Whey protein isolate-pectin embedded probiotic microcapsule as well as preparation method and application thereof in preparation of honey product for relieving constipation

Abstract

The invention discloses a whey protein isolate-pectin embedded probiotic microcapsule as well as a preparation method and application thereof in preparation of a honey product for relieving constipation, belongs to the technical field of microorganism application, and particularly relates to a preparation method of the whey protein isolate-pectin embedded probiotic microcapsule. Comprising the following steps: dissolving pectin in a water bath, mixing with whey protein isolate, and hydrating to obtain a whey protein isolate-pectin mixed solution; adjusting the pH value to 8.8-9.2, and heating in a water bath to obtain a whey protein isolate-pectin compound; the preparation method comprises the following steps: inoculating lactobacillus paracasei L21 into a culture medium, culturing, resuspending in a whey protein isolate-pectin compound, and spray-drying to obtain the whey protein isolate-pectin embedded probiotic microcapsule. A whey protein isolate-pectin composite wall material is adopted, and probiotics are embedded through spray drying, so that the probiotic microcapsule which is suitable for a hypertonic honey system and has a good protection effect and a controllable release characteristic is constructed.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, and particularly relates to whey protein isolate-pectin encapsulated probiotic microcapsules, their preparation method, and their application in the preparation of honey products for relieving constipation. Background Technology

[0002] Functional constipation is a disease characterized by reduced bowel movement frequency and difficulty in defecation, accompanied by symptoms such as abdominal pain and bloating. Severe cases can also cause systemic symptoms such as anal fissures and bleeding. Currently, constipation is mainly treated with medication, but long-term use can lead to dependence and significant side effects. Therefore, there is an urgent need to develop safer and more effective alternative therapies. Honey, as a natural food, is rich in monosaccharides, organic acids, and various bioactive substances. It can not only provide the body with rapid energy but also has the potential to regulate intestinal flora and improve the intestinal environment. However, as a hyperosmolar food, honey's high sugar and low water content limits its application in processing, resulting in insufficient development of its derivatives and low product added value. Furthermore, the specific dose-response relationship of honey in relieving constipation is not yet clear, often resulting in limited therapeutic effects for single honey products. Probiotics are live microorganisms that, when ingested in sufficient quantities, can produce health benefits to the host, with functions such as regulating intestinal flora, enhancing immune defense, and improving constipation. However, probiotics are extremely sensitive to the external environment and are easily inactivated by factors such as high temperature, gastric acid, hyperosmolarity, and bile salts during processing, storage, and digestive tract transport, which severely restricts their application in functional foods. The survival and homeostasis of probiotics face even greater challenges, especially in the high-osmolarity honey system.

[0003] Microencapsulation technology provides a physical barrier for probiotics through wall material encapsulation, significantly enhancing their resistance to adverse environments. Spray drying and freeze drying are commonly used methods for microcapsule preparation, but changes in heat, oxygen, and osmotic pressure during the drying process can still damage bacterial activity. Therefore, suitable protectants are needed to improve the protective effect on probiotics. Whey protein isolate (WPI) has good film-forming and emulsifying properties, forming a dense structure on the surface of microcapsules, effectively encapsulating active ingredients and controlling their release. Pectin (Citrus pectin, CP), as a natural anionic polysaccharide, not only has good gelling and stability but also enhances the adhesion and colonization ability of probiotics in the intestine. However, using single proteins or polysaccharides as wall materials often has limitations in encapsulation efficiency, mechanical strength, or environmental responsiveness. Therefore, there is an urgent need to develop probiotic microcapsules suitable for hypertonic honey systems. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes whey protein isolate-pectin encapsulated probiotic microcapsules, their preparation method, and their application in the preparation of constipation-relieving honey products. This invention utilizes the Maillard reaction to prepare a whey protein isolate-pectin (WPIC) composite wall material, and encapsulates probiotics through spray drying, constructing probiotic microcapsules suitable for hypertonic honey systems with good protective effects and controllable release characteristics. This provides a theoretical basis and technical support for developing probiotic honey products with constipation-relieving functions.

