Preparation of auricularia auricular polysaccharide / casein emulsion gel, and preparation method and application thereof

Abstract

The application discloses a kind of preparation auricularia auricular polysaccharide / casein emulsion gel and its preparation method and application.The preparation method of the application includes the following steps: 1) auricularia auricular polysaccharide aqueous solution is mixed with casein aqueous solution, to obtain protein-polysaccharide complex solution;2) water phase is mixed into oil phase, the water phase is the protein-polysaccharide complex solution, and the obtained mixture is ultrasonically homogenized, to obtain emulsion;3) gluconic acid-delta-lactone is added to the emulsion and mixed, incubated, to obtain auricularia auricular polysaccharide / casein emulsion gel.The auricularia auricular polysaccharide / casein emulsion gel disclosed in the application is applied to curcumin encapsulation.After adding auricularia auricular polysaccharide, the gel performance of casein emulsion gel is significantly improved;After adding auricularia auricular polysaccharide, the loading rate of curcumin loaded in casein emulsion gel is significantly increased.

Description

Technical Field

[0001] This invention belongs to the technical field of emulsion gel materials and sustained-release drug formulations, and relates to a method for preparing black fungus polysaccharide / casein emulsion gel and its application. Background Technology

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

[0003] Emulsion gels, also known as latex or gel emulsions, are complex colloidal materials containing both emulsion droplets and gels, exhibiting both emulsifying and gelling properties. Typically, a continuous phase forms a continuous gel matrix, within which oil droplets are embedded. The dispersed oil droplets in the system are not only stabilized by the emulsifier but also trapped within the continuous gel network, endowing the oil with excellent rheological and textural properties. Furthermore, due to the presence of a continuous phase and oil droplets, emulsion gels can encapsulate both hydrophilic and hydrophobic active ingredients for the transport of bioactive substances. The emulsion preparation process generally involves two steps: first, the emulsifier is mixed with the oil phase using various methods such as high-speed shearing or homogenizing ultrasound to prepare the emulsion; then, a series of induction methods are used to induce gelation of the continuous phase to form a gel network that can stabilize the oil droplets. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a black fungus polysaccharide / casein emulsion gel, and its application. The addition of black fungus polysaccharide significantly improves the gel performance of the casein emulsion gel; it also significantly increases the loading rate of curcumin on the casein emulsion gel.

[0005] This 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;

[0006] 2) Add an aqueous phase to the oil phase and mix, wherein the aqueous phase is the protein-polysaccharide complex solution, and homogenize the resulting mixture by ultrasonication to obtain an emulsion;

[0007] 3) Add gluconate-δ-lactone to the emulsion and mix, then incubate to obtain black fungus polysaccharide / casein emulsion gel.

[0008] 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;

[0009] The concentration of the casein aqueous solution can be 100~200 mg / ml, specifically 160 mg / ml, 160~200 mg / ml, 100~160 mg / ml or 150~200 mg / ml.

[0010] In the above method, the aqueous solution of black fungus polysaccharide is prepared by dispersing black fungus polysaccharide powder in ultrapure water, dispersing, and then hydrating.

[0011] The casein aqueous solution is prepared by dispersing casein powder in deionized water, adding alkali (specifically NaOH), dispersing, and then hydrating.

[0012] 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.

[0013] In the above method, the volume ratio of the aqueous phase to the oil phase can be 0.1 to 9:1, specifically 2:3 or 0.1 to 5:1.

[0014] In the above method, the ultrasonic homogenization process is as follows: 1)-2) The power is 100~300 W, and the process is carried out at a frequency of 1~10 s on and 1~10 s off for 1~5 min to obtain a crude emulsion; specifically, it can be carried out at a power of 200 W for 3 s on and 3 s off for 3 min.

[0015] 2) With a power of 100~300 W, process for 1~5 min at a frequency of 1~10 s on and 1~10 s off to obtain a fine emulsion; specifically, at a power of 250 W, process for 3 min at a frequency of 5 s on and 5 s off.

[0016] The ultrasonic homogenization was performed in an ice-water bath.

[0017] In the above method, the final concentration of gluconate-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.

[0018] In the above method, the incubation temperature can be 1~7℃, specifically 4℃, and the incubation time can be 10~15h, specifically 12h.

[0019] The present invention also provides a black fungus polysaccharide / casein emulsion gel prepared by the above method.

