Probiotic-loaded protein fiber gel and preparation method thereof

Abstract

The invention discloses protein fiber gel loaded with probiotics and a preparation method of the protein fiber gel, and relates to the technical field of gel embedding. The method comprises the following steps: S1, mixing whey protein isolate powder with ultrapure water, and stirring at room temperature for 2-4 hours to obtain a whey protein isolate solution; s2, adjusting the pH value of the whey protein isolate solution, heating and stirring, and cooling in an ice-water bath to obtain a whey protein isolate fiber solution; s3, adjusting the pH value of the whey protein isolate fiber solution, adding the probiotic suspension and the protective solution, and stirring to obtain a whey protein isolate fiber pregel solution; and S4, standing the whey isolated protein fiber pre-gel solution at 4-40 DEG C for 6-24 hours to obtain the protein fiber gel loaded with the probiotics. The probiotic-loaded protein fiber gel prepared by the invention has the advantages of stomach low-swelling protection and efficient intestinal release.

Description

Technical Field

[0001] The invention belongs to the technical field of gel embedding, and particularly relates to a probiotic-loaded protein fiber gel and a preparation method thereof. Background Art

[0002] Probiotics are beneficial bacteria found in the human intestines, known for their beneficial effects on host health. They can regulate the balance of intestinal flora, enhance immunity, and promote nutrient absorption. In recent years, as people's awareness of health has increased, probiotics have been widely used in food, health products, and pharmaceuticals, such as yogurt, probiotic beverages, and dietary supplements.

[0003] Despite their numerous health benefits, probiotics face the following major challenges in practical application: Probiotics are strongly damaged by gastric acid (low pH) and bile salts as they pass through the human gastrointestinal tract, significantly reducing their survival rate. Studies have shown that the survival rate of unprotected probiotics in the acidic environment of the stomach is typically less than 10%.

[0004] To improve the survival rate of probiotics in gastric acid, researchers have begun to focus on using natural or synthetic polymers (such as sodium alginate, chitosan, gelatin, etc.) to encapsulate and protect probiotics. Although existing technologies using natural or synthetic polymers to encapsulate probiotics have improved their survival rate in gastric acid to a certain extent, the survival improvement effect remains unsatisfactory. To this end, this application proposes a probiotic-loaded protein fiber gel that significantly improves the survival rate of probiotics in the gastrointestinal environment and a method for preparing the gel. Summary of the Invention

[0005] In order to overcome the above-mentioned defects, the present application provides a probiotic-loaded protein fiber gel and a preparation method thereof.

[0006] A method for preparing a probiotic-loaded protein fiber gel comprises the following steps: S1, preparing whey protein isolate solution; Mixing whey protein isolate powder and ultrapure water, stirring at room temperature for 2 to 4 hours, and preparing a whey protein isolate solution with a mass concentration of 3% to 7%; S2, preparing a whey protein isolate fiber solution; The whey protein isolate solution prepared in step S1 is adjusted to a pH of 2.0 to 3.0, magnetically stirred at 80 to 85° C. for 10 to 12 hours, and then cooled in an ice-water bath for 20 to 30 minutes to obtain a whey protein isolate fiber solution; S3, preparing a whey protein isolate fiber pregel solution; Adjusting the pH of the whey protein isolate fiber solution prepared in step S2 to 2.0-7.0, adding the probiotic suspension and the protective solution, stirring for 15-20 minutes for premixing, and obtaining a whey protein isolate fiber pregel solution; wherein the volume ratio of the probiotic suspension, the protective solution, and the pH-adjusted whey protein isolate fiber solution is 1:(1.5-2.5):(45-55); S4, preparing protein fiber gel loaded with probiotics; The whey protein isolate fiber pregel solution is allowed to stand at 4-40° C. for 6-24 hours to obtain a protein fiber gel loaded with probiotics.

[0007] Preferably, in step S2, magnetic stirring is performed at 80° C. for 10 h.

[0008] Preferably, in step S3, the protective solution is a sucrose solution, and the mass percentage concentration of the sucrose solution is 5% to 10%.

[0009] Preferably, in step S3, the probiotic suspension is prepared as follows: the probiotic strain is inoculated into a modified liquid culture medium mMRS, cultured at 37-39°C for 24-48 hours to complete the primary culture and obtain the primary culture solution; then 4-8 mL of the primary culture solution is transferred to 100-150 mL of fresh modified liquid culture medium mMRS, and cultured at 37-39°C for 24-48 hours to complete the secondary culture; centrifuged at a temperature of 4-8°C for 10-20 minutes to obtain cells; the cells are washed with phosphate buffer solution, and the washed cells are resuspended in phosphate buffer solution to obtain 1×10 7 ~1×10 9 CFU / ml of probiotic suspension.