[0005] To achieve the above objectives, this invention provides a method for preparing whey protein isolate-pectin encapsulated probiotic microcapsules, comprising the following steps: 1) dissolving pectin in a water bath, mixing it with whey protein isolate, and hydrating it to obtain a whey protein isolate-pectin mixture; 2) adjusting the pH of the whey protein isolate-pectin mixture obtained in step 1) to 8.8~9.2, and heating it in a water bath to obtain a whey protein isolate-pectin complex; 3) Lactobacillus paracasei (… Lacticaseibacillus paracasei L21 was inoculated into the culture medium, cultured, centrifuged to collect the precipitate, and resuspended in the whey protein isolate-pectin complex obtained in step 2). The mixture was then spray-dried to obtain whey protein isolate-pectin encapsulated probiotic microcapsules.

[0006] Preferably, the water bath dissolution temperature in step 1) is 35~45℃.

[0007] Preferably, the mass ratio of pectin to whey protein isolate in step 1) is 1:1 to 4.

[0008] Preferably, the hydration temperature in step 1) is 4°C, and the hydration time is 10~14h.

[0009] Preferably, the water bath heating temperature in step 2) is 70~90℃, and the water bath heating time is 90~150min.

[0010] Preferably, the inoculation amount in step 3) is 1.5~2.5% (v / v), the culture temperature in step 3) is 37℃, the culture time is 20~28h, the centrifugation temperature in step 3) is 4℃, the centrifugation speed is 7000~9000g, and the centrifugation time is 3~7min.

[0011] Preferably, in step 3), the inlet temperature of the spray dryer is 110~130℃, the outlet temperature is 60~80℃, the feed rate is 400~600mL / h, and the air flow rate is 4~6m³ / h. 3 / h.

[0012] The present invention also provides whey protein isolate-pectin encapsulated probiotic microcapsules prepared by the above preparation method.

[0013] This invention also provides the application of the whey protein isolate-pectin encapsulated probiotic microcapsules in the preparation of honey products for relieving constipation.

