Preparation method of balanced polyunsaturated fatty acid pumpkin seed oil-multivariate probiotic composite microcapsule

Abstract

The invention discloses a preparation method of a balanced polyunsaturated fatty acid pumpkin seed oil-multi-element probiotic composite microcapsule, 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. According to the preparation method of the microcapsule, probiotics are delivered to a target position, and the microcapsule is helpful for establishing a stable bacterial colony of an intestinal tract.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and particularly relates to a method for preparing balanced polyunsaturated fatty acid pumpkin seed oil-multi-probiotic composite microcapsules. Background Art

[0002] Probiotics are generally defined as live microorganisms that, when ingested in adequate amounts, confer a positive effect on the health of the host. According to the Food and Agriculture Organization, probiotics must remain in the product for at least 10 days during its shelf life and in the digestive system. 6 ~10 7 CFU / g can achieve its expected effect. Probiotics have many health benefits such as protecting the natural microflora of the intestine, enhancing the immune system and lowering blood cholesterol. However, probiotics are sensitive to oxygen, temperature, gastrointestinal tract, and adverse conditions during processing and transportation. Therefore, in the food industry, it is of great significance to maintain the vitality of probiotics during food processing, production and storage, as well as in food and the digestive tract, and to release them safely in the intestine.

[0003] Microcapsule delivery technology has been extensively studied, but there is still a lack of specialized microcapsule delivery vehicles to deliver probiotics to specific locations while maintaining good activity.

[0004] Therefore, in order to solve the above technical problems, it is necessary to provide a new technical solution to solve this technical problem, especially a preparation method of a balanced polyunsaturated fatty acid pumpkin seed oil-multi-probiotic composite microcapsule. Summary of the invention

[0005] The purpose of the present invention is to solve the technical problem of how to deliver probiotics to a specific location for release while ensuring that the probiotics have good activity, and to provide a method for preparing balanced polyunsaturated fatty acid pumpkin seed oil-multi-probiotic composite microcapsules.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing a composite microcapsule of pumpkin seed oil-multiple probiotics with balanced polyunsaturated fatty acids, characterized in that the composite microcapsule comprises a composite microcapsule, wherein the composite microcapsule uses mixed pressed oil and probiotics as core materials and a mixture of whey protein and gum arabic as wall materials; the core material is wrapped in the wall material; the mixed pressed oil comprises pumpkin seed oil and linseed oil;

[0008] The composite microcapsule preparation method comprises the following steps:

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

[0010] S11. Weigh pumpkin seeds and flax seeds in a mass ratio of 4:1 to 6:1, respectively, mix the weighed pumpkin seeds and flax seeds, and then press them to obtain a mixed pressed oil;

[0011] S2. Preparation of microcapsule wall materials;

[0012] S21. Select whey protein and gum arabic as microcapsule wall materials;

[0013] S22. Weigh whey protein and stir it in distilled water at room temperature to obtain a whey protein solution after complete hydration; and then pretreat the whey protein solution to obtain a whey protein stock solution;

[0014] The pretreatment method comprises: dropping 1 mol / L NaOH solution into the whey protein solution to adjust the pH to 12, heating the whey protein solution to 65°C after standing for 30 minutes to unfold the protein and then refold it, then cooling it to room temperature, dropping 1 mol / L HCl solution to adjust the pH to 7, and centrifuging to obtain the supernatant to obtain the whey protein reserve solution;

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

[0016] S24. Weigh the whey protein solution and the gum arabic solution, mix them, homogenize them, add 10% citric acid solution to adjust the pH to 3.6, and place them at 4°C overnight to fully hydrate the complex;

[0017] S3. Activation and preparation of probiotics;

[0018] S31. Select one or more of Bifidobacterium pseudolongum, Bifidobacterium pseudosmall chain, Lactobacillus and Akkermansia mucinosa as probiotic strains;

[0019] S32. The frozen probiotics were inoculated on a sterile culture medium, and after growth, they were inoculated again on a liquid culture medium for batch culture. Cell pellets were obtained by centrifugation during the logarithmic growth phase, and the precipitate was washed with sterile saline after centrifugation to obtain a concentrated bacterial solution;

[0020] S33. Divide the concentrated bacterial solution 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 homogenized;

[0023] S42. The gum arabic stock solution was gradually added under a homogenizing environment; and then 10% citric acid solution was added to adjust the pH to 3.6 to induce complexation between the whey protein stock solution and the gum arabic stock solution;

[0024] S43. Then add 20 U / g of glutamine transaminase, heat and stir at 37°C for 2 h to fully solidify, and finally the emulsion is vacuum freeze-dried to prepare solid composite microcapsules.

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

[0026] Preferably, the step S22 is specifically as follows: dissolving whey protein in distilled water, stirring at room temperature for 1 hour to completely hydrate, and obtaining a 3% whey protein solution.

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

[0028] Preferably, the step of S32 is specifically as follows: taking out the Lactobacillus rhamnosus stored in a freezer at -80°C, streaking and inoculating on a sterile solid culture medium, culturing at 37°C for 48h, picking a single colony and placing it in a sterile liquid culture medium and anaerobic culturing at 37°C for 16h, inoculating the activated bacterial solution into the liquid culture medium at an inoculum rate of 1%, and carrying out batch culture, harvesting the cell mass by centrifugation at 4°C during the logarithmic growth phase, discarding the supernatant, and washing the precipitate twice with sterile saline to prepare a concentrated bacterial solution, at which the bacterial solution concentration is approximately 10 10 CFU / mL; when multiple strains are selected, the above resurrection operations are performed separately, and the obtained concentrated bacterial liquids are mixed to prepare a mixed concentrated bacterial liquid, and the concentration of the mixed bacterial liquid is 10 10 CFU / mL.

