Preparation method and application of myofibrillar protein-sunflower seed oil-high M type alginic acid ternary composite probiotic microcapsule

Abstract

The invention specifically discloses a myofibrillar protein-sunflower seed oil-high M type alginic acid ternary composite probiotic microcapsule and a preparation method thereof, and belongs to the technical field of food. Myofibrillar protein and sodium alginate are used as inner wall materials, gelatin and sunflower seed oil are used as outer wall materials, and lactobacillus acidophilus is embedded in a double-layer mode through electrostatic interaction. The specific preparation method comprises the following steps: mixing a bacterial suspension with the composite wall material, dropwise adding the mixture into pure water, and carrying out ultrasonic curing, dewatering and vacuum freeze drying to obtain the microcapsule. The microcapsule prepared by the invention has a good embedding rate, can play a role in protection and slow release in a simulated digestion process, improves the thermal stability and storage stability of probiotics, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of food technology, specifically to a method for preparing and applying a ternary probiotic microcapsule composed of myofibrillar protein, sunflower seed oil, and high M-type alginate. Background Technology

[0002] Lactobacillus acidophilus, belonging to the genus Lactobacillus, is an important probiotic in the intestines of humans and animals. It has functions such as improving intestinal microecological balance, inhibiting pathogenic bacteria, enhancing immunity, and promoting growth. However, Lactobacillus acidophilus cells are relatively fragile, requiring strict storage conditions, and exhibit poor heat and acid resistance. After ingestion, it is easily inactivated by the highly acidic environment of the stomach, reducing its activity in the intestines. The beneficial effects of probiotics depend on a sufficient quantity reaching the appropriate locations in the body; therefore, improving its survival rate in food production and through the gastrointestinal tract remains a challenging issue.

[0003] To address this issue, numerous studies have demonstrated that microencapsulation technology can provide effective isolation and protection for Lactobacillus acidophilus, significantly enhancing its resistance to adverse environments in the digestive tract and maximizing its probiotic effects. This technology maintains bacterial stability and bioactivity by encapsulating active ingredients in micron-sized capsules, blocking adverse external factors. Conventional preparation methods include spray drying, extrusion, coating, and freeze-drying.

[0004] However, existing microencapsulation technologies still have the following problems: traditional wall materials cannot effectively resist stomach acid and bile salts, causing a large number of probiotics to be inactivated before reaching the intestines; common spray drying methods can cause mechanical damage to the bacteria, significantly affecting their subsequent beneficial effects; microcapsules produced by extrusion methods have a strong granular texture and low yield, making large-scale production difficult; spray coating technology requires cumbersome multi-layer coatings, increasing experimental complexity and reducing production efficiency. Furthermore, microcapsules are prone to inactivation during freeze-drying and exhibit poor storage stability. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a myofibrillar protein-sunflower seed oil-high M-type alginate ternary complex probiotic microcapsule and its preparation method, which improves the stability of the microcapsule and the survival rate of the probiotics in many ways: On the one hand, the combination of plant oil and its oil gel with myofibrillar protein in the prepared material can help inhibit the initial digestion of protein in the stomach and intestines, thereby delaying the exposure time of probiotics and achieving targeted release of probiotics into the intestines. This solves the problem that existing microcapsule wall materials are easily digested quickly in gastric juice and cannot effectively protect probiotics from crossing the highly acidic environment of the stomach.

[0006] On the other hand, meat protein is used as the inner wall material, and an optimal myofibril protein-sunflower seed oil-high M-type alginate ternary composite intestinal targeted delivery system is constructed based on sodium alginate with different M / G contents and different types of oils. By increasing viscosity, the encapsulation rate and stability are improved.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The first objective of this invention is to provide a myofibrillar protein-sunflower seed oil-high M-type alginate ternary complex probiotic microcapsule, comprising a core material and a double-layer composite wall material. The core material is probiotics, the inner composite wall material comprises myofibrillar protein and high M-type sodium alginate, and the outer composite wall material comprises gelatin and sunflower seed oil. Preferably, the probiotics are Lactobacillus acidophilus.

[0008] Furthermore, the high-M type alginate is high-mannuronic acid sodium alginate with an M content of over 60%.

[0009] Furthermore, the mass ratio of myofibrillar protein: high M-type sodium alginate: gelatin: sunflower seed oil is 2:2:1:2.

