Preparation method and application of hyaluronic acid-concentrated whey protein encapsulated probiotic microcapsule

Abstract

The invention belongs to the technical field of preparation of probiotic microcapsules, and particularly relates to a preparation method and application of hyaluronic acid-concentrated whey protein encapsulated probiotic microcapsules. According to the invention, the hyaluronic acid and the concentrated whey protein are jointly used as the encapsulating material to microencapsulate the probiotics, and the particles formed by mixing the hyaluronic acid and the concentrated whey protein can enhance the protection effect of the encapsulating material on the probiotics; according to the hyaluronic acid, the concentrated whey protein is modified, so that the formed microcapsule has a more uniform and compact surface morphology, the condensation of surface milk powder after the microcapsule is dried is prevented, and the porosity of the surface of the microcapsule is reduced due to the interaction of the concentrated whey protein and the hyaluronic acid; the influence of harmful environmental factors such as high temperature and high acidity on microorganisms is reduced, the protection effect of the microcapsule is improved, and the problem of probiotic leakage caused by only using concentrated whey protein as an encapsulating material is avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic microcapsule preparation, and particularly relates to a preparation method and application of probiotic microcapsules encapsulated by hyaluronic acid-concentrated whey protein. Background Art

[0002] Probiotics are a type of active microorganisms that are beneficial to the host by colonizing in the host and changing the composition of the bacterial flora in a certain part of the host. They have many benefits for humans and animals, such as alleviating lactose intolerance, enhancing the immune system, preventing and treating cancer, lowering cholesterol levels, reducing gastrointestinal diseases including antibiotic-associated diarrhea, increasing the eradication rate of the gastric pathogen Helicobacter pylori, and regulating brain function. They are widely used in the food and pharmaceutical fields. In order for these active microorganisms that are beneficial to the host to play a positive role to the maximum extent, they must maintain their activity and stable metabolic capacity in food or medicine. However, during processing, transportation, storage and digestion, probiotics are often affected by adverse factors such as processing conditions, storage temperature, and the host digestive system (gastric acid, bile salts, enzymes), resulting in reduced activity, so that the number of live bacteria that eventually colonize the human intestine is less than 10. 6 CFU / g or CFU / g, it cannot play a probiotic role. In order to improve the survival rate of probiotics during storage and digestion, researchers in this field have proposed various solutions, such as selecting acid-resistant and bile-resistant strains, stress adaptation, application of cell protectants and microencapsulation, among which microencapsulation is currently considered to be the most effective method, which can not only protect probiotics from the influence of external adverse environment, but also achieve controlled release at the target location.

[0003] Microencapsulation refers to the process of using natural or synthetic polymer materials to encapsulate tiny solid particles, droplets or gases to form particles with a particle size of several micrometers to several millimeters and semi-permeable or sealed capsules. Among them, the material embedded inside the microcapsule is called the core material, and the external capsule is called the wall material. In the food industry, the selection of suitable microcapsule wall materials and methods to obtain probiotic microcapsule products with high probiotic activity, economic benefits and application potential is still one of the research hotspots in the field of probiotic food. A variety of materials such as prebiotics and dairy products can be used as protective encapsulation materials for microcapsules, which can selectively stimulate the growth and activity of probiotics in the gastrointestinal tract. In addition, the combination of microcapsules with other materials with emulsification can further enhance the stability of embedded microorganisms, such as concentrated whey protein, which is the main wall material for probiotic encapsulation. However, concentrated whey protein microcapsules are easily decomposed in a highly acidic environment, which is not conducive to the preservation of probiotics and maintaining their activity and metabolic capacity. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method and application of hyaluronic acid-concentrated whey protein encapsulated probiotic microcapsules. The preparation method uses hyaluronic acid and concentrated whey protein as encapsulation materials to microencapsulate probiotics, which can reduce the porosity of the microcapsules, reduce the leakage of probiotics, and ensure the activity of the probiotics.

[0005] The present invention provides a method for preparing probiotic microcapsules encapsulated by hyaluronic acid-concentrated whey protein, comprising the following steps: mixing and heating a concentrated whey protein solution with hyaluronic acid to obtain a colloid mixture;

[0006] The colloidal mixture is mixed with a probiotic suspension and frozen, and the obtained frozen product is freeze-dried to obtain the probiotic microcapsules.

[0007] Preferably, the concentration of the concentrated whey protein solution is 8% to 12% w / v, and the volume ratio of the mass of the hyaluronic acid to the concentrated whey protein solution is 3 to 5 g:100 mL.

[0008] Preferably, the molecular weight of the hyaluronic acid is (3-170)×10 4 Da.

[0009] Preferably, the molecular weight of the hyaluronic acid is (100-170)×10 4 da, (20~40)×10 4 da or (3~10)×10 4 Da.

[0010] Preferably, the volume ratio of the colloidal mixture to the probiotic suspension is 1 to 2:1; the effective live bacteria concentration in the probiotic suspension is not less than 10 10 CFU / mL.

[0011] Preferably, the probiotics include lactic acid bacteria.

[0012] Preferably, the lactic acid bacteria include one or more of Lactobacillus plantarum, Lactococcus lactis, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.

[0013] Preferably, the mixing process is accompanied by stirring, and the stirring speed is 450-1200 r / min; the heating temperature is 70-80° C., and the heating time is 15-20 min.

[0014] Preferably, the freezing temperature is -80°C and the time is 12 hours; the freeze-drying temperature is -45°C, the pressure is 5.33 Pa, and the time is 48 hours.