[0010] Preferably, in step S3, the probiotic strain is Limosilactobacillus reuteri TMW1.656.

[0011] Preferably, in step S3, the modified liquid culture medium mMRS is obtained by uniformly mixing the following components, by weight: 1000 parts of sterile water, 10-15 parts of maltose, 10-15 parts of peptone, 10-15 parts of malt extract, 5-10 parts of fructose, 5-10 parts of glucose, 5-10 parts of fermentation extract, 5-10 parts of bovine extract, 2.6-5 parts of potassium dihydrogen phosphate, 4-10 parts of dipotassium hydrogen phosphate, 3-10 parts of ammonium chloride, 1-5 parts of Tween 80, 0.5-1 part of L-cysteine-HCL, 0.1-1 part of magnesium sulfate heptahydrate and 0.05-0.1 part of manganese sulfate tetrahydrate.

[0012] Preferably, in step S1, the mass ratio of whey protein isolate powder to ultrapure water is (3-7):(97-93).

[0013] Compared with the prior art, the present invention has the following beneficial effects: In the present application, in the process of preparing the protein fiber gel loaded with probiotics in Examples 1 to 3, the whey protein isolate forms whey protein isolate fibers under the action of pH regulation and heat induction in step S2, and then, by adjusting the pH value of the whey protein isolate fiber solution in step S3, the aggregation behavior of the whey protein isolate fibers can be effectively regulated. Specifically: in step S3, the pH value of the whey protein isolate fiber solution is regulated to be close to the isoelectric point of the whey protein isolate fibers. At this time, the net surface charge of the whey protein isolate fibers approaches zero, the electrostatic repulsion between the whey protein isolate fibers is greatly reduced, and the whey protein isolate fibers are interwoven through hydrogen bonds and hydrophobic interactions to form a dense fiber network. The dense fiber network composed of these whey protein isolate fibers is manifested as large particles of whey protein isolate fiber aggregates, and the whey protein isolate fiber aggregates are large particles. large particles; when these large particles of whey protein isolate fiber aggregates are left to stand at the temperature described in step S4, the whey protein isolate fibers on different large particles of whey protein isolate fiber aggregates and the different whey protein isolate fibers on the same large particle of whey protein isolate fiber aggregates are interwoven through hydrogen bonds and hydrophobic interactions to form a denser three-dimensional fiber network, and in the formation process of the large particles of whey protein isolate fiber aggregates and the three-dimensional fiber network, the amino acid residues on the surfaces of the whey protein isolate fibers are regularly arranged, and the probiotics are attached to the fiber network under the action of the hydrophobicity of the whey protein isolate fibers in the fiber network, so that the probiotics are tightly wrapped in the fiber network, forming a protein fiber gel loaded with probiotics; In the process of preparing the protein fiber gel loaded with probiotics in Examples 4 to 7, in step S3, the pH of the whey protein isolate fiber solution is adjusted to deviate from the isoelectric point of the whey protein isolate fiber. At this time, the whey protein isolate fiber is negatively charged, the electrostatic repulsion between the whey protein isolate fibers is enhanced, the aggregation between the whey protein isolate fibers is effectively suppressed, and it is difficult to form large particles of whey protein isolate fiber aggregates. At this time, the whey protein isolate fibers are mainly small particles of whey protein isolate fiber aggregates generated by the aggregation between the whey protein isolate fibers and whey protein isolate fibers that have not aggregated. The whey protein isolate fiber exists in the form of fibers. During the standing process in step S4, these small particles of whey protein isolate fiber aggregates and the unaggregated whey protein isolate fibers are lightly cross-linked through hydrogen bonding and hydrophobic interactions to form a three-dimensional loose network. During the formation of the small particles of whey protein isolate fiber aggregates and the loose network, the amino acid residues on the surface of the whey protein isolate fibers are regularly arranged. The probiotics are attached to the fiber network under the action of the hydrophobicity of the whey protein isolate fibers in the fiber network, so that the probiotics are wrapped in the fiber network, forming a protein fiber gel loaded with probiotics. The denser fiber network present in the probiotic-loaded protein fiber gel prepared in Examples 1 to 3 of the present application and the three-dimensional loose network present in Examples 4 to 7 can form a physical barrier under the simulated gastric fluid and simulated intestinal fluid environment, effectively limiting the penetration of simulated gastric fluid and simulated intestinal fluid into the probiotic-loaded protein fiber gel and affecting the probiotic bacteria.