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

[0030] Preferably, it also includes mixed pressed oil microcapsules and probiotic microcapsules; the core material of the mixed pressed oil microcapsules includes the mixed pressed oil in step S1, and the core material of the probiotic microcapsules includes the probiotics in step S3; the wall materials in the mixed pressed oil microcapsules and the probiotic microcapsules are both the microcapsule wall materials in step S2; the mixed pressed oil microcapsules and the probiotic microcapsules are used as references for the embedding rate and viable cell count parameters of the composite microcapsules.

[0031] Preferably, the preparation method of the mixed pressed oil microcapsules is to disperse the mixed pressed oil into a whey protein stock solution and homogenize at 7000 rpm for 3 minutes; then gradually add the gum arabic stock solution under continuous stirring at 400 rpm; add 10% citric acid solution dropwise to adjust the pH to 3.6 to induce complexation between whey protein and gum arabic; then add 20 U / g of glutamine transaminase, heat and stir at 37°C for 2 hours to fully solidify, and obtain an emulsion.

[0032] Preferably, the preparation method of the probiotic microcapsules is as follows: disperse the single or mixed concentrated bacterial solution into the whey protein stock solution, homogenize at 7000 rpm for 3 minutes; then gradually add the gum arabic stock solution under continuous stirring at 400 rpm; add 10% citric acid solution dropwise to adjust the pH to 3.6 to induce complexation between the whey protein stock solution and the gum arabic stock solution; then add 20 U / g of glutamine transaminase, heat and stir at 37°C for 2 hours to fully solidify, and finally vacuum freeze-dry the emulsion to prepare solid microcapsules.

[0033] The beneficial effects of the present invention are:

[0034] The present invention uses two nutrients, mixed squeezed oil and probiotics, as core materials and embedded in the microcapsule wall material. The probiotics are selected from Bifidobacterium pseudolongum, Bifidobacterium pseudosmall chain, Lactobacillus and Akkermansia mucinosa. The probiotics can tolerate the animal digestive tract environment and successfully colonize in the intestines of humans and animals. They have the functional characteristics of regulating the balance of intestinal flora, preventing and treating diarrhea, promoting toxin elimination, enhancing the body's overall immunity and preventing dental caries. The mixed squeezed oil is a balanced polyunsaturated fatty acid oil, which is produced by squeezing after mixing pumpkin seeds and flax seeds. The mass ratio of pumpkin seeds and flax seeds is 5 to 7.5, so that the ω-6 and ω-3 unsaturated fatty acids in the squeezed mixed oil meet the dietary recommended ratio of 4 to 6:1.

[0035] The fat can only be digested by lipase in the intestines before the probiotics are released near the site of action in the intestines, ensuring that the delivered probiotics are released in the intestinal appendages.

[0036] In addition to the nutritional components, the two embedded materials also have a synergistic effect. The present invention further improves the embedding rate and activity of the probiotics by adding mixed pressed oil as a load for the probiotics and then embedding it in the microcapsule.

[0037] The pseudo-bifidobacterium longum, pseudo-bifidobacterium small chain and mucin Akkermansia are the newly discovered probiotic strains, which are less used in China. The probiotics of the present invention are preferably multivariate mixed strains, which have the functions of regulating intestinal microorganisms, enhancing immunity, preventing intestinal inflammation, repairing and renewing intestinal mucin, and the multivariate mixed probiotics can also increase the effects of survival and mutual promotion. The mixing of pseudo-bifidobacterium longum and pseudo-bifidobacterium small chain enhances the mediation effect on intestinal microbial flora, enhances immunity, has a certain preventive effect on allergic diseases and inflammatory bowel disease, and helps to establish a stable and healthy intestinal flora environment. The activities of pseudo-bifidobacterium longum and pseudo-bifidobacterium small chain have a mutually promoting effect on lactobacillus and mucin Akkermansia, and the pH environment produced by pseudo-bifidobacterium longum and pseudo-bifidobacterium small chain is usually beneficial to Akkermansia, especially in the acidic range (such as pH 5.5-6.5), while pseudo-bifidobacterium longum and pseudo-bifidobacterium small chain produce organic acids and lactic acid that can promote the survival and growth of lactobacillus.

[0038] Bifidobacterium pseudospermum can produce organic acids to help maintain the pH balance in the intestine and prevent the reproduction of pathogens. Bifidobacterium pseudospermum can produce short-chain fatty acids such as lactic acid and acetic acid, which help inhibit the growth of harmful bacteria. At the same time, the organic acids and lactic acid produced by the two bifidobacteria can promote the growth of Lactobacillus and Akkermansia mucinensis. Akkermansia mucinensis can utilize mucin in the intestinal mucus layer. This process promotes the renewal and repair of the intestinal mucosa and maintains the integrity of the intestinal barrier. A healthy intestinal barrier helps prevent harmful substances and pathogens from entering the blood, thereby reducing the risk of inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is the relative content of the fatty acids of pumpkin seed oil and linseed oil of the present invention.

[0040] Figure 2 This is a verification comparison data chart of ω-6 / ω-3 fatty acids of the mixed pressed oil of the present invention.

[0041] Figure 3 It is the Fourier infrared spectrum of the composite microcapsule (WP-LO-GA 65) of the present invention.

[0042] Figure 4 It is a scanning electron microscope picture of the composite microcapsule (WP-LO-GA 65) of the present invention.