[0010] The second objective of this invention is to provide a method for preparing a ternary probiotic microcapsule composed of myofibrillar protein, sunflower seed oil, and high M-type alginate, comprising the following steps: S1. Dissolve myofibrillar protein in phosphate buffer, add high-M sodium alginate and dissolve it completely, then shear thoroughly to obtain solution 1; S2. Dissolve gelatin in pure water, add sunflower seed oil, and shear thoroughly to obtain solution 2; S3. Disperse the probiotic suspension into solution 1 and stir to obtain solution 3; preferably, the probiotic is Lactobacillus acidophilus; S4. Add solutions 2 and 3 dropwise to pure water simultaneously and then perform ultrasonic coagulation. S5. Add calcium chloride solution, sonicate to solidify, remove water layer to obtain wet microcapsules, vacuum freeze dry and grind into powder to obtain myofibrillar protein-sunflower seed oil-high M-type alginic acid ternary complex probiotic microcapsules.

[0011] Furthermore, in step S1, the mass fraction of myofibrillar protein and sodium alginate is 2%, the shearing rate is 7000 r / min, and the shearing time is 2 min.

[0012] Furthermore, in step S2, the mass fraction of gelatin in pure water is 1%, the mass fraction of sunflower seed oil in the gelatin solution is 2%, the shearing speed is 7000 r / min, and the shearing time is 1 min.

[0013] Furthermore, the probiotic suspension is obtained by resuspending probiotics in 0.85% sterile physiological saline; In a specific embodiment, the probiotic suspension was prepared as follows: a single colony was inoculated into 5 mL of liquid culture medium and cultured at 37°C until the bacteria covered the bottom of the test tube. After the culture was completed, the culture was centrifuged at 4°C and 4000 rpm for 6 min, the supernatant was discarded, the bacterial precipitate was collected, and the precipitate was resuspended in 3 mL of 0.85% sterile physiological saline.

[0014] Furthermore, in step S3, the mass ratio of the probiotic suspension to solution 1 is 1:9, the stirring speed is 200 r / min, and the stirring time is 20 min.

[0015] Furthermore, in step S4, the mass ratio of solution 2 to solution 3 is 1:1, the volume ratio of solution 2 and solution 3 to pure water is 1:10, and the ultrasonic time is 20 min.

[0016] Furthermore, in step S5, the concentration of the calcium chloride solution is 3%, the volume of the calcium chloride solution is 10% of the total system volume, and the ultrasonic time is 40 min.

[0017] Furthermore, in step S5, the vacuum freeze-drying temperature is -60~80℃, and the time is 48 h. Beneficial effects

[0018] (1) This invention employs microencapsulation technology to encapsulate probiotics layer by layer, constructing a ternary targeted delivery system with high encapsulation rate and good delivery effect through myofibrillar protein, sunflower seed oil, and high M-type sodium alginate. Myofibrillar protein and high M-type sodium alginate are combined as the inner wall material of the microcapsule, while sunflower seed oil and gelatin are combined as the outer wall material of the microcapsule. The double-layer encapsulation provides a good isolation and protection effect for probiotics, effectively enhancing the resistance of probiotics to adverse environments in the digestive tract, thereby delaying the exposure time of probiotics and achieving targeted release of probiotics into the intestine.

[0019] (2) The addition of high-M-type sodium alginate, sunflower seed oil, and gelatin increased the probiotic encapsulation rate from 62% to 91%. The microcapsules encapsulated with high-M-type sodium alginate and sunflower seed oil had a particle size of 515 μm, which is 6.7 times the particle size of protein monolayer encapsulation. Furthermore, the myofibrillar protein-sunflower seed oil-high-M-type alginate ternary complex probiotic microcapsules significantly improved the heat resistance and storage properties of the probiotics. In particular, after heating in an 80℃ water bath for 2 min, the number of viable probiotics increased from 3.7 Log CFU / mL to 8.0 Log CFU / mL, and after storage at 4℃ for 35 days, the number of viable probiotics increased from 4.2 Log CFU / mL to 6.7 Log CFU / mL. This invention significantly improved the probiotic encapsulation rate and formed larger particle sizes, laying a foundation for efficient carrier delivery to the intestines.

[0020] (3) This invention effectively solves the problem of probiotic inactivation in gastric juice. Compared with free bacteria and myofibrillar protein encapsulation alone, the introduction of high-M sodium alginate and sunflower seed oil increases the survival rate of probiotics in the gastric stage of the microcapsule samples from 0% to 87%, and the survival rate after gastrointestinal infection from 0% to 82%. In addition, this invention is simpler than spray drying, extrusion, and spraying technologies, and is suitable for large-scale production. Attached Figure Description

[0021] Figure 1 This is a comparison of the encapsulation rate of probiotic microcapsules in Example 1 and Comparative Examples 2-9.