[0014] In addition, the process for preparing the probiotic-loaded protein fiber gel of the present application is simple, does not require the addition of chemical cross-linking agents, and can form a solid gel at a relatively low temperature and after a short period of standing, making the production process green and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The appearance of the probiotic-loaded protein fiber gel samples prepared in Examples 1 to 7; Figure 2 These are laser confocal microscopy images of the corresponding probiotic-loaded protein fiber gel microscopic test samples of Examples 1 to 3; Figure 3 These are laser confocal microscopy images of the corresponding probiotic-loaded protein fiber gel microscopic test samples of Examples 4 to 7; Figure 4 Graph showing the change in storage modulus G' of the probiotic-loaded protein fiber gel samples prepared in Examples 1 to 7 as a function of frequency; Figure 5 Graph showing the change in loss modulus G″ of the probiotic-loaded protein fiber gel samples prepared in Examples 1 to 7 as a function of frequency. DETAILED DESCRIPTION

[0016] Example 1: A method for preparing a probiotic-loaded protein fiber gel comprises the following steps: S1, preparing whey protein isolate solution; Whey protein isolate and ultrapure water were mixed at a mass ratio of 1:19, and magnetically stirred at 25° C. and 500 rpm for 2 h to prepare a whey protein isolate solution with a mass concentration of 5%; S2, preparing a whey protein isolate fiber solution using the whey protein isolate solution; The whey protein isolate solution prepared in step S1 was adjusted to pH 2.0, magnetically stirred at 80° C. and 500 rpm for 2 h, and then cooled in an ice-water bath for 20 min to obtain a whey protein isolate fiber solution; S3, using the whey protein isolate fiber solution to prepare a whey protein isolate fiber pregel solution; After adjusting the pH of the whey protein isolate fiber solution prepared in step S2 to 5.0, the probiotic suspension and the sucrose solution (the sucrose solution is a protective solution) are added, and the mixture is stirred for 15 minutes at a stirring speed of 10,000 g for pre-mixing to obtain a whey protein isolate fiber pregel solution; wherein the volume ratio of the probiotic suspension, the sucrose solution, and the pH-adjusted whey protein isolate fiber solution is 1:2:50, and the mass concentration of the sucrose solution is 5%; In Example 1, the probiotic suspension is prepared by the following probiotic suspension method, which comprises the following steps: Limosilactobacillus reuteri TMW1.656 was inoculated into modified liquid medium mMRS and cultured at 37°C for 24 hours to complete the primary culture and obtain the primary culture fluid. Then, 4 mL of the primary culture fluid was transferred to 100 mL of fresh modified liquid medium mMRS and cultured at 37°C for 24 hours to complete the secondary culture. Then, the cells were centrifuged at 4°C and a centrifugal force of 10,000 g for 10 minutes to obtain the cells. The cells were then washed with phosphate buffer solution (0.02 mM, pH 7.4) and resuspended in 3.0 mL of phosphate buffer solution to obtain a probiotic suspension. The cell count of the probiotic suspension was approximately 1.76 × 10 9 (CFU / ml); in this application, bacterial species Limosilactobacillus reuteri TMW1.656 is the strain published in the study "Effects of whey protein isolate fiber and typical antioxidants on the survival of Lactobacillus reuteri TMW 1.656 under room temperature storage stress." Limosilactobacillus reuteri TMW1.656; In this Example 1, the improved liquid culture medium mMRS was obtained by uniformly mixing the following components: 1000 g sterile water, 10 g maltose, 10 g peptone, 10 g malt extract, 5 g fructose, 5 g glucose, 5 g fermentation extract, 5 g bovine extract, 2.6 g potassium dihydrogen phosphate, 4 g dipotassium hydrogen phosphate, 3 g ammonium chloride, 1 g Tween 80, 0.5 g L-cysteine-HCL, 0.1 g magnesium sulfate heptahydrate, and 0.05 g manganese sulfate tetrahydrate; S4, preparing a probiotic-loaded protein fiber gel using a whey protein isolate fiber pregel solution; The whey protein isolate fiber pregel solution was placed in a constant temperature box at 4° C. for 24 hours to obtain a protein fiber gel loaded with probiotics.

[0017] Example 2: The difference between Example 2 and Example 1 is that: in step S4, the mixture is left to stand in a constant temperature box at 25° C. for 12 hours.

[0018] Example 3: The difference between Example 3 and Example 1 is that: in step S4, the mixture is left to stand in a constant temperature box at 40° C. for 6 hours.

[0019] Embodiment 4: The difference between Example 4 and Example 1 is that: in step S3, the pH of the whey protein isolate fiber solution prepared in step S2 is adjusted to 7.0; and in step S4, it is allowed to stand in a constant temperature box at 25°C for 12 hours.