[0043] Figure 5 Schematic diagram of the oil PV value (A) and p-AV value (B) of the composite microcapsule (WP-LO-GA 65) of the present invention during storage.

[0044] Figure 6It is a schematic diagram of the number of viable probiotic bacteria during storage of the composite microcapsule (WP-LO-GA 65) of the present invention.

[0045] Figure 7 It is a schematic diagram of the release rate of oil in the composite microcapsule (WP-LO-GA 65) of the present invention during the simulated digestion process in vitro.

[0046] Figure 8 It is the number of viable probiotics in the composite microcapsule (WP-LO-GA 65) of the present invention during the in vitro simulated digestion process.

[0047] Fig. 9 It is a schematic diagram of the particle size of the composite microcapsule (WP-LO-GA 65) of the present invention during the simulated digestion process in vitro. DETAILED DESCRIPTION

[0048] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0049] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0050] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0051] The present invention provides a method for preparing a composite microcapsule of pumpkin seed oil-multiple probiotics with balanced polyunsaturated fatty acids: the composite microcapsule comprises a core material of mixed pressed oil and probiotics and a wall material of a mixture of whey protein and gum arabic; the core material is wrapped in the wall material; the mixed pressed oil comprises pumpkin seed oil and linseed oil. The composite microcapsule preparation method comprises the following steps:

[0052] S1. Preparation of balanced polyunsaturated fatty acid mixed squeezed oil: pumpkin seeds and flax seeds were weighed in a mass ratio of 5:1 to 7.5:1, and the weighed pumpkin seeds and flax seeds were mixed and squeezed to obtain a mixed squeezed oil;

[0053] S2. Preparation of microcapsule wall materials;

[0054] S21. Select whey protein and gum arabic as microcapsule wall materials;

[0055] S22. Weigh whey protein, dissolve 9 g of whey protein in 300 mL of distilled water, and stir at room temperature for 1 h to completely hydrate to obtain a 3% whey protein solution; then pretreat the whey protein solution to obtain a whey protein stock solution, named WP;

[0056] The pretreatment method comprises the following steps: dropping 1 mol / L NaOH solution into a whey protein solution to adjust the pH to 12, heating the whey protein solution to 65°C after standing for 30 minutes to unfold and refold the protein, and then cooling the solution to room temperature, dropping 1 mol / L HCl solution to adjust the pH to 7, and centrifuging to obtain a supernatant to obtain a whey protein reserve solution.

[0057] By pre-treatment, the protein structure of whey protein is destroyed and the water solubility of the protein is improved, thereby enhancing the embedding property of the microcapsule of the present invention.

[0058] S23. Preparation of Gum Arabic Stock Solution

[0059] The order of steps S22 and S23 may be interchanged.

[0060] 6 g of gum arabic was dissolved in 200 mL of distilled water, stirred at 50 °C for 3 h, and then hydrated at 4 °C overnight to obtain a 3% gum arabic stock solution, named GA;

[0061] S24. 300 ml of whey protein solution and 200 ml of gum arabic solution were weighed and mixed, homogenized at 7000 rpm for 3 min, and 10% citric acid solution was added dropwise to adjust the pH to 3.6. The mixture was placed at 4°C overnight to allow the complex to be fully hydrated and named WP-GA65;

[0062] S3. Activation and preparation of probiotics

[0063] S31. Select one or more of Bifidobacterium pseudolongum, Bifidobacterium pseudosmall chain, Lactobacillus and Akkermansia mucinosa as probiotic strains;

[0064] In this embodiment, a mixture of pseudo-Bifidobacterium longum, pseudo-Bifidobacterium spp., Lactobacillus and Akkermansia mucinensis is preferably used as the probiotic strain. Lactobacillus is selected from one or more of Lactobacillus salivarius, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri and Lactobacillus johnsonii.

[0065] S32. The frozen probiotics were inoculated on a sterile culture medium, and after growth, they were inoculated again on a liquid culture medium for batch culture. Cell pellets were obtained by centrifugation during the logarithmic growth phase, and the precipitate was washed with sterile saline after centrifugation to obtain a concentrated bacterial solution;

[0066] The specific steps are as follows: take out the probiotics stored in -80℃ freezer, streak inoculate on sterile solid culture medium, culture at 37℃ for 48h, pick a single colony and place it in sterile liquid culture medium and culture it anaerobically at 37℃ for 16h, inoculate the activated bacterial solution into the liquid culture medium at a 1% inoculum, culture in batches, harvest the cell mass by centrifugation at 4℃ during the logarithmic growth phase, discard the supernatant, wash the precipitate twice with sterile saline, and prepare a single concentrated bacterial solution. At this time, the bacterial solution concentration is about 10 10 CFU / mL; In the preferred embodiment, when multiple strains are selected, the above resurrection operation is performed separately, and the obtained concentrated bacterial liquids are mixed to prepare a mixed concentrated bacterial liquid, and the concentration of the mixed bacterial liquid is 10 10 CFU / mL.

[0067] S33. Divide the concentrated bacterial solution into multiple equal portions, preferably two portions in this embodiment;

[0068] The order of steps S2 and S3 can be interchanged.