[0022] Figure 2 This is a comparison of the particle size of probiotic microcapsules in Example 1 and Comparative Examples 2-9.

[0023] Figure 3 This is a comparison of the rheological properties of probiotic microcapsules in Example 1 and Comparative Examples 2-9.

[0024] Figure 4 This is a comparison of the thermal stability of the probiotic microcapsules in Example 1 and Comparative Examples 1-9.

[0025] Figure 5 This is a comparison of the storage stability of probiotic microcapsules in Example 1 and Comparative Examples 1-9.

[0026] Figure 6 This is a comparison of the digestion survival rate of probiotic microcapsules in Example 1 and Comparative Examples 1-9.

[0027] Figure 7 These are laser confocal microscopy images of the probiotic microcapsule digests from Example 1 and Comparative Examples 7-9. Detailed Implementation

[0028] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0029] The following examples and comparative examples use: The probiotic is Lactobacillus acidophilus, strain number BNCC336636.

[0030] High-M sodium alginate is defined as sodium alginate with an M content of over 60% (Manufacturer: Shanghai Maclean Biochemical Technology Co., Ltd., Model: S749906-100g).

[0031] High-G sodium alginate has a G content of over 60% (manufacturer: Shanghai Maclean Biochemical Technology Co., Ltd., model: S749909-100g).

[0032] Sunflower seed oil (manufacturer: Yihai Kerry Arawana Food Group Co., Ltd.).

[0033] The sunflower seed oil oil gel was prepared by dissolving sitosterol (manufacturer: Shanghai Aladdin Biochemical Technology Co., Ltd., model: R014997) and lecithin (manufacturer: Shanghai Aladdin Biochemical Technology Co., Ltd., model: R002341-25g) in sunflower seed oil (manufacturer: Yihai Kerry Arawana Food Group Co., Ltd.) and stirring at 90℃ for 40 minutes. The sunflower seed oil oil gel was then stored at 4℃ overnight before use. The mass ratio of sitosterol:lecithin:sunflower seed oil was 16:4:105. Example

[0034] Myofibrillar protein was extracted, and 2% of myofibrillar protein was dissolved in phosphate buffer. 2% high-M sodium alginate was added and dissolved completely. The solution was then sheared at 7000 r / min for 2 min using a high-speed disperser to obtain solution 1.

[0035] Dissolve 1% gelatin in pure water, add 2% sunflower seed oil, and shear at high speed for 1 min at 7000 r / min to obtain solution 2.

[0036] Preparation of probiotic suspension: Inoculate a single colony into 5 mL of liquid culture medium and incubate at 37°C until the bacteria cover the bottom of the test tube. After incubation, centrifuge the culture solution at 4°C and 4000 rpm for 6 min, discard the supernatant, collect the bacterial precipitate, and resuspend it in 3 mL of 0.85% sterile physiological saline.

[0037] The bacterial suspension was dispersed in solution 1 (1:9) and stirred at 200 rpm for 20 min to obtain solution 3.

[0038] Place a beaker containing 200 mL of distilled water in an ultrasonic instrument, and simultaneously add solution 2 and solution 3 (1:1) to the beaker. The amount of solution 2 and solution 3 added is 20 mL each. Perform ultrasonic coagulation for 20 min.

[0039] Add 10% of the total volume of the system to a 3% calcium chloride solution, cure under ultrasonic conditions for 40 min, remove the water layer to obtain wet microcapsules, freeze-dry them and grind them into powder. Vacuum freeze-drying temperature is -60~80℃ and time is 48 h.

[0040] Comparative Example 1: Free bacteria Inoculate a single colony into 5 mL of liquid culture medium and incubate at 37°C until the bacteria cover the bottom of the test tube. After incubation, centrifuge the culture solution at 4°C and 4000 rpm for 6 min, discard the supernatant, collect the bacterial precipitate, and resuspend it in 3 mL of 0.85% sterile physiological saline to prepare a probiotic suspension.

[0041] Comparative Example 2: Myofibrillar protein encapsulation alone Myofibrillar protein was extracted, and 2% of myofibrillar protein was dissolved in phosphate buffer. The solution was then sheared at 7000 r / min for 2 min using a high-speed disperser to obtain solution 1.