[0020] Example 5: The difference between Example 5 and Example 1 is that: in step S3, the pH of the whey protein isolate fiber solution prepared in step S2 is adjusted to 7.0; and in step S4, it is allowed to stand in a constant temperature box at 40° C. for 6 hours.

[0021] Example 6: The difference between Example 6 and Example 1 is that: in step S1, whey protein isolate and ultrapure water are mixed in a mass ratio of 3:97; in step S3, the pH of the whey protein isolate fiber solution prepared in step S2 is adjusted to 7.0; in step S4, the solution is allowed to stand in a constant temperature box at 25°C for 12 hours.

[0022] Example 7: The difference between Example 6 and Example 1 is that: in step S1, whey protein isolate and ultrapure water are mixed in a mass ratio of 7:93; in step S3, the pH of the whey protein isolate fiber solution prepared in step S2 is adjusted to 2.0; in step S4, the solution is allowed to stand in a constant temperature box at 40°C for 6 hours.

[0023] Product performance test: 1. Gel whiteness test and gel microstructure analysis of probiotic-loaded protein fiber gels prepared in different embodiments: The surface color of the top of the gel was measured using a colorimeter. At least three points of each sample were randomly selected to estimate the average value. The whiteness value of the gel was calculated using the following equation to estimate the color difference between the probiotic-loaded protein fiber gels prepared in different examples.

[0024] (1) In formula (1), L * 、a * and b * is the color parameter of the sample, L * Indicates the brightness of the sample, a * Indicates the position of the sample on the red-green axis (positive value indicates red, negative value indicates green) and b *Indicates the position of the sample on the red-green axis (positive value indicates yellow, negative value indicates blue). The appearance of the probiotic-loaded protein fiber gel prepared in different embodiments of the present application is as follows: Figure 1 The gel whiteness test results of the probiotic-loaded protein fiber gels prepared in different embodiments are shown in Table 1. When the gel whiteness value of the probiotic-loaded protein fiber gel is ≥80.00, the gel whiteness of the probiotic-loaded protein fiber gel is pale white.

[0025] Table 1

[0026] Depend on Figure 1 As can be seen from Table 1, the order of the gel whiteness of the protein fiber gel loaded with probiotics is mainly: the gel whiteness of the protein fiber gel loaded with probiotics prepared in Example 3 > the gel whiteness of the protein fiber gel loaded with probiotics prepared in Example 2 > the gel whiteness of the protein fiber gel loaded with probiotics prepared in Example 1 > the gel whiteness of the protein fiber gel loaded with probiotics prepared in Example 5 > the gel whiteness of the protein fiber gel loaded with probiotics prepared in Example 4 > the gel whiteness of the protein fiber gel loaded with probiotics prepared in Example 6 > the gel whiteness of the protein fiber gel loaded with probiotics prepared in Example 7.

[0027] In the process of preparing the protein fiber gel loaded with probiotics in Examples 1 to 3, the whey protein isolate forms whey protein isolate fibers under the action of pH control (i.e., pH setting) and thermal induction (i.e., temperature setting) in step S2. Then, the pH value of the whey protein isolate fiber solution is adjusted in step S3 to effectively regulate the aggregation behavior of the whey protein isolate fibers. Specifically, in step S3, the pH value of the whey protein isolate fiber solution is adjusted to be close to the isoelectric point of the whey protein isolate fibers. At this time, the net surface charge of the whey protein isolate fibers approaches zero, the electrostatic repulsion between the whey protein isolate fibers is greatly reduced, and the whey protein isolate fibers are interwoven through hydrogen bonds and hydrophobic interactions to form a dense fiber network. The dense fiber network composed of these whey protein isolate fibers is manifested as large whey protein isolate fiber aggregates. When these large particles of whey protein isolate fiber aggregates are left to stand at the temperature described in step S4, the whey protein isolate fibers on different large particles of whey protein isolate fiber aggregates and the different whey protein isolate fibers on the same large particle of whey protein isolate fiber aggregates will be interwoven through hydrogen bonds and hydrophobic interactions to form a denser three-dimensional fiber network. In the process of forming the large particles of whey protein isolate fiber aggregates and the three-dimensional fiber network, the amino acid residues on the surface of the whey protein isolate fibers are arranged regularly, and the probiotics are attached to the fiber network under the action of the hydrophobicity of the whey protein isolate fibers in the fiber network, so that the probiotics are tightly wrapped in the fiber network to form a protein fiber gel loaded with probiotics.

