihydroflavonoid compound in nature and belongs to the vitamin PP family. Epigallocatechin gallate (EGCG) is the most abundant polyphenol in green tea, accounting for 50% to 80% of the total catechins in tea. Both have a wide range of biological activities and health functions, such as antioxidant, antibacterial, anti-inflammatory, antiviral, and cancer prevention.

[0003] The inventor has found new information, including literature on the preparation of dihydroquercetin into nanoemulsions and literature on the preparation of antioxidant microemulsions with epigallocatechin gallate. Will the combination of the two exert a more efficient synergistic effect? ​​There are currently few literature reports on the combined use of them. However, a key problem currently exists is that due to the large difference in solubility between hydrophilic EGCG and lipophilic DHQ, they cannot be directly combined for application, resulting in low bioavailability of EGCG and DHQ in practical applications.

[0004] Therefore, it is necessary to use certain technical means to solve the problem that hydrophilic EGCG and lipophilic DHQ cannot be directly combined and applied due to their large solubility differences. In addition, it is also necessary to explore the specific characteristics of the emulsion loaded with these two functional substances and in what areas they can be applied. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for preparing a water-in-oil-in-water double emulsion loaded with dihydroquercetin and epigallocatechin gallate by a two-step emulsification method; also provides the application of the double emulsion in edible fungi such as black-skinned chicken mushroom and dried product preservative materials such as strawberry and yam, as well as a specific application method.

[0006] This application is built on a stable W 1 / O / W 2 The double emulsion system is used to load both hydrophilic and lipophilic functional substances, solving the problem that the hydrophilic EGCG and lipophilic DHQ cannot be directly combined due to the large difference in solubility, thereby improving the stability and bioavailability of these two functional substances in different environments.

[0007] The preparation method of the double emulsion provided in this application is as follows:

[0008] S1: internal water phase W 1 Preparation: 0.5 mg / mL epigallocatechin gallate, 1 mg / mL sodium chloride, 0.1 mg / mL ascorbic acid, and 0.5 mg / mL gelatin were dissolved in deionized water, and stirred at 500 rpm and 60°C for 20 min in a magnetic stirrer until fully dissolved, and this was used as the internal aqueous phase W. 1 ;

[0009] S2: Colostrum W 1 Preparation of W / O: a certain amount of dihydroquercetin was dissolved in olive oil to make it saturated, and then 1-5% of polyglycerol ricinoleate was placed in olive oil based on the volume of the oil phase, and magnetically stirred for 30 minutes at room temperature until it was fully dissolved, and this was used as the oil phase O. The inner water phase was added to the oil phase at a water / oil volume ratio of 10-50%, and high-speed dispersion was performed for 3 minutes to prepare W. 1 / Ocolostrum;

[0010] S3: External water phase W 2 Preparation: weigh sodium hyaluronate, add deionized water to dissolve to prepare a 1 mg / mL sodium hyaluronate aqueous solution; weigh whey protein, add deionized water to dissolve to prepare a 3 mg / mL whey protein aqueous solution;

[0011] Sodium hyaluronate aqueous solution and whey protein aqueous solution were respectively prepared into a polysaccharide / protein complex with a mass ratio of 1:1 to 9:1 and a pH of 4 as the external aqueous phase W. 2 ;

[0012] S4: Preparation of W 1 / O / W 2 Double breasts: Add the W prepared in S2 1 / O Colostrum is slowly added to the external water phase W in S3 at a volume ratio of 10-90% at a speed of 4000-20000 r / min within 1 minute. 2 Then continue to disperse at the same speed for 2 minutes to obtain W 1 / O / W 2 Breasts.

[0013] The present application also intends to protect the use of the water-in-oil-in-water double emulsion loaded with dihydroquercetin and epigallocatechin gallate as a fresh-keeping material, such as in the preservation of fruits or vegetables or edible fungi such as black-skinned chicken fir, strawberry, and yam. The above are only examples, not limited to the above categories, and can also be other categories of fruits or vegetables or edible fungi.

[0014] The application method of the double emulsion is to dilute the double emulsion by 5-30 times with water or ultrapure water, and then soak the fruits, vegetables or edible fungi to be preserved in the diluted emulsion for 20-40 seconds, take them out, drain and dry them.

[0015] The beneficial effects of the present invention are:

[0016] (1) This application uses a two-step emulsification method to construct a W that is synergistically stabilized by a polysaccharide-protein complex. 1 / O / W 2 The double emulsion system reduces the amount of traditional emulsifiers by using a polysaccharide-protein complex, and achieves the loading of two functional substances, hydrophilic and lipophilic, solving the problem that the hydrophilic EGCG and lipophilic DHQ cannot be directly combined due to the large difference in solubility, and effectively improves the stability and bioavailability of the double emulsion loaded with these two active ingredients in different environments;

[0017] (2) The present application evaluated the structural characteristics, antioxidant properties, and stability of the double emulsion. Based on the above characteristics, it was applied to the preservation of black-skinned chicken fir, strawberry, and yam. The results showed that the prepared emulsion had W 1 / O / W 2 Double emulsion has a typical two-layer three-phase structure and has good antioxidant and stability properties. It has a significant color protection effect on black-skinned chicken mushroom, strawberry, yam, etc., and effectively realizes the efficient and stable utilization of two functional substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 W prepared at different PGPR concentrations 1 / O Colostrum appearance (left 0 day, right Fig.10 Days); from left to right: 1%, 2%, 3%, 4%, 5% ( Figure 2-3 same);

[0019] Figure 2 The left picture shows W prepared at different PGPR concentrations. 1 / OComparison of colostrum before and after centrifugation. The left picture is before centrifugation, and the right picture is after centrifugation.

