Preparation method of pumpkin seed oil-probiotic composite microcapsule capable of balancing polyunsaturated fatty acids

Abstract

The invention discloses a preparation method of a pumpkin seed oil-probiotic composite microcapsule capable of balancing polyunsaturated fatty acids, and relates to the technical field of biological materials. Comprising a composite microcapsule, and the composite microcapsule takes mixed pressed oil and probiotics as a core material and takes a whey protein and Arabic gum mixture as a wall material; the core material is embedded in the wall material; the mixed pressed oil comprises pumpkin seed oil and linseed oil; the preparation method comprises the following steps: S1, preparing balanced polyunsaturated fatty acid mixed pressed oil; s2, preparing a microcapsule wall material; s3, activating and preparing the probiotics; and S4, preparing the microcapsule. The invention provides the preparation method of the composite microcapsule capable of accurately delivering the probiotics to a target position.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological materials, and particularly relates to a preparation method of balanced polyunsaturated fatty acid pumpkin seed oil-probiotic compound microcapsules. BACKGROUND

[0002] Probiotics are generally defined as live microorganisms that have a positive effect on the health of the host when ingested in sufficient quantities. According to the provisions of the Food and Agriculture Organization, probiotics need to exceed the minimum limit of 10 6 ~ 10 7 CFU / g to achieve their intended effect in the product shelf life and in the intestinal digestive system. Probiotics have many health benefits, such as protecting the natural microbial community of the intestinal tract, enhancing the immune system, and reducing blood cholesterol. However, probiotics are very sensitive to oxygen, temperature, the gastrointestinal tract, and adverse conditions during processing and transportation. Therefore, in the food industry, it is of great significance to maintain the viability of probiotics during food processing, production, and storage, and to safely release them in the intestine.

[0003] There has been a lot of research on microcapsule delivery technology, but there is still a lack of specialized microcapsule delivery carriers for delivering probiotics to specific locations while maintaining good activity.

[0004] Therefore, in order to solve the above technical problems, a new technical solution is needed to solve this technical problem, especially a preparation method of balanced polyunsaturated fatty acid pumpkin seed oil-probiotic compound microcapsules. SUMMARY

[0005] The present application aims to solve the technical problem of how to deliver probiotics to specific locations for release while ensuring good activity of probiotics, and provides a preparation method of balanced polyunsaturated fatty acid pumpkin seed oil-probiotic compound microcapsules.

[0006] In order to achieve the above-mentioned purpose, the present application provides the following technical solution:

[0007] The present application provides a preparation method of balanced polyunsaturated fatty acid pumpkin seed oil-probiotic compound microcapsules: characterized in that: it comprises compound microcapsules, the compound microcapsules take mixed pressed oil and probiotics as core material, and take whey protein and gum arabic mixture as wall material; the core material is wrapped in the wall material; the mixed pressed oil comprises pumpkin seed oil and flaxseed oil;

[0008] The preparation method of the compound microcapsules comprises the following steps:

[0009] S1. Preparation of balanced polyunsaturated fatty acid mixed pressed oil;

[0010] S11. Pumpkin seeds and flax seeds are weighed according to a mass ratio of 4:1-6:1, the weighed pumpkin seeds and flax seeds are mixed, and mixed pressed oil is prepared by pressing;

[0011] S2. Preparation of microcapsule wall material;

[0012] S21. Whey protein and gum arabic are selected as the microcapsule wall material;

[0013] S22. The whey protein is weighed and placed in distilled water for stirring at room temperature, and a whey protein solution is obtained after complete hydration; the whey protein solution is pretreated to prepare a whey protein stock solution;

[0014] The pretreatment method is as follows: 1Mol / L NaOH solution is added dropwise to the whey protein solution to adjust the pH to 12, the whey protein solution is heated to 65℃ after being static for 30 min, the protein is unfolded and refolded, and then cooled to room temperature, 1Mol / L HCl solution is added dropwise to adjust the pH to 7, and the whey protein stock solution is obtained by centrifugation and taking the supernatant;

[0015] S23. Preparation of gum arabic stock solution;

[0016] S24. The whey protein solution and the gum arabic solution are weighed, mixed, homogenized, and 10% citric acid solution is added dropwise to adjust the pH to 3.6, and the compound is placed at 4℃ overnight to fully hydrate;

[0017] S3. Activation and preparation of probiotics;

[0018] S31. Lactobacillus rhamnosus is selected as the probiotic strain;

[0019] S32. The frozen Lactobacillus rhamnosus is inoculated on a sterile culture medium, and after culture and growth, it is inoculated again on the liquid culture medium for batch culture, and the cell mass is prepared by centrifugation in the logarithmic growth phase, and the concentrated bacterial solution is prepared by washing the precipitate with sterile normal saline after centrifugation;

[0020] S33. The concentrated bacterial solution is divided into two equal parts;

[0021] S4. Preparation of microcapsules;

[0022] S41. The concentrated bacterial solution and the mixed pressed oil are dispersed into the whey protein stock solution, and after homogenization;

[0023] S42. The gum arabic stock solution is gradually added under the homogenization environment; 10% citric acid solution is further added to adjust the pH to 3.6, so as to induce the complexation between the whey protein stock solution and the gum arabic stock solution;

[0024] S43. Add glutamine transaminase 20 U / g, heat and stir at 37℃ for 2h to fully solidify, and then freeze-dry the final emulsion to obtain solid composite microcapsules.

[0025] Preferably, the mass ratio of pumpkin seeds to flax seeds is 5-7.5:1.

[0026] Preferably, the step S22 specifically comprises: dissolving whey protein in distilled water, stirring at room temperature for 1h to fully hydrate, and obtaining a 3% whey protein solution.