[0028] In Examples 1 to 3 of the present application, the large particles of whey protein isolate fiber aggregates present in the whey protein fiber pregel solution prepared in step S3 will scatter light, making the pregel solution milky white and turbid. During the standing process of step S4, the whey protein isolate fibers on different large particles of whey protein isolate fiber aggregates and the different whey protein isolate fibers on the same large particle of whey protein isolate fiber aggregates will be interwoven through weak hydrogen bonds and hydrophobic interactions to form a three-dimensional, denser fiber network. The above-mentioned denser fiber network will also scatter light, so that the protein fiber gel loaded with probiotics prepared in Examples 1 to 3 appears white and opaque, as shown in FIG. Figure 1 shown.

[0029] In the process of preparing the protein fiber gel loaded with probiotics in Examples 4 to 7, in step S3, the pH of the whey protein isolate fiber solution is adjusted to deviate from the isoelectric point of the whey protein isolate fiber. At this time, the whey protein isolate fiber is negatively charged, the electrostatic repulsion between the whey protein isolate fibers is enhanced, the aggregation between the whey protein isolate fibers is effectively suppressed, and it is difficult to form large particles of whey protein isolate fiber aggregates. At this time, the whey protein isolate fibers are mainly small particles of whey protein isolate fiber aggregates generated by the aggregation between the whey protein isolate fibers and whey protein isolate fibers that have not aggregated. The whey protein isolate fiber exists in the form of fibers. During the standing process in step S4, these small particles of whey protein isolate fiber aggregates and the unaggregated whey protein isolate fibers are lightly cross-linked through hydrogen bonding and hydrophobic interactions to form a three-dimensional loose network. During the formation of the small particles of whey protein isolate fiber aggregates and the loose network, the amino acid residues on the surface of the whey protein isolate fibers are regularly arranged. The probiotics are attached to the fiber network under the action of the hydrophobicity of the whey protein isolate fibers in the fiber network, so that the probiotics are wrapped in the fiber network, forming a protein fiber gel loaded with probiotics. In Examples 4 to 7 of the present application, the small particles of whey protein isolate fiber aggregates present in the whey protein fiber pregel solution prepared in step S3 have weak light scattering, making the pregel solution transparent or translucent. During the standing process of step S4, the whey protein isolate fibers on different whey protein isolate fiber aggregate small particles and the different whey protein isolate fibers on the same whey protein isolate fiber aggregate small particles are interwoven to form a loose fiber network under the action of hydrogen bonds and hydrophobic interactions. The loose fiber network also has weak light scattering, making the protein fiber gel loaded with probiotics prepared in Examples 4 to 7 appear transparent or translucent, as shown in FIG. Figure 1 shown.

[0030] Gel micromorphology: Prepare the corresponding probiotic-loaded protein fiber gel microscopic test samples of Examples 1 to 7; wherein, the method of preparing the probiotic-loaded protein fiber gel microscopic test samples of Examples 1 to 7 is different from the preparation method of the probiotic-loaded protein fiber gel described in Examples 1 to 7 in that: in the process of preparing the probiotic-loaded protein fiber gel microscopic test samples of Examples 1 to 7, in step S3, while adding the probiotic suspension and sucrose solution, 1 ml of rhodamine B solution is also added, and the mass percentage of rhodamine B solution is 0.001 wt%; rhodamine B solution is used to mark the whey isolate protein fibers and large particles of whey isolate protein fiber aggregates in the probiotic-loaded protein fiber gel microscopic test samples prepared in Examples 1 to 7.

[0031] 100 μL of the corresponding probiotic-loaded protein fiber gel microscopic test samples of Examples 1 to 7 were transferred to a concave glass slide with a lid, sealed, and stored in the dark at room temperature overnight. Subsequently, the present application used a laser confocal microscope to observe the microstructure of the corresponding probiotic-loaded protein fiber gel microscopic test samples of Examples 1 to 7. The oil objective lens had a magnification of 63 times, an excitation wavelength of 488 nm, an emission wavelength of 570-651 nm, a scanning rate of 200 Hz, and a resolution of 1024×1024 px. Digital images were obtained from different positions of each specimen (i.e., the probiotic-loaded protein fiber gel microscopic test sample).