[0020] The black fungus polysaccharide / casein emulsion gel described in this invention is used in the encapsulation of curcumin.

[0021] Casein, due to its amphiphilic properties, polymer structure, and charge characteristics, is widely used as a natural emulsifier to stabilize emulsions, exhibiting strong emulsifying performance. It can be homogenized and adsorbed onto the droplet interface, maintaining emulsion stability. Black fungus polysaccharide, a macromolecule, can act as an effective emulsifier in oil-in-water emulsions due to its hydrophobic groups, which promote rapid adsorption onto the oil droplet surface. Furthermore, the hydrophilic regions, side chains, and main chain of black fungus polysaccharide can extend into the continuous phase, providing steric hindrance and electrostatic repulsion to inhibit or slow down oil droplet aggregation, thereby maintaining emulsion stability.

[0022] The present invention has the following beneficial effects:

[0023] 1. This invention uses ultrasonic emulsification technology to prepare a stable emulsion, which has the advantages of being environmentally friendly, efficient and non-toxic.

[0024] 2. Compared with other emulsification methods, this invention uses ultrasound to generate cavitation bubbles through physical actions such as shear force, mechanical force, and high pressure, thereby reducing the droplet size during the emulsification process, promoting the formation of tiny or even nano-sized droplets, and improving the stability of the emulsion.

[0025] 3. The emulsion prepared by the ultrasonic method of this invention has a more uniform droplet distribution, thereby improving the stability of the emulsion.

[0026] 4. After adding black fungus polysaccharide, the gel performance of casein emulsion gel is significantly improved; after adding black fungus polysaccharide, the loading rate of curcumin on casein emulsion gel is significantly increased, which can be used as an encapsulation material to improve the bioavailability of curcumin. Attached Figure Description

[0027] Figure 1 These are optical microscope images of the emulsion gels from Examples 1 to 3 and the comparative examples of the present invention.

[0028] Figure 2 The images shown are from laser confocal microscopy observations of the emulsion coagulations in Examples 1 to 3 and the comparative example of the present invention.

[0029] Figure 3 The changes in apparent viscosity of emulsion coagulants in Examples 1 to 3 and the comparative examples of the present invention with increasing shear rate are shown.

[0030] Figure 4 The changes in the G′ and G′′ values ​​of the emulsion gels in Examples 1 to 3 and the comparative example of the present invention as the frequency increases are shown.

[0031] Figure 5The images show the emulsion coagulation temperature scans for Examples 1 to 3 and the comparative example of this invention.

[0032] Figure 6 Examples 1 to 3 and the comparative example show the oscillation step of emulsion coagulation in this invention.

[0033] Figure 7 Examples 1 to 3 and the comparative example show the creep recovery of the emulsion coagulation in this invention.

[0034] Figure 8 Examples 1 to 3 and comparative examples of the water-holding capacity of emulsion coagulation in this invention.

[0035] Figure 9 Examples 1 to 3 and the comparative emulsion coagulant of the present invention illustrate the intermolecular interactions.

[0036] Figure 10 The encapsulation efficiency of curcumin by emulsion gel in Examples 1 to 3 and the comparative example of the present invention is shown. Detailed Implementation

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] In the following examples, the black fungus polysaccharide was provided by the edible fungi team of Shanxi Agricultural University;

[0040] Casein (total nitrogen ≥13.5%) was purchased from Solarbio Science & Technology Co., Ltd.

[0041] Curcumin (BR, 98%), Nile Red and Nile Blue were purchased from Yuanye Biotechnology Co., Ltd.

[0042] Glucono-δ-lactone (GDL, 99%) was purchased from Aladdin Biochemical Technology Co., Ltd.

[0043] Example 1

[0044] A method for preparing black fungus polysaccharide / casein emulsion gel by ultrasound includes the following steps:

[0045] 1. Preparation of stock solutions of black fungus polysaccharide (AAP), casein (CA), and polysaccharide-protein complex solutions

[0046] 50 mg of black fungus polysaccharide powder was dispersed in ultrapure water to a volume of 50 mL. The dispersion was carried out at room temperature (~25℃) for 6 h, and finally completely hydrated overnight at 4℃ to prepare a 1 mg / mL AAP stock solution.