[0027] Preferably, the step S23 specifically comprises: dissolving gum arabic in an appropriate amount of distilled water, stirring at 50℃ for 3h, and then hydrating overnight at 4℃ to obtain a 3% gum arabic stock solution.

[0028] Preferably, the step S32 specifically comprises: taking Lactobacillus rhamnosus frozen at -80℃, streaking on sterile solid medium, and culturing at 37℃ for 48h; picking a single colony and placing it in sterile liquid medium for anaerobic culture at 37℃ for 16h; inoculating the activated bacterial solution into liquid medium at a 1% inoculation amount for batch culture; centrifuging the cell mass at 4℃ during the logarithmic growth phase to discard the supernatant and wash the precipitate twice with sterile normal saline to obtain a concentrated bacterial solution, which has a concentration of about 10 10 CFU / mL.

[0029] Preferably, the concentrated bacterial solution of S33 is divided into two equal parts.

[0030] Preferably, it further comprises mixing pressed oil microcapsules and Lactobacillus rhamnosus microcapsules; the core material of the mixed pressed oil microcapsules comprises the mixed pressed oil in step S1, and the core material of the Lactobacillus rhamnosus microcapsules comprises the probiotics in step S3; the wall material in the mixed pressed oil microcapsules and the Lactobacillus rhamnosus microcapsules is the microcapsule wall material in step S2; and the mixed pressed oil microcapsules and the Lactobacillus rhamnosus microcapsules are used as a control for the embedding rate and viable bacterial count parameters of composite microcapsules.

[0031] Preferably, the preparation method of the mixed pressed oil microcapsules comprises: dispersing the mixed pressed oil into the whey protein stock solution, homogenizing at 7000rpm for 3min; then gradually adding the gum arabic stock solution under continuous stirring at 400rpm; adding 10% citric acid solution drop by drop to adjust the pH to 3.6 to induce complexation between whey protein and gum arabic; and adding glutamine transaminase 20 U / g, heating and stirring at 37℃ for 2h to fully solidify to obtain an emulsion.

[0032] Preferably, the preparation method of the Lactobacillus rhamnosus microcapsule is that the concentrated bacterial solution is dispersed into a whey protein stock solution, homogenized at 7000 rpm for 3 min; then the gum arabic stock solution is gradually added under continuous stirring at 400 rpm; a 10% citric acid solution is added dropwise to adjust the pH to 3.6 to induce complexation between the whey protein stock solution and the gum arabic stock solution; 20 U / g of transglutaminase is added, and heating and stirring are carried out at 37°C for 2 h for sufficient solidification, and finally the solid microcapsules are prepared by vacuum freeze-drying of the final milk emulsion.

[0033] The present application has the following advantages:

[0034] The present application uses mixed pressed oil and probiotics as core materials embedded in the microcapsule wall material, and the probiotics are Lactobacillus rhamnosus. The probiotics can tolerate the environment of the animal digestive tract and successfully colonize in the intestines of humans and animals, and have the functional properties of regulating intestinal flora balance, preventing and treating diarrhea, promoting toxin elimination, improving overall immunity, and preventing dental caries. The mixed pressed oil is a balanced polyunsaturated fatty acid oil, which is produced by pressing after mixing pumpkin seeds and flaxseeds. The mass ratio of pumpkin seeds to flaxseeds is 5 to 7.5, so that the ω-6 and ω-3 unsaturated fatty acids in the mixed oil meet the recommended ratio of 4-6:1.

[0035] The oil and fat can only be digested by lipase in the intestine to allow the probiotics to be released near the action site in the intestine, ensuring that the delivered probiotics are released in the vicinity of the intestine.

[0036] In addition to the nutritional ingredients, the two embedded materials also have a synergistic effect. The present application further improves the embedding rate of probiotics and the activity of probiotics by increasing the mixed pressed oil as the load of probiotics and embedding it in the microcapsule. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is the relative content of fatty acids of pumpkin seed oil and flaxseed oil of the present application.

[0038] Figure 2 is a verification comparison data graph of ω-6 / ω-3 fatty acids of the mixed pressed oil of the present application.

[0039] Figure 3 is the Fourier infrared spectrum of the composite microcapsule (WP-L-O-GA 65) of the present application.

[0040] Figure 4 is the scanning electron microscope graph of the composite microcapsule (WP-L-O-GA 65) of the present application.

[0041] Figure 5 is the laser confocal scanning microscope graph of the composite microcapsule (WP-L-O-GA 65) of the present application.

[0042] Figure 6 Figure 1 is a schematic diagram of the PV value (A) and p-AV value (B) of the oil during storage of the composite microcapsule (WP-L-O-GA 65) of the present application.

[0043] Figure 7 Figure 2 is a schematic diagram of the viable count of probiotics during storage of the composite microcapsule (WP-L-O-GA 65) of the present application.

[0044] Figure 8 Figure 3 is a schematic diagram of the release rate of the oil in the composite microcapsule (WP-L-O-GA 65) of the present application during in vitro simulated digestion.

[0045] Figure 9 Figure 4 is a schematic diagram of the viable count of probiotics in the composite microcapsule (WP-L-O-GA 65) of the present application during in vitro simulated digestion.

[0046] Figure 10 Figure 5 is a schematic diagram of the particle size of the composite microcapsule (WP-L-O-GA 65) of the present application during in vitro simulated digestion. DETAILED DESCRIPTION

[0047] In order to make the above objectives, features and advantages of the present application more apparent and comprehensible, the specific embodiments of the present application will be described in detail below.