[0015] The present invention also provides the preparation method described in the above technical solution or the use of the probiotic microcapsules prepared by the preparation method described in the above technical solution in one or more of food preparation, drug preparation, probiotic storage, probiotic transportation and probiotic delivery.

[0016] Beneficial effects:

[0017] The present invention provides a method for preparing probiotic microcapsules encapsulated by hyaluronic acid-concentrated whey protein, comprising the following steps: mixing and heating a concentrated whey protein solution with hyaluronic acid to obtain a colloidal mixture; mixing and freezing the colloidal mixture with a probiotic suspension, and freeze-drying the obtained frozen product to obtain the probiotic microcapsules. The present invention uses hyaluronic acid and concentrated whey protein as encapsulation materials to microencapsulate probiotics, and the microparticles formed after the hyaluronic acid and concentrated whey protein are mixed can enhance the protective effect of the encapsulation material on the probiotics. The hyaluronic acid can modify the concentrated whey protein to make the formed microcapsules have a more uniform and dense surface morphology, prevent the coagulation of milk powder on the surface of the microcapsules after drying, and the interaction between the concentrated whey protein and the hyaluronic acid reduces the porosity of the microcapsule surface, reduces the influence of harmful environmental factors such as high temperature and high acidity on microorganisms, increases the protective effect of the microcapsules, and avoids the problem of leakage of probiotics caused by using only concentrated whey protein as an encapsulation material. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0019] Figure 1 The flowchart of the preparation of probiotic microcapsules encapsulated by hyaluronic acid-concentrated whey protein in Example 1;

[0020] Figure 2 This is a SEM image of the probiotic microcapsules encapsulated by hyaluronic acid-concentrated whey protein prepared in Examples 2 to 13 in Test Example 1;

[0021] Figure 3 The survival rate of the four probiotics in the simulated gastrointestinal fluid in test example 3 is measured, mean±SD, n=3;

[0022] Figure 4 The release test results of the probiotic microcapsules encapsulated by hyaluronic acid-concentrated whey protein in the simulated intestinal fluid in Test Example 5, mean±SD, n=3;

[0023] Figure 5 The results of the viability test of the probiotic microcapsules encapsulated by hyaluronic acid-concentrated whey protein in Test Example 6 after continuous incubation in simulated gastric fluid and simulated intestinal fluid, mean±SD, n=3;

[0024] Figure 6 These are the stability test results of free probiotics and probiotic microcapsules stored at different temperatures in Test Example 7, mean±SD, n=3. DETAILED DESCRIPTION

[0025] The present invention provides a method for preparing probiotic microcapsules encapsulated by hyaluronic acid-concentrated whey protein, comprising the following steps: mixing and heating a concentrated whey protein solution with hyaluronic acid to obtain a colloid mixture;

[0026] The colloidal mixture is mixed with a probiotic suspension and frozen, and the obtained frozen product is freeze-dried to obtain the probiotic microcapsules.

[0027] The present invention mixes and heats a concentrated whey protein solution with hyaluronic acid to obtain a colloidal mixture. As an embodiment, the preparation method of the concentrated whey protein solution of the present invention comprises: mixing the concentrated whey protein with distilled water, stirring evenly at room temperature, and placing at 4°C for 10 to 12 hours to obtain the whey protein solution. As an embodiment, the stirring time can be 2 hours. As an embodiment, the concentration of the concentrated whey protein solution of the present invention can be 8% to 12% w / v; as another embodiment, the concentration of the concentrated whey protein solution can be 10% w / v. The present invention does not specifically limit the source of the concentrated whey protein, and it can be purchased through conventional commercial channels.

[0028] As an embodiment, the mass ratio of the hyaluronic acid of the present invention to the volume ratio of the concentrated whey protein solution can be 3-5 g:100 mL; as another embodiment, the mass ratio of the hyaluronic acid to the volume ratio of the concentrated whey protein solution can be 4 g:100 mL. As an embodiment, the molecular weight of the hyaluronic acid of the present invention can be (3-170)×10 4 da; As another embodiment, the molecular weight of the hyaluronic acid may be (100-170)×10 4 da, (20~40)×10 4 da or (3~10)×10 4 da; As another embodiment, the molecular weight of the hyaluronic acid may be (100-170)×10 4 Da.

[0029] As an embodiment, the mixing process of the present invention is accompanied by stirring, and the stirring speed can be 450-1200r / min; as another embodiment, the stirring speed can be 450r / min. As an embodiment, the heating temperature can be 70-80°C; as another embodiment, the heating temperature can be 80°C. As an embodiment, the heating time of the present invention can be 15-20min; as another embodiment, the heating time can be 20min.

[0030] After obtaining the colloidal mixture, the present invention mixes and freezes the colloidal mixture with a probiotic suspension, and freeze-dries the obtained frozen product to obtain the probiotic microcapsules. As an embodiment, the volume ratio of the colloidal mixture to the probiotic suspension of the present invention can be 1 to 2:1; as another embodiment, the volume ratio of the colloidal mixture to the probiotic suspension of the present invention can be 2:1. As an embodiment, the effective live bacteria concentration in the probiotic suspension of the present invention is not less than 10 10 CFU / mL. As an embodiment, the probiotics of the present invention may be lactic acid bacteria; as another embodiment, the lactic acid bacteria of the present invention may be one or more of Lactobacillus plantarum, Lactococcus lactis, Lactobacillus delbrueckii and Streptococcus thermophilus. In a specific embodiment, the Lactobacillus plantarum of the present invention may be Lactobacillus plantarum LP01; the Lactococcus lactis may be Lactococcus lactis MG1363; the Lactobacillus delbrueckii may be Lactobacillus delbrueckii ATCC 11842; the Streptococcus thermophilus may be Streptococcus thermophilus ST-1.