[0047] Preparation of casein stock solution: 8.0 g of casein powder was dispersed in deionized water to a volume of 49 mL, then 1 mL of 5M NaOH was added. The dispersion was continued at room temperature (~25℃) for 6 h, and finally, it was completely hydrated overnight at 4℃ to obtain a CA stock solution with a concentration of 160 mg / mL. A 50 g / mL CA stock solution was prepared by diluting the above 160 mg / mL CA stock solution.

[0048] 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.

[0049] 2. Preparation of CA-AAP emulsions and emulsion gels

[0050] Emulsion preparation: The above polysaccharide-protein complex solution (aqueous phase) was added to corn oil (oil phase) at a volume ratio of 4:6. The mixture was ultrasonically homogenized using an ultrasonic homogenizer (JY92-IIN, Ningbo Scientz Biotechnology Co., China). The sample was first treated at 200 W (on for 3 s, off for 3 s) for 3 min to obtain a crude emulsion. Then, it was treated at 250 W (on for 5 s, off for 5 s) for 3 min to obtain a fine emulsion. To maintain a constant temperature, the above emulsion preparation process was carried out in an ice-water bath.

[0051] Emulsion gel preparation: Glucono-δ-lactone (GDL) was added to the freshly prepared emulsion (final concentration 6 mg / mL), stirred and mixed, and finally the sample was incubated at 4℃ for 12 h, denoted as CA-AAP. 0.1% .

[0052] Example 2

[0053] The black fungus polysaccharide / casein emulsion gel was prepared according to the method in Example 1 of this invention, except that 150 mg of black fungus polysaccharide powder was used to prepare a 3 mg / ml AAP reserve. The final emulsion gel was denoted as CA-AAP. 0.3% .

[0054] Example 3

[0055] The black fungus polysaccharide / casein emulsion gel was prepared according to the method in Example 1 of this invention, except that 250 mg of black fungus polysaccharide powder was used to prepare a 5 mg / ml AAP reserve. The final emulsion gel was denoted as CA-AAP. 0.5% .

[0056] Comparative Example

[0057] A fungus polysaccharide / casein emulsion gel was prepared according to the method in Example 1 of this invention, except that no fungus polysaccharide powder was added (i.e., the amount added was 0 mg). The final emulsion gel was denoted as CA-AAP. 0% .

[0058] Example 4

[0059] 1. Microstructure observation

[0060] The morphology of the emulsion gels prepared in Examples 1-3 and the comparative examples of this invention was observed using an optical microscope (ECLIPSE E100, Nikon, Japan) at 400x magnification. The results are as follows: Figure 1 As shown, by Figure 1 The results show that the droplets in Example 2 of the present invention are smaller and the coagulation is more stable.

[0061] The microstructure was observed using a 20× objective confocal laser scanning microscope (TCS SP8, Leica, Germany). During emulsion preparation, 50 μL of Nile Red (1 mg / mL, excitation wavelength 488 nm) and 50 μL of Nile Blue A (1 mg / mL, excitation wavelength 633 nm) were mixed with the oil and aqueous phases, respectively. The results are as follows: Figure 2 As shown, Figure 2 In the image, the oil phase is green and the aqueous phase is red, indicating that the emulsions and gels prepared by this method are all oil-in-water emulsions; Figure 2 The results show that the droplets in Example 2 are smaller and the condensation is more stable.

[0062] 2. Texture determination

[0063] Before testing, the emulsion gels prepared in Examples 1-3 and the comparative example of this invention were stored at room temperature for 2 hours, and then analyzed using a texture analyzer (TMS-PRO, Food Technology Corporation, USA). This analyzer was equipped with a cylindrical measuring probe with a diameter of 36 mm. The experimental parameters used were as follows: test speed 1 mm / s, post-test speed 2 mm / s, trigger force 5 g, deformation 50%, and interval time 2 s. Each sample was tested at least three times. The results of the texture determination are shown in Table 1. As can be seen from the results in Table 1, Example 2 has relatively higher hardness and better overall gel performance.

[0064] 3. Rheological Analysis

[0065] The rheological properties of the emulsion gels prepared in Examples 1-3 and the comparative examples of this invention were determined using an MCR 102 rheometer (Anton Paar, Austria). 50 mm parallel plates with a 1 mm gap were used for testing.