[0032] In this application, the corresponding probiotic-loaded protein fiber gel microscopic test samples of Examples 1 to 7 were observed using a laser confocal microscope to observe the distribution of large particles of whey isolate protein fiber aggregates, small particles of whey isolate protein fiber aggregates, and whey isolate protein fibers in the probiotic-loaded protein fiber gel microscopic test samples. Figure 2 and Figure 3 As shown, Figure 2 and Figure 3 In the figure, the red area indicates the large particles of whey protein isolate fiber aggregates, the small particles of whey protein isolate fiber aggregates, and the dispersed whey protein isolate fibers stained with rhodamine B). Figure 3 It can be seen from the CLSM images of the corresponding probiotic-loaded protein fiber gel microscopic test samples of Examples 4 to 7 that the CLSM images are a relatively uniform red as a whole, which is related to the fact that the morphology of the whey isolate protein fibers in the corresponding probiotic-loaded protein fiber gel microscopic test samples of Examples 4 to 7 is mainly in the form of small particles of whey isolate protein fiber aggregates and non-aggregated whey isolate protein fibers, while there is no large particle structure of whey isolate protein fiber aggregates; and there are relatively obvious red areas in the CLSM images of the corresponding probiotic-loaded protein fiber gel microscopic test samples of Examples 1 to 3, which is related to the presence of large particles of whey isolate protein fiber aggregates in the corresponding probiotic-loaded protein fiber gel microscopic test samples of Examples 1 to 3, and from Figure 2 It can be seen from the figure that the particle size of large particles of whey protein isolate fiber aggregates is generally 2 to 25 μm.

[0033] 2. Test of the change of storage modulus G' and loss modulus G'' of protein fiber gel samples loaded with probiotics with frequency The storage modulus (G') of the probiotic-loaded protein fiber gel samples prepared in Examples 1 to 7 was tested using a rheometer. The test results are as follows: Figure 4 As shown; This application also uses a rheometer to test the loss modulus (G'') of the protein fiber gel samples loaded with probiotics prepared in Examples 1 to 7. The test results are as follows Figure 4 As shown; wherein, the storage modulus G' value reflects the elastic strength of the gel, and the loss modulus G'' reflects the viscous strength of the gel. When the storage modulus G' and the loss modulus G'' were tested, the whey isolate protein fiber pregel solution obtained in step S3 of the probiotic-loaded protein fiber gel prepared in Examples 1 to 7 was added to the flat plate, and the gap distance between different solutions was ensured to be 1 mm, and then covered with paraffin oil to prevent water evaporation. The scanning frequency was 0.1-100 Hz, and the scanning temperature was set in stages at 4°C, 25°C and 40°C. The linear viscoelastic curve was obtained by performing strain scanning at a frequency of 1 Hz in the strain range of 0.1-100%, as shown in FIG. Figure 4 and Figure 5 shown.

[0034] The storage modulus (G') is greater than the loss modulus (G''), indicating that the material (i.e., the whey protein fiber gel loaded with probiotics prepared in the present application) has solid-like elastic characteristics; and the storage modulus G' value directly reflects the elastic strength of the whey protein fiber gel loaded with probiotics. The size of the storage modulus G' value is related to the intermolecular interaction force of whey isolate protein fibers in the whey protein fiber gel loaded with probiotics (such as hydrophobic interaction and hydrogen bonding) and the fiber network structure formed in the whey protein fiber gel loaded with probiotics.

[0035] By comparison Figure 4 and Figure 5 The test results show that the order of storage modulus (G') and loss modulus (G'') is: Example 3 > Example 2 > Example 1 > Example 5 > Example 4 > Example 6 > Example 7. Figure 4 and Figure 5 It can also be seen that the storage modulus G' and storage modulus G'' values of the probiotic-loaded protein fiber gel samples prepared in Examples 1 to 7 increase with frequency in the frequency range of 0.1-100 Hz, and the storage modulus G' is always higher than the loss modulus G'', which indicates that the probiotic-loaded protein fiber gel prepared in the present application has a continuous strong cross-linked network, which can effectively transfer stress and maintain elastic dominant behavior; and the solid-state feature of storage modulus G'> loss modulus G'' is the core sign of gel network continuity and structural integrity, which can directly reflect the anti-deformation ability of the probiotic-loaded protein fiber gel prepared in the present application.

[0036] 3. Gel hardness test of probiotic-loaded protein fiber gels prepared in different examples: The gel hardness of the probiotic-loaded protein fiber gels prepared in Examples 1 to 7 was measured using a P 50 cylindrical probe of a texture analyzer and TPA mode. During the measurement, the compression degree was 30%, the trigger force was 5 g, the test speed was 5 mm / s, and the gel hardness was compressed to 30% of the original height. Taking the gel hardness test method of the probiotic-loaded protein fiber gel prepared in Example 1 as an example: during the test, three parallel test samples of the probiotic-loaded protein fiber gel prepared in Example 1 were taken for testing, and the hardness average value was calculated based on the hardness of the three parallel test samples obtained in the test as the gel hardness of the probiotic-loaded protein fiber gel prepared in Example 1, wherein the hardness of the three parallel test samples was obtained by the first peak of the texture curve obtained by the test; The gel hardness test method of the probiotic-loaded protein fiber gel prepared in Examples 2 to 7 is the same as the gel hardness test method of the probiotic-loaded protein fiber gel prepared in Example 1; The gel hardness test results of the probiotic-loaded protein fiber gels prepared in Examples 1 to 7 are shown in Table 2.