[0031] As an embodiment, the freezing temperature of the present invention can be -80°C, and the time can be 12 to 24 hours; as another embodiment, the freezing temperature can be -80°C, and the time can be 12 hours. As an embodiment, the freeze-drying temperature can be -45°C, the pressure can be 5.33 Pa, and the time can be 48 hours.

[0032] As an embodiment, the probiotic microcapsules obtained by the preparation method of the present invention can be stored at 4°C.

[0033] The present invention also provides the use of the preparation method described in the above technical solution or the probiotic microcapsules prepared by the preparation method described in the above technical solution in one or more of food preparation, drug preparation, probiotic preservation, probiotic transportation and probiotic delivery. The probiotic microcapsules prepared by the preparation method of the present invention are more adaptable to highly acidic environments, such as having a higher survival rate in simulated gastric fluid; and can be slowly released in simulated intestinal fluid, etc., which is conducive to the colonization of probiotics in intestinal tissues, and can maintain good stability under -4 to 25 ° C. At the same time, the probiotic microcapsules have no pores, have a better protective effect on probiotics, and can maintain good activity of probiotics during transportation, in vivo delivery, etc. Based on the above advantages, the preparation method of the present invention or the probiotic microcapsules prepared by the preparation method can be used in food preparation, drug preparation, probiotic preservation, probiotic transportation and probiotic delivery.

[0034] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] All glassware used in the following examples were sterilized in an autoclave at 121°C for 20 min.

[0036] Example 1

[0037] A method for preparing probiotic microcapsules encapsulated by hyaluronic acid-concentrated whey protein, the preparation process is as follows Figure 1 As shown, the steps are as follows:

[0038] 1. Preparation of bacterial suspension: Inoculate the lactic acid bacteria into the culture medium at an inoculum rate of 2% (v / v) for expansion culture. After the bacterial suspension is transferred twice, the lactic acid bacteria are cultured under suitable conditions for 28 hours until the lactic acid bacteria reach the logarithmic phase to obtain a lactic acid bacteria suspension;

[0039] Different lactic acid bacteria use different culture media and culture conditions, as follows:

[0040] The culture medium for Lactobacillus plantarum is MRS broth medium, and the culture conditions are static culture at 37°C; the culture medium for Streptococcus thermophilus is LM17 medium, and the culture conditions are static culture at 42°C; the culture medium for Lactobacillus delbrueckii is MRS medium, and the culture conditions are static culture at 37°C; the culture medium for Lactococcus lactis is GM17 medium, and the culture conditions are static culture at 30°C.

[0041] The lactic acid bacteria liquid was centrifuged at 2600g and 4°C for 10 min to collect the bacteria;

[0042] After washing the cells twice with 0.85% (w / v) saline, resuspend them in 0.85% (w / v) saline and adjust the concentration of the bacterial suspension to no less than 10 10 CFU / mL, that is, the effective concentration of bacteria in the bacterial suspension is not less than 10 10 CFU / mL.

[0043] 2. Dissolve whey protein concentrate (purchased from Fonterra Cooperative Group Limited, CAS No. 281-998-7) in distilled water to prepare a 10% (w / v) whey protein concentrate (WPC) solution, stir magnetically at room temperature for 2 h, and then store at 4°C overnight to fully hydrate.

[0044] 4% (w / v, g / mL) hyaluronic acid (HA) was added to the WPC solution, and the mixture was stirred at a constant speed of 450 r / min with a mechanical stirrer to make it uniformly mixed, and then heated in a water bath at 80°C for 20 minutes to form a gel.

[0045] 3. The bacterial suspension prepared in step 1 was mixed evenly with the gel in a ratio of 1:2 (v:v), pre-frozen at -80°C for 12 h, and freeze-dried for 48 h at -45°C and a pressure of 5.33 Pa using a vacuum freeze dryer (SCIENTZ-10N, Ningbo Xinzhi Biotechnology Co., Ltd.) to obtain hyaluronic acid-coated concentrated whey protein microparticles ( Figure 1 ), i.e., whey protein concentrate coated with hyaluronic acid and stored at 4°C.

[0046] Example 2

[0047] The preparation method in Example 1 was used to prepare probiotic microcapsules encapsulated with hyaluronic acid-concentrated whey protein, except that the lactic acid bacteria was Lactobacillus plantarum LP01 (referred to as LP01, disclosed in the following literature: [Ting Wang, Zhipeng Xue, Haoran Liu, Ruiting Zhao, Jiahui Peng, Siling Huang, Nan Li, Xueping Guo, Zhenshang Xu Food-grade expression system of Lactobacillus plantarum using β-galactosidase small subunit as selection marker and lactoseas screening condition LWT-Food Science and Technology (IF 6.0) Pub Date: 2023-05-26, DOI: 10.1016 / j.lwt.2023.114922], and the hyaluronic acid was a high molecular weight hyaluronic acid with a molecular weight of (100-170)×10 4 da (high molecular weight hyaluronic acid was supplied by Bloomage Biotech Co., Ltd. (Jinan, China)).