[0014] Preferably, the method involves adding whey protein isolate-pectin-encapsulated probiotic microcapsules to honey and treating it in a water bath at 25-35℃ for 10-30 minutes to obtain a honey product that relieves constipation.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: The present invention provides whey protein isolate-pectin encapsulated probiotic microcapsules, their preparation method and their application in the preparation of honey products for relieving constipation. The present invention uses Maillard reaction to prepare whey protein isolate-pectin (WPIC) composite wall material, and encapsulates probiotics by spray drying, constructing probiotic microcapsules with good protective effect and controllable release characteristics suitable for hypertonic honey systems, providing a theoretical basis and technical support for the development of probiotic honey products with constipation relief function. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Figure 1 shows the standard curve of lysine and the change in WPIC grafting degree during the Maillard reaction. In the figure, A is the standard curve of lysine and B is the change in WPIC grafting degree. Different letters in the figure indicate significant differences. Figure 2 The pepsin and trypsin digestibility of WPI and WPIC are shown in the figure. A represents WPIC 1:1 and B represents WPIC 4:1. Different letters in the figure indicate significant differences. Figure 3 The survival rate of *Lactobacillus paracasei* L21 in microcapsules is shown in the figure. Different letters in the figure indicate significant differences. This represents 0.01 < P ≤ 0.05. This represents 0.001 < P ≤ 0.01. This means P ≤ 0.001. This means P ≤ 0.0001; Figure 4Figure 1 shows the particle size and zeta potential of the microencapsulated particles, where A represents the particle size and B represents the zeta potential. Different letters in the figure indicate significant differences. P ≤ 0.0001, ns indicates no significant difference; Figure 5 SEM images of microcapsules prepared by WPIC via spray drying and freeze drying are shown below. In the images, A represents WPIC microcapsules spray-dried using the wall material method; B represents WPIC / 0 microcapsules spray-dried using the wall material method; C represents WPIC / 30 microcapsules spray-dried using the wall material method; D represents WPIC / 60 microcapsules spray-dried using the wall material method; E represents WPIC / 90 microcapsules spray-dried using the wall material method; F represents WPIC / 120 microcapsules spray-dried using the wall material method; G represents WPIC / 150 microcapsules spray-dried using the wall material method; and H represents WPIC / 180 microcapsules spray-dried using the wall material method. Freeze-dried microcapsules, I for WPIC / 0 for wall material, J for WPIC / 30 for wall material, L for WPIC / 60 for wall material, M for WPIC / 90 for wall material, N for WPIC / 120 for wall material, O for WPIC / 150 for wall material, P for WPIC / 180 for wall material, scale bar is 5μm, red arrow represents Lactobacillus paracasei L21; Figure 6 The viable count and survival rate of Lactobacillus paracasei L21 in microcapsules under simulated gastrointestinal conditions are shown in the figure. In the figure, A represents viable count and B represents survival rate. Different lowercase letters in the figure indicate significant differences between groups, and different uppercase letters indicate significant differences within groups. Figure 7 To determine the viable count and survival rate of *Lactobacillus paracasei* L21 in spray-dried microcapsules and freeze-dried microcapsules under simulated pasteurization conditions, where A represents the viable count of spray-dried microcapsules, B represents the survival rate of spray-dried microcapsules, C represents the viable count of freeze-dried microcapsules, and D represents the survival rate of freeze-dried microcapsules, different lowercase letters in the figure indicate significant differences between groups, and different uppercase letters indicate significant differences within groups; Figure 8 The viable counts of *Lactobacillus paracasei* L21 in spray-dried microcapsules stored at 4℃ and 25℃ for 8 weeks and in freeze-dried microcapsules are given. Where A represents the viable count of microcapsules spray-dried at 4℃, B represents the viable count of microcapsules spray-dried at 25℃, C represents the viable count of microcapsules freeze-dried at 4℃, and D represents the viable count of microcapsules freeze-dried at 25℃. Figure 9The osmotic properties of Lactobacillus paracasei L21 microcapsules and their survival rates under different concentrations of sodium chloride stress are shown in the figure. In the figure, A represents the osmotic properties of the microcapsules and B represents the survival rate. Different lowercase letters in the figure indicate significant differences between groups, and different uppercase letters indicate significant differences within groups. Figure 10 The figures show the changes in body weight and fecal water content of mice in each group. In the figure, A represents mouse body weight, B represents fecal water content, and different letters indicate significant differences