[0069] S4. Preparation of microcapsules

[0070] S41. The single or mixed concentrated bacterial solution and the mixed pressed oil were dispersed into the whey protein stock solution and homogenized by centrifugation at 7000rpm for 3min;

[0071] S42. The gum arabic stock solution was gradually added under centrifugation at 400 rpm; and then 10% citric acid solution was added to adjust the pH to 3.6 to induce complexation between the whey protein stock solution and the gum arabic stock solution;

[0072] S43. Then add 20 U / g of glutamine transaminase, heat and stir at 37°C for 2 hours to fully solidify, and finally the emulsion is vacuum freeze-dried to form solid composite microcapsules, which are sealed and placed in a 4°C high and low temperature test box for storage and named WP-LO-GA65.

[0073] In this embodiment, the mass ratio of the pumpkin seeds to the flax seeds is 5:1.

[0074] Determination of the ratio of pumpkin seeds to flax seeds is one of the key points of the present invention. Balanced polyunsaturated fatty acids are obtained by determining the ratio of pumpkin seeds to flax seeds. The present invention takes the recommended dietary intake ratio of PUFAs as a target and establishes a suitable mathematical model to determine the specific mixing ratio of pumpkin seeds to flax seeds.

[0075]

[0076] W p , W f —The percentage of the mass of the two raw materials, pumpkin seeds and flax seeds, in the total raw material formula to be determined;

[0077] Op , O f —Oil yield of pumpkin seeds and flax seeds (%);

[0078] H p , H f —Percentage of ω-6 PUFAs in pumpkin seed oil and flax seed oil;

[0079] h p 、h f —Percentage of ω-3 PUFAs in pumpkin seed oil and flax seed oil;

[0080] Y=4:1~6:1, this ratio is the recommended dietary intake ratio of ω-6 / ω-3 PUFAs.

[0081] The oil yield ratio of pumpkin seeds and flax seeds is as follows based on statistical data:

[0082] Table 1-1 Oil extraction rate of pumpkin seed oil and flaxseed oil

[0083]

[0084] As shown in Table 1-1, the oil yields of pumpkin seeds and flax seeds are 26.72±0.05% and 33.45±0.04% respectively. Vegetable oil is an indispensable substance for human life activities and body metabolism. Fatty acid is one of the important indicators for determining the quality of edible oil. Its composition and ratio largely determine the nutritional value of edible oil. The contents of five main fatty acids in pumpkin seed oil (PO) and flax seed oil (FO), namely palmitic acid (PA), stearic acid (SA), oleic acid (OA), linoleic acid (LA), and linolenic acid (ALA) are as follows: Figure 1As shown. The fatty acids in PO are mainly composed of two unsaturated fatty acids, OA (32.20±0.25%) and LA (50.32±0.13%), while its ALA content (0.11±0.06%) is relatively low. It can be seen that the ω-6 / ω-3PUFAs ratio in PO does not meet the dietary intake ratio recommended by the Chinese Nutrition Society for polyunsaturated fatty acids (PUFAs) (ω-6 / ω-3PUFAs=4:1~6:1). Long-term consumption is likely to cause fatty liver, hyperlipidemia, atherosclerosis and other diseases. The unsaturated fatty acid content in FO is 88.19%, mainly including ALA (51.76±0.25%)>OA (20.48±0.07%)>LA (15.95±0.27%). Its ALA content is one of the highest ω-3PUFAs content in vegetable oils known so far. ALA is not only an essential fatty acid for the human body, but can also be synthesized into metabolites such as eicosapentaenoic acid and docosahexaenoic acid through dehydrogenation and carbon chain elongation in the body. It has the effects of anti-thrombosis, lowering blood lipids, and preventing and treating ischemic cardiovascular diseases.

[0085] Determining the mass ratio of pumpkin seeds and flax seeds is a necessary prerequisite for the preparation of mixed pressed oil, which affects the proportion of PUFAs (polyunsaturated fatty acids) in the oil. Figure 1 The polyunsaturated fatty acids (PUFAs) analysis results of pumpkin seed oil (PO) and flax seed oil (FO) were substituted into the mathematical model established by formula 1-1. It was calculated that when the ω-6 / ω-3PUFAs ratio of the mixed pressed oil sample met the 4:1 to 6:1, the mass ratio range (Wp / Wf) of pumpkin seed and flax seed oil crops was 4.84 to 7.49. The predicted value is calculated by the mathematical model established in Table 1-1, and the experimental value is the value measured in the actual experiment.

[0086] Pumpkin seeds and flax seeds were mixed and pressed in a mass ratio of 4:1 to 8:1 to obtain mixed pressed oil, and its fatty acid content was determined to further verify the reliability of the theoretical results. Figure 2As shown in the figure, the predicted and experimental values ​​of the ratio of ω-6 / ω-3 polyunsaturated fatty acids (PUFAs) in mixed pressed oil samples obtained from different oil crop ratios are shown. The results show that when Wp / Wf is less than 7, the experimental value of ω-6 / ω-3PUFAs in mixed pressed oil is slightly lower than the predicted value. The high viscosity extraction system is not conducive to the extraction of oil lipid components, resulting in a decrease in the fatty acid content of mixed cold pressed oil. It may also be because after the oil crops enter the press, the raw materials are broken and easily block the press cage, thereby affecting the oil output. When Wp / Wf is greater than 7, the experimental value of ω-6 / ω-3 polyunsaturated fatty acids (PUFAs) is almost close to the predicted value. In general, the predicted value curve and the experimental value curve within the calculation result range obtained by this mathematical model show a good fit, which is a fast way to determine the quality ratio of oilseeds in the mixed pressing process. When Wp / Wf is 4, the ratio of ω-6 / ω-3 polyunsaturated fatty acids (PUFAs) in the mixed squeezed oil sample is 3.26±0.02, which is lower than the recommended dietary ratio. The proportion of pumpkin seeds in the mixed oil crops continues to increase gradually. As the Wp / Wf gradient increases, the ω-6 / ω-3PUFAs ratio shows an increasing state. When Wp / Wf is 5 and 7.5, the ω-6 / ω-3PUFAs ratio in the mixed squeezed oil sample is 4.08±0.09 and 6.11±0.04, respectively, which are slightly higher than the upper and lower limits of the recommended dietary ratio. Therefore, the quality ratio of the two oil crops is finally determined to be 5 to 7.5, and the corresponding mixed squeezed oil oil yield is 28.90% to 28.56%. Since flax seeds have a lower market price than pumpkin seeds, in this embodiment, the mass ratio of pumpkin seeds and flax seeds is preferably 5 to save production costs.