[0048] In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present application, but the present application can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without departing from the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0049] Secondly, "one embodiment" or "embodiment" referred to herein means that a specific feature, structure or characteristic can be included in at least one implementation of the present application. "In one embodiment" appearing in different places in the specification does not mean the same embodiment, nor is it an embodiment that is separate or alternative to other embodiments.

[0050] The present application provides a preparation method of a polyunsaturated fatty acid pumpkin seed oil-probiotic composite microcapsule, which comprises a composite microcapsule, wherein the composite microcapsule takes mixed pressed oil and probiotics as core material, takes a mixture of whey protein and gum arabic as wall material; the core material is wrapped in the wall material; the mixed pressed oil comprises pumpkin seed oil and flaxseed oil. The preparation method of the composite microcapsule comprises the following steps:

[0051] S1. Preparation of the balanced polyunsaturated fatty acid mixed expeller oil 5:1-7.5:1 mass ratio of pumpkin seeds and flax seeds were weighed respectively, and the weighed pumpkin seeds and flax seeds were mixed and then pressed to obtain a mixed expeller oil;

[0052] S2. Preparation of the microcapsule wall material;

[0053] S21. Whey protein and gum arabic were selected as the microcapsule wall material;

[0054] S22. Whey protein was weighed, 9 g of whey protein was dissolved in 300 mL of distilled water, and the mixture was stirred at room temperature for 1 h to completely hydrate, to obtain a 3% whey protein solution; the whey protein solution was pretreated to obtain a whey protein stock solution, which was named WP;

[0055] The pretreatment method was as follows: 1 mol / L NaOH solution was added dropwise to the whey protein solution to adjust the pH to 12, the whey protein solution was heated to 65℃ after standing for 30 min, the protein was unfolded and refolded, and then cooled to room temperature, 1 mol / L HCl solution was added dropwise to adjust the pH to 7, and the supernatant was obtained by centrifugation to obtain the whey protein stock solution.

[0056] By the pretreatment method, the protein structure of the whey protein was destroyed, the water solubility of the protein was improved, and thus the embedding property of the microcapsule was enhanced.

[0057] S23. Preparation of gum arabic stock solution

[0058] The order of steps S22 and S23 can be interchanged.

[0059] 6 g of gum arabic was dissolved in 200 mL of distilled water, stirred at 50℃ for 3 h, and then hydrated at 4℃ overnight to obtain a 3% gum arabic stock solution, which was named GA;

[0060] S24. 300 ml of the whey protein solution and 200 ml of the gum arabic solution were mixed, homogenized at 7000 rpm for 3 min, 10% citric acid solution was added dropwise to adjust the pH to 3.6, and the mixture was placed at 4℃ overnight to fully hydrate the complex, which was named WP-GA65;

[0061] S3. Activation and preparation of probiotics

[0062] S31. Lactobacillus rhamnosus was selected as the probiotic strain;

[0063] S32. The frozen Lactobacillus rhamnosus was inoculated on a sterile culture medium, and after culture and growth, it was inoculated again on the liquid culture medium for batch culture, and the cell mass was prepared by centrifugation in the logarithmic growth phase; the precipitate was washed with sterile normal saline after centrifugation to obtain a concentrated bacterial solution;

[0064] The specific steps are: taking out Lactobacillus rhamnosus frozen at -80°C, inoculating on a sterile solid culture medium, culturing at 37°C for 48h, picking single colonies and placing them in a sterile liquid culture medium for anaerobic culture at 37°C for 16h, inoculating the activated bacterial liquid into the liquid culture medium at an inoculation amount of 1%, carrying out batch culture, centrifuging the cell mass at the logarithmic growth phase at 4°C, discarding the supernatant, washing the precipitate twice with sterile normal saline, and preparing a concentrated bacterial liquid, at this time the concentration of the bacterial liquid is about 10 10 CFU / mL.

[0065] S33. The concentrated bacterial liquid is divided into multiple dry aliquots, preferably two aliquots in this embodiment;

[0066] The order of steps S2 and S3 can be interchanged.

[0067] S4. Preparation of microcapsules

[0068] S41. The concentrated bacterial liquid and mixed pressed oil are dispersed into the whey protein stock solution, and centrifuged for homogenization at 7000rpm for 3min;

[0069] S42. The gum arabic stock solution is gradually added under the condition of centrifugation at 400rpm; then a 10% citric acid solution is added to adjust the pH to 3.6, so as to induce the complexation between the whey protein stock solution and the gum arabic stock solution;

[0070] S43. 20U / g of transglutaminase is further added, and heating and stirring are carried out at 37°C for 2h to fully solidify; finally, the whey emulsion is vacuum freeze-dried to prepare solid composite microcapsules, which are sealed and placed in a high-low temperature test box at 4°C for storage, and are named WP-L-O-GA65.

[0071] In this embodiment, the mass ratio of pumpkin seeds and flax seeds is 5:1.

[0072] The determination of the ratio of pumpkin seeds and flax seeds is one of the focuses of the present application, and the balanced polyunsaturated fatty acids are prepared by determining the ratio of pumpkin seeds and flax seeds. The present application takes the recommended dietary intake ratio of PUFAs as the target, establishes a suitable mathematical model to determine the specific mixing ratio of pumpkin seeds and flax seeds.

[0073]

[0074] W p , W f —The mass of the two raw materials of pumpkin seeds and flax seeds accounts for the percentage of the total raw material formula;

[0075] O p , O f —The oil yield of the two raw materials of pumpkin seeds and flax seeds (%);

[0076] H p , Hf —Percentage of ω-6PUFAs in pumpkin seed oil and flaxseed oil;

[0077] h p h f —Percentage of ω-3 PUFAs in pumpkin seed oil and flaxseed oil;

[0078] Y = 4:1 to 6:1, which represents the ratio of recommended dietary intake of ω-6 / ω-3 PUFAs.