between groups: "D" represents the blank group, "M" represents the model group, "Y" represents the mosapride group, "W" represents the whey protein isolate-pectin group, "F" represents the honey group, "L21" represents the free probiotic group, "WC" represents the encapsulated probiotic group, and "FL" represents the probiotic honey group. Figure 11 The changes in the time of first black feces excretion in mice of each group are shown in the figure. Different letters in the figure indicate significant differences between groups: "D" is the blank group, "M" is the model group, "Y" is the mosapride group, "W" is the whey protein isolate-pectin group, "F" is the honey group, "L21" is the free probiotic group, "WC" is the encapsulated probiotic group, and "FL" is the probiotic honey group. Figure 12 The graph shows the changes in small intestinal propulsion rate in mice of different groups. Different letters in the graph indicate significant differences between groups: "D" is the blank group, "M" is the model group, "Y" is the mosapride group, "W" is the whey protein isolate-pectin group, "F" is the honey group, "L21" is the free probiotic group, "WC" is the encapsulated probiotic group, and "FL" is the probiotic honey group. Figure 13 The figure shows the changes in fecal SCFAs in each group of mice. A represents the acetic acid level in the intestinal contents of mice, B represents the propionic acid level in the intestinal contents of mice, C represents the butyric acid level in the intestinal contents of mice, and D represents the total acid level in the intestinal contents of mice. Different letters in the figure indicate significant differences between groups: "D" represents the blank group, "M" represents the model group, "Y" represents the mosapride group, "W" represents the whey protein isolate-pectin group, "F" represents the honey group, "L21" represents the free probiotic group, "WC" represents the encapsulated probiotic group, and "FL" represents the probiotic honey group. Figure 14 The graph shows the changes in the gut microbiota phylum level of mice in each group. In the graph, "D" is the blank group, "M" is the model group, "Y" is the mosapride group, "W" is the whey protein isolate-pectin group, "F" is the honey group, "L21" is the free probiotic group, "WC" is the encapsulated probiotic group, and "FL" is the probiotic honey group. Figure 15 The graph shows the changes in the gut microbiota genera of mice in each group. In the graph, "D" represents the blank group, "M" represents the model group, "Y" represents the mosapride group, "W" represents the whey protein isolate-pectin group, "F" represents the honey group, "L21" represents the free probiotic group, "WC" represents the encapsulated probiotic group, and "FL" represents the probiotic honey group. Figure 16 LefSe analysis of gut microbiota in mice of each group was performed. A is the evolutionary branching diagram of LEfSe analysis for groups D, M and FL, and B is the LDA score diagram for groups D, M and FL. In the figure, "D" is the blank group, "M" is the model group and "FL" is the probiotic honey group. Figure 17 The histopathological changes in the colon tissue of mice in each group are shown below. A represents group D, B represents group M, C represents group Y, D represents group W, E represents group F, F represents group L21, G represents group WC, and H represents group FL. In the figure, "D" represents the blank group, "M" represents the model group, "Y" represents the mosapride group, "W" represents the whey protein isolate-pectin group, "F" represents the honey group, "L21" represents the free probiotic group, "WC" represents the encapsulated probiotic group, and "FL" represents the probiotic honey group. Figure 18 The changes in serum gastrointestinal regulatory peptides in mice in each group are shown. In the figure, A represents the serum 5-HT level, B represents the serum Ach level, C represents the serum SP level, and D represents the serum MTL level. "D" represents the blank group, "M" represents the model group, "Y" represents the mosapride group, "W" represents the whey protein isolate-pectin group, "F" represents the honey group, "L21" represents the free probiotic group, "WC" represents the encapsulated probiotic group, and "FL" represents the probiotic honey group. Figure 19 The expression levels of constipation-related genes in each group of mice are shown. A represents the colonic 5-HT4R gene expression level, B represents the colonic 5-HT3R gene expression level, C represents the colonic SERT gene expression level, D represents the colonic AQP4 gene expression level, E represents the colonic AQP8 gene expression level, F represents the colonic MAOA gene expression level, G represents the colonic TPH1 gene expression level, and H represents the colonic GPR43 gene expression level. Different letters in the figure indicate significant differences between groups: "D" represents the blank group, "M" represents the model group, "Y" represents the mosapride group, "W" represents the whey protein isolate-pectin group, "F" represents the honey group, "L21" represents the free probiotic group, "WC" represents the encapsulated probiotic group, and "FL" represents the probiotic honey group. Detailed Implementation