[0087] The specific production method of mixed pressed oil is as follows: firstly, pumpkin seeds and flax seeds are weighed and mixed evenly in a mass ratio of 5:1, cold pressed and centrifuged, and the upper layer of clear oil is taken to obtain balanced mixed pressed oil. At the same time, a certain mass of pumpkin seeds and flax seeds are weighed and cold pressed according to the same parameters to obtain oil, centrifuged, and the upper layer of clear oil is taken, and mixed to obtain direct blended oil (BO) as a control.

[0088] Compared with the direct blending oil which is pressed separately and then mixed, the mixed pressed oil has stronger oxidation stability.

[0089] Preparation of control:

[0090] The present invention also includes mixed squeezed oil microcapsules and probiotic microcapsules, which are used as reference materials for the embedding rate and viable bacterial count of composite microcapsules. The core material of the mixed squeezed oil microcapsules includes the mixed squeezed oil in step S1, and the core material of the probiotic microcapsules includes the single probiotic concentrate or the mixed probiotic concentrate in step S3; the wall materials in the mixed squeezed oil microcapsules and the Lactobacillus rhamnosus microcapsules are the microcapsule wall materials in step S2.

[0091] 1. Preparation of mixed pressed oil microcapsules

[0092] The preparation method of mixed squeezed oil microcapsules is as follows: 10.0g of mixed squeezed oil is dispersed into the whey protein stock solution and homogenized at 7000rpm for 3min. Then, the gum arabic stock solution is gradually added under continuous stirring at 400rpm. 10% citric acid solution is added dropwise to adjust the pH to 3.6 to induce the complexation between the whey protein stock solution and the gum arabic stock solution. Then 180U of glutamine transaminase is added, and the mixture is heated and stirred at 37℃ for 2h to fully solidify to obtain an O / W emulsion. It is named WP-O-GA 65. The final solution is stored at 4℃ for future use. The emulsion is placed in a vacuum freeze dryer and dried to form solid microcapsules, which are then sealed and placed in a 4℃ high and low temperature test chamber for future use.

[0093] 2. Preparation of probiotic microcapsules

[0094] The preparation method of probiotic microcapsules is as follows:

[0095] Disperse the single or mixed concentrated bacterial solution into the whey protein stock solution and homogenize at 7000rpm for 3min. Then, gradually add the gum arabic stock solution under continuous stirring at 400rpm. Add 10% citric acid solution dropwise to adjust the pH to 3.6 to induce the complexation between whey protein and gum arabic. Then add 180U of glutamine aminotransferase, heat and stir at 37℃ for 2h to fully solidify, and finally the emulsion is vacuum freeze-dried to form solid microcapsules, which are sealed and placed in a 4℃ high and low temperature test box for storage and are named WP-L-GA 65.

[0096] The single concentrated bacterial liquid and the mixed concentrated bacterial liquid in the embodiments of the present invention are named for the convenience of distinction according to the different selected bacterial species. The only difference is the selected bacterial species. The concentrated bacterial liquid (or probiotics) can also be used to directly refer to the single concentrated bacterial liquid and the mixed concentrated bacterial liquid.

[0097] Parameter analysis of the product prepared by the present invention:

[0098] 1. Analysis of embedding rate and viable bacteria count

[0099] The embedding rate is a key parameter of oil microcapsules, which can determine the ability of the wall material to embed the core material. The embedding rates of mixed pressed oil samples in mixed pressed oil microcapsules (WP-O-GA 65) and composite microcapsules (WP-LO-GA 65) were 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 embedding rate of oil increased significantly by 4.43% (p<0.05). The embedding rate is usually closely related to factors such as wall materials, embedding process, and drying method. Under the same conditions, mixed pressed oil (MPO), probiotics alone, and both co-encapsulation are more advantageous than co-encapsulation. It is very important to minimize the loss of cell viability during the microencapsulation process to ensure the maximum number of living cells in the matrix. Table 2-1 shows the number of viable probiotics in probiotic microcapsules (WP-L-GA65) and composite microcapsules (WP-LO-GA 65). It can be observed from the table that the number of viable probiotics in the composite microcapsules (WP-LO-GA65) (8.33±0.17log CFU / g) is higher than that in the control group probiotic microcapsules (WP-L-GA65). The increase in the number of viable bacteria may be related to the structure of the microcapsules. The presence of mixed pressed oil (MPO) provides an additional protective barrier for probiotics, weakening the impact of the external adverse environment. In addition, mixed pressed oil (MPO) adheres to the phospholipid protein membrane of probiotics, limiting the migration of probiotic cells. Therefore, the oil-fat composite microcapsules loaded with probiotics of the present invention are a promising encapsulation technology that can improve the survival rate of probiotics.