[0079] The oil yield ratio of pumpkin seeds and flax seeds, based on statistical data, is as follows:

[0080] Table 1-1 Oil extraction rate of pumpkin seed oil and flaxseed oil

[0081]

[0082] Table 1-1 shows that the oil yields of pumpkin seeds and flax seeds are 26.72±0.05% and 33.45±0.04%, respectively. Vegetable oils are indispensable substances for human life activities and metabolism. Fatty acids are one of the important indicators determining the quality of edible oils, and their composition and ratio largely determine their nutritional value. The contents of the five main fatty acids in pumpkin seed oil (PO) and flaxseed oil (FO), namely palmitic acid (PA), stearic acid (SA), oleic acid (OA), linoleic acid (LA), and linolenic acid (ALA), are as follows: Figure 1 As shown in the figure, the fatty acids in PO are mainly composed of two types of unsaturated fatty acids: OA (32.20±0.25%) and LA (50.32±0.13%). However, its ALA content (0.11±0.06%) is relatively low. Therefore, the ω-6 / ω-3 PUFAs ratio in PO does not conform to the dietary recommended intake ratio of polyunsaturated fatty acids (PUFAs) by the Chinese Nutrition Society (ω-6 / ω-3 PUFAs = 4:1 to 6:1). Long-term consumption may lead to diseases such as fatty liver, hyperlipidemia, and atherosclerosis. FO contains 88.19% unsaturated fatty acids, mainly comprising ALA (51.76±0.25%) > OA (20.48±0.07%) > LA (15.95±0.27%). Its ALA content is one of the highest among known vegetable oils in terms of ω-3 PUFAs. ALA is not only an essential fatty acid for the human body, but it can also synthesize metabolites such as eicosapentaenoic acid and docosahexaenoic acid in the body through dehydrogenation and carbon chain elongation. It has the effects of antithrombosis, lowering blood lipids, and preventing and treating ischemic cardiovascular diseases.

[0083] Determining the optimal mass ratio of pumpkin seeds to flax seeds is a necessary prerequisite for preparing blended pressed oil, as it affects the proportion of PUFAs (polyunsaturated fatty acids) in the oil. This is determined by considering the respective oil yields of pumpkin seeds and flax seeds (Table 1-1) and... Figure 1 The analysis results of polyunsaturated fatty acids (PUFAs) in pumpkin seed oil (PO) and flaxseed oil (FO) were used to establish a mathematical model based on formula 1-1. The calculations showed that when the ω-6 / ω-3 PUFAs ratio of the mixed pressed oil sample met the range of 4:1 to 6:1, the mass ratio (Wp / Wf) of the two oilseed crops (p and f) was 4.84 to 7.49. The predicted values ​​were calculated using the mathematical model established in Table 1-1, while the experimental values ​​were obtained from actual experiments.

[0084] Pumpkin seeds and flax seeds were mixed and pressed in a mass ratio of 4:1 to 8:1 to obtain blended pressed oil. The fatty acid content was determined to further verify the reliability of the theoretical results. Figure 2 The figure shows the predicted and experimental values ​​of the ω-6 / ω-3 polyunsaturated fatty acid (PUFA) ratio in mixed-pressed oil samples obtained with different oilseed crop ratios. The results indicate that when Wp / Wf is less than 7, the experimental value of ω-6 / ω-3 PUFAs in the mixed-pressed oil is slightly lower than the predicted value. A high-viscosity pressing system is not conducive to the extraction of lipid components from oils, leading to a decrease in the fatty acid content of the mixed cold-pressed oil. This may also be because the raw materials are easily broken and clogged in the press after entering the press, thus affecting the oil yield. When Wp / Wf is greater than 7, the experimental and predicted values ​​of ω-6 / ω-3 polyunsaturated fatty acids (PUFAs) are almost identical. Overall, the predicted value curve and the experimental value curve show a good fit within the calculation range obtained by this mathematical model, making it a rapid method for determining the oilseed mass ratio in mixed pressing processes. When Wp / Wf is 4, the ω-6 / ω-3 polyunsaturated fatty acid (PUFA) ratio in the mixed pressed oil sample is 3.26±0.02, which is lower than the dietary recommendation ratio. As the proportion of pumpkin seeds in the mixed oilseed crops is gradually increased, the ω-6 / ω-3 PUFA ratio increases with the increase of Wp / Wf. When Wp / Wf is 5 and 7.5, the ω-6 / ω-3 PUFA ratio in the mixed pressed oil sample is 4.08±0.09 and 6.11±0.04, respectively, both slightly higher than the upper and lower limits of the dietary recommendation ratio. Therefore, the final mass ratio of the two oilseed crops is determined to be 5–7.5, corresponding to an oil yield of 28.90%–28.56% for the mixed pressed oil. Since flaxseed is cheaper than pumpkin seeds in the market, in this embodiment, a mass ratio of pumpkin seeds to flaxseed is preferred to be 5 to save production costs.

[0085] The specific preparation method of the mixed expeller oil is as follows: first, the pumpkin seeds and flax seeds are uniformly mixed according to a mass ratio of 5:1, centrifuged after cold pressing, and the upper clear oil is taken to obtain the balanced mixed expeller oil. At the same time, the pumpkin seeds and flax seeds are weighed according to the same parameters, cold-pressed to obtain oil, centrifuged, and the upper clear oil is taken, and then mixed to obtain a directly blended oil (BO) as a control.

