Auricularia auricula polysaccharide / casein emulsion gel as well as preparation method and application thereof

Abstract

The invention discloses black fungus polysaccharide / casein emulsion gel as well as a preparation method and application thereof. The preparation method comprises the following steps: 1) mixing an aqueous solution of auricularia auricula polysaccharide with an aqueous solution of casein to obtain a protein-polysaccharide compound solution; (2) a water phase is added into the oil phase and mixed, the water phase is the protein-polysaccharide compound solution, the obtained mixture is subjected to ultrasonic homogenization, and emulsion is obtained; and 3) adding gluconic acid-delta-lactone into the emulsion, mixing, and incubating to obtain the auricularia auricula polysaccharide / casein emulsion gel. The auricularia auricula polysaccharide / casein emulsion gel disclosed by the invention is applied to curcumin encapsulation. After the black fungus polysaccharide is added, the gel performance of the casein emulsion gel is obviously improved; after the auricularia auricula polysaccharide is added, the loading rate of the casein emulsion gel loaded with curcumin is obviously increased.

Description

Technical Field

[0001] The invention belongs to the technical field of emulsion gel materials and drug sustained-release preparations, and relates to a preparation method and application of black fungus polysaccharide / casein emulsion gel. Background Art

[0002] Curcumin (Cur) is a hydrophobic natural polyphenol extracted mainly from the rhizome of turmeric, and has been widely used as a colorant in food. It is well known for its good anti-inflammatory, antioxidant and anti-tumor properties. However, curcumin has the characteristics of chemical instability, poor bioavailability and rapid metabolism, which limits its application in food and pharmaceutical preparations.

[0003] Emulsion gel, also known as latex or gel emulsion, is a complex colloidal material in which both emulsion droplets and gel exist, and it has the properties of emulsification and gel. Usually, the continuous phase forms a continuous gel matrix, and the oil droplets are embedded in the gel matrix. The dispersed oil droplets in the system are not only stabilized by the emulsifier, but also captured in the continuous gel network, giving the oil excellent rheological and texture properties. In addition, due to the continuous phase and oil droplets, the emulsion gel can encapsulate hydrophilic and hydrophobic active ingredients for the transport of bioactive substances. The emulsion preparation process can usually be divided into two steps. First, the emulsion is prepared by mixing the emulsifier with the oil phase through various methods such as high-speed shearing or homogenization ultrasound; then the continuous phase is induced to gel through a series of induction methods to form a gel network that can stabilize the oil droplets. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method and application of black fungus polysaccharide / casein emulsion gel. After adding black fungus polysaccharide, the gel performance of the casein emulsion gel is significantly improved; after adding black fungus polysaccharide, the loading rate of curcumin loaded on the casein emulsion gel is significantly increased.

[0005] The present invention provides a method for preparing a black fungus polysaccharide / casein emulsion gel, comprising the following steps: 1) mixing a black fungus polysaccharide aqueous solution with a casein aqueous solution to obtain a protein-polysaccharide complex solution; 2) adding a water phase to the oil phase and mixing, wherein the water phase is the protein-polysaccharide complex solution, and performing ultrasonic homogenization on the obtained mixture to obtain an emulsion; 3) adding glucono-δ-lactone to the emulsion, mixing, and incubating to obtain a black fungus polysaccharide / casein emulsion gel.

[0006] In the above method, the concentration of the black fungus polysaccharide aqueous solution can be 0.5-10 mg / ml, specifically 1, 3, 5 mg / ml or 1-5 mg / ml; The concentration of the casein aqueous solution may be 100-200 mg / ml, specifically 160 mg / ml, 160-200 mg / ml, 100-160 mg / ml or 150-200 mg / ml.

[0007] In the above method, the black fungus polysaccharide aqueous solution is prepared as follows: black fungus polysaccharide powder is dispersed in ultrapure water, dispersed, and then hydrated to obtain; The casein aqueous solution is prepared as follows: casein powder is dispersed in deionized water, alkali (specifically NaOH) is added, dispersed, and then hydrated to obtain the casein aqueous solution.