[0066] 3.1 Shear Rate Scan

[0067] At 25℃, using 0.1 ~ 100 s −1 The shear rate was measured using a frequency scanning mode, and the apparent viscosity (η) was recorded. The results are as follows: Figure 3 As shown, by Figure 3 The results show that the apparent viscosity of the emulsion gel decreases with increasing shear rate, and all emulsion gels exhibit shear thinning. At the same shear rate, the viscosity of the emulsion gel increases with increasing AAP content, with Example 2 showing the highest apparent viscosity.

[0068] 3.2 Frequency Scan

[0069] At 25℃, 0.1 ~ 100 s -1 The elastic modulus (G′) and viscous modulus (G′′) of the sample were measured within the frequency range. The results are as follows: Figure 4 As shown, by Figure 4 The results show that all samples exhibited viscoelastic solid behavior with a gel-like structure across the entire frequency range (G′>G′′). The dynamic rheological properties (G′ and G′′) of the examples were superior to those of the comparative emulsion gels. With increasing AAP content, both G′ and G′′ of the emulsion gels showed a trend of first increasing and then decreasing, reaching their optimal values ​​in Example 2.

[0070] 3.3 Temperature Scan

[0071] The thermal stability of the emulsion gel was determined in the range of 5–80 °C. The heating rate was set to 1.5 °C / min, and the shear strain and frequency were fixed at 0.1% and 5 Hz, respectively. The results are as follows: Figure 5 As shown, by Figure 5 The results show that within the temperature testing range, none of the emulsion gels exhibited a sol-gel transition. At 80°C, the elastic modulus (G′) was still slightly higher than the viscous modulus (G′′), indicating that the emulsion gels remained in a gel state. Example 2 exhibited the highest modulus and the greatest gel strength after being affected by temperature.

[0072] 3.4 Oscillating Step Test

[0073] At 25°C, strain was increased to 0.1% and 1.5 Hz for 60 s, then increased to 500% strain for 20 s, and then reduced to 0.1% strain for 60 s. The strain increase and decrease were repeated three times in a single measurement. Results are as follows: Figure 6 As shown, by Figure 6 The results show that all emulsion gels exhibited rapid shear recovery. In the low-shear phase, the G′ of the samples gradually increased with increasing AAP addition, reaching its maximum in Example 2. The comparative example showed the highest recovery rate after initial shear (90.50%). The recovery rate of the examples was relatively lower than that of the comparative example; however, after secondary and tertiary shearing, the recovery rate of the examples increased significantly, indicating rapid emulsion gel network reconstruction. The recovery rate after secondary shearing was greater than 87%, and the recovery rate after tertiary shearing was greater than 92%. This indicates that each emulsion gel exhibits good self-healing ability after multiple shearing cycles.

[0074] 3.5 Creep Recovery

[0075] A constant shear stress of 1 Pa was applied to the sample for 5 min. After removing the applied stress, creep recovery was recorded for 5 min. The results are as follows. Figure 7 As shown, by Figure 7 The results show that the creep strain of the emulsion gel decreased after the addition of AAP, indicating that AAP can enhance the gel's resistance to external forces. Example 2 of this invention exhibits a high recovery rate. This indicates that it possesses characteristics such as rapid deformation, good recovery rate, and good gelling properties.

[0076] 4. Water Holding Capacity (WHC)

[0077] Place 10 ml of sample into a 50 ml centrifuge tube, and then centrifuge at 8000 g for 30 min at 4°C. The WHC calculation formula is as follows:

[0078] WHC=(1-(W1-W2) / W1)×100%

[0079] Where W1 represents the mass (g) of the sample before centrifugation, and W2 represents the mass (g) of the sample after centrifugation. The results are as follows: Figure 8 As shown, by Figure 8 The results show that the WHC values ​​of all emulsion gels are above 85%. Compared with the comparative emulsion gels, the WHC of the emulsion gels with added AAP is significantly different and improved, especially the emulsion gel of Example 2.

[0080] 5. Intermolecular interactions in emulsion gels

[0081] 1g of the emulsion gel samples prepared in Examples 1-3 and the comparative examples of this invention were dispersed in 20 mL of four different solutions:

[0082] (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, centrifuge at 10000g for 15 min. The protein content in the five solutions was determined using the Bradford method.