[0086] Compared with the mixed expeller oil which is mixed first and then pressed, and the directly blended oil which is pressed separately and then mixed, the oxidation stability of the mixed expeller oil is stronger.

[0087] Preparation of the control:

[0088] The present application also includes mixed expeller oil microcapsules and lactobacillus rhamnosus microcapsules, and the mixed expeller oil microcapsules and lactobacillus rhamnosus microcapsules are used as control of the embedding rate and viable count parameters of the composite microcapsules. The core material of the mixed expeller oil microcapsules includes the mixed expeller oil in step S1, and the core material of the lactobacillus rhamnosus microcapsules includes the probiotics in step S3; and the wall material in the mixed expeller oil microcapsules and the lactobacillus rhamnosus microcapsules is the microcapsule wall material in step S2.

[0089] I. Preparation of mixed expeller oil microcapsules

[0090] The preparation method of the mixed expeller oil microcapsules is as follows: 10.0 g of mixed expeller oil is dispersed into a whey protein stock solution, and homogenized at 7000 rpm for 3 min. Then, the gum arabic stock solution is gradually added under continuous stirring at 400 rpm. A 10% citric acid solution is added dropwise to adjust the pH to 3.6 to induce complexation between the whey protein stock solution and the gum arabic stock solution. Then, 180 U of glutamine transaminase is added, and the mixture is heated and stirred at 37℃ for 2 h to fully solidify, to obtain an O / W emulsion. The emulsion is named WP-O-GA65. The final solution is stored at 4℃ for standby. The emulsion is placed in a vacuum freeze dryer to dry into solid microcapsules, which are sealed and stored in a 4℃ high-low temperature test box for standby.

[0091] II. Preparation of lactobacillus rhamnosus microcapsules

[0092] The preparation method of the lactobacillus rhamnosus microcapsules is as follows:

[0093] The concentrated bacterial solution is dispersed into a whey protein stock solution, and homogenized at 7000 rpm for 3 min. Then, the gum arabic stock solution is gradually added under continuous stirring at 400 rpm. A 10% citric acid solution is added dropwise to adjust the pH to 3.6 to induce complexation between the whey protein and the gum arabic. Then, 180 U of glutamine transaminase is added, and the mixture is heated and stirred at 37℃ for 2 h to fully solidify. The final emulsion is vacuum freeze-dried to form solid microcapsules, which are sealed and stored in a 4℃ high-low temperature test box for standby, and named WP-L-GA65.

[0094] Analysis of parameters of the product prepared according to the present application:

[0095] 1. Analysis of encapsulation efficiency and viable bacteria count

[0096] Encapsulation efficiency is a key parameter of oil microcapsules, which can determine the ability of wall material to encapsulate core material. The encapsulation efficiency of mixed pressed oil in mixed pressed oil microcapsules (WP-O-GA65) and composite microcapsules (WP-L-O-GA 65) was 77.48±1.27% and 81.91±0.67%, respectively (Table 2-1). When mixed pressed oil microcapsules were loaded with Lactobacillus rhamnosus (LGG), the encapsulation efficiency of oil increased significantly by 4.43% (p<0.05). Encapsulation efficiency is usually closely related to wall material, encapsulation process, drying method and other factors. Under the same conditions, mixed pressed oil (MPO), Lactobacillus rhamnosus (LGG) alone and their co-encapsulation showed that co-encapsulation had more advantages. It is very important to minimize the loss of cell viability during microencapsulation to ensure the maximum number of viable cells in the matrix. Table 2-1 shows the viable bacteria count of Lactobacillus rhamnosus (LGG) in Lactobacillus rhamnosus microcapsules (WP-L-GA 65) and composite microcapsules (WP-L-O-GA 65). It can be observed from the table that the viable bacteria count of Lactobacillus rhamnosus (8.33±0.17 log CFU / g) in composite microcapsules (WP-L-O-GA 65) was higher than that in Lactobacillus rhamnosus microcapsules (WP-L-GA 65). The increase in viable bacteria count may be related to the microcapsule structure. The presence of mixed pressed oil (MPO) provides an additional protective barrier for Lactobacillus rhamnosus (LGG), weakening the impact of the external adverse environment. In addition, mixed pressed oil (MPO) adheres to the phospholipid protein membrane of Lactobacillus rhamnosus (LGG), limiting the migration of probiotic cells. Therefore, the oil composite microcapsules of the present application loaded with probiotics are a promising encapsulation technology that can improve the survival rate of probiotics.

[0097] Table 2-1 Encapsulation of oils and active bacteria number in WP-L-O-GA microcapsule

[0098]

[0099] Note: The results are expressed as mean ± standard deviation. Values with different letters for the same index are significantly different (p<0.05).