[0042] The bacterial suspension was dispersed in solution 1 (1:9) and stirred at 200 rpm for 20 min to obtain solution 2.

[0043] Place a beaker containing 200 mL of distilled water in an ultrasonic instrument, add 20 mL of solution 2 to the beaker, and sonicate for 20 min. Add 10% of the total volume of the system with 3% calcium chloride solution, and solidify under ultrasonic conditions for 40 min. Remove the water layer to obtain wet microcapsules, freeze-dry them, and then grind them into powder.

[0044] Comparative Example 3: Myofibrillar protein encapsulated with high-G sodium alginate Myofibrillar protein was extracted, and 2% of myofibrillar protein was dissolved in phosphate buffer. 2% high-G sodium alginate was added and dissolved completely. The solution was then sheared at 7000 r / min for 2 min using a high-speed disperser to obtain solution 1.

[0045] The bacterial suspension was dispersed in solution 1 (1:9) and stirred at 200 rpm for 20 min to obtain solution 2.

[0046] Place a beaker containing 200 mL of distilled water in an ultrasonic instrument, add 20 mL of solution 2 to the beaker, and sonicate for 20 min. Add 10% of the total volume of the system with 3% calcium chloride solution, and solidify under ultrasonic conditions for 40 min. Remove the water layer to obtain wet microcapsules, freeze-dry them, and then grind them into powder.

[0047] Comparative Example 4: Myofibrillar protein encapsulated with high-M sodium alginate Myofibrillar protein was extracted, and 2% of myofibrillar protein was dissolved in phosphate buffer. 2% high-M sodium alginate was added and dissolved completely. The solution was then sheared at 7000 r / min for 2 min using a high-speed disperser to obtain solution 1.

[0048] The bacterial suspension was dispersed in solution 1 (1:9) and stirred at 200 rpm for 20 min to obtain solution 2.

[0049] Place a beaker containing 200 mL of distilled water in an ultrasonic instrument, add 20 mL of solution 2 to the beaker, and sonicate for 20 min. Add 10% of the total volume of the system with 3% calcium chloride solution, and solidify under ultrasonic conditions for 40 min. Remove the water layer to obtain wet microcapsules, freeze-dry them, and then grind them into powder.

[0050] Comparative Example 5: Comparative Example without Sunflower Seed Oil and Replaced with High-G Sodium Alginate Myofibrillar protein was extracted, and 2% of myofibrillar protein was dissolved in phosphate buffer. 2% high-G sodium alginate was added and dissolved completely. The solution was then sheared at 7000 r / min for 2 min using a high-speed disperser to obtain solution 1.

[0051] Dissolve 1% gelatin in pure water and shear at high speed at 7000 r / min for 1 min to obtain solution 2.

[0052] The bacterial suspension was dispersed in solution 1 (1:9) and stirred at 200 rpm for 20 min to obtain solution 3.

[0053] Place a beaker containing 200 mL of distilled water in an ultrasonic instrument, and simultaneously add 20 mL each of solution 2 and solution 3 (1:1) to the beaker. Sonicate for 20 min. Add 10% of the total volume of the system with 3% calcium chloride solution, and solidify under ultrasonic conditions for 40 min. Remove the water layer to obtain wet microcapsules, freeze-dry them, and then grind them into powder.

[0054] Comparative Example 6: Comparative Example without Sunflower Seed Oil Myofibrillar protein was extracted, and 2% of myofibrillar protein was dissolved in phosphate buffer. 2% high-M sodium alginate was added and dissolved completely. The solution was then sheared at 7000 r / min for 2 min using a high-speed disperser to obtain solution 1.

[0055] Dissolve 1% gelatin in pure water and shear at high speed at 7000 r / min for 1 min to obtain solution 2.

[0056] The bacterial suspension was dispersed in solution 1 (1:9) and stirred at 200 rpm for 20 min to obtain solution 3.

[0057] Place a beaker containing 200 mL of distilled water in an ultrasonic instrument, and simultaneously add 20 mL each of solution 2 and solution 3 (1:1) to the beaker. Sonicate for 20 min. Add 10% of the total volume of the system with 3% calcium chloride solution, and solidify under ultrasonic conditions for 40 min. Remove the water layer to obtain wet microcapsules, freeze-dry them, and then grind them into powder.