[0020] Figure 3 W prepared at different PGPR concentrations 1 / OPhoto of colostrum under a microscope;

[0021] Figure 4 W prepared under different water-oil ratios 1 / O colostrum emulsion appearance diagram; the water-oil volume ratio from left to right is: 10%, 20%, 30%, 40%, 50% ( Figure 5-6 same);

[0022] Figure 5 W prepared under different water-oil ratios 1 / OComparison of colostrum before and after centrifugation;

[0023] Figure 6 W prepared under different water-oil ratios 1 / OPhoto of colostrum under a microscope;

[0024] Figure 7 W prepared at different mass ratios of sodium hyaluronate and whey protein 1 / O / W 2 Bi-breast appearance, particle size and potential map;

[0025] Figure 8 W prepared with different volume ratios of sodium hyaluronate and whey protein 1 / O / W 2 Bi-breast appearance, particle size and potential map;

[0026] Fig. 9 W is prepared by sodium hyaluronate and whey protein at different dispersion speeds 1 / O Colostrum appearance, particle size and potential diagram;

[0027] Fig.10 W is prepared by sodium hyaluronate and whey protein at different dispersion times 1 / O Colostrum appearance, particle size and potential diagram;

[0028] Fig.11 Prepare W for the single factor test results 1 / O Colostrum particle size, potential diagram;

[0029] Fig.12 For the different numbers of W in Table 7 1 / O / W 2 Bilateral breast appearance map;

[0030] Fig.13 The particle size, potential-3D effect surface diagram, contour map and W prepared under the optimal process in Example 2 are shown in FIG. 1 / O / W 2 Bilateral breast appearance map;

[0031] Fig.14 is the W obtained in Example 1 1 / O Colostrum appearance and CLSM images;

[0032] Fig.15 is W obtained in Example 2 1 / O / W 2 Breast appearance and ultra-depth microscopy

[0033] image;

[0034] Fig.16 is W obtained in Example 2 1 / O / W 2 CLSM images of both breasts;

[0035] Fig.17 CLSM images of sodium hyaluronate and whey protein;

[0036] Fig.18 is W under the optimal formula 1 / O / W 2 Fourier transform infrared spectra of both breasts;

[0037] Fig.19 W 1 / O / W 2 Three-phase contact angle diagram of double breasts;

[0038] Fig. 20 The middle left picture is W 1 / O / W 2 The DPPH free radical scavenging rate graph of both breasts; the right graph is the ABTS free radical scavenging rate graph;

[0039] Fig.21 W at different pH 1 / O / W 2 Investigation of breast stability;

[0040] Fig. 22 W under different salt ion strengths 1 / O / W 2 Investigation of breast stability;

[0041] Fig.23 W at different temperatures 1 / O / W 2 Investigation of breast stability;

[0042] Fig.24 W 1 / O / W 2 Investigation of thermal stability of breasts;

[0043] Fig.25 This is the appearance and color diagram of strawberry under different treatments;

[0044] Fig.26 This is the appearance and color diagram of yam under different treatments;

[0045] Fig. 27 This is the appearance and color diagram of the black-skinned Alpinia fasciata under different treatments;

[0046] Fig.28 The scanning electron microscope images of the black-skinned Alpinia fasciata stems under different treatments;

[0047] Fig.29 The scanning electron microscope images of the cap of Alpinia fasciata under different treatments;

[0048] Fig.30 This is the rehydration rate diagram of black-skinned fir under different treatments; DETAILED DESCRIPTION

[0049] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0050] Example 1

[0051] 1.1 Preparation of W 1 and W 1 / Ocolostrum

[0052] (1)W 1 Preparation: EGCG (0.5 mg / mL), sodium chloride (1 mg / mL), ascorbic acid (0.1 mg / mL), and gelatin (0.5 mg / mL) were dissolved in deionized water and stirred at 60°C with a magnetic stirrer at 500 rpm for 20 min until fully dissolved. This was used as the internal aqueous phase (W 1 ).

[0053] (2)Colostrum W 1 Preparation of water / oil: a certain amount of dihydroquercetin was dissolved in olive oil to make it saturated, and then 1%, 2%, 3%, 4% and 5% of polyglycerol ricinoleate were placed in olive oil, and magnetically stirred at room temperature for 30 minutes until fully dissolved, which was used as the oil phase (O). The internal water phase (W) was prepared according to different water / oil volume ratios (10%, 20%, 30%, 40%, 50%). 1 ) was added to the oil phase (O) and dispersed at high speed for 3 minutes to prepare W 1 / Ocolostrum.

[0054] ① Regarding the optimal PGPR concentration, the appearance and microstructure of colostrum were investigated by storage and centrifugation. Figure 1-2 As shown, when the water-oil volume ratio is fixed at 20%, when the PGPR concentration is 1%-3%, W 1 / O colostrum showed obvious phase separation after storage for 10 days and centrifugation, which indicates that this concentration range is not enough to stabilize the oil-water interface for a long time. When the PGPR concentration is ≥4%, the emulsion remains a homogeneous system after storage for 10 days and centrifugation without stratification. In addition, the microscopic images (attached) Figure 3 The comparison between the average particle size of the emulsion (Table 1) and the average particle size of the emulsion (Table 1) can also reflect the W prepared at different PGPR concentrations. 1 Differences in W / O emulsions. 1 / O emulsion droplet average particle size varies greatly, ranging from 3.21-2.87μm, and decreases with the increase of PGPR concentration. Among them, the particle size of emulsion droplets with 1%-3% PGPR concentration is significantly larger than that of emulsion droplets with 4%-5% concentration. When the PGPR concentration is increased to more than 4%, the droplet particle size decreases less. Considering the safety and cost of the emulsifier addition, 4% PGPR concentration is selected as the subsequent experimental condition.