[0100] Table 2-1 Encapsulation of oils and active bacteria number in WP-LO-GA microcapsule

[0101]

[0102] Note: The test results are expressed as mean ± standard deviation. The values ​​of the same index with different letters in different samples are significantly different (p<0.05).

[0103] 2. Fourier transform infrared spectroscopy analysis

[0104] Depend on Figure 3 It can be seen that the whey protein storage solution is at 3367.45cm -1 There is a characteristic absorption peak of OH or NH stretching vibration, 2927.5cm -1 The absorption peak of is related to CH stretching vibration. The absorption peak of amide I band (C=O stretching vibration) is located at 1651.01 cm-1 The absorption peak of amide II band (NH bending vibration) is at 1544.72 cm -1 The absorption peak of amide III band (CN stretching vibration) is located at 1248.44 cm -1 With the introduction of gum arabic storage solution, the peak of the amide I band of whey protein storage solution increased from 1651.01 cm -1 Towards the lower wave number 1645.69cm -1 The typical bands of the storage solution of gum arabic include 3384.94 cm -1 OH stretching vibration at 2930.43 cm -1 CH stretching vibration at 1612 cm -1 Asymmetric vibration of -COO, 1421.17 cm -1 OH bending vibration at 1142.16cm -1 and 1067.75cm -1 The CO stretching vibration and COH bending vibration are located at 3009.34cm. -1 Corresponding to the stretching vibration of the unsaturated carbon CH bond, 2926.04 cm -1 The 2855.89 cm -1 The position is related to the CH stretching vibration of the aldehyde group, 1746.67 cm -1 The stretching vibration of C=O is 1162.08cm -1 、1098.31cm -1 The characteristic peak at 1612 cm is related to CO stretching vibration. The infrared spectrum peak intensity related to mixed pressed oil (MPO) in mixed pressed oil microcapsules (WP-O-GA65) and composite microcapsules (WP-LO-GA65) is significantly weakened, indicating that mixed pressed oil (MPO) is embedded in the microcapsules. It is worth noting that in the spectra of probiotic microcapsules (WP-L-GA65) and composite microcapsules (WP-LO-GA 65), the original gum arabic storage solution has a peak at 1612 cm -1 The absorption peak at 1725 cm -1 A new absorption peak appears nearby.

[0105] 3. Scanning Electron Microscope Physical Shape Analysis

[0106] Depend on Figure 4It can be seen that no free probiotics were observed on the surface of the probiotic microcapsules (WP-L-GA65) and the composite microcapsules (WP-LO-GA 65), indicating that the freeze-drying process of the emulsion did not cause the microcapsule structure to rupture and the cells were still fixed in the core area. The surfaces of all microcapsules were dented and wrinkled, which may be due to the rapid evaporation of water during the vacuum freeze-drying process, which caused the microcapsules to form porous aggregates and lose their spherical structure. This is one of the typical results of the vacuum freeze-drying process. The surface of the probiotic microcapsule (WP-L-GA 65) is relatively smooth, but there are still obvious aggregates of whey protein and gum arabic (WP-GA) on its surface. The mixed pressed oil microcapsules (WP-O-GA 65) and the composite microcapsules (WP-LO-GA 65) showed similar morphology, showing blocky shapes with wrinkled surfaces. The structure of the composite microcapsules (WP-LO-GA 65) seemed to be more compact than that of the mixed pressed oil microcapsules (WP-O-GA 65), which was consistent with the higher oil encapsulation rate and the number of viable probiotics in the composite microcapsules (WP-LO-GA 65).

[0107] 4. Analysis of PV and p-AV values ​​of oil during storage of composite microcapsules

[0108] like Figure 5 The changes in PV values ​​(A) and p-AV values ​​(B) of mixed pressed oil (MPO) (control), mixed pressed oil microcapsules (WP-O-GA 65) and composite microcapsules (WP-LO-GA 65) stored at 60°C for 28 days are shown. As expected, the PV values ​​and p-AV values ​​of both microcapsules during storage were significantly higher than those of unencapsulated mixed pressed oil (MPO) (p < 0.05). After being stored at 60℃ for 28 days, the PV values ​​of mixed pressed oil (MPO), probiotic microcapsules (WP-L-GA 65) and composite microcapsules (WP-LO-GA 65) increased to 45.98, 24.76 and 22.38 mmol / kg, respectively, which were 63.76, 33.87 and 30.52 times higher than the initial PV values, respectively. The PV value of mixed pressed oil (MPO) (17.17 mmol / kg) on ​​the 7th day exceeded the limit of GB / T2716-2018 "National Standard of the People's Republic of China for Vegetable Oil" (≤9.85 mmol / kg), while that of WP-L-GA (13.34 mmol / kg) and WP-LO-GA (11.65 mmol / kg) microcapsules occurred on the 14th day. The p-AV values ​​of the three samples increased to 12.98, 7.82, and 7.41, respectively, after the storage ended, which were 132.81, 76.62, and 75.39 times higher than the initial p-AV values.

[0109] pass Figure 6From the data analysis, it can be seen that during the entire storage period, the composite microcapsule (WP-LO-GA65) showed the lowest PV value and p-AV value, indicating that the addition of Lactobacillus rhamnosus (LGG) promoted the oxidative stability of oils.

[0110] 5. Analysis of the number of viable probiotic bacteria during storage of composite microcapsules

[0111] To maximize the health benefits of probiotics, it is important to reduce the loss of their vitality before they are consumed. Studies have shown that low-temperature storage is an effective way to maintain the vitality of probiotics in microcapsules. Figure 6 As shown in the figure, the initial viable bacterial counts of the probiotic microcapsules (WP-L-GA 65) and composite microcapsules (WP-LO-GA 65) (7.58 log CFU / g and 8.33 log CFU / g, respectively) decreased after being stored at 4°C for 28 days (reduced by 1.23 and 1.03 log CFU / g, respectively), but both remained above 6 log CFU / g. The composite microcapsules (WP-LO-GA 65) showed the highest probiotic activity throughout the storage period.