[0058] Comparative Example 7 was replaced with a comparative example containing high-G sodium alginate. Myofibrillar protein was extracted, and 2% of myofibrillar protein was dissolved in phosphate buffer. 2% high-G sodium alginate was added and dissolved completely. The solution was then sheared at 7000 r / min for 2 min using a high-speed disperser to obtain solution 1.

[0059] Dissolve 1% gelatin in pure water, add 2% sunflower seed oil, and shear at high speed for 1 min at 7000 r / min to obtain solution 2.

[0060] The bacterial suspension was dispersed in solution 1 (1:9) and stirred at 200 rpm for 20 min to obtain solution 3.

[0061] Place a beaker containing 200 mL of distilled water in an ultrasonic instrument, and simultaneously add 20 mL each of solution 2 and solution 3 (1:1) to the beaker. Sonicate for 20 min. Add 10% of the total volume of the system with 3% calcium chloride solution, and solidify under ultrasonic conditions for 40 min. Remove the water layer to obtain wet microcapsules, freeze-dry them, and then grind them into powder.

[0062] Comparative Example 8: The comparative example where high-G sodium alginate was replaced and sunflower seed oil oil gel was used instead of sunflower seed oil. Myofibrillar protein was extracted, and 2% of myofibrillar protein was dissolved in phosphate buffer. 2% high-G sodium alginate was added and dissolved completely. The solution was then sheared at 7000 r / min for 2 min using a high-speed disperser to obtain solution 1.

[0063] Dissolve 1% gelatin in pure water, add 2% sunflower seed oil oleogel, and shear at high speed for 1 min at 7000 r / min to obtain solution 2.

[0064] The bacterial suspension was dispersed in solution 1 (1:9) and stirred at 200 rpm for 20 min to obtain solution 3.

[0065] Place a beaker containing 200 mL of distilled water in an ultrasonic instrument, and simultaneously add 20 mL each of solution 2 and solution 3 (1:1) to the beaker. Sonicate for 20 min. Add 10% of the total volume of the system with 3% calcium chloride solution, and solidify under ultrasonic conditions for 40 min. Remove the water layer to obtain wet microcapsules, freeze-dry them, and then grind them into powder.

[0066] Comparative Example 9: A comparative example where sunflower seed oil oil gel was used to replace sunflower seed oil. Myofibrillar protein was extracted, and 2% of myofibrillar protein was dissolved in phosphate buffer. 2% high-M sodium alginate was added and dissolved completely. The solution was then sheared at 7000 r / min for 2 min using a high-speed disperser to obtain solution 1.

[0067] Dissolve 1% gelatin in pure water, add 2% sunflower seed oil oleogel, and shear at high speed for 1 min at 7000 r / min to obtain solution 2.

[0068] The bacterial suspension was dispersed in solution 1 (1:9) and stirred at 200 rpm for 20 min to obtain solution 3.

[0069] Place a beaker containing 200 mL of distilled water in an ultrasonic instrument, and simultaneously add 20 mL each of solution 2 and solution 3 (1:1) to the beaker. Sonicate for 20 min. Add 10% of the total volume of the system with 3% calcium chloride solution, and solidify under ultrasonic conditions for 40 min. Remove the water layer to obtain wet microcapsules, freeze-dry them, and then grind them into powder.

[0070] Performance testing After preparing the probiotic microcapsules, the encapsulation efficiency, particle size, and rheological properties of Comparative Examples 2-9 were compared with those of Examples 1 after different encapsulation methods. Thermal stability and digestibility were also measured. Laser confocal microscopy images of the digestate from Comparative Examples 7-9 were compared with those from Examples 1.

[0071] Experimental data are presented as mean ± standard deviation and analyzed using analysis of variance (Duncan's test) in SPSS software (IBM SPSS Statistics 25.0, America). A p-value less than 0.05 was considered statistically significant. All experimental indicators were performed three times.

[0072] The detection method for the aforementioned indicator is as follows: 1. Encapsulation rate Microcapsules were added to a certain volume of phosphate buffer solution, and then sheared at 5000 r / min for 2 min to completely release the encapsulated probiotics. The mixture was then diluted and inoculated onto MRS agar, and the viable count was calculated after incubation at 37℃ for 48 h. The encapsulation efficiency was calculated using the following formula: Encapsulation rate = N / N0 × 100% Where: N is the number of viable bacteria embedded in the microcapsule (Log CFU / mL); N0 is the total number of viable bacteria in the bacterial suspension before embedding (Log CFU / mL).