[0055] ② Regarding the optimal water / oil volume ratio, the appearance and microstructure of colostrum were also investigated by storage and centrifugation. Figure 4-5 As shown in Figure 2, when the PGPR concentration is fixed at 4% (v / v) of the oil phase volume, the emulsion with a water-to-oil ratio of 10% shows stratification after centrifugation and storage at room temperature for 10 days, and the emulsion with a water-to-oil ratio of 50% shows demulsification after centrifugation, while the emulsions with other water-to-oil ratios remain a homogeneous system. 1 / O emulsion has poor stability, which is because the Brownian motion of water droplets in the emulsion will cause the droplets to flocculate in a short time. Figure 6 ) and the average particle size of the emulsion (Table 1) show that the W prepared at a water-oil ratio of 10%-50% 1 / O emulsion droplet size is between 2.96-3.13nm, which is generally uniform. The emulsion particle size is smallest when the water-oil ratio is 1:9. As the water-oil ratio increases, the droplet size gradually increases. Since the emulsion with a water-oil ratio of 1:9 shows stratification, 20% (v / v) is selected as the optimal water-oil ratio.

[0056] Based on the above results, W 1 / O colostrum was formed by mixing 20% ​​(v / v) internal water phase and 80% (v / v) oil phase (containing 4% PGPR, v / v) at 8000 rpm for 3 min. This ratio and conditions were used in the subsequent preparation of double emulsions.

[0057] Table 1 Preparation of W at different PGPR concentrations and different water-oil volume ratios 1 / OThe average particle size of colostrum

[0058]

[0059] In Table 1, (A) W prepared with different PGPR concentrations (1%-5%, v / v) when the water-oil volume ratio was 20% 1 / O colostrum average particle size; (B) W prepared with different water-oil ratios (10%-50%, v / v) when PGPR concentration was 4% (v / v) 1 / O Average colostrum particle size.

[0060] 1.2W 1 / O / W 2 Preparation of breasts

[0061] Preparation of W 1 / O / W 2 Double breast, single factor experimental design

[0062] A certain amount of sodium hyaluronate and whey protein were weighed, respectively, and dissolved in deionized water to prepare 1 mg / mL sodium hyaluronate aqueous solution and 3 mg / mL whey protein aqueous solution, and polysaccharide / protein complexes with different mass ratios (1:1, 3:1, 5:1, 7:1, 9:1, m / m) were prepared, and the pH was adjusted to 4, which was used as the external aqueous phase (W 2 ). The prepared W 1 Colostrum was slowly added to the external aqueous phase (W / O) at different volume ratios (10%, 30%, 50%, 70%, 90%, v / v) at a dispersion speed of 12000 r / min within 1 min. 2 ) and then continued to disperse at the same speed for 2 minutes to obtain W 1 / O / W 2 Breasts.

[0063] Prepare W according to the above method 1 / O / W 2 For double emulsion, the particle size and Zeta potential of the emulsion were used as evaluation indicators, and the mass ratio of SH to WP was set to 1:1, 3:1, 5:1, 7:1, and 9:1 (m / m), the colostrum volume ratio was 70%, the high-speed dispersion was 12000rpm, and the dispersion time was 3min; the colostrum volume ratio was set to 10%, 30%, 50%, 70%, and 90% (v / v), the mass ratio of SH to WP was 5:1, the high-speed dispersion was 12000rpm, and the dispersion time was 3min; the high-speed dispersion speed was set to 4000rpm, 8000rpm, 12000rpm, 16000rpm, and 20000rpm, the mass ratio of SH to WP was 5:1, the colostrum volume ratio was 70%, and the dispersion time was 3min; the dispersion time was set to 1min, 2min, 3min, 4min, and 5min, the mass ratio of SH to WP was 5:1, the colostrum volume ratio was 70%, and the high-speed dispersion was 12000rpm. Single factor process investigations were conducted on the mass ratio of SH / WP, colostrum volume ratio, dispersion speed and dispersion time.

[0064] (1) The relationship between the different mass ratios of SH / WP and the particle size and potential of double emulsions was investigated. The results are shown in the attached figure. Figure 7As shown in the figure, with the increase of SH / WP mass ratio, the particle size of the emulsion shows a trend of first decreasing and then increasing, and the emulsion potential first increasing and then decreasing. When the mass ratio of SH / WP complex is relatively low, the complex is not enough to completely wrap the oil phase, making the oil phase easy to aggregate, resulting in a larger oil droplet volume and a higher particle size; when the mass ratio of SH / WP complex is too high, the stable system of the emulsion will be destroyed, and the particle size will increase and the potential will decrease. When the mass ratio of SH / WP complex is 5, the particle size value of the emulsion is the smallest, the potential value is the highest, and the emulsion system is uniform and stable.

[0065] (2) The relationship between the volume ratio of sodium hyaluronate to whey protein aqueous solution and the particle size and potential of double emulsion was investigated. The results are shown in the attached figure. Figure 8 As shown in the figure, with the increase of colostrum volume, the particle size of the emulsion shows a trend of gradually decreasing and the potential gradually increasing. When the proportion of colostrum is too low, the external water phase is not enough to wrap all the colostrum, and the unadsorbed oil droplets gather together, accelerating the Ostwald ripening of the emulsion, which makes it easy for the oil droplets to aggregate, the particle size becomes larger, and the potential decreases. When the colostrum volume ratio is 70%, the inspection results are optimal.

[0066] (3) The relationship between particle size and potential of double emulsions prepared by sodium hyaluronate and whey protein aqueous solution at different rotation speeds was investigated. The results are shown in the attached figure. Fig. 9 As shown. The particle size of the emulsion after high-speed dispersion is significantly reduced. This is mainly because high-pressure dispersion can fully break up the emulsion into small droplets, thereby reducing its particle size. When the speed is 4000rpm, the prepared emulsion shows obvious stratification. When the speed reaches 20000rpm, the excessively high speed will damage the internal structure of the emulsion, thereby increasing the particle size and decreasing the potential. Considering the energy consumption of the homogenizing equipment and the damage to the emulsion, 12000rpm is the best speed.