[0008] In the above method, the volume ratio of the black fungus polysaccharide aqueous solution to the casein aqueous solution can be 0.1-10:1, specifically 1:1, 1-10:1 or 0.1-5:1.

[0009] In the above method, the volume ratio of the water phase to the oil phase may be 0.1-9:1, specifically 2:3 or 0.1-5:1.

[0010] In the above method, the ultrasonic homogenization treatment is as follows 1)-2): 1) the power is 100-300 W, with a frequency of on for 1-10 s and off for 1-10 s, and the treatment is carried out for 1-5 min to obtain a crude emulsion; specifically, the power can be 200 W, with a frequency of on for 3 s, off for 3 s, and treatment for 3 min; 2) The power is 100-300 W, with a frequency of 1-10 s on and 1-10 s off, for 1-5 min to obtain a fine emulsion; specifically, the power can be 250 W, with a frequency of 5 s on and 5 s off, for 3 min; The ultrasonic homogenization is performed in an ice water bath.

[0011] In the above method, the final concentration of the glucono-δ-lactone added to the emulsion can be 1-10 mg / mL, specifically 6 mg / mL, 1-6 mg / mL, 6-10 mg / mL or 4-8 mg / mL.

[0012] In the above method, the incubation temperature may be 1-7°C, specifically 4°C, and the incubation time may be 10-15h, specifically 12h.

[0013] The present invention also provides a black fungus polysaccharide / casein emulsion gel prepared by the above method.

[0014] The black fungus polysaccharide / casein emulsion gel of the invention is used in curcumin encapsulation.

[0015] Casein is widely used as a natural emulsifier to stabilize emulsions due to its amphiphilic properties, polymer structure and charge characteristics, and has strong emulsifying properties. It can be adsorbed on the droplet interface through homogenization to keep the emulsion stable. Black fungus polysaccharide is a macromolecular substance that can act as an effective emulsifier in oil-in-water emulsions. This is because it has hydrophobic groups that promote its rapid adsorption to the surface of oil droplets. In addition, the hydrophilic regions, side chains and main chains of black fungus polysaccharides can extend into the continuous phase, providing steric resistance and electrostatic repulsion to inhibit or slow down the aggregation of oil droplets, thereby maintaining the stability of the emulsion protein.

[0016] The present invention has the following beneficial effects: 1. The present invention adopts ultrasonic emulsification technology to prepare a stable emulsion, which has the advantages of being environmentally friendly, highly efficient and non-toxic.

[0017] 2. Compared with other emulsification methods, the present invention uses ultrasound to generate cavitation bubbles through physical effects such as shear force, mechanical force, and high pressure, thereby reducing the size of droplets during the emulsification process, promoting the formation of tiny or even nano-scale droplets, and improving the stability of the emulsion.

[0018] 3. The emulsion prepared by the ultrasonic method of the present invention has a more uniform droplet distribution, thereby being able to better improve the stability of the emulsion.

[0019] 4. After adding black fungus polysaccharide, the gel performance of the casein emulsion gel is significantly improved; after adding black fungus polysaccharide, the loading rate of curcumin loaded by the casein emulsion gel is significantly increased, and it can be used as an encapsulation material to improve the bioavailability of curcumin. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 These are images of emulsion gels observed under an optical microscope in Examples 1 to 3 of the present invention and a comparative example.

[0021] Figure 2 These are images of emulsions of Examples 1 to 3 of the present invention and a comparative example observed using a laser confocal microscope.

[0022] Figure 3 The figure shows the change of apparent viscosity of the emulsion gels of Examples 1 to 3 of the present invention and the comparative example with increasing shear rate.

[0023] Figure 4 The graph shows the changes of G′ and G″ values ​​of the emulsion gels of Examples 1 to 3 of the present invention and the comparative example as the frequency increases.