[0037] Table 2

[0038] It can be seen from Table 2 that the order of gel hardness of the protein fiber gel loaded with probiotics prepared in Examples 1 to 7 is: the gel hardness of the protein fiber gel loaded with probiotics prepared in Example 3 > the gel hardness of the protein fiber gel loaded with probiotics prepared in Example 2 > the gel hardness of the protein fiber gel loaded with probiotics prepared in Example 1 > the gel hardness of the protein fiber gel loaded with probiotics prepared in Example 5 > the gel hardness of the protein fiber gel loaded with probiotics prepared in Example 4 > the gel hardness of the protein fiber gel loaded with probiotics prepared in Example 6 > the gel hardness of the protein fiber gel loaded with probiotics prepared in Example 7.

[0039] The difference in gel hardness of the probiotic-loaded protein fiber gel is also related to the intermolecular interaction force of whey isolate protein fibers in the probiotic-loaded protein fiber gel (such as hydrophobic interaction and hydrogen bonding) and the fiber network structure formed in the probiotic-loaded whey protein fiber gel.

[0040] 4. Test of the survival rate of probiotics after the probiotic-loaded protein fiber gel samples prepared in Examples 1 to 7 were digested in an in vitro simulated digestive system model (1) Preparation of simulated gastric fluid (SGF): Mix 0.32 g of pepsin, 0.2 g of NaCl and 100 mL of deionized water to obtain simulated gastric fluid with a pH of 2.0.

[0041] (2) Preparation of simulated intestinal fluid (SIF): Mix 0.02 g NaCl, 1.0 g trypsin, 4.5 g bile salts and 100 mL phosphate buffer solution (0.02 M, pH 7.4) evenly.

[0042] (3) Sterilize the simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) by passing them through a 0.22 μm filter membrane respectively and set aside.

[0043] Taking the probiotic-loaded protein fiber gel prepared in Example 1 as an example, the survival rate test method of the probiotics after digestion in an in vitro simulated digestive system model is as follows: 0.9 g of phosphate buffer solution and 0.1 g of the probiotic-loaded protein fiber gel prepared in Example 1 were mixed at 37° C. for 10 minutes to obtain a mixture test solution; Then, the mixed test solution was divided into two parts and added to 9 g of sterilized simulated gastric fluid and 9 g of sterilized simulated intestinal fluid, respectively, to obtain liquid phase A and liquid phase B. Then, liquid phase A and liquid phase B were preliminarily incubated at 100 rpm and 37°C for 2 hours. Then, the preliminarily incubated liquid phase A was transferred to 9 times the volume of sterilized simulated gastric fluid and incubated for a second time for 4 hours, and the preliminarily incubated liquid phase B was transferred to 9 times the volume of sterilized simulated intestinal fluid and incubated for a second time for 4 hours. Subsequently, probiotic-loaded protein fiber gels were obtained from liquid phase A and liquid phase B of the secondary incubation, and each probiotic-loaded protein fiber gel was pulverized using a cell disruptor for 5 minutes. The gels were then serially diluted. After serial dilution, 100 μl of the liquid phase was pipetted into a modified solid culture medium. The modified solid culture medium was then incubated at 37°C for 48 hours, and surviving cells were counted using the plate count method. The survival rate (%) = number of viable cells after treatment with simulated gastrointestinal fluid / number of viable cells before treatment × 100%. The results are shown in Table 3.

[0044] After the probiotic-loaded protein fiber gels prepared in Examples 2 to 7 were digested in an in vitro simulated digestive system model, the survival rate of the probiotics was tested in the same manner as the survival rate of the probiotics after the probiotic-loaded protein fiber gels prepared in Example 1 were digested in an in vitro simulated digestive system model. The test results are also shown in Table 3.

[0045] The comparative test refers to testing the survival rate of free bacteria after digesting 1 ml of free bacteria in an in vitro simulated digestive system model, where the free bacteria refer to the probiotics described in this application; Table 3

[0046] As can be seen from Table 3, after the probiotic-loaded protein fiber gel samples prepared in Examples 1 to 7 were digested in an in vitro simulated digestive system model, the survival rates of the probiotics were as follows from large to small: Example 3 (pH 5.0, 40°C, 6h) > Example 2 > Example 1 > Example 5 > Example 4 > Example 6 > Example 7; this order is completely consistent with the storage modulus (G') and gel hardness of the probiotic-loaded protein fiber gels prepared in Examples 1 to 7. Moreover, it can be seen from Table 3 that the survival rate of free bacteria after digestion in the in vitro simulated digestive system model is 0%, that is, the free bacteria are completely inactivated, while the probiotics in the probiotic-loaded protein fiber gel prepared by the present application are still relatively high after digestion in the in vitro simulated digestive system model. From the protein fiber gel samples loaded with probiotics prepared in Example 1 to Example 7, after digestion in the in vitro simulated digestive system model, the survival rate of probiotics can reach 68.25% to 84.26% after digestion in gastric juice, and the survival rate of probiotics after digestion in intestinal fluid can reach 59.45% to 74.65%. This shows that the probiotic survival rate of the probiotic-loaded protein fiber gel samples prepared by the present application is relatively high after digestion in the in vitro simulated digestive system model.