[0067] (4) The relationship between particle size and potential of double emulsions prepared by sodium hyaluronate and whey protein aqueous solution at different reaction times was investigated. The results are shown in the attached figure. Fig.10 As shown in the figure, with the increase of homogenization time, the particle size of the emulsion first decreases and then increases, and the potential first increases and then decreases. When the averaging time is 3 minutes, the droplet size is the smallest and the potential value is the best.

[0068] Combined with Fig.11 The results of the single factor experiment showed that the SH / WP mass ratio was 5, the colostrum volume ratio was 70%, the high-speed dispersion was 12000rpm, and the dispersion time was 3min.

[0069] 1.3 Response surface experimental design

[0070] The results of single factor were adopted, SH / WP mass ratio was 5, colostrum volume ratio was 70%, high speed dispersion was 12000rpm, and dispersion time was 3min. With SH / WP mass ratio (X1) and colostrum volume ratio (X2) as independent variables, and emulsion particle size (Y1) and potential (Y2) as dependent variables, a two-factor five-level star point design was conducted to predict the optimal process, and the results were verified. The levels of each factor are shown in Table 2. The results of 13 measurements conducted by the star point design test are shown in Table 3. The double emulsion products numbered 1-13 in Table 3 are shown in the attached Fig.12 .

[0071] Table 2 Star point design factor level table

[0072]

[0073] Table 3 Results of central composite design-response surface methodology experiment

[0074]

[0075]

[0076] The centroid design method (CCD) in Design-Expert 11 software was used to fit the experimental data in Table 3, and the model was fitted with a quadratic polynomial. In the average particle size model, the linear term B and the quadratic term B 2 The influence on the average particle size reached an extremely significant level, and the first-order term A had a significant effect (see Table 4); in the potential model, the first-order term B and the second-order term B 2 The influence on Zeta potential is very significant, the first-order term A and the second-order term A 2 The influence is significant (see Table 5). After regression fitting, the quadratic polynomial is as follows:

[0077] Y1=43-0.0538A+0.1418B-0.0275AB+0.0393A 2 +0.1268B 2 , R 2 =0.9150,

[0078] P<0.01

[0079] Y2=43.55-0.3478A-0.4873B-0.1400AB-0.3394A 2 -0.8319B 2 , R 2 =0.9338,

[0080] P<0.01

[0081] W 1 / O / W 2The differences in average particle size and Zeta potential between the two models were extremely significant (P < 0.01), and the differences in lack of fit terms were not significant (P > 0.05). The goodness of fit of the two models was R 2 are 0.9150 and 0.9338 respectively, and the model fit is high, so this model can be used to analyze W 1 / O / W 2 Prediction and analysis of the preparation process of double breasts.

[0082] Table 4 ANOVA results of regression model based on particle size

[0083]

[0084]

[0085] Note: * indicates significant difference, P<0.05; ** indicates extremely significant difference, P<0.01; the same below.

[0086] Table 5 ANOVA results of the potential-based regression model

[0087]

[0088]

[0089] Use software to optimize the response surface and draw the response surface curve of each factor to the response value, see Fig.13 According to the above prediction results, the minimum value of Y1 is the best, and the optimal W is determined 1 / O / W 2 The process was SH / WP compound mass ratio of 3, colostrum volume ratio of 63.32%, homogenization pressure of 12000rpm, homogenization time of 3min; 2 The maximum value is the best, determine the optimal W 1 / O / W 2 The process was SH / WP compound mass ratio of 6.9, colostrum volume ratio of 66.24%, homogenization pressure of 12000 rpm, and homogenization time of 3 min.

[0090] Table 6 Confirmation experiment of the best formula

[0091]

[0092] According to the optimal process modified by Y1 and Y2, three groups of parallel W 1 / O / W 2 Double emulsion, particle size prediction value is 43.04 (μm), Zeta prediction value is 43.03, deviation = (predicted value - measured value) / predicted value, the results are shown in Table 6, the deviations of both are <5.0%, indicating that the established model has good predictive power and reliable values. Fig.13 ), due to the flocculation phenomenon of double milk in the Y2 process, and the deviation value was higher than that of Y1, therefore, the Y1 process with a SH / WP composite mass ratio of 3, a colostrum volume ratio of 63.32%, a homogenization pressure of 12000rpm, and a homogenization time of 3min was used as the preparation process of W 1 / O / W 2 Double lotion is the best choice.

[0093] In order to verify the formation of the water-in-oil-in-water double emulsion structure, the microscopic morphology of the emulsion was observed using an ultra-depth-of-field three-dimensional microscope and a confocal laser scanning microscope.

[0094] For the W of 1.1 in Example 1 1 / O Colostrum appearance and microscopic images are analyzed. Fig.14 As shown, W 1 / O Colostrum appearance, CLSM image (using Nile red to stain the oil phase), the internal water phase W 1 It was successfully embedded in the oil phase and the prepared colostrum had good homogeneity.

[0095] For W in Example 1.3 1 / O / W 2 Analyze the appearance and microscopic images of both breasts, as shown in the attached Fig.15 , 16 , 17: W 1 / O / W 2 The surface system of the double emulsion is uniform and stable, milky white, and there is no aggregation or stratification. The CLSM image shows a typical two-layer three-phase structure, that is, the oil phase is stained with Nile red, the internal water phase (W 1 ) is wrapped by the oil phase (O), while the oil phase is also wrapped by the external water phase (W 2 ) are tightly packed, confirming that the emulsion is a water-in-oil-in-water double emulsion.