[0024] Figure 5 The figure shows the gelation temperature scan of the emulsions of Examples 1 to 3 of the present invention and the comparative example.

[0025] Figure 6The oscillation step conditions of the emulsion gelation of Examples 1 to 3 of the present invention and the comparative example are shown.

[0026] Figure 7 The creep recovery of the emulsions in Examples 1 to 3 of the present invention and the comparative example is shown.

[0027] Figure 8 The water holding capacity of the emulsions of Examples 1 to 3 of the present invention and the comparative example is shown.

[0028] Fig. 9 The intermolecular interactions of the emulsion gels of Examples 1 to 3 of the present invention and the comparative example are shown.

[0029] Fig.10 The encapsulation efficiency of curcumin by the emulsion gel of Examples 1 to 3 of the present invention and the comparative example is shown. DETAILED DESCRIPTION

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] In the following examples, black fungus polysaccharide was provided by the edible fungus team of Shanxi Agricultural University; Casein (Casein, total nitrogen ≥13.5%) was purchased from Solebow Technology Co., Ltd.; Curcumin (BR, 98%), Nile Red, and Nile Blue were purchased from Yuanye Biotechnology Co., Ltd.; Glucono-δ-lactone (GDL, 99%) was purchased from Aladdin Biochemical Technology Co., Ltd.; Example 1 A method for preparing black fungus polysaccharide / casein emulsion gel by ultrasonication specifically comprises the following steps: 1. Preparation of Auricularia auricula polysaccharide (AAP) stock solution, casein (CA) stock solution, and polysaccharide-protein complex solution 50 mg of black fungus polysaccharide powder was dispersed in ultrapure water to reach 50 mL, and the dispersion was continued at room temperature (~25°C) for 6 h. Finally, it was completely hydrated at 4°C overnight to prepare a 1 mg / ml AAP stock solution.

[0033] Preparation of casein stock solution: 8.0 g of casein powder was dispersed in deionized water to a volume of 49 mL, and then 1 mL of 5M NaOH was added. The dispersion was continued at room temperature (~25°C) for 6 h, and finally completely hydrated at 4°C overnight to prepare a 160 mg / ml CA stock solution. The 50 g / ml CA stock solution was prepared by diluting the above 160 mg / ml CA stock solution.

[0034] Preparation of polysaccharide-protein complex solution: The above 50 mg / ml CA stock solution and 1 mg / ml AAP stock solution were mixed at a volume ratio of 1:1 to prepare a protein-polysaccharide complex solution sample.

[0035] 2. Preparation of CA-AAP emulsions and emulsion gels Emulsion preparation: The polysaccharide-protein complex solution (water phase) was added to corn oil (oil phase) at a volume ratio of 4:6. The mixture was homogenized using an ultrasonic homogenizer (JY92-IIN, Ningbo Scientz Biotechnology Co., China). The sample was first treated at 200 W (on 3 s, off 3 s) for 3 min to obtain a coarse emulsion. Then, the sample was treated at 250 W (on 5 s, off 5 s) for 3 min to obtain a fine emulsion. In order to maintain a constant temperature, the emulsion preparation process was carried out in an ice-water bath.

[0036] Preparation of emulsion gel: Glucono-δ-lactone (GDL) was added to the newly prepared emulsion (final concentration was 6 mg / mL), stirred and mixed, and finally the sample was incubated at 4°C for 12 h, which was recorded as CA-AAP. 0.1% .

[0037] Example 2 Auricularia auricularia polysaccharide / casein emulsion gel was prepared according to the method in Example 1 of the present invention, except that 150 mg of auricularia auricularia polysaccharide powder was used to prepare a 3 mg / ml AAP reserve. The final emulsion gel was recorded as CA-AAP 0.3% .

[0038] Example 3 Auricularia auricularia polysaccharide / casein emulsion gel was prepared according to the method in Example 1 of the present invention, except that 250 mg of auricularia auricularia polysaccharide powder was used to prepare a 5 mg / ml AAP reserve. The final emulsion gel was recorded as CA-AAP. 0.5% .