[0047] The denser fiber network present in the probiotic-loaded protein fiber gel prepared in Examples 1 to 3 of the present application and the three-dimensional loose network present in Examples 4 to 7 can form a physical barrier under the simulated gastric fluid and simulated intestinal fluid environment, effectively limiting the penetration of simulated gastric fluid and simulated intestinal fluid into the probiotic-loaded protein fiber gel and affecting the probiotic bacteria; thereby, the probiotic-loaded protein fiber gel prepared in the present application can better protect the probiotics, allowing more active probiotics to enter the intestine, and further allowing the probiotic-loaded protein fiber gel to responsively swell in the intestinal environment and be triggered by pancreatic enzyme hydrolysis to release more active probiotics in a targeted manner.

[0048] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modification, equivalent replacement, improvement, etc. made by any technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention.

Claims

1. A method for preparing a probiotic-loaded protein fiber gel, characterized by: The steps include: S1, mixing whey protein isolate powder with ultrapure water, stirring at room temperature for 2 to 4 hours, to obtain a whey protein isolate solution with a mass concentration of 3% to 7%; S2, adjusting the pH of the whey protein isolate solution to 2.0-3.0, stirring at 80° C.-85° C. for 10-12 h, and cooling in an ice-water bath for 20-30 min to obtain a whey protein isolate fiber solution; S3, regulating the pH of the whey protein isolate fiber solution to 2.0-7.0, then adding the probiotic suspension and the protective solution, stirring for 15-20 min to obtain the whey protein isolate fiber pregel solution; wherein the volume ratio of the probiotic suspension, the protective solution and the whey protein isolate fiber solution after regulating the pH is 1:(1.5-2.5):(45-55); S4. The whey protein isolate fiber pregel solution is allowed to stand at 4-40° C. for 6-24 hours to obtain a protein fiber gel loaded with probiotics.

2. The method for preparing a probiotic-loaded protein fiber gel according to claim 1, characterized in that: In step S3, the protective solution is a sucrose solution, and the mass percentage concentration of the sucrose solution is 5% to 10%.

3. The method for preparing a probiotic-loaded protein fiber gel according to claim 1, characterized in that: In step S3, the probiotic suspension is prepared as follows: the probiotic strain is inoculated into a modified liquid culture medium mMRS, cultured at 37-39°C for 24-48 hours to complete the primary culture and obtain the primary culture solution; then 4-8 mL of the primary culture solution is transferred to 100-150 mL of fresh modified liquid culture medium mMRS, and cultured at 37-39°C for 24-48 hours to complete the secondary culture; centrifuged at a temperature of 4-8°C for 10-20 minutes to obtain cells; the cells are washed with phosphate buffer solution, and the washed cells are resuspended in phosphate buffer solution to obtain 1×10 7 ~1×10 9 CFU / ml of probiotic suspension.

4. The method for preparing a probiotic-loaded protein fiber gel according to claim 2 or 3, characterized in that: In step S3, the probiotic strain is Limosilactobacillus reuteri TMW1.

656.

5. The method for preparing a probiotic-loaded protein fiber gel according to claim 3, characterized in that: In step S3, the modified liquid culture medium mMRS is obtained by uniformly mixing the following components, by weight: 1000 parts of sterile water, 10-15 parts of maltose, 10-15 parts of peptone, 10-15 parts of malt extract, 5-10 parts of fructose, 5-10 parts of glucose, 5-10 parts of fermentation extract, 5-10 parts of bovine extract, 2.6-5 parts of potassium dihydrogen phosphate, 4-10 parts of dipotassium hydrogen phosphate, 3-10 parts of ammonium chloride, 1-5 parts of Tween 80, 0.5-1 part of L-cysteine-HCL, 0.1-1 part of magnesium sulfate heptahydrate and 0.05-0.1 part of manganese sulfate tetrahydrate.

6. The method for preparing a probiotic-loaded protein fiber gel according to claim 1, characterized in that: In step S1, the mass ratio of whey protein isolate powder to ultrapure water is (3-7):(93-97).

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