[0096] In the following examples, unless otherwise specified, all experiments are conducted using double emulsions obtained under the conditions of the best ratio.

[0097] Example 2

[0098] 2.1 Characterization of sodium hyaluronate / whey protein mixed particles

[0099] 2.1.1 Fourier transform infrared spectroscopy (FTIR) analysis (Appendix Fig.18 )

[0100] The structures of SH, WP, and SH / WP complexes were analyzed by FTIR. The freeze-dried samples and dried KBr were mixed in a ratio of 1:100 and pressed into sample-KBr sheets. The samples were scanned in transmission mode by attenuated total reflection method in the wave number range of 4000–400 cm -1 , the interval is 0.5cm -1 . .

[0101] When proteins and sugar molecules are covalently bonded, the typical feature on the infrared spectrum is between 3700 and 3200 cm -1 A broad peak appears in the range of 1260-1000cm -1 There are also absorptions in the range of 2 and 4, which are caused by the stretching vibrations of the hydroxyl group and the carbon-oxygen bond. Fig.18 It can be seen that the whey protein and sodium hyaluronate complex is at 3700-3200 cm -1 The peak width in the range of 1260-1000 cm -1 The absorption intensity in the range is also higher than that of the original protein. The main peaks of WP are as follows: 3275.42 cm -1 (Amide A band, representing NH stretching and hydrogen bonding), 1628.56 cm -1 (amide I band, representing CO stretching / hydrogen bonding and COO-) and 1531.45 cm -1 (amide II band, representing CN stretching and NH bending modes), 1394.8 cm -1 (amide III band, representing CN bending mode) and 1240.28 cm -1 (Amide III band, representing NH stretching), 2925.46 cm -1 The band at is due to CH 2 The WP is at 1628.56cm -1 、1531.45cm -1 When SH was added, a red shift occurred, and WP+SH was at 1240.28cm -1 The NH deformation (amide III) disappears at 1149.22 cm -1 The generation of new peaks indicated that the addition of SH could change the structure of WP and there existed a certain degree of hydrogen bonding and electrostatic adsorption between WP and SH.

[0102] 2.1.2 Three-phase contact angle analysis

[0103] The contact angles of sodium hyaluronate, whey protein and mixed particles were measured using a JY-82C video contact angle meter. SH, WP and SH / WP complex were freeze-dried into powders, pressed into cylindrical tablets with a diameter of 10 mm and a thickness of about 1 mm using a mold, and immersed in a quartz dish filled with olive oil. 3 μL of distilled water was squeezed out from the tip of the syringe and dropped on the surface of the circular sheet to form a droplet. After standing for 30 seconds, the change in the droplet shape was recorded using a camera equipped with the contact angle meter.

[0104] The three-phase contact angle (θ) is a common parameter for evaluating the wettability of solid particles. Particles with moderate wettability (close to 90°) can stabilize both the O / W and W / O interfaces, promote the effective adsorption and accumulation of solid particles at the two-phase interface, and then form an effective spatial physical barrier on the droplet surface to resist coalescence. Fig.19 In the figure, the three-phase contact angles of WP and SH alone are 114.243° and 69.33°, respectively, which indicates that the surface wettability of the two particles is poor, either too hydrophobic or too hydrophilic. In contrast, the composite aggregates formed by electrostatic adsorption between the positively charged WP and the negatively charged SH significantly improved the surface wettability of the individual WP and SH to 99.375°, which is closest to 90°. The lipophilic and hydrophilic balance is achieved, thus effectively stabilizing the W 1 / O / W 2 Double lotion.

[0105] 2.2SH / WP synergistically stabilizes W 1 / O / W 2 Double emulsion characterization

[0106] 2.2.1 Ultra-depth microscopy and confocal laser scanning microscopy (CLSM)

[0107] Microscope and confocal laser scanning microscopy (CLSM) observations confirmed that the milk was a typical W 1 / O / W 2 Double emulsion structure, rather than being carried into the oil phase to form particle aggregates. Figure 14-17 As shown, Fig.14 W 1 / O Colostrum appearance and CLSM images; Fig.15 The middle left picture shows the appearance and microstructure of the double emulsion under the optimal ratio, and the right picture is a partial enlarged picture of the left picture; Fig.16 , 17 W 1 / O / W 2 CLSM images of SH / WP synergistic compound synergistically stabilized double emulsion. The oil phase was stained with Nile red (red), and the inner aqueous phase W 1 (non-fluorescent and black) is wrapped by the oil phase O and appears black-red, indicating that W 1 / O structure formation; external water phase W 2 (No fluorescence, black) Successfully matched W 1 / O colostrum is wrapped and the outer water phase does not penetrate into the inner water phase, showing a typical W 1 / O / W 2 Double emulsion two-layer three-phase structure. Fig.17 Figure 2 is the CLSM image of the SH / WP composite. Sodium hyaluronate (blue) and whey protein (red) were stained with fluorescent white and Nile blue, respectively. The overlap of blue fluorescence and red fluorescence is clearly visible. This phenomenon shows that the interface adsorption layer formed by SH / WP with opposite charges is firmly adsorbed on the oil-water interface and effectively inhibits the aggregation between the inner and outer droplets, making the prepared W 1 / O / W 2 Has good stability.

[0108] 2.2.2 Antioxidant activity studies

[0109] The antioxidant activity of the emulsions was evaluated by scavenging DPPH and ABTS free radicals.