[0039] Comparative Example Auricularia auricularia polysaccharide / casein emulsion gel was prepared according to the method in Example 1 of the present invention, except that no auricularia auricularia polysaccharide powder was added (i.e., the added amount was 0 mg). The final emulsion gel was recorded as CA-AAP 0% .

[0040] Example 4 1. Microstructure observation The morphology of the emulsion gels prepared in Examples 1-3 of the present invention and the comparative example was observed by using an optical microscope (ECLIPSE E100, Nikon, Japan) with the image magnified 400 times. Figure 1 As shown in Figure 1 It can be seen from the results that the droplets in Example 2 of the present invention are smaller and the condensation is more stable.

[0041] The microstructure was observed using a confocal laser scanning microscope (TCS SP8, Leica, Germany) with a 20× objective lens. During the emulsion preparation process, 50 μL of Nile red (1 mg / mL, excitation wavelength of 488 nm) and 50 μL of Nile blue A (1 mg / mL, excitation wavelength of 633 nm) were mixed with the oil phase and the water phase, respectively. Figure 2 As shown in Figure 2 In the figure, the oil phase is green and the water phase is red, indicating that the emulsion gels prepared by this method are all oil-in-water type; Figure 2 It can be seen from the results that the droplets in Example 2 are smaller and the condensation is more stable.

[0042] 2. Texture determination Before testing, the emulsion gels prepared in Examples 1-3 of the present invention and the comparative example were stored at room temperature for 2 h, and then analyzed using a texture analyzer (TMS-PRO, Food Technology Corporation, USA), which was equipped with a cylindrical measuring probe with a diameter of 36 mm. The test parameters used were set as follows: test speed of 1 mm / s, post-test speed of 2 mm / s, trigger force of 5 g, deformation of 50%, and interval time of 2 s. Each sample was tested at least three times. Through the above texture determination, the results are shown in Table 1. It can be seen from the results in Table 1 that Example 2 has a relatively large hardness and better overall gel performance.

[0043] 3. Rheological analysis The rheological properties of the emulsion gels prepared in Examples 1 to 3 of the present invention and the comparative example were measured by an MCR 102 rheometer (Anton Paar, Austria), using 50 mm parallel plates and a gap of 1 mm.

[0044] 3.1 Shear rate scan At 25°C, 0.1 ~ 100 s −1 The shear rate was measured in frequency sweep mode and the apparent viscosity (η) was recorded. Figure 3 As shown in Figure 3 The results show that when the shear rate increases, the apparent viscosity of the emulsion gel decreases, and all emulsion gels show shear thinning. At the same shear rate, as the amount of AAP added increases, the viscosity of the emulsion gel increases, among which Example 2 has the largest apparent viscosity.

[0045] 3.2 Frequency Sweep At 25°C, 0.1 ~ 100 s -1 The elastic modulus (G') and viscous modulus (G'') of the sample were measured within the frequency range of Figure 4 As shown in Figure 4 It can be seen from the results that all samples exhibit viscoelastic solid behavior of gel-like structure in the entire frequency range (G′>G′′). The dynamic rheological properties (G′ and G′′) of the embodiments are better than those of the emulsion gel of the comparative example. With the increase of the amount of AAP added, G′ and G′′ of the emulsion gel both show a trend of first increasing and then decreasing, and reach the best in Example 2.

[0046] 3.3 Temperature Scan The thermal stability of the latex gel was measured in the range of 5 ~ 80℃. The heating rate was set at 1.5℃ / min, and the shear strain and frequency were fixed at 0.1% and 5 Hz, respectively. Figure 5 As shown in Figure 5 The results show that within the temperature test range, all emulsion gels did not undergo sol-gel transition. At 80°C, the elastic modulus (G′) was still slightly higher than the viscous modulus (G′′), indicating that the emulsion gel was still in a gel state. Example 2 had the largest modulus, and the gel strength was the largest after being affected by temperature.