[0073] The results showed the effect of layer-by-layer encapsulation on the encapsulation rate of probiotic microcapsules: The encapsulation rates of probiotic microcapsules in Example 1 and Comparative Examples 2-9 are as follows: Figure 1 As shown. Compared to the 62% encapsulation rate of Comparative Example 2 (encapsulation of myofibrillar protein alone), the encapsulation rate of Comparative Example 3 (with high G-type sodium alginate) increased to 78%, while that of Comparative Example 4 (with high M-type sodium alginate) further increased to 88%. Adding gelatin to this further increased the encapsulation rate of Comparative Example 5 to 84% and Comparative Example 6 to 89%. Further addition of sunflower seed oil resulted in an encapsulation rate of 89% for Comparative Example 7, while the encapsulation rate of Example 1 reached the highest value of 91%. Furthermore, Comparative Examples 8 and 9 replaced sunflower seed oil with sunflower seed oil oleogel, achieving encapsulation rates of 71% and 88%, respectively. Therefore, the microcapsules prepared using high M-type sodium alginate in Example 1 showed significantly better encapsulation performance than those prepared using high G-type sodium alginate in Comparative Example 7. Moreover, with the same type of sodium alginate in Example 1, the microcapsules prepared using sunflower seed oil increased the encapsulation rate from 88% to 91% compared to the microcapsules prepared using sunflower seed oil oleogel in Comparative Example 9. This indicates that the addition of high-M sodium alginate and sunflower seed oil has a synergistic enhancing effect, which is more conducive to the construction of a high encapsulation rate delivery system.

[0074] 2. Particle size The particle size of the microcapsules was evaluated using a laser particle size analyzer. The sample was added to the measuring cell with an opacity between 2% and 8%. The refractive index and absorptivity were 1.436 and 0.001, respectively. The dispersant was deionized water with a refractive index of 1.33, and the temperature was 25°C. The size was denoted as D4.3.

[0075] The results showed that layer-by-layer encapsulation had an effect on the particle size of probiotic microcapsules: The probiotic microcapsule particle sizes in Example 1 and Comparative Examples 2-9 are as follows: Figure 2As shown. Compared to Example 1, with a particle size of 515.00 μm, Comparative Example 2, using only myofibrillar protein monolayer encapsulation, had a particle size of 76.30 μm; Comparative Example 7, using high-G-type sodium alginate instead, had a particle size of 394.50 μm; and Comparative Example 9, using sunflower seed oil oleoglucon instead of sunflower seed oil, had a particle size of 371.00 μm. The particle size of Example 1 was approximately 6.7 times that of Comparative Example 2, and significantly higher than Comparative Examples 3-9, indicating that the composite encapsulation strategy of high-M-type sodium alginate and sunflower seed oil helps to form microcapsules with larger particle sizes. To a certain extent, the larger the microcapsule particle size, the better the protective effect on probiotics. This suggests that microcapsules with high-M-type sodium alginate and sunflower seed oil can significantly improve the protective effect on probiotics.

[0076] 3. Rheological properties The measurements were performed using a rotational rheometer with a 50 mm parallel plate, a 1 mm gap, and the sample was kept isothermally at 25°C. The measurement mode and parameters were: strain scan range 0.01–100 s. -1 The apparent viscosity was measured at a scanning frequency of 1 Hz.

[0077] The results showed the effect of layer-by-layer encapsulation on the rheological properties of probiotic microcapsules: The rheological properties of the probiotic microcapsules in Example 1 and Comparative Examples 2-9 are as follows: Figure 3 As shown, the apparent viscosity of all samples tended to decrease with increasing shear rate, exhibiting shear thinning characteristics. Compared to Comparative Example 2, the apparent viscosity of Examples 1 and Comparative Examples 3-9 increased with the addition of sodium alginate, gelatin, and oil. This indicates that the addition of sodium alginate, gelatin, and sunflower seed oil helps improve the storage stability of the microcapsules.

[0078] 4. Laser confocal The oil phase of the samples was stained with Nile Red, the protein with Nile Blue, and the probiotics with Syto9. 20 μL of each dye was added to each sample (200 μL) and reacted in the dark for 5 min. A small amount of sample was evenly spread on a glass slide, immediately covered with a coverslip, and imaged at 20x magnification to observe the distribution of oil, protein, and probiotics within the microcapsules.