[0048] Example 3

[0049] The preparation method in Example 1 was used to prepare probiotic microcapsules encapsulated with hyaluronic acid-concentrated whey protein, except that the lactic acid bacteria was Lactococcus lactis MG1363 (abbreviated as MG1363, disclosed in the following literature: [Lemay ML, Otto A, MaaβS, Plate K, Becher D, Moineau S. Investigating Lactococcus lactis MG1363 Response to Phage p2 Infection at the Proteome Level. Mol Cell Proteomics. 2019Apr;18(4):704-714.doi:10.1074 / mcp.RA118.001135.Epub 2019Jan 24.PMID:30679258;PMCID:PMC6442364.]), and the hyaluronic acid was a high molecular weight hyaluronic acid with a molecular weight of (100-170)×10 4 Da.

[0050] Example 4

[0051] The preparation method in Example 1 was used to prepare probiotic microcapsules encapsulated with hyaluronic acid-concentrated whey protein, except that the lactic acid bacteria was Lactobacillus delbrueckii subsp. bulgaricus ATCC11842 (referred to as ATCC 11842, disclosed in the following literature: [Wang, T., Xu, Z., Lu, S., Xin, M., and Kong, J. (2016). Effects of glutathione on acid stress resistance and symbiosis between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. International Dairy Journal 61, 22-28.], and the hyaluronic acid was a high molecular weight hyaluronic acid with a molecular weight of (100-170)×10 4 Da.

[0052] Example 5

[0053] The preparation method in Example 1 was used to prepare probiotic microcapsules encapsulated with hyaluronic acid-concentrated whey protein, except that the lactic acid bacteria was Streptococcus thermophilus ST-1 (referred to as ST-1, disclosed in the following literature: [Wang, T., Xu, Z., Lu, S., Xin, M., and Kong, J. (2016). Effects of glutathione onacid stress resistance and symbiosis between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. International Dairy Journal 61, 22-28.]), and the hyaluronic acid was a high molecular weight hyaluronic acid with a molecular weight of (100-170)×10 4 Da.

[0054] Example 6

[0055] The hyaluronic acid-concentrated whey protein encapsulated probiotic microcapsules were prepared by the preparation method in Example 2, the only difference being that the high molecular weight hyaluronic acid in Example 2 was replaced by medium molecular weight hyaluronic acid with a molecular weight of (20-40)×10 4da (medium molecular weight hyaluronic acid was supplied by Bloomage Biotech Co., Ltd. (Jinan, China)).

[0056] Example 7

[0057] The hyaluronic acid-concentrated whey protein encapsulated probiotic microcapsules were prepared by the preparation method in Example 2, the only difference being that the high molecular weight hyaluronic acid in Example 2 was replaced by low molecular weight hyaluronic acid with a molecular weight of (3-10)×10 4 Da.

[0058] Example 8

[0059] The hyaluronic acid-concentrated whey protein encapsulated probiotic microcapsules were prepared by the preparation method in Example 3, the only difference being that the high molecular weight hyaluronic acid in Example 2 was replaced by medium molecular weight hyaluronic acid with a molecular weight of (20-40)×10 4 Da.

[0060] Example 9

[0061] The hyaluronic acid-concentrated whey protein encapsulated probiotic microcapsules were prepared by the preparation method in Example 3, the only difference being that the high molecular weight hyaluronic acid in Example 2 was replaced by low molecular weight hyaluronic acid with a molecular weight of (3-10)×10 4 Da.

[0062] Example 10

[0063] The hyaluronic acid-concentrated whey protein encapsulated probiotic microcapsules were prepared by the preparation method in Example 4, the only difference being that the high molecular weight hyaluronic acid in Example 2 was replaced by medium molecular weight hyaluronic acid with a molecular weight of (20-40)×10 4 Da.

[0064] Embodiment 11

[0065] The hyaluronic acid-concentrated whey protein encapsulated probiotic microcapsules were prepared by the preparation method in Example 4, the only difference being that the high molecular weight hyaluronic acid in Example 2 was replaced by low molecular weight hyaluronic acid with a molecular weight of (3-10)×10 4 da (low molecular weight hyaluronic acid was supplied by Bloomage Biotech Co., Ltd. (Jinan, China)).

[0066] Example 12

[0067] The hyaluronic acid-concentrated whey protein encapsulated probiotic microcapsules were prepared by the preparation method in Example 5, the only difference being that the high molecular weight hyaluronic acid in Example 2 was replaced by medium molecular weight hyaluronic acid with a molecular weight of (20-40)×10 4 Da.

[0068] Embodiment 13

[0069] The hyaluronic acid-concentrated whey protein encapsulated probiotic microcapsules were prepared by the preparation method in Example 5, the only difference being that the high molecular weight hyaluronic acid in Example 2 was replaced by low molecular weight hyaluronic acid with a molecular weight of (3-10)×10 4 Da.

[0070] Test Example 1

[0071] SEM analysis can provide a good understanding of the uniformity and microstructure of the microcapsules. The hyaluronic acid-concentrated whey protein encapsulated probiotic microcapsules prepared in Examples 2 to 13 were subjected to SEM analysis. The SEM images of the hyaluronic acid-concentrated whey protein encapsulated probiotic microcapsules in different examples are shown in FIG. Figure 2 shown.