[0047] 3.4 Oscillation step test At 25°C, the shear was applied at 0.1% and 1.5 Hz for 60 s, then increased to 500% strain for 20 s, and then returned to 0.1% strain for 60 s. The strain increase and decrease were repeated three times in one measurement. Figure 6 As shown in Figure 6It can be seen from the results that all emulsion gels show rapid shear recovery. In the low shear stage, with the increase in the amount of AAP added, the G' of the sample gradually increases and reaches a maximum value in Example 2. The recovery rate of the comparative example after initial shear is the highest (90.50%). The recovery rate of the embodiment is lower than that of the comparative example. However, after secondary and tertiary shearing, the recovery rate of the embodiment increases significantly, indicating that the emulsion gel network is rebuilt rapidly. The secondary shear recovery rate is greater than 87%, and the tertiary shear recovery rate is greater than 92%. This shows that after multiple shearing, each emulsion gel exhibits good self-healing ability.

[0048] 3.5 Creep recovery A constant shear stress of 1 Pa was applied to the sample for 5 min. After the applied stress was removed, the creep recovery was recorded for 5 min. Figure 7 As shown in Figure 7 The results show that the creep strain of the emulsion gel is reduced after adding AAP, indicating that AAP can enhance the gel's ability to resist external forces. Example 2 of the present invention has a higher recovery rate. This shows that it has the characteristics of fast deformation, good recovery rate, and good gelling performance.

[0049] 4. Water Holding Capacity (WHC) Place 10 ml of sample in a 50 ml centrifuge tube and centrifuge at 8000 g for 30 min at 4 °C. The WHC calculation formula is as follows: WHC=(1-(W1-W2) / W1)×100% Where W1 represents the mass of the sample before centrifugation (g), and W2 represents the mass of the sample after centrifugation (g). Figure 8 As shown in Figure 8 The results show that the WHC values ​​of all emulsion gels are above 85%. Compared with the emulsion gels of the comparative example, the WHC of the emulsion gels with the addition of AAP is significantly different and improved, especially the emulsion gel of Example 2.

[0050] 5. Interactions between molecules in emulsion gel 1 g of the milky gel samples prepared in Examples 1-3 of the present invention and the comparative examples were dispersed in 20 mL of the four solutions: (S1) 0.05 M NaCl, (S2) 0.6 M NaCl, (S3) 0.6 M NaCl + 1.5 M urea, (S4) 0.6 M NaCl + 8 M urea, (S5) 0.6 M NaCl + 8 M urea + 0.5 M β-mercaptoethanol. After continuous stirring, centrifugation was performed at 10,000 g for 15 min. The protein content of the five solutions was determined by Bradford method.

[0051] The differences in protein solubility between S2 solution and S1 solution, S3 solution and S2 solution, S4 solution and S3 solution, and S5 solution and S4 solution represent electrostatic interactions, hydrogen bonds, hydrophobic interactions, and disulfide bonds, respectively. Fig. 9 As shown in Fig. 9 The results show that the main interaction force of all emulsion gels is electrostatic interaction, and the interaction force increases with the increase of AAP addition.

[0052] 6. Curcumin Encapsulation Efficiency (EE) Encapsulation process: 1. First, mix curcumin with corn oil so that the final concentration of curcumin is 1-3 mg / mL (specifically 2 mg / mL).

[0053] 2. Then stir at 1000 rpm at 60°C for 1 to 3 h (specifically 2 h).

[0054] 3. Finally, according to the method described in "2. Preparation of CA-AAP emulsion and emulsion gel" in Example 1 above, corn oil containing curcumin was used as the oil raw material to prepare an emulsion gel loaded with curcumin.