[0100] 2. Fourier infrared spectroscopy analysis

[0101] FromFigure 3 The whey protein stock solution showed a characteristic absorption peak at 3367.45 cm -1 corresponding to O-H or N-H stretching vibration. The absorption peak at 2927.5 cm -1 was associated with C-H stretching vibration. The amide I band (C=O stretching vibration) absorption peak was observed at 1651.01 cm -1 , the amide II band (N-H bending vibration) absorption peak was at 1544.72 cm -1 , and the amide III band (C-N stretching vibration) absorption peak was at 1248.44 cm -1 . With the introduction of gum arabic stock solution, the amide I band of whey protein stock solution shifted from 1651.01 cm -1 to a lower wave number of 1645.69 cm -1 . The typical spectral bands of gum arabic stock solution included O-H stretching vibration at 3384.94 cm -1 , C-H stretching vibration at 2930.43 cm -1 , -COO asymmetric vibration at 1612 cm -1 , O-H bending vibration at 1421.17 cm -1 , C-O stretching vibration and C-O-H bending vibration at 1142.16 cm -1 and 1067.75 cm -1 . Mixed pressed oil (MPO) was mainly composed of compounds such as fatty acids, aldehydes and ketones. In the infrared spectrum, 3009.34 cm -1 corresponded to the stretching vibration of unsaturated carbon C-H bond, while 2926.04 cm -1 was attributed to the C-H stretching vibration of methylene, 2855.89 cm -1 was associated with the C-H stretching vibration of aldehyde group, 1746.67 cm -1 was C=O stretching vibration, and the characteristic peaks at 1162.08 cm -1 and 1098.31 cm -1 were associated with C-O stretching vibration. The intensity of the infrared spectral peaks associated with mixed pressed oil (MPO) in the mixed pressed oil microcapsule (WP-O-GA65) and the composite microcapsule (WP-L-O-GA65) was significantly weakened, indicating that the mixed pressed oil (MPO) was embedded in the microcapsule. It is worth noting that in the spectrum of Lactobacillus rhamnosus microcapsule (WP-L-GA65) and composite microcapsule (WP-L-O-GA65), the original gum arabic stock solution absorption peak at 1612 cm -1 was not detected, but a new absorption peak appeared near 1725 cm -1 .

[0102] 3. Physical shape analysis using scanning electron microscopy

[0103] Depend on Figure 4 As can be seen, no free probiotics were observed on the surface of either the *Lactobacillus rhamnosus* microcapsules (WP-L-GA65) or the composite microcapsules (WP-LO-GA65), indicating that the freeze-drying process of the emulsion did not cause the microcapsule structure to rupture, and the cells remained fixed in the core region. The depressions and wrinkles on the surface of all microcapsules are likely due to the rapid evaporation of moisture during the vacuum freeze-drying process, which caused the microcapsules to form porous aggregates and lose their spherical structure—a typical result of the vacuum freeze-drying process. The surface of the *Lactobacillus rhamnosus* microcapsules (WP-L-GA65) was relatively smooth, but it still showed obvious aggregates of whey protein and gum arabic (WP-GA). The mixed pressed oil microcapsules (WP-O-GA 65) and the composite microcapsules (WP-LO-GA 65) exhibited similar morphologies, appearing as lumps with wrinkled surfaces. The structure of the composite microcapsules (WP-LO-GA 65) appeared to be more compact than that of the mixed pressed oil microcapsules (WP-O-GA 65), which is consistent with the higher oil encapsulation rate and probiotic viable count in the composite microcapsules (WP-LO-GA 65).

[0104] 4. Laser confocal scanning microscopy analysis

[0105] like Figure 5 As shown, the microstructure of the composite microcapsules (WP-LO-GA 65) was observed using laser scanning confocal microscopy (CLSM). Image a presents the results of double staining of whey protein storage solution (WP) and Lactobacillus rhamnosus (LGG). FITC binds to WP and emits green fluorescence (b). The emulsion after microcapsule reconstitution contains many small green particles, which are relatively uniformly distributed throughout the system by the whey protein storage solution (WP). The purple particles in images c and f are the result of PI binding to the DNA within Lactobacillus rhamnosus (LGG). As can be seen from image a, the green color is significantly lighter because the purple in image c overlaps with the green in image b, indicating that most of Lactobacillus rhamnosus (LGG) is present in the aqueous phase. Image d presents the results of double staining of mixed pressed oil (MPO) and Lactobacillus rhamnosus (LGG). d contains some relatively large green spherical particles (corresponding to mixed pressed oil (MPO) droplets), which are surrounded by the aqueous phase. The aqueous phase contains some purple particles of Lactobacillus rhamnosus (LGG) aggregates, but some green and purple overlap. This indicates that after the emulsion forms, most of the Lactobacillus rhamnosus (LGG) is distributed between the aqueous and interfacial phases, with only a small amount remaining in the oil phase.

[0106] 5. Analysis of PV and p-AV values ​​of oils during storage of composite microcapsules

[0107] As Figure 6 The changes of PV value (A) and p-AV value (B) of mixed pressed oil (MPO) (control), mixed pressed oil microcapsules (WP-O-GA 65) and complex microcapsules (WP-L-O-GA 65) stored at 60℃ for 28d are shown. As expected, the PV value and p-AV value of both microcapsules during storage were significantly higher than that of un-embedded mixed pressed oil (MPO) (p<0.05). After 28d storage at 60℃, the PV value of mixed pressed oil (MPO) and Lactobacillus rhamnosus microcapsules (WP-L-GA 65), complex microcapsules (WP-L-O-GA 65) increased to 45.98, 24.76, 22.38 mmol / kg, respectively, which was 63.76, 33.87, 30.52 times higher than the initial PV value, respectively. Among them, the PV value of mixed pressed oil (MPO) (17.17 mmol / kg) far exceeded the limit value of GB / T 2716—2018 “People’s Republic of China National Standard Vegetable Oil” (≤9.85 mmol / kg) at the 7th day, while the PV value of WP-L-GA (13.34 mmol / kg) and WP-L-O-GA (11.65 mmol / kg) occurred at the 14th day. The p-AV value of the three samples increased to 12.98, 7.82, 7.41, respectively, at the end of storage, which was 132.81, 76.62, 75.39 times higher than the initial p-AV value, respectively.

[0108] By Figure 6 and data analysis, it can be seen that during the entire storage period, the complex microcapsules (WP-L-O-GA 65) showed the lowest PV value and p-AV value, indicating that the addition of Lactobacillus rhamnosus (LGG) had a promoting effect on the oxidative stability of oil.