[0079] Laser confocal microscopy images of the digestive contents of myofibrillar protein-sunflower seed oil-high M-type alginic acid ternary complex probiotic microcapsules show: Laser confocal microscopy images of the probiotic microcapsule digests in Example 1 and Comparative Examples 7-9 are shown below. Figure 7As shown in the figure. Compared with Comparative Examples 8-9, the microcapsules made with sunflower seed oil in Examples 1 and 7 had a larger volume after gastric digestion, maintained better structural integrity, and had a better encapsulation effect; compared with Comparative Example 7, the microcapsules made with high-M sodium alginate in Example 1 had a higher survival rate of probiotics after gastrointestinal digestion. This indicates that the microcapsules constructed from high-M sodium alginate and sunflower seed oil have the best protective effect on probiotics.

[0080] 5. Thermal stability The bacteria or microcapsules were added to test tubes and incubated in water baths at 60℃, 70℃, and 80℃ for 2 min. After cooling, the microcapsules were subjected to high-speed shearing at 5000 r / min for 2 min to evaluate the effect of temperature on the activity of probiotics.

[0081] The results showed the effect of layer-by-layer encapsulation on the thermal stability of probiotic microcapsules: The thermal stability of the probiotic microcapsules in Example 1 and Comparative Examples 1-9 is as follows: Figure 4 As shown, the multilayered encapsulation of probiotics exhibited higher survival rates under different temperature conditions. Compared to Comparative Example 1, in Example 1, the number of viable probiotics increased from 6.76 Log CFU / mL to 8.61 Log CFU / mL after heating in a 60°C water bath, from 6.75 Log CFU / mL to 8.17 Log CFU / mL after heating in a 70°C water bath, and from 3.73 Log CFU / mL to 8.02 Log CFU / mL after heating in an 80°C water bath, indicating that the multilayered encapsulation structure effectively enhanced the thermal stability of the probiotic microcapsules. Compared to Comparative Example 7, in Example 1, the number of viable probiotics increased from 7.40 Log CFU / mL to 8.02 Log CFU / mL after heating in a 80°C water bath, indicating that the microcapsules made with high-M type sodium alginate had a significantly higher survival rate after heat treatment than those made with high-G type sodium alginate. The results indicate that the microcapsules constructed from high-M sodium alginate and sunflower seed oil have superior heat resistance.

[0082] Storage stability Free probiotics and microcapsules containing probiotics were aliquoted into sterile plastic centrifuge tubes (containing 10 mL of sterile saline) and stored at 4°C for 35 days, with samples taken every 7 days. The viable count of probiotics was calculated to assess the storage stability of the microcapsules.

[0083] The results showed the effect of layer-by-layer encapsulation on the storage stability of probiotic microcapsules: The storage stability of the probiotic microcapsules in Example 1 and Comparative Examples 1-9 is as follows: Figure 5As shown, with the increase in the number of encapsulation layers, the difference in viable bacterial count before and after 35 days of storage gradually decreased, indicating that layer-by-layer encapsulation helps improve storage stability. Compared with Comparative Example 7, the viable bacterial count in Example 1 decreased by only 0.57 Log CFU / mL after 35 days of storage, equivalent to 34% of the decrease in Comparative Example 7, indicating that the microcapsules made with high-M type sodium alginate have significantly better storage stability at 4°C than those made with high-G type sodium alginate. Compared with Comparative Example 9, after 7 days of storage, the viable bacterial count in the microcapsules made with sunflower seed oil was significantly higher than that in the microcapsules made with sunflower seed oil gel. In summary, the microcapsules constructed with high-M type sodium alginate and sunflower seed oil exhibit excellent storage stability.

[0084] 7. Digestion survival rate Simulated gastric juice (SGF) and simulated intestinal juice (SIF) were prepared first. A sample (1.0 g) was mixed with 8 mL of SGF. Each sample was mixed with hydrochloric acid (6 M) to adjust the pH to 2.0, and then 1 mL of pepsin solution was added, achieving a pepsin activity of 2000 U / mL. The mixture was then transferred to a shaking incubator to simulate gastric digestion. After completion, it was mixed with an equal volume of SIF to inactivate pepsin, and viable bacteria were counted. The remaining 5 mL of digestive fluid from each group was mixed with 4 mL of SIF, and sodium hydroxide solution (2 M) was added to adjust the pH to 7.0. Then, 0.5 mL of pancreatic enzyme solution was added, followed by 0.5 mL of bile salt solution (124.2 mg / mL), for a total volume of 10 mL, achieving a pancreatic enzyme activity of 100 U / mL. These mixtures were incubated in a shaking incubator for 2 hours to simulate intestinal digestion, and viable bacteria were counted.