[0110] 2.2.2.1 DPPH free radical scavenging rate

[0111] DPPH is a free radical that is more stable than hydroxyl and superoxide radicals and is widely used in vitro to evaluate the scavenging activity of antioxidant components (Zhou K, Yu L. Antioxidant Properties of Bran Extracts from TregoWheat Grown at Different Locations [J]. Journal of Agricultural and Food Chemistry, 2004, 52: 1112-1117.). The scavenging activity of samples on DPPH free radicals was determined by referring to the method described by Woranuch et al. (Woranuch S, Yoksan R. Preparation, characterization and antioxidant property of water-soluble ferulic acid grafted chitosan [J]. Carbohydrate Polymers, 2013, 96: 495-502.). Specifically, W 1 / Ocolostrum, W 1 / O / W 2The double emulsion was dispersed in deionized water to form a series of sample solutions with a concentration gradient of 1.25 mg / mL, 2.5 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL and 30 mg / mL. Then, 1 mL of the sample solution was mixed with 3 mL of DPPH-ethanol solution (0.2 mmol / L), and after standing in the dark at room temperature for 30 min, the absorbance of the mixture at 517 nm was measured, and anhydrous ethanol was used as a blank to adjust the zero. The calculation formula for the scavenging rate of DPPH free radicals is as follows:

[0112]

[0113] In the formula, A 1 , A 2 and A 0 Represent the absorbance of the sample, sample control (ethanol instead of DPPH solution) and blank control (ethanol instead of sample), respectively.

[0114] The DPPH removal rate of double emulsion is shown in the attached figure. Fig. 20 The specific results of the test are shown in Table 7 below. 1 / Ocolostrum, W 1 / O / W 2 Both milks have good DPPH free radical scavenging ability. 1 The clearance capacity of colostrum was 10.84% ​​to 87.09%, W 1 / O / W 2 The scavenging ability of W was 7.48%86.49, slightly higher than that of double milk, but with the increase of concentration, 1 / O / W 2 The free radical scavenging ability of W 1 / O colostrum, this is because the polysaccharide has a certain antioxidant activity, and the prepared SH / WP complex effectively enhances the scavenging activity of DPPH free radicals.

[0115] 2.2.2.2 ABTS free radical scavenging rate

[0116] W was determined according to the method reported by Xie et al. (Xie MH, Hu B, Wang Y, Zeng XX. Grafting of gallic acid ontochitosan enhances antioxidant activities and alters rheological properties of the copolymer [J]. Journal of Agricultural and Food Chemistry, 2014, 62: 9128-9136.) 1 / Ocolostrum, W 1 / O / W 2 The scavenging rate of ABTS free radicals by both breasts. + Solution and 2.45mmol / LK 2 S 2 O 8 The solutions were mixed and kept at room temperature and away from light for 12 hours to obtain ABTS. + The mother solution was diluted with 0.2 mol / L phosphate buffer solution (pH 7.4) to make its absorbance at 734 nm 0.7, thus obtaining ABTS. + Then, 0.5 mL of sample solution with different concentrations (1.25-30 mg / mL) was mixed with 5 mL of ABTS + The working solution was mixed and incubated at room temperature in the dark for 6 min, and then its absorbance at 734 nm was measured. + The free radical scavenging rate was calculated as follows:

[0117]

[0118] In the formula, A 1 , A 2 and A 0 Represent the sample and sample control (phosphate buffer instead of ABTS + The absorbance of the blank control (deionized water instead of sample solution) was measured.

[0119] The results are attached Fig. 20 As shown in the right figure. As can be seen from the figure, W 1 / Ocolostrum, W 1 / O / W 2Both emulsions have good scavenging ability for ABTS free radicals, and their free radical scavenging rate shows the same trend as the above-mentioned DPPH free radical, and is positively correlated with the concentration of the emulsion. When the concentration is greater than 5 mg / mL, the free radical scavenging ability of both emulsions is significantly improved, and is higher than the DPPH free radical scavenging ability at this concentration, which may be due to the different affinities of the antioxidant active substances in the emulsion with different free radicals. When the concentration is greater than 10 mg / mL, W 1 / O / W 2 Both breasts showed better clearance.

[0120] Table 7 Antioxidant test data W of double emulsion 1 / O Antioxidant activity of colostrum

[0121] Mass concentration (mg / mL) 1.25 2.5 5 10 15 20 25 30 DPPH clearance rate (%) 10.84 17.17 32.30 57.70 78.08 87.09 94.49 99.50 ABTS clearance rate (%) 32.50 44.79 65.21 102.56 110.99 114.22 118.67 124.74

[0122] W 1 / O / W 2 Antioxidant activity of double milk

[0123] Mass concentration (mg / mL) 1.25 2.5 5 10 15 20 25 30 DPPH clearance rate (%) 7.48 15.45 29.79 54.32 69.51 86.49 101.35 114.90 ABTS clearance rate (%) 11.22 29.44 55.16 113.06 119.26 126.41 127.20 131.46

[0124] It can be seen from the above table and the attached figure that both W1 / O colostrum and W1 / O / W2 double emulsion have good antioxidant activity. In comparison, as the emulsion concentration increases, the antioxidant effect of the double emulsion is more excellent.

[0125] 2.2.3 Stability study

[0126] 2.2.3.1 pH stability study: The stability of the double emulsion at the following pH values ​​was analyzed by using 0.1 mM hydrochloric acid or sodium hydroxide to 2 ) was adjusted to the following pH values: 3, 4, 5, 6, 7.5, 9, and double emulsions were prepared according to the above optimal ratio to observe the appearance, particle size potential and microstructure of the emulsions. Fig.21 As shown. When pH ≤ 5, the prepared emulsion is white in appearance, the system is uniform and there is no stratification phenomenon; the microstructure of the emulsion is basically unchanged, its particle size is small, the Zeta potential value is high, and the droplets remain evenly dispersed. As the pH increases, the emulsion begins to stratify. When the pH value increases to above 7.5, its microstructure is severely damaged, the droplet size increases rapidly, and a large cluster of floccules appears inside. This is mainly due to the reduction of the net charge of the complex at high pH values, the weakening of the complexation, and the limited aggregation of the droplets, which reduces the electrostatic repulsion between the droplets and weakens the interface stability. In summary, the SH / WP complex can better enhance the stability of the emulsion under acidic conditions.