[0018] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail. The terms “comprising,” “including,” “having,” and “containing” as used herein are open-ended, meaning they include but are not limited to.

[0019] The materials used in this invention were sourced from: pectin (purchased from Shanghai Yuanye Biotechnology Co., Ltd.), whey protein isolate (purchased from Fonterra, New Zealand), and Lactobacillus paracasei (…). Lacticaseibacillus paracasei L21 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.31130. MRS liquid culture medium was purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., honey was purchased from Xinjiang Saiwai Bencao Bee Industry Co., Ltd., PBS was purchased from Beijing Solarbio Biotechnology Co., Ltd., and BCA protein quantification kit was purchased from Beijing Solarbio Biotechnology Co., Ltd.

[0020] Example 1 1) Pectin (CP) was dissolved in a water bath at 40°C, then mixed with whey protein isolate (WPI) at a mass ratio of 1:4, and hydrated at 4°C for 12 hours to obtain a whey protein isolate-pectin mixture; 2) Using 5 mol / L The pH of the whey protein isolate-pectin mixture was adjusted to 9.0 with NaOH, and heated in a water bath at 80℃ for 120 min to obtain the whey protein isolate-pectin complex (WPIC); 3) Frozen *Lactobacillus paracasei* L21 was inoculated into MRS liquid medium at an inoculation rate of 2% (v / v) and cultured at 37℃ for 24 h. After activation, it was inoculated again into new MRS liquid medium at an inoculation rate of 2% (v / v) and cultured at 37℃ for 24 h to obtain *Lactobacillus paracasei* L21 that had been activated twice. The *Lactobacillus paracasei* L21 that had been activated twice was inoculated into MRS liquid medium at an inoculation rate of 2% (v / v) and cultured at 37℃ for 24 h. After centrifugation at 4℃ and 8000g for 5 min, the precipitate was collected, washed twice with PBS (0.1 mol / L, pH 7.4), resuspended in WPIC, and spray-dried (spray drying conditions: inlet temperature 120℃, outlet temperature 70℃, feed rate 500 mL / h, air flow rate 5 m³ / h). 3 / h), to obtain whey protein isolate-pectin encapsulated probiotic microcapsules.

[0021] Probiotic microcapsules encapsulated in whey protein isolate and pectin were added to honey (17%–19% water content, pH 2.99±0.02) to ensure that the number of live bacteria in the honey product was ≥10. 7 CFU / g, treated in a 30℃ water bath for 20 minutes, yields a honey product that relieves constipation.

[0022] Example 2 1) Pectin (CP) was dissolved in a water bath at 40°C, then mixed with whey protein isolate (WPI) at a mass ratio of 1:1 and hydrated at 4°C for 12 hours to obtain a whey protein isolate-pectin mixture; 2) Using 5 mol / L The pH of the whey protein isolate-pectin mixture was adjusted to 9.0 with NaOH, and heated in a water bath at 80℃ for 120 min to obtain the whey protein isolate-pectin complex (WPIC); 3) Frozen *Lactobacillus paracasei* L21 was inoculated into MRS liquid medium at an inoculation rate of 2% (v / v) and cultured at 37℃ for 24 h. After activation, it was inoculated again into new MRS liquid medium at an inoculation rate of 2% (v / v) and cultured at 37℃ for 24 h to obtain *Lactobacillus paracasei* L21 that had been activated twice. The *Lactobacillus paracasei* L21 that had been activated twice was inoculated into MRS liquid medium at an inoculation rate of 2% (v / v) and cultured at 37℃ for 24 h. After centrifugation at 4℃ and 8000g for 5 min, the precipitate was collected, washed twice with PBS (0.1 mol / L, pH 7.4), resuspended in WPIC, and spray-dried (spray drying conditions: inlet temperature 120℃, outlet temperature 70℃, feed rate 500 mL / h, air flow rate 5 m³ / h). 3 / h), to obtain whey protein isolate-pectin encapsulated probiotic microcapsules.

[0023] Probiotic microcapsules encapsulated in whey protein isolate and pectin were added to honey (17%–19% water content, pH 2.99±0.02) to ensure that the number of live bacteria in the honey product was ≥10. 7 CFU / g, treated in a 30℃ water bath for 20 minutes, yields a honey product that relieves constipation.