[0109] 6. Analysis of probiotic viable count during storage of complex microcapsules

[0110] To maximize the health benefits of probiotics, it is crucial to minimize the loss of their viability before they are consumed. Studies have shown that low-temperature storage is an effective method to maintain the viability of probiotics in microcapsules. As Figure 7 shown, the initial viable count of Lactobacillus rhamnosus microcapsules (WP-L-GA 65) and complex microcapsules (WP-L-O-GA 65) (7.58 log CFU / g and 8.33 log CFU / g, respectively) decreased by 1.23 and 1.03 log CFU / g, respectively, after 28d storage at 4℃, but remained above 6 log CFU / g. Complex microcapsules (WP-L-O-GA 65) showed the highest probiotic viability during the entire storage period.

[0111] 7. In vitro simulated digestion analysis of composite microcapsules

[0112] 7.1 Analysis of oil release rate

[0113] The release rates of two microcapsules, the mixed pressed oil microcapsule (WP-O-GA 65) and the composite microcapsule (WP-LO-GA65), during in vitro simulated digestion were compared, such as... Figure 8 As shown, there were significant differences in the percentage of oil release from the two microcapsules during the gastric digestion stage (SGF) and the enteric digestion stage (SIF) (p>0.05). After 2 hours of digestion in the gastric digestion stage (SGF), the release rates of mixed pressed oil (MPO) from the mixed pressed oil microcapsules (WP-O-GA 65) and the composite microcapsules (WP-LO-GA 65) were 17.7% and 15.9%, respectively. The composite microcapsule (WP-LO-GA 65) showed the lowest oil release rate, indicating that it was better able to prevent premature release of active substances. After entering the enteric digestion stage (SIF), the microcapsules were rapidly hydrolyzed, and the release rates of mixed pressed oil (MPO) from the two microcapsules reached 76.7% and 79.5%, respectively, significantly higher than the oil release rate in the gastric digestion stage (SGF) (p<0.05). These results indicate that microencapsulated mixed pressed oil (MPO) can slow down its release rate, achieving a sustained-release effect and improving its bioavailability.

[0114] 7.2 Analysis of viable bacteria count of probiotics

[0115] The survival of two probiotic microcapsules, Lactobacillus rhamnosus microcapsules (WP-L-GA65) and composite microcapsules (WP-LO-GA 65), under simulated gastrointestinal conditions, such as... Figure 9 As shown in the experiment, the microcapsules did not rupture after passing through the gastric digestion stage (SGF), but rather expanded slightly. After exposure to both the gastric digestion stage (SGF) and the intestinal digestion stage (SIF), the probiotic cells in the *Lactobacillus rhamnosus* microcapsules (WP-L-GA 65) decreased by 1.57 log CFU / g. Unlike the *Lactobacillus rhamnosus* microcapsules (WP-L-GA 65), the presence of the oil phase in the composite microcapsules (WP-LO-GA 65) showed an effective protective effect. After exposure to both the gastric digestion stage (SGF) and the intestinal digestion stage (SIF), the probiotics in the composite microcapsules (WP-LO-GA 65) decreased by 1.01 log CFU / g. The high survival rate of the probiotics may be related to the release of the oil phase. When the oil-containing probiotic microcapsules are digested, the wall material of the microcapsules is disrupted, leading to the release of the oil phase. In conclusion, these experimental findings strongly demonstrate that loading probiotics onto oil microcapsules can more effectively improve the survival rate of probiotics in the human digestive system.

[0116] 7.3 Analysis of Particle Size Variation

[0117] Three microcapsules underwent particle size changes during in vitro simulated digestion, such as Figure 10 As shown. Before digestion, the average particle size (D) of the mixed pressed oil microcapsules (WP-O-GA 65), Lactobacillus rhamnosus microcapsules (WP-L-GA 65), and composite microcapsules (WP-LO-GA 65) is... 4,3 The average particle sizes (D4,3) of the microcapsules were 12.54, 10.12, and 13.14 μm, respectively. After simulating the gastric digestion stage, the droplet size significantly increased (p<0.05), reaching 15.38, 14.35, and 16.86 μm, respectively. This may be due to the decrease in pH and increase in ionic strength in the simulated gastric juice, which altered the electrostatic interactions between droplets, causing particle aggregation and flocculation, thus leading to an increase in microcapsule size. After the intestinal digestion stage, the microcapsule size significantly decreased (p<0.05). The average particle size (D4,3) values ​​of the mixed pressed oil microcapsules (WP-O-GA65), Lactobacillus rhamnosus microcapsules (WP-L-GA 65), and composite microcapsules (WP-LO-GA 65) were 5.38, 4.89, and 5.85 μm, respectively.

[0118] This invention uses *Lactobacillus rhamnosus* as a model probiotic and employs mixed pressed oil microcapsules (WP-O-GA 65) as a carrier to deliver *Lactobacillus rhamnosus*, constructing a multilayer encapsulation system with good stability, high encapsulation efficiency, and protective effect against *Lactobacillus rhamnosus*. The composite microcapsule system allows for slow release; upon reaching the intestines, the microcapsule wall material is digested and destroyed, the oil is broken down into fatty acids and absorbed by the intestines, completely releasing the probiotics and ensuring their release near specific sites.