[0072] from Figure 2 It can be seen that the surface morphology of the three microcapsules formed by different probiotics has certain regularities. The shape and surface morphology of the concentrated whey protein particles coated with hyaluronic acid are mostly irregular, and after freeze-drying, the particles are mainly 2μm in size.

[0073] The surface morphology of microcapsules produced by encapsulation with low molecular weight hyaluronic acid is as follows Figure 2 As shown in Figure A, the surface of this microcapsule is porous, the texture is uneven, and the internal structure is rough and partially collapsed. The reason for this surface morphology may be the loss of water during the freeze-drying process. Since this microcapsule has an uneven gel mechanism with a dense surface layer and a loose core, it shrinks after freeze-drying, resulting in partial collapse of the structure.

[0074] The surface morphology of the microcapsules produced by the preparation of medium molecular weight hyaluronic acid is a network structure composed of some particles adhered together. Figure 2 As shown in Figure B. The surface of this microcapsule also has certain pores, but the pores are smaller than those of the microcapsules formed by low molecular weight hyaluronic acid, and the structure becomes more compact.

[0075] The electron microscopic structure of microcapsules encapsulated with high molecular weight hyaluronic acid is shown in Figure 2. Figure 2 As shown in Figure C, the surface of this microcapsule is relatively continuous and uniform, with a dense structure and no pores, indicating that high molecular weight hyaluronic acid has a certain compatibility with concentrated whey protein. Hyaluronic acid encapsulation covers the strains remaining on the surface of the microcapsule, so encapsulation can improve the survival ability of the strains when exposed to adverse environments.

[0076] In summary, it can be concluded that as the molecular weight of hyaluronic acid increases, the integrity of the formed microcapsules becomes higher. Figure 2It can be seen that the four probiotics encapsulated with high molecular weight hyaluronic acid ( Figure 2 In C), the surface of Lactobacillus plantarum LP01 is smoother and denser. Bacteria and whey protein can adhere together and interact with each other through glycoprotein and protein. Different strains have different degrees of adhesion to whey protein, resulting in differences in surface morphology. In addition, whey protein and hyaluronic acid have important gelling properties, which can cause complex coagulation through electrostatic interaction under certain conditions.

[0077] Test Example 2

[0078] Determination of encapsulation efficiency of hyaluronic acid-whey protein concentrate encapsulated probiotic microcapsules after freeze-drying:

[0079] The viable counts of probiotics in the hyaluronic acid microcapsules prepared in Examples 2 to 13 were detected using a standard plate count method.

[0080] Packaging efficiency = (N×M) / N0×100%

[0081] Wherein, N is the logarithmic value of live cells in 1g microcapsule, M is the total mass of collected microcapsules, and N0 is the logarithmic value of free cells before microencapsulation. The measurement results are shown in Table 1.

[0082] Table 1 Results of viable bacterial count and encapsulation efficiency of probiotic microcapsules encapsulated by hyaluronic acid-concentrated whey protein in Examples 2 to 13 (log cfu / mL, n=3)

[0083]

[0084] It can be concluded from Table 1 that the encapsulation efficiency has reached the level of active probiotics recommended by the International Dairy Federation. The probiotics encapsulated with high molecular weight hyaluronic acid in the same strain all have a high encapsulation efficiency, and the encapsulation rate is higher than 83.8±0.3%, and significantly higher than the probiotics encapsulated with medium molecular weight hyaluronic acid (P<0.05). After the four probiotics were encapsulated with high molecular weight hyaluronic acid, the encapsulation efficiency of Lactobacillus plantarum (98.35±0.55%) was significantly higher than that of Lactococcus lactis (92.3±0.11%), Lactobacillus delbrueckii (83.8±0.3%) and Streptococcus thermophilus (89.9±0.7%). It shows that using high molecular weight hyaluronic acid as encapsulation wall material can improve the activity of microorganisms during freeze-drying.

[0085] The probiotic with the lowest encapsulation rate in this test case is Lactobacillus delbrueckii. The encapsulation rates of this bacterium with low molecular weight, medium molecular weight and high molecular weight hyaluronic acid are 77±1.9%, 65.1±1.7% and 83.8±0.3% respectively. The more compact gel structure can encapsulate the probiotic cells, with a higher encapsulation rate and better protection. In addition, more hydrophilic groups can reduce interfacial tension and increase the encapsulation rate of probiotic cells. The wall material after embedding with high molecular weight hyaluronic acid becomes more hydrophobic and the structure is more compact, so the encapsulation efficiency becomes higher.

[0086] In this test example, the encapsulation effect of Lactobacillus plantarum was the best, with the highest encapsulation rate reaching 98.4±0.5%. Figure 2 In C), after the concentrated whey protein is wrapped by high molecular weight hyaluronic acid, the surface structure is tight and has no gaps, so there is almost no probiotics exposed outside. The combination of plant lactobacillus and whey protein makes the encapsulation effect better.

[0087] Test Example 3

[0088] Survival ability of four probiotics in simulated gastrointestinal fluid:

[0089] The preparation method of simulated gastric fluid (SGF, pH 2.0) is as follows: pepsin (purchased from Sinopharm Chemical Reagent Co., Ltd., 9001-75-6) is filtered and sterilized, dissolved in a sterilized 0.5% NaCl solution to a final concentration of 3 g / L, and the pH is adjusted to 2.0 with concentrated hydrochloric acid;

[0090] Simulated intestinal fluid (SIF, pH 7.4) was prepared by dissolving trypsin (1 g / L, purchased from Van der Biotech Co., Ltd. 9002-07-7) and porcine bile salt (1 g / L) in sterile 0.5% NaCl solution and adjusting the pH to 8.0.