[0112] 6. In vitro simulated digestion analysis of composite microcapsules

[0113] 6.1 Analysis of oil release rate

[0114] The release rates of two microcapsules, mixed pressed oil microcapsules (WP-O-GA 65) and composite microcapsules (WP-LO-GA 65), during simulated digestion in vitro, such as Figure 7 As shown. There was a significant difference in the percentage of oil released from the two microcapsules in the gastric digestion stage (SGF) and the intestinal 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) in the two microcapsules of mixed pressed oil microcapsules (WP-O-GA 65) and composite microcapsules (WP-LO-GA 65) were 17.7% and 15.9%, respectively. Among them, the release rate of oil in the composite microcapsule (WP-LO-GA 65) was the lowest, indicating that it could better prevent the premature release of active substances. After entering the intestinal digestion stage (SIF) for digestion, the microcapsules were rapidly hydrolyzed, and the release rates of mixed pressed oil (MPO) in the two microcapsules reached 76.7% and 79.5%, which were significantly higher than the release of oil in the gastric digestion stage (SGF) (p<0.05). The above results show that microencapsulated mixed pressed oil (MPO) can slow down its release rate, achieve a sustained release effect, and improve its bioavailability.

[0115] 6.2 Analysis of viable count of probiotics

[0116] The survival of two probiotic microcapsules, probiotic microcapsules (WP-L-GA 65) and composite microcapsules (WP-LO-GA 65), under simulated gastrointestinal conditions, such as Figure 8 As shown. The experiment found that after passing through the gastric digestion stage (SGF), the microcapsules did not break, but slightly expanded. After the probiotic microcapsules (WP-L-GA 65) were exposed to the gastric digestion stage (SGF) and the intestinal digestion stage (SIF), the probiotic cells decreased by 1.57log CFU / g. Unlike the probiotic 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 the gastric digestion stage (SGF) and the intestinal digestion stage (SIF), the probiotics in the composite microcapsules (WP-LO-GA 65) decreased by 1.01log CFU / g. The high survival rate retention of probiotics may be related to the release of the oil phase. When the probiotic microcapsules containing oil are digested, the wall material of the microcapsules is destroyed, resulting in the release of the oil phase. In summary, these experimental conclusions strongly prove that loading probiotics with oil microcapsules can more effectively improve the survival rate of probiotics in the human digestive system.

[0117] 6.3 Analysis of particle size change

[0118] The particle size changes of the three microcapsules during in vitro simulated digestion, such as Fig. 9 Before digestion, the average particle size (D 4,3 ) values ​​were 12.54, 10.12, and 13.14 μm, respectively. After the simulated gastric digestion stage, the droplet size increased significantly (p<0.05), increasing to 15.38, 14.35, and 16.86 μm, respectively. This may be due to the decrease in pH value and increase in ionic strength in the simulated gastric fluid, which changed the electrostatic interaction between the droplets and caused particle aggregation and flocculation, thereby increasing the particle size of the microcapsules. After the intestinal digestion stage, the particle size of the microcapsules decreased significantly (p<0.05), and the average particle size (D4,3) values ​​of the mixed pressed oil microcapsules (WP-O-GA65), probiotic microcapsules (WP-L-GA 65), and composite microcapsules (WP-LO-GA 65) were 5.38, 4.89, and 5.85 μm, respectively.

[0119] The present invention uses a mixture of pseudo-bifidobacterium longum, pseudo-bifidobacterium small chain, lactobacillus and mucin Akkermansia as probiotic strains as model probiotics, uses mixed squeeze oil microcapsules (WP-O-GA 65) as carrier loads to deliver probiotics, and constructs a multi-layer embedding system with good stability, high embedding rate and protective effect on probiotics. The composite microcapsule system can be slowly released, and the wall material of the microcapsule is digested and destroyed after reaching the intestinal tract, and the grease is decomposed into fatty acids and then absorbed by the intestinal tract, and the probiotics are completely released, so that the probiotics are released near a specific part. Multi-mixed probiotic strains have the advantages of regulating intestinal microorganisms, enhancing immunity, preventing intestinal inflammation, repairing and renewing intestinal mucin, and the several multi-mixed probiotics can also increase survival and promotion between each other, which helps to establish intestinal flora and establish a healthy and stable intestinal micro-ecological system.