Claims

1. A method for preparing an equalized polyunsaturated fatty acid pumpkin seed oil-probiotic composite microcapsule, characterized in that: The composite microcapsule comprises mixed pressed oil and probiotics as core material, whey protein and gum arabic mixture as wall material; the core material is embedded in the wall material; the mixed pressed oil comprises pumpkin seed oil and flaxseed oil; The preparation method of the composite microcapsule comprises the following steps: S1. Preparation of mixed pressed oil of equalized polyunsaturated fatty acids; S11. Pumpkin seeds and flaxseeds are weighed according to a mass ratio of 4:1-6:1, and the weighed pumpkin seeds and flaxseeds are mixed and pressed to obtain mixed pressed oil; S2. Preparation of microcapsule wall material; S21. Whey protein and gum arabic are selected as the microcapsule wall material; S22. Whey protein is weighed and placed in distilled water for stirring at room temperature, and whey protein solution is obtained after complete hydration; the whey protein solution is appropriately pretreated to prepare whey protein stock solution; The pretreatment method is: 1Mol / L NaOH solution is added dropwise in the whey protein solution to adjust the pH to 12, the whey protein solution is heated to 65℃ after being static for 30min, the protein is unfolded and refolded, and then cooled to room temperature, 1Mol / L HCl solution is added dropwise to adjust the pH to 7, and the whey protein stock solution is obtained by centrifugation and taking the supernatant; S23. Preparation of gum arabic stock solution; S24. The whey protein solution and gum arabic solution are weighed and mixed, then centrifuged and homogenized, and 10% citric acid solution is added dropwise to adjust the pH to 3.6, and the mixture is placed at 4℃ overnight to fully hydrate the complex; S3. Activation and preparation of probiotics; S31. Lactobacillus rhamnosus is selected as the probiotic strain; S32. The frozen Lactobacillus rhamnosus is inoculated on a sterile culture medium, and after culture and growth, it is inoculated again on the liquid culture medium for batch culture, and the cell mass is prepared by centrifugation at the logarithmic growth phase, and the concentrated bacterial solution is prepared by washing the precipitate with sterile normal saline after centrifugation; S33. The concentrated bacterial solution is divided into two equal parts; S4. Preparation of microcapsules; S41. The concentrated bacterial solution and the mixed pressed oil are dispersed into the whey protein stock solution, and after homogenization for a certain time; S42. The gum arabic stock solution is gradually added under the homogenization environment; 10% citric acid solution is further added to adjust the pH to 3.6 to induce the complexation between the whey protein stock solution and the gum arabic stock solution; S43. 20U / g of transglutaminase is further added, heated and stirred at 37℃ for 2h to fully solidify, and the final emulsion is vacuum freeze-dried to prepare solid composite microcapsules.

2. The method for preparing the composite microcapsules as described in claim 1, characterized in that: The mass ratio of the pumpkin seeds and the flaxseeds is 5-7.5:

1.

3. The method for preparing composite microcapsules as described in claim 1, characterized in that: The step of S22 is specifically: the whey protein is dissolved in distilled water, stirred at room temperature for 1h to completely hydrate, and 3% whey protein solution is obtained.

4. The method for preparing the composite microcapsules as described in claim 1, characterized in that: The step of S23 is specifically: the gum arabic is dissolved in distilled water, stirred at 50℃ for 3h, then hydrated at 4℃ overnight, and 3% gum arabic stock solution is obtained.

5. The method for preparing composite microcapsules as described in claim 1, characterized in that: The step of S32 is specifically: taking out the Lactobacillus rhamnosus frozen at -80℃, inoculating on a sterile solid culture medium, culturing at 37℃ for 48h, picking a single colony and placing it in a sterile liquid culture medium to culture anaerobically at 37℃ for 16h, inoculating the activated bacterial liquid into a liquid culture medium with an inoculation amount of 1%, carrying out batch culture, centrifuging the cell mass at the logarithmic growth phase at 4℃, discarding the supernatant, washing the precipitate with sterile normal saline twice, and preparing a concentrated bacterial liquid, at this time the concentration of the bacterial liquid is about 10 10 CFU / mL.

6. The method for preparing composite microcapsules as described in claim 1, characterized in that: The equal parts of the concentrated bacterial solution of S33 are two.

7. The method for preparing composite microcapsules as described in claim 1, characterized in that: Also included are mixed expeller oil microcapsules and lactobacillus rhamnosus microcapsules; the core material of the mixed expeller oil microcapsules comprises the mixed expeller oil in step S1, and the core material of the lactobacillus rhamnosus microcapsules comprises the probiotic bacteria in step S3; the wall material in the mixed expeller oil microcapsules and the lactobacillus rhamnosus microcapsules is the microcapsule wall material in step S2; the mixed expeller oil microcapsules and the lactobacillus rhamnosus microcapsules are used as a control for the embedding rate and viable bacterial count parameters of the composite microcapsules.

8. The method for preparing the composite microcapsules as described in claim 7, characterized in that: The preparation method of the mixed expeller oil microcapsules is as follows: the mixed expeller oil is dispersed into a whey protein stock solution, and homogenized at 7000 rpm for 3 min; then, under continuous stirring at 400 rpm, an acacia stock solution is gradually added; a 10% citric acid solution is added dropwise to adjust the pH to 3.6 to induce complexation between the whey protein and the acacia; 20 U / g of transglutaminase is further added, and heating and stirring are performed at 37°C for 2 h to fully solidify, to obtain a milk emulsion.

9. The method for preparing the composite microcapsules as described in claim 7, characterized in that: The preparation method of the lactobacillus rhamnosus microcapsules is as follows: the concentrated bacterial solution is dispersed into a whey protein stock solution, and homogenized at 7000 rpm for 3 min; then, under continuous stirring at 400 rpm, an acacia stock solution is gradually added; a 10% citric acid solution is added dropwise to adjust the pH to 3.6 to induce complexation between the whey protein stock solution and the acacia stock solution; 20 U / g of transglutaminase is further added, and heating and stirring are performed at 37°C for 2 h to fully solidify, and the final milk emulsion is vacuum freeze-dried to form solid microcapsules.

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