[0127] 2.2.3.2 Salt ion strength stability study

[0128] Take the double emulsion with the best ratio and add different concentrations of NaCl, namely: 0mM, 50mM, 100mM, 150mM, 200mM, 250mM, and observe its appearance, particle size potential and microstructure. Fig. 22 Except for the emulsion without salt ions, all emulsions showed obvious stratification; the particle size of the emulsions was relatively uniform, and the microstructure did not change significantly, which may be due to the ionized Cl - The electrostatic repulsion between droplets is strengthened, which helps to inhibit the aggregation of droplets and form an interfacial layer that is not easily deformed. The NaCl concentration is negatively correlated with the emulsion potential. This may be because as the NaCl concentration increases, the electrostatic repulsion between droplets weakens and aggregation occurs. At this time, the surface charge of the droplets is relatively sufficient, and the electrostatic repulsion can effectively hinder the partial aggregation of droplets.

[0129] 2.2.3.3 Temperature stability study

[0130] The double emulsion with the best ratio was treated at different temperatures. The results were: kept at 4°C for 30 minutes, and kept in a constant temperature water bath at 25°C, 37°C, 50°C, 65°C, and 80°C for 30 minutes respectively. The appearance, particle size potential, and microstructure were observed. Fig.23 As shown. The emulsions treated at different temperatures all maintained a uniform and stable state, without stratification and no obvious changes in the microstructure. When the ambient temperature increased from 4°C to 50°C, the droplet size increased slightly, and the increasing trend became obvious when the temperature was further increased. This may be because the increase in temperature increased the Brownian motion of the complex and the oil droplets; the potential value of the emulsion increased with the increase in temperature, and when the temperature was greater than 50°C, the potential began to decrease. This may be because the excessively high temperature caused the droplets to undergo violent thermal motion, and the strength of the secondary bonds between molecules was less than the electrostatic attraction between particles, which caused the potential to decrease and the stability of the emulsion to decrease.

[0131] 2.2.3.4 Thermal stability analysis

[0132] By DSC detection, Fig.24 As shown, the DSC test obtained W 1 / O / W 2 Phase transition starting temperature, phase transition peak temperature and phase transition enthalpy of double emulsion. During the cooling process, only one endothermic peak was observed, and there was no extra phase transition signal, which indicates that the double emulsion only experienced one phase transition process during the cooling process, indicating that the internal oil phase did not leak and the emulsion structure was intact. During the heating process, the emulsion did not break or leak the internal oil phase, indicating that the prepared W 1 / O / W 2 Double breasts are suitable for higher temperature environments and can be used in daily logistics processes such as food, medicine and cosmetics.

[0133] Example 3

[0134] The double emulsion (optimal ratio) in Example 1 was applied to the preservation of strawberries, yam slices, and black-skinned Alpinia fasciata. The specific effects are shown in the analysis of 3.1-3.3. Among them, 5X represents a treatment group diluted 5 times, and 30X represents a treatment group diluted 30 times.

[0135] The application method is to dilute the double emulsion, soak strawberries, Chinese yam and black-skinned Alpinia fasciata for about 30 seconds, take them out and drain them. The black-skinned Alpinia fasciata is dried at 60℃ for 12 hours in a heat pump; the strawberries are freeze-dried from -30℃ to 25℃ in a freeze dryer for 48 hours; and the Chinese yam slices are frozen in a -40℃ refrigerator for 30 days. Observe and take pictures.

[0136] 3.1(1) Investigation of the appearance and color of strawberries

[0137] As attached Fig.25 As shown in the figure, from top to bottom are: no treatment (two strawberries per group), double emulsion diluted 30 times treatment group, double emulsion diluted 5 times treatment group. Fig.25 It can be seen that the strawberry fruits in the untreated group faded severely after freeze-drying, the fruit color was whitish and the glossiness was low. The strawberries treated with double emulsion still maintained a bright red color, showing a good color protection effect.

[0138] right Fig.25 The chromaticity value in the middle right should be explained. As shown in the figure, the smaller the △E is, the smaller the color difference is, and the closer it is to the chromaticity of the freshest strawberry. The following is the same for Chinese yam and black-skinned Alpinia fasciata.

[0139] 3.2(1) Investigation of the appearance and color of yam slices

[0140] The yam slices treated with double emulsion and the untreated yam slices were directly frozen in a -40℃ refrigerator for 30 days, and then taken out and photographed. Fig.26 As shown in the figure, from top to bottom are the results of the untreated group, the double emulsion diluted 30 times, and the yam slices diluted 5 times. The right figure shows the effect of different treatment groups on the color of the yam slices. The yam slices in the untreated group will oxidize and turn yellow after long-term storage, while the yam slices treated with the emulsion still maintain a white color and the yellowing phenomenon is not significant.

[0141] 3.3(1) Investigation on the appearance and color of the black-skinned Alpinia fasciata

[0142] Attached Fig. 27From top to bottom, the results of black-skinned Alpinia chorii without treatment, double emulsion diluted 30 times, and 5 times dilution are shown. The right picture shows the effect of different treatment groups on the color of black-skinned Alpinia chorii dried products. The black-skinned Alpinia chorii in the untreated group showed obvious fading after drying, and the color of the fungus turned white. After drying, the black-skinned Alpinia chorii treated with emulsion was close to its original color (in the fresh state), and the black-skinned Alpinia chorii treated with high concentration (5X) had a tendency to darken in color, showing an excellent color protection effect.