Claims

1. A method for preparing balanced polyunsaturated fatty acid pumpkin seed oil-multi-probiotic composite microcapsules: characterized in that: The composite microcapsule comprises a mixed squeezed oil and probiotics as a core material and a mixture of whey protein and gum arabic as a wall material; the core material is embedded in the wall material; the mixed squeezed oil comprises pumpkin seed oil and linseed oil; The composite microcapsule preparation method comprises the following steps: S1. Preparation of balanced polyunsaturated fatty acid mixed pressed oil; S11. Weigh pumpkin seeds and flax seeds in a mass ratio of 4:1 to 6:1, respectively, mix the weighed pumpkin seeds and flax seeds, and then press them to obtain a mixed pressed oil; S2. Preparation of microcapsule wall materials; S21. Select whey protein and gum arabic as microcapsule wall materials; S22. Weigh whey protein and place it in distilled water and stir at room temperature to obtain a whey protein solution after complete hydration; and then prepare a whey protein stock solution after appropriate pretreatment of the whey protein solution; The pretreatment method comprises: dropping 1 mol / L NaOH solution into the whey protein solution to adjust the pH to 12, heating the whey protein solution to 65°C after standing for 30 minutes to unfold the protein and then refold it, then cooling it to room temperature, dropping 1 mol / L HCl solution to adjust the pH to 7, and centrifuging to obtain the supernatant to obtain the whey protein reserve solution; S23. Prepare gum arabic stock solution; S24. After weighing the whey protein solution and the gum arabic solution, the mixture was centrifuged and homogenized, and a 10% citric acid solution was added dropwise to adjust the pH to 3.6, and the mixture was placed at 4° C. overnight to allow the complex to be fully hydrated; S3. Activation and preparation of probiotics; S31. Select one or more of Bifidobacterium pseudolongum, Bifidobacterium pseudosmall chain, Lactobacillus and Akkermansia mucinosa as probiotic strains; S32. The frozen probiotics were inoculated on a sterile culture medium, and after growth, they were inoculated again on a liquid culture medium for batch culture. Cell pellets were obtained by centrifugation during the logarithmic growth phase, and the precipitate was washed with sterile saline after centrifugation to obtain a concentrated bacterial solution; S33. Divide the concentrated bacterial solution into two equal parts; S4. Preparation of microcapsules; S41. The concentrated bacterial solution and the mixed squeezed oil are dispersed into the whey protein reserve solution and homogenized for a certain period of time; S42. The gum arabic stock solution was gradually added under a homogenizing environment; and then 10% citric acid solution was added to adjust the pH to 3.6 to induce complexation between the whey protein stock solution and the gum arabic stock solution; S43. Then add 20 U / g of glutamine transaminase, heat and stir at 37°C for 2 h to fully solidify, and finally the emulsion is vacuum freeze-dried to prepare solid composite microcapsules.

2. The method for preparing the composite microcapsule according to claim 1, characterized in that: The mass ratio of the pumpkin seeds to the flax seeds is 5 to 7.5:

1.

3. The method for preparing the composite microcapsule according to claim 1, characterized in that: The step S22 is specifically as follows: dissolving whey protein in distilled water, stirring at room temperature for 1 hour to completely hydrate, and obtaining a 3% whey protein solution.

4. The method for preparing the composite microcapsule according to claim 1, characterized in that: The step of S23 is specifically as follows: dissolving gum arabic in distilled water, stirring at 50° C. for 3 hours, and then hydrating at 4° C. overnight to obtain a 3% gum arabic stock solution.

5. The method for preparing the composite microcapsule according to claim 1, characterized in that: The steps of S32 are as follows: taking out the probiotic strains stored at -80°C; streaking and inoculating on a sterile solid culture medium, culturing at 37°C for 48 hours, picking a single colony and placing it in a sterile liquid culture medium and anaerobic culturing at 37°C for 16 hours, inoculating the activated bacterial solution into the liquid culture medium at a 1% inoculum, and culturing in batches, harvesting the cell mass by centrifugation at 4°C during the logarithmic growth phase, discarding the supernatant, and washing the precipitate twice with sterile saline to prepare a single concentrated bacterial solution, at which the bacterial solution concentration is approximately 10 10 CFU / mL; when multiple bacterial species are selected, the above resurrection operations are performed separately, and the obtained concentrated bacterial liquids are mixed to prepare a mixed concentrated bacterial liquid, and the concentration of the mixed bacterial liquid is 1010 CFU / mL.

6. The method for preparing the composite microcapsule according to claim 1, characterized in that: The concentrated bacterial solution of S33 is divided into two equal parts.

7. The method for preparing the composite microcapsule according to claim 1, characterized in that: It also includes mixed pressed oil microcapsules and probiotic microcapsules; the core material of the mixed pressed oil microcapsules includes the mixed pressed oil in step S1, and the core material of the probiotic microcapsules includes the probiotics in step S3; the wall materials in the mixed pressed oil microcapsules and the probiotic microcapsules are both the microcapsule wall materials in step S2; the mixed pressed oil microcapsules and the probiotic microcapsules are used as reference objects for the embedding rate and viable bacteria count parameters of the composite microcapsules.

8. The method for preparing the composite microcapsule according to claim 7, characterized in that: The preparation method of the mixed pressed oil microcapsules comprises the following steps: dispersing the mixed pressed oil into a whey protein reserve solution, homogenizing at 7000 rpm for 3 minutes; then gradually adding a gum arabic reserve solution under continuous stirring at 400 rpm; adding a 10% citric acid solution dropwise to adjust the pH to 3.6 to induce complexation between the whey protein and the gum arabic; and then adding 20 U / g of glutamine transaminase, heating and stirring at 37° C. for 2 hours to fully solidify, and obtaining an emulsion.

9. The method for preparing the composite microcapsule according to claim 7, characterized in that: The preparation method of the probiotic microcapsules comprises the following steps: dispersing the single or mixed concentrated bacterial solution into a whey protein stock solution, homogenizing at 7000 rpm for 3 minutes; then gradually adding a gum arabic stock solution under continuous stirring at 400 rpm; adding a 10% citric acid solution dropwise to adjust the pH to 3.6 to induce complexation between the whey protein stock solution and the gum arabic stock solution; then adding 20 U / g of glutamine transaminase, heating and stirring at 37° C. for 2 hours to fully solidify, and finally vacuum freeze-drying the emulsion to prepare solid microcapsules.

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