[0055] The emulsion gels (1 g) with different AAP contents in Examples 1-3 of the present invention and the comparative example were mixed with 5 mL of 95% (v / v) ethanol, ultrasonicated for 30 min, centrifuged at 5000 rpm for 10 min, and the absorbance of the supernatant was measured at 423 nm. The curcumin content was calculated using the standard curve method. EE was calculated by the following formula: EE (%) = (total curcumin content - curcumin content in supernatant) / (total curcumin content) × 100%. The results are shown in Fig.10 As shown in Fig.10 The results show that the encapsulation efficiency of curcumin gradually increases with the increase of AAP addition. The encapsulation efficiency of Example 2 is the highest (83.18%). In summary, CA-AAP emulsion gel can be used as an encapsulation material to improve the bioavailability of curcumin.

[0056] Table 1 Texture characteristics of the emulsion gels of Examples 1 to 3 of the present invention and the comparative example Comparative Example Example 1 Example 2 Example 3 Hardness (N) 1.14±0.33 b 1.03±0.14 b 3.64±0.6 a 3.30±0.58 a Adhesion (mJ) 0.65±0.07 c 0.5±0.08 c 1.69±0.1 b 2.03±0.12 a Elasticity (mm) 1.37±0.12 b 1.24±0.26 b 1.93±0.26 a 2.29±0.20 a Cohesion 0.42±0.06 b 0.41±0.07 b 0.57±0.03 ab 0.6±0.14 a Adhesiveness (N) 0.52±0.11 b 0.41±0.03 b 2.22±0.33 a 2.03±0.82 a Chewing property (mJ) 0.78±0.11 c 0.61±0.05 c 3.85±0.58 b 7.30±0.62 a

Claims

1. A method for preparing a black fungus polysaccharide / casein emulsion gel, comprising the following steps: 1) mixing a black fungus polysaccharide aqueous solution with a casein aqueous solution to obtain a protein-polysaccharide complex solution; 2) adding a water phase to the oil phase and mixing, wherein the water phase is the protein-polysaccharide complex solution, and performing ultrasonic homogenization on the obtained mixture to obtain an emulsion; 3) adding glucono-δ-lactone to the emulsion, mixing, and incubating to obtain a black fungus polysaccharide / casein emulsion gel.

2. The method according to claim 1, characterized in that The concentration of the black fungus polysaccharide aqueous solution is 0.5-10 mg / ml; The concentration of the casein aqueous solution is 100-200 mg / ml.

3. The method according to claim 1 or 2, characterized in that: The black fungus polysaccharide aqueous solution is prepared by the following method: dispersing black fungus polysaccharide powder in ultrapure water, dispersing, and then hydrating; The casein aqueous solution is prepared by the following method: casein powder is dispersed in deionized water, alkali is added, dispersed, and then hydrated to obtain the solution.

4. The method according to any one of claims 1 to 3, characterized in that The volume ratio of the black fungus polysaccharide aqueous solution to the casein aqueous solution is 0.1-10:

1.

5. The method according to any one of claims 1 to 4, characterized in that The volume ratio of the water phase to the oil phase is 0.1-9:

1.

6. The method according to any one of claims 1 to 5, characterized in that The ultrasonic homogenization process is as follows 1)-2): 1) the power is 100-300 W, the frequency is on for 1-10 s and off for 1-10 s, and the process is performed for 1-5 min to obtain a crude emulsion; 2) The power is 100-300 W, with a frequency of 1-10 s on and 1-10 s off for 1-5 min to obtain a fine emulsion; The ultrasonic homogenization is performed in an ice water bath.

7. The method according to any one of claims 1 to 6, characterized in that The final concentration of the glucono-δ-lactone added to the emulsion is 1-10 mg / mL.

8. The method according to any one of claims 1 to 7, characterized in that The incubation temperature is 1-7°C and the incubation time is 10-15h.

9. Auricularia auricularia polysaccharide / casein emulsion gel prepared by the method according to any one of claims 1 to 8.

10. Use of the black fungus polysaccharide / casein emulsion gel according to claim 9 in curcumin encapsulation.

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