[0143] (2) Investigation of the microstructure of the black-skinned Gallus galliformis

[0144] Attached Fig.28 , 29 These are the pictures of the stem and cap of the black-skinned Alpinia solani under different treatments.

[0145] The microstructure of the black-skinned chicken mushroom in different treatment groups was investigated by scanning electron microscopy. The black-skinned chicken mushroom in the untreated group had small pores, dense tissue, poor three-dimensionality, and collapse, which may be due to the high drying temperature causing serious damage to the internal tissue structure of the mushroom. The black-skinned chicken mushroom treated with 5 times and 30 times dilution showed a porous network structure with large pores and good structural integrity, indicating that the double milk has a good protective effect on the internal tissue structure of the black-skinned chicken mushroom during the drying process.

[0146] (3) The nutritional components of the black-skinned Alpinia fasciata are investigated, as shown in the following table:

[0147] Table 8 Effects of different treatment groups on the nutritional components of black-skinned chicken mushroom

[0148]

[0149]

[0150] As can be seen from Table 8, compared with the untreated group, the black-skinned chicken fir dried products after double emulsion dipping have a good effect in maintaining quality.

[0151] The rehydration rate of black-skinned chicken mushroom is as follows Fig.30 The rehydration rate of the black-skinned chicken mushroom treated with the 5-fold treatment group was not high, and the rehydration rate of the black-skinned chicken mushroom treated with the 30-fold treatment group was basically the same as that of the black-skinned chicken mushroom without treatment group at the second hour of rehydration, with no significant difference, which indicates that the double emulsion treatment will not have an adverse effect on the rehydration ability of the black-skinned chicken mushroom.

[0152] The results of the determination of the opening rate of black-skinned chicken fir are shown in Table 9. Fresh black-skinned chicken fir was dipped in double emulsion and then dried, and stored in a -4°C constant temperature box for 7 days (not dried). The opening rate is shown in Table 9. The opening rate of black-skinned chicken fir after double emulsion dipping decreased significantly during storage, and with the increase of emulsion concentration, it decreased from 100% of the untreated to 25%, which shows that the double emulsion has a good inhibitory effect on the opening phenomenon of black-skinned chicken fir and has a certain preservation effect.

[0153] Table 9-Opening rate of black-skinned chicken mushroom (fresh) stored for 10 days at 4℃

[0154]

Claims

1. A method for preparing a water-in-oil-in-water double emulsion loaded with dihydroquercetin and epigallocatechin gallate, comprising the following steps: S1: Preparation of internal aqueous phase W1: Dissolve 0.5 mg / mL epigallocatechin gallate, 1 mg / mL sodium chloride, 0.1 mg / mL ascorbic acid, and 0.5 mg / mL gelatin in deionized water, and stir in a magnetic stirrer at 500 rpm at 60°C for 20 min until fully dissolved, to serve as the internal aqueous phase W1; S2: Preparation of colostrum W1 / O: a certain amount of dihydroquercetin was dissolved in olive oil to make it saturated, and then 1-5% of polyglycerol ricinoleate was placed in olive oil based on the volume of the oil phase, and magnetically stirred at room temperature for 30 min until fully dissolved, and this was used as the oil phase O. The inner water phase W1 was added to the oil phase at a water / oil volume ratio of 10-50%, and dispersed at high speed for 3 min to prepare W1 / O colostrum; S3: Preparation of the external aqueous phase W2: Weigh sodium hyaluronate and add deionized water to dissolve to prepare a 1 mg / mL sodium hyaluronate aqueous solution; weigh whey protein and add deionized water to dissolve to prepare a 3 mg / mL whey protein aqueous solution; Sodium hyaluronate aqueous solution and whey protein aqueous solution are respectively prepared into a polysaccharide / protein complex with a mass ratio of 1:1 to 9:1 and a pH of 4, which is used as the external aqueous phase W2; S4: Preparation of W1 / O / W2 double emulsion: Slowly add the W1 / O colostrum prepared in S2 at a volume ratio of 10-90% at a dispersion speed of 4000-20000 r / min within 1 min to the external water phase W2 in S3, and then continue to disperse at the same speed for 2 min to obtain W1 / O / W2 double emulsion.

2. The method for preparing the water-in-oil-in-water double emulsion loaded with dihydroquercetin and epigallocatechin gallate according to claim 1, characterized in that: In S2, the volume ratio of polyglycerol ricinoleate to the oil phase is 4%, and the inner water phase is added to the oil phase at a water / oil volume ratio of 20%.

3. The method for preparing the water-in-oil-in-water double emulsion loaded with dihydroquercetin and epigallocatechin gallate according to claim 1, characterized in that: In S3, a sodium hyaluronate aqueous solution and a whey protein aqueous solution are respectively prepared into a polysaccharide / protein complex with a mass ratio of 3:

1.

4. The method for preparing the water-in-oil-in-water double emulsion loaded with dihydroquercetin and epigallocatechin gallate according to claim 1, characterized in that: In S4, the W1 / O colostrum prepared in S2 was slowly added to the external aqueous phase W2 in S3 at a dispersion speed of 12000 r / min within 1 min at a volume ratio of 63.32%, and then dispersed at the same speed for 2 min to obtain W1 / O / W2 double emulsion.

5. Application of water-in-oil-in-water double emulsion loaded with dihydroquercetin and epigallocatechin gallate as a fresh-keeping material.

6. The use according to claim 5, characterized in that The water-in-oil-in-water double emulsion loaded with dihydroquercetin and epigallocatechin gallate is used in the preservation of black-skinned chicken mushroom, strawberry and yam.

7. The use according to claim 6, characterized in that Dilute the double emulsion 5 to 30 times with water or ultrapure water, then soak the fruits, vegetables or edible fungi to be preserved in the diluted emulsion for 20 to 40 seconds, take them out, drain and dry.

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