[0024] Example 3 1) Pectin (CP) was dissolved in a water bath at 35°C, then mixed with whey protein isolate (WPI) at a mass ratio of 1:4, and hydrated at 4°C for 10 hours to obtain a whey protein isolate-pectin mixture; 2) Using 5 mol / L The pH of the whey protein isolate-pectin mixture was adjusted to 8.8 with NaOH, and heated in a 70℃ water bath for 150 min to obtain the whey protein isolate-pectin complex (WPIC); 3) Frozen *Lactobacillus paracasei* L21 was inoculated into MRS liquid medium at an inoculation rate of 1.5% (v / v) and cultured at 37℃ for 28 h. After activation, it was inoculated again into new MRS liquid medium at an inoculation rate of 1.5% (v / v) and cultured at 37℃ for 28 h to obtain *Lactobacillus paracasei* L21 that had been activated twice. The activated *Lactobacillus paracasei* L21 was inoculated into MRS liquid medium at an inoculation rate of 1.5% (v / v) and cultured at 37℃ for 28 h. After centrifugation at 7000g for 7 min at 4℃, the precipitate was collected and treated with PBS (0.1 mol / L, pH 5.5). 7.4) Wash twice, resuspend in WPIC, and spray dry (spray drying conditions: inlet temperature 110℃, outlet temperature 60℃, feed rate 400mL / h, air flow rate 4m). 3 / h), to obtain whey protein isolate-pectin encapsulated probiotic microcapsules.

[0025] Probiotic microcapsules encapsulated in whey protein isolate and pectin were added to honey (17%–19% water content, pH 2.99±0.02) to ensure that the number of live bacteria in the honey product was ≥10. 7 CFU / g, treated in a 25℃ water bath for 30 minutes, yields a honey product that relieves constipation.

[0026] Example 4 1) Pectin (CP) was dissolved in a water bath at 45°C, then mixed with whey protein isolate (WPI) at a mass ratio of 1:1 and hydrated at 4°C for 14 hours to obtain a whey protein isolate-pectin mixture; 2) Using 5 mol / L Adjust the pH of the whey protein isolate-pectin mixture to 9.2 with NaOH, and heat in a 90℃ water bath for 90 min to obtain whey protein isolate-pectin complex (WPIC); 3) Inoculate the frozen *Lactobacillus paracasei* L21 into MRS liquid medium at an inoculation rate of 2.5% (v / v) and incubate at 37℃ for 20 h. After activation, inoculate again into new MRS liquid medium at an inoculation rate of 2.5% (v / v) and incubate at 37℃ for 20 h to obtain twice-activated *Lactobacillus paracasei* L21. Inoculate the twice-activated *Lactobacillus paracasei* L21 into MRS liquid medium at an inoculation rate of 2.5% (v / v) and incubate at 37℃ for 20 h. Centrifuge at 4℃ and 9000g for 3 min, collect the precipitate, and use PBS (0.1mol / L, pH 10) to extract the precipitate. 7.4) Wash twice, resuspend in WPIC, and spray dry (spray drying conditions: inlet temperature 130℃, outlet temperature 80℃, feed rate 600mL / h, air flow rate 6m). 3 / h), to obtain whey protein isolate-pectin encapsulated probiotic microcapsules.

[0027] Probiotic microcapsules encapsulated in whey protein isolate and pectin were added to honey (17%–19% water content, pH 2.99±0.02) to ensure that the number of live bacteria in the honey product was ≥10. 7 CFU / g, treated in a 35℃ water bath for 10 minutes, yields a honey product that relieves constipation.

[0028] Experimental Example 1 I. Physicochemical properties of whey protein isolate-pectin complex (WPIC).

[0029] Pectin (CP) was dissolved in a water bath at 40°C and then mixed with whey protein isolate (WPI) at a mass ratio of 1:4 (WPIC 4:1) or 1:1 (WPIC 1:1). The mixture was then hydrated in a refrigerator at 4°C for 12 hours to obtain a whey protein isolate-pectin mixture. The pH of the whey protein isolate-pectin mixture was adjusted to 9.0 using 5 mol / L NaOH, and the mixture was heated in a water bath at 80°C for 180 minutes. Samples were taken every 30 minutes and named WPIC / 0, WPIC / 30, WPIC / 60, WPIC / 90, WPIC / 120, WPIC / 150, and WPIC / 180.