[0085] The results showed the effect of layer-by-layer encapsulation on the digestibility and survival rate of probiotic microcapsules: The digestion survival rate of probiotic microcapsules in Example 1 and Comparative Examples 1-9 is as follows: Figure 6 As shown in the figure. Compared with Comparative Examples 1-2, the number of surviving probiotics in Example 1 increased from 0 Log CFU / mL during gastric digestion to a maximum of 6.41 Log CFU / mL, and after gastrointestinal digestion, the number of surviving probiotics increased from 0 Log CFU / mL to a maximum of 6.04 Log CFU / mL, indicating that layer-by-layer encapsulation significantly improved the survival ability of probiotics in the gastrointestinal digestive environment. With the increase of the number of encapsulation layers, compared with Comparative Examples 3-9, Example 1 showed the highest digestive survival rate, with the number of viable bacteria decreasing by only 1.34 Log CFU / mL after gastrointestinal digestion. The survival rate of probiotics during gastric digestion increased from 0% to 87%, and the survival rate after gastrointestinal digestion increased from 0% to 82%. This indicates that the microcapsules constructed using high-M sodium alginate and sunflower seed oil are most effective in enhancing digestive fluid tolerance.

[0086] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A ternary complex probiotic microcapsule of myofibrillar protein-sunflower seed oil-high M-type alginate, characterized in that, It includes a core material and a double-layer composite wall material. The core material is probiotics, the inner composite wall material includes myofibrillar protein and high-M sodium alginate, and the outer composite wall material includes gelatin and sunflower seed oil. Preferably, the probiotics are Lactobacillus acidophilus.

2. The myofibrillar protein-sunflower seed oil-high M-type alginate ternary complex probiotic microcapsule according to claim 1, characterized in that, High-M type alginate is high-mannuronic acid type sodium alginate with an M content of over 60%.

3. The myofibrillar protein-sunflower seed oil-high M-type alginate ternary complex probiotic microcapsule according to claim 1, characterized in that, The mass ratio of myofibrillar protein, high-M sodium alginate, gelatin, and sunflower seed oil is 2:2:1:

2.

4. The preparation method of the myofibrillar protein-sunflower seed oil-high M-type alginate ternary complex probiotic microcapsule according to claim 1, characterized in that, Includes the following steps: S1. Dissolve myofibrillar protein in phosphate buffer, add high-M sodium alginate and dissolve it completely, then shear thoroughly to obtain solution 1; S2. Dissolve gelatin in pure water to prepare a gelatin solution, add sunflower seed oil, and shear thoroughly to obtain solution 2; S3. Disperse the probiotic suspension into solution 1 and stir to obtain solution 3; preferably, the probiotic is Lactobacillus acidophilus; S4. Add solutions 2 and 3 dropwise to pure water simultaneously and then perform ultrasonic coagulation. S5. Add calcium chloride solution, sonicate to solidify, remove water layer to obtain wet microcapsules, vacuum freeze dry and grind into powder to obtain myofibrillar protein-sunflower seed oil-high M-type alginic acid ternary complex probiotic microcapsules.

5. The preparation method according to claim 2, characterized in that, In step S1, the mass fraction of myofibrillar protein and high-M sodium alginate is 2%, the shearing rate is 7000 r / min, and the shearing time is 2 min.

6. The preparation method according to claim 2, characterized in that, In step S2, the mass fraction of gelatin in pure water is 1%, the mass fraction of sunflower seed oil in the gelatin solution is 2%, the shearing speed is 7000 r / min, and the shearing time is 1 min.

7. The preparation method according to claim 2, characterized in that, In step S3, the probiotic suspension is obtained by resuspending probiotics in 0.85% sterile saline; the stirring speed is 200 r / min and the stirring time is 20 min.

8. The preparation method according to claim 2, characterized in that, In step S4, the mass ratio of solution 2 to solution 3 is 1:1, the volume ratio of solution 2 and solution 3 to pure water is 1:10, and the ultrasonic time is 20 min.

9. The preparation method according to claim 2, characterized in that, In step S5, the concentration of the calcium chloride solution is 3%, the volume of the calcium chloride solution is 10% of the total volume of the system, and the ultrasonic time is 40 min.

10. The preparation method according to claim 2, characterized in that, In step S5, the vacuum freeze-drying temperature is -60~80℃ and the time is 48 h.

Images