[0091] Four bacteria, Lactobacillus plantarum LP01, Lactococcus lactis MG1363, Lactobacillus delbrueckii ATCC 11842, and Streptococcus thermophilus ST-1, equivalent to 2% of the volume of the culture medium, were transferred to fresh culture medium and cultured until OD 600 =1.0, collect the cells by centrifugation, discard the culture medium, add an equal volume of simulated gastric fluid (SGF, pH 2.0) or simulated intestinal fluid (SIF, pH 7.4), and culture at 37°C. Take out 1.0 mL aliquots from the centrifuge tube every half an hour, dilute them 8 times in 0.85% (w / v) saline, and then take 5 μL of the dilution to spot on the solid culture medium, incubate at temperature, and count after 48 hours. The test results are as follows Figure 3As shown, A is the survival results of four probiotics in simulated gastric fluid, and B is the survival results of four probiotics in simulated intestinal fluid.

[0092] Depend on Figure 3 It can be concluded that when the four probiotics were exposed to SGF (pH 2.0), Lactobacillus plantarum LP01 had the best tolerance, and Lactobacillus delbrueckii ATCC 11842 had no microorganisms surviving after 120 min ( Figure 3 However, the populations of the four probiotics remained basically unchanged in SIF (pH 7.4) ( Figure 3 (middle B).

[0093] There are differences in the survival of probiotic strains in acidic environments, and Lactobacillus delbrueckii was observed to be the most acid-sensitive strain in this test case. In particular, free Lactobacillus plantarum had a greater viable count than other free probiotics, which may be due to its higher tolerance to acid, and ingestion of unprotected probiotics will lead to reduced viability. Therefore, these four probiotics can be microencapsulated to ensure greater survival in the gastric environment.

[0094] Test Example 4

[0095] Survival of probiotic microcapsules encapsulated by hyaluronic acid-whey protein concentrate in simulated gastric fluid

[0096] For probiotics to have a positive impact on health, they must reach their site of action alive and establish themselves in sufficient numbers, however the main barrier to the survival of ingested microorganisms is the acidic environment of the stomach (pH 2.0). To determine the potential of microencapsulated and free probiotics to survive through the stomach after oral administration, the stability of microencapsulated or free probiotics in simulated gastric fluid was tested.

[0097] The determination method is:

[0098] Transfer 2% of the overnight cultured bacteria into 5 ml of fresh medium and grow until OD 600 =1.0, centrifuge to collect bacteria, discard the culture medium, add an equal volume of simulated gastric juice (simulated intestinal juice), and culture at 37°C. Take out 1.0mL aliquots from the centrifuge tube every half an hour, then immediately centrifuge at 4°C (6000×g, 2min) to terminate the reaction and separate bacteria from the simulated gastric juice (simulated intestinal juice). Take out 1.0mL aliquots from the centrifuge tube, dilute 8 times in 0.85% (w / v) physiological saline, then take 5μL of the dilution and place on a solid culture medium, incubate at a specific temperature, and count after 48h. The results are shown in Table 2.

[0099] Table 2 Survival of four microencapsulated and free probiotics in simulated gastric fluid at pH 2.0 and temperature 37°C (log cfu / mL, n=3)

[0100]

[0101]

[0102] Table 2 shows the survival of four probiotics in simulated gastric fluid in the unencapsulated state and after encapsulation with three molecular weight hyaluronic acids. The results showed that in the SGF test, compared with free probiotics, encapsulation with hyaluronic acid and concentrated whey protein increased the survival rate of probiotics (P<0.05). It was found that the survival rate of the same strain microcapsules encapsulated with high molecular weight hyaluronic acid was higher than that of the other two hyaluronic acids, and the most significant effect was the Lactobacillus delbrueckii microcapsules. The survival rate of the unencapsulated Lactobacillus delbrueckii after the reaction in gastric fluid was 0, and the survival rate of the Lactobacillus delbrueckii microcapsules formed by encapsulation with high molecular weight hyaluronic acid was 97.97±1.01%, and the number of viable bacteria was 8.51±0.21%, reaching the recommended level.

[0103] According to test example 3 Figure 3 Experiments in Zhong A demonstrated that four probiotics were sensitive to low pH conditions. It was reported that the addition of WPC alone to a liquid medium (pH 2.0) containing probiotics could increase the buffering capacity and reduce the negative effects of the acidic environment on the strains. Therefore, whey protein concentrate can act as a buffer to reduce the damage of gastric juice to probiotics and form a physical barrier between cells and acidic gastric juice due to its hydrogel properties. In addition, the additional coating step using hyaluronic acid further enhances the protection of bacteria. It will provide a thicker shell around the whey protein concentrate microcapsules, keeping the probiotics in a protected microenvironment and preventing contact between bacteria and acid solutions.

[0104] The survival rate of WPC and high molecular weight hyaluronic acid in this test case is above 96%, and the microcapsules prepared with WPC and low molecular weight hyaluronic acid seem to be easily penetrated by gastric juice, with a survival rate of about 70% to 80%. In general, the added coating provides better protection for the probiotics than the uncoated microcapsules.