[0030] 1. Grafting Degree Determination: The grafting degree of WPIC was determined using the o-phthalaldehyde (OPA) method. 80 mg of OPA was dissolved in 2 mL of methanol and mixed with 50 mL of 0.1 M tetraborate buffer (pH 9.7), 200 μL of β-thiol, and 5 mL of 20% (w / w) sodium dodecyl sulfate solution (SDS). After mixing, the solution volume was brought to 100 mL with distilled water. 200 μL of sample solution (2 mg / mL) was added to 4 mL of the mixed solution, and the mixture was incubated at 35 °C for 2 min. The absorbance of the sample was measured at 340 nm. Grafting degree (%) = (A0 - A t ) / A0, where A0 and A t These represent the content of free amino acids in whey protein isolate-pectin mixtures (WPIC / 0) and (WPIC / 30, WPIC / 60, WPIC / 90, WPIC / 120, WPIC / 150, WPIC / 180) (WPIC conjugates), respectively.

[0031] The degree of WPI grafting to CP can be assessed by quantitatively analyzing the free amino acid content of the protein. The linear regression equation for lysine concentration on absorbance is y = 0.8052x + 0.1245 (where y is the absorbance at 340 nm, x is the lysine concentration, and R0 is the absorbance). 2 =0.9991). For example Figure 1 China A and Figure 1 As shown in Figure B, the grafting degree of both mass ratio (WPIC) samples increased significantly with heating time in the early stage of the reaction (P<0.05), indicating that WPI and CP rapidly covalently bonded in the early stage of the reaction. The grafting degree reached its peak at 120 min of reaction, with maximum values ​​of 19.77±0.27% (WPIC 1:1) and 22.39±0.72% (WPIC 4:1), respectively, and then decreased. This may be because the initial reaction can change the spatial conformation of the protein, exposing the amino groups on the WPI chain and allowing them to interact with CP, thereby increasing the DG value of WPIC. However, with the extension of Maillard reaction time, the grafting degree of WPIC showed a slight decrease. This may be because the cross-linking rate of WPI increased under prolonged heat treatment, leading to an increase in aggregate content, while the glycosylation sites did not increase accordingly, failing to react with more CP, thus hindering the Maillard reaction process. The grafting degree of WPIC 4:1 was generally higher than that of WPIC 1:1. This may be because the CP used in the experiment, with a degree of esterification (DE) of 58-62%, may produce more free galacturonic acid through β-elimination during heat treatment. However, galacturonic acid is not a reducing sugar and cannot participate in the Maillard reaction. Furthermore, the presence of CP lowers the pH of the system, and an acidic environment is unfavorable for the Maillard reaction.

[0032] 2. In vitro gastrointestinal digestion: First, simulated gastric juice (SGF) and simulated intestinal juice (SIF) were prepared. SGF contained pepsin (enzyme activity concentration of 2000 U / mL) and sodium chloride (47.2 mmol / L), and the pH was adjusted to 3.0 using 1 mol / L hydrochloric acid. SIF contained trypsin (based on trypsin activity, 100 U / mL), bile salts (47.2 mmol / L), sodium chloride (38.4 mmol / L), and calcium chloride (0.6 mmol / L), and the pH was adjusted to 7.0 using 1 mol / L sodium hydroxide solution. The digestion process consisted of two phases: the gastric phase and the intestinal phase. 5 mL of the sample to be tested was mixed with an equal volume of SGF solution and incubated at 37°C with shaking for 2 h to simulate the gastric digestive environment. Then, 10 mL of SIF solution was added, and the reaction was continued at 37°C for 4 h to simulate the intestinal digestive process. After the reaction, the mixture was immediately placed in an ice-water bath to stop enzyme activity. After centrifugation, the sample was centrifuged at 10000×g for 10 min at 4°C. The precipitate was collected, resuspended in pre-cooled PBS buffer, and washed twice to thoroughly remove digestion products and soluble components. Finally, the protein content was determined using a BCA protein quantification kit. Digestibility (%) = [1-(P1 / P0)]×100%, where P1 represents the protein content after pepsin or trypsin digestion, and P0 represents the protein content before digestion.