[0083] The differences in protein solubility between solutions S2 and S1, S3 and S2, S4 and S3, and S5 and S4 represent electrostatic interactions, hydrogen bonds, hydrophobic interactions, and disulfide bonds, respectively. The results are as follows: Figure 9 As shown, by Figure 9 The results show that the main interaction force in all emulsion gels is electrostatic interaction. The interaction force increases with the increase of AAP addition.

[0084] 6. Curcumin encapsulation efficiency (EE)

[0085] Encapsulation process:

[0086] 1. First, mix curcumin with corn oil to achieve a final curcumin concentration of 1-3 mg / mL (specifically, 2 mg / mL).

[0087] 2. Then stir at 1000 rpm at 60℃ for 1~3 h (2 h specifically).

[0088] 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.

[0089] The emulsion gels (1 g) with different AAP contents from Examples 1-3 and the comparative examples of this invention were mixed with 5 mL of 95% (v / v) ethanol, sonicated 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 as follows: Figure 10 As shown, by Figure 10 The results show that the encapsulation efficiency of curcumin gradually increases with the increase of AAP addition. Example 2 showed the highest encapsulation efficiency (83.18%). In conclusion, CA-AAP emulsion gel can be used as an encapsulation material to improve the bioavailability of curcumin.

[0090] Table 1. Texture properties of emulsion gels from Examples 1-3 and comparative examples of the present invention.

[0091] Comparative Example Example 1 Example 2 Example 3 Hardness (N) <![CDATA[1.14±0.33 b ]]> <![CDATA[1.03±0.14 b ]]> <![CDATA[3.64±0.6 a ]]> <![CDATA[3.30±0.58 a ]]> Adhesion (mJ) <![CDATA[0.65±0.07 c ]]> <![CDATA[0.5±0.08 c ]]> <![CDATA[1.69±0.1 b ]]> <![CDATA[2.03±0.12 a ]]> Elasticity (mm) <![CDATA[1.37±0.12 b ]]> <![CDATA[1.24±0.26 b ]]> <![CDATA[1.93±0.26 a ]]> <![CDATA[2.29±0.20 a ]]> cohesion <![CDATA[0.42±0.06 b ]]> <![CDATA[0.41±0.07 b ]]> <![CDATA[0.57±0.03 ab ]]> <![CDATA[0.6±0.14 a ]]> Adhesiveness (N) <![CDATA[0.52±0.11 b ]]> <![CDATA[0.41±0.03 b ]]> <![CDATA[2.22±0.33 a ]]> <![CDATA[2.03±0.82 a ]]> Chewing power (mJ) <![CDATA[0.78±0.11 c ]]> <![CDATA[0.61±0.05 c ]]> <![CDATA[3.85±0.58 b ]]> <![CDATA[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; 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; The volume ratio of the black fungus polysaccharide aqueous solution to the casein aqueous solution is 0.1~10:1; 2) Add an aqueous phase to the oil phase and mix, wherein the aqueous phase is the protein-polysaccharide complex solution, and homogenize the resulting mixture by ultrasonication to obtain an emulsion; The volume ratio of the aqueous phase to the oil phase is 0.1 to 9:1; The ultrasonic homogenization process is as follows: First, the power is 100-300 W, and the process is carried out at a frequency of 1-10 seconds on and 1-10 seconds off for 1-5 minutes to obtain a crude emulsion; second, the power is 100-300 W, and the process is carried out at a frequency of 1-10 seconds on and 1-10 seconds off for 1-5 minutes to obtain a fine emulsion; the ultrasonic homogenization is carried out in an ice-water bath. 3) Add gluconate-δ-lactone to the emulsion and mix, then incubate to obtain black fungus polysaccharide / casein emulsion gel.

2. The method according to claim 1, characterized in that, The aqueous solution of black fungus polysaccharide was prepared by dispersing black fungus polysaccharide powder in ultrapure water, and then hydrating it. The casein aqueous solution is prepared by dispersing casein powder in deionized water, adding alkali, dispersing, and then hydrating.

3. The method according to claim 1 or 2, characterized in that, The final concentration of gluconate-δ-lactone added to the emulsion is 1~10 mg / mL.

4. The method according to claim 1 or 2, characterized in that, The incubation temperature is 1~7℃, and the time is 10~15h.

5. The black fungus polysaccharide / casein emulsion gel prepared by the method of any one of claims 1-4.

6. The application of the black fungus polysaccharide / casein emulsion gel according to claim 5 in curcumin encapsulation.

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