[0105] Test Example 5

[0106] Release of probiotic microcapsules encapsulated by hyaluronic acid-whey protein concentrate in simulated intestinal fluid

[0107] The release of microcapsules in simulated artificial intestinal fluid is an important indicator for evaluating whether microcapsules can be released at a specific point in the animal intestine, and is also a criterion for judging whether probiotics can survive in the intestine for colonization and proliferation. It is necessary to ensure that the encapsulated cells are released in the medium SIF.

[0108] 1 g of the hyaluronic acid-concentrated whey protein encapsulated probiotic microcapsules in Examples 2 to 13 were weighed and added to a conical flask containing 200 mL of simulated intestinal fluid (pH 7.4), mixed evenly, and incubated at 37° C. Every half an hour, 1.0 mL of aliquots were taken out from the conical flask, and then immediately centrifuged at 4° C. (6000×g, 2 min) to terminate the reaction, and bacteria were separated from the simulated intestinal fluid to determine the survival of the bacteria. The results are shown in FIG. Figure 4 As shown, the four figures A, B, C and D represent LP01, MG1363, ATCC11842 and ST-1 respectively, and A, B and C in each figure represent microcapsules encapsulated by low molecular weight hyaluronic acid, medium molecular weight hyaluronic acid and high molecular weight hyaluronic acid respectively.

[0109] from Figure 4 It can be concluded that when the encapsulated probiotics were directly placed in SIF at pH 7.4, the release of live cells in the four probiotic samples was basically similar, and the release quantity of all encapsulated probiotics in 120 minutes could meet the specified requirements (the number of live cells of ready-to-eat probiotic foods should be ≥10 in accordance with the provisions of the Probiotic Foods). 7 CFU / g). The low molecular weight hyaluronic acid encapsulated microcapsule A completely dissolved within 30-60 min, which enabled the encapsulated probiotics to be released quickly. The high molecular weight hyaluronic acid encapsulated microcapsule C could be slowly released in the intestinal fluid and completely released in about 90-120 min. This release rate was more conducive to the colonization of the intestinal tract by probiotics and played a beneficial role. In addition, the probiotic activity in the high molecular weight hyaluronic acid encapsulated microcapsule A and the medium molecular weight hyaluronic acid encapsulated microcapsule B decreased with the extension of the culture time, because as the cross-linking density of the microcapsule hydrogel structure decreased, more and more cells came into contact with the intestinal fluid, resulting in the death of the probiotics. However, the number of probiotics in the high molecular weight hyaluronic acid encapsulated microcapsule C did not decrease, indicating that the probiotic microcapsule prepared with the high molecular weight hyaluronic acid C material had a tighter structure and effectively protected the encapsulated probiotics. The results showed that WPC and high molecular weight hyaluronic acid encapsulated probiotics played a vital role in the survival and colonization of the intestine.

[0110] Test Example 6

[0111] Survival of probiotic microcapsules encapsulated by hyaluronic acid-whey protein concentrate after continuous incubation in simulated gastric fluid and simulated intestinal fluid

[0112] In order for probiotics to play a positive role in health, they must be able to survive in gastric juice and reach the intestines so that they can colonize in large quantities in the colon. The survival ability of the hyaluronic acid-whey protein concentrate encapsulated probiotic microcapsules prepared in Examples 2 to 13 after continuous incubation in simulated gastric juice and simulated intestinal juice was determined as follows:

[0113] Weigh 1 g of the hyaluronic acid-concentrated whey protein encapsulated probiotic microcapsules prepared in Examples 2 to 13 respectively, add them to a conical flask containing 200 mL of simulated gastric fluid, mix well, and incubate at 37°C for 180 min. After incubation, filter out the bacteria through a sieve, then put them into 200 mL of simulated intestinal fluid, mix well, and incubate again at 37°C for 180 min. During the continuous incubation in simulated gastric fluid and simulated intestinal fluid, it is necessary to take out 1.0 mL aliquots from the conical flask every half an hour, then immediately centrifuge at 4°C (6000×g, 2 min) to terminate the reaction, and separate the bacteria from the simulated intestinal fluid, and measure the survival of the bacteria. The results are as follows: Figure 5 As shown, the four figures A, B, C and D represent LP01, MG1363, ATCC11842 and ST-1 respectively, and A, B and C in each figure represent microcapsules encapsulated by low molecular weight hyaluronic acid, medium molecular weight hyaluronic acid and high molecular weight hyaluronic acid respectively.

[0114] Depend on Figure 5 It can be concluded that after continuous culture in simulated gastrointestinal fluid for 240 minutes, the diffusion and release trends of different probiotics in the same microcapsules are similar. In simulated gastric fluid, the number of probiotics in microcapsule A encapsulated by low molecular weight hyaluronic acid increases rapidly with the culture time, but the number of probiotics tends to decrease when incubated with simulated intestinal fluid at the end. It seems that gastrointestinal fluid enters the poorly protected particles through the surface pinholes, resulting in a decrease in the number of probiotics. Probiotics encapsulated by medium molecular weight hyaluronic acid B are also gradually released in simulated gastric fluid, and the number of viable bacteria decreases after incubation in simulated intestinal fluid. The number of viable bacteria released by microcapsules encapsulated by high molecular weight hyaluronic acid in simulated gastric fluid is less than that of the other two microcapsules. After being transferred to simulated intestinal fluid, the activity of probiotics slowly increases and there is no downward trend. This shows that the wall material of microcapsules with high molecular weight hyaluronic acid is more stable in an acidic environment, and can be disintegrated when transferred to a weak alkaline environment, resulting in the release of strains. In low molecular weight hyaluronic acid encapsulated microcapsules A, the number of probiotics decreased by about 1 log cycle, and in medium molecular weight hyaluronic acid encapsulated microcapsules B, the number of probiotics decreased by 0.56 log cycles. However, in high molecular weight hyaluronic acid encapsulated microcapsules C, the number of probiotics increased to 8 log cycles. These results show that WPC and high molecular weight hyaluronic acid are promising materials for protecting probiotics from harsh gastrointestinal tract damage.

[0115] Test Example 7

[0116] Stability of free probiotics and probiotic microcapsules during storage at -20°C, 4°C and 25°C

[0117] Microencapsulation of various bacterial cultures, including probiotics, is a common practice to extend their shelf life, based on sublimation and low water activity in microcapsules, which can prolong the survival of probiotics by reducing their metabolism. This is also a favorable way to promote the transportation, storage and utilization of probiotic microcapsules.

[0118] Free bacterial liquids of Lactobacillus plantarum LP01, Lactococcus lactis MG1363, Lactobacillus delbrueckii ATCC11842 and Streptococcus thermophilus ST-1 and the probiotic microcapsules encapsulated with hyaluronic acid-concentrated whey protein prepared in Examples 2 to 13 were stored in refrigerators at -20°C, 4°C and 25°C for 30 days, respectively, and the stability of the free strains and the encapsulated strains in the microcapsules was determined. The results are shown in FIG. Figure 6 As shown, PC, A, B and C represent free strains, microcapsules encapsulated by low molecular weight hyaluronic acid, microcapsules encapsulated by medium molecular weight hyaluronic acid and microcapsules encapsulated by high molecular weight hyaluronic acid, respectively.

[0119] Depend on Figure 6 It can be concluded that the stability of the same microcapsule wall material of different strains is similar, and the cell viability of embedded and free probiotics at three different temperatures decreases to varying degrees with the extension of storage time. After 30 days of storage, the number of live bacteria of free probiotics at -20℃, 4℃ and 25℃ decreased rapidly compared with encapsulated probiotics, indicating that the protective film formed by hyaluronic acid and WPC around the probiotics provides protection for the strains. During the 30-day storage at -20℃ and 4℃, the stability of the three microencapsulated probiotics was very good. However, at 25℃, the probiotic activity in the three microcapsules decreased to varying degrees. This is because the storage temperature can affect the stability of microencapsulated probiotics. Low temperature can reduce the rate of reactions such as fat oxidation and reduce the formation of free radicals, which is beneficial to maintain cell vitality.

[0120] During the 30-day storage period at the three temperatures, the probiotics loaded in the concentrated whey protein microparticles coated with high molecular weight hyaluronic acid had the best stability, followed by the microcapsules encapsulated with medium molecular weight hyaluronic acid, and the probiotics in the microcapsules encapsulated with low molecular weight had the worst stability, indicating that the three microcapsule wall materials caused differences in cell viability. Cracks were observed on the surface of the microcapsules encapsulated with low molecular weight hyaluronic acid and medium molecular weight hyaluronic acid ( Figure 2A to B), which may help oxygen diffuse through the capsule, leading to cell death. The density and structure of the microcapsules encapsulated by high molecular weight hyaluronic acid are stable, and a relatively dense and continuous surface morphology is obtained by using microcapsules, which provides a protective barrier for probiotics during storage and reduces the diffusion of oxygen to the strains.

[0121] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing probiotic microcapsules encapsulated by hyaluronic acid-concentrated whey protein, characterized in that: The method comprises the following steps: mixing and heating a concentrated whey protein solution and hyaluronic acid to obtain a gelatinous mixture; The colloidal mixture is mixed with a probiotic suspension and frozen, and the obtained frozen product is freeze-dried to obtain the probiotic microcapsules.

2. The preparation method according to claim 1, characterized in that: The concentration of the concentrated whey protein solution is 8% to 12% w / v, and the volume ratio of the mass of the hyaluronic acid to the concentrated whey protein solution is 3 to 5 g:100 mL.

3. The preparation method according to claim 1, characterized in that: The molecular weight of the hyaluronic acid is (3-170)×10 4 Da.

4. The preparation method according to claim 3, characterized in that: The molecular weight of the hyaluronic acid is (100-170)×10 4 da, (20~40)×10 4 da or (3~10)×10 4 Da.

5. The preparation method according to claim 1, characterized in that: The volume ratio of the colloidal mixture to the probiotic suspension is 1 to 2:1; the effective live bacteria concentration in the probiotic suspension is not less than 10 10 CFU / mL.

6. The preparation method according to claim 1 or 5, characterized in that: The probiotics include lactic acid bacteria.

7. The preparation method according to claim 6, characterized in that: The lactic acid bacteria include one or more of Lactobacillus plantarum, Lactococcus lactis, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.

8. The preparation method according to claim 1, characterized in that: The mixing process is accompanied by stirring, and the stirring speed is 450-1200 r / min; the heating temperature is 70-80° C., and the heating time is 15-20 min.

9. The preparation method according to claim 1, characterized in that: The freezing temperature is -80°C and the time is 12 hours; the freeze-drying temperature is -45°C, the pressure is 5.33 Pa and the time is 48 hours.

10. Use of the preparation method according to any one of claims 1 to 9 or the probiotic microcapsules prepared by the preparation method according to any one of claims 1 to 9 in one or more of food preparation, drug preparation, probiotic storage, probiotic transportation and probiotic delivery.

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