[0033] The results of free amino content measured after in vitro simulated gastrointestinal digestion are as follows: Figure 2 China A and Figure 2 As shown in Figure B. After digestion with pepsin and trypsin, the digestibility of WPI was 73.08 ± 0.54%. In a WPI 4:1 system (e.g., Figure 2 The digestibility of WPIC / 0, WPIC / 30, WPIC / 60, WPIC / 90, WPIC / 120, WPIC / 150, and WPIC / 180 was 62.47±0.99%, 50.53±2.01%, 45.66±0.58%, 42.86±0.84%, 26.79±1.09%, 31.98±1.10%, and 36.83±1.12%, respectively. In a WPIC 1:1 system (such as...), Figure 2The digestibility of the corresponding complexes in the WPIC 4:1 and WPIC 1:1 systems was 64.84±1.11%, 52.54±1.88%, 49.73±0.78%, 44.23±1.97%, 32.31±1.35%, 35.65±1.71%, and 40.67±1.33%, respectively. The digestibility of all complexes in the WPIC 4:1 and WPIC 1:1 systems was significantly lower than that of the WPI group (P<0.05). These results indicate that the Maillard reaction helps improve the digestibility of the WPIC complex. This may be because CP and WPI form a covalent complex resistant to hydrolysis by pepsin and trypsin through the Maillard reaction. Furthermore, the Maillard reaction increases the steric hindrance of amino acid residues on the WPI surface, thereby masking the action sites of digestive enzymes and reducing protein digestibility. Among them, the WPIC / 120 sample showed the best digestibility, indicating that the 120 min reaction time allowed WPI and CP to achieve sufficient covalent cross-linking, forming a structurally stable complex system with a suitable spatial conformation. Insufficient reaction time results in inadequate cross-linking, leading to a loose composite structure that is easily degraded by digestive enzymes. Excessive reaction time, on the other hand, can cause excessive protein aggregation or pectin chain degradation, compromising the integrity of the protective structure. Therefore, WPIC / 120 exhibits the strongest resistance to digestion.

[0034] II. Physicochemical properties of whey protein isolate-pectin encapsulated probiotic microcapsules.

[0035] Frozen *Lactobacillus paracasei* L21 was inoculated into MRS liquid medium at a 2% (v / v) inoculum and cultured at 37°C for 24 h. After activation, it was again inoculated into fresh MRS liquid medium at a 2% (v / v) inoculum and cultured at 37°C for 24 h, resulting in twice-activated *Lactobacillus paracasei* L21. The twice-activated *Lactobacillus paracasei* L21 was then inoculated into MRS liquid medium at a 2% (v / v) inoculum and cultured at 37°C for 24 h. After centrifugation at 4°C and 8000g for 5 min, the precipitate was collected, washed twice with PBS (0.1 mol / L, pH 7.4), resuspended in WPIC prepared in Example 1, and then spray-dried (spray drying conditions: inlet temperature 120°C, outlet temperature 70°C, feed rate 500 mL / h, air flow rate 5 m³ / h). 3 Alternatively, pre-freeze at -80℃ for 12 hours and freeze-dry at -50℃ for 20 hours to obtain whey protein isolate-pectin-encapsulated probiotic microcapsules. A single WPI wall material group was used as a control.

[0036] 1. Encapsulation efficiency of microcapsules: Dissolve 1g of spray-dried or freeze-dried powder in 9mL of PBS, dilute and spread on solid MRS medium, and calculate the survival rate of *Lactobacillus paracasei* L21. Encapsulation efficiency (%) = N1 / N0 × 100%, where N1 and N0 represent the number of colonies in the microcapsules after drying and the number of colonies before drying, respectively.

[0037] like Figure 3 The survival rate of *Lactobacillus paracasei* L21 after microencapsulation is shown in the figure. The results showed that the survival rates of probiotic microcapsules prepared by spray drying using WPIC / 0, WPIC / 30, WPIC / 60, WPIC / 90, WPIC / 120, WPIC / 150, and WPIC / 180 as wall materials were 15.02±1.67%, 35.54±3.23%, 45.81±1.13%, 51.24±2.79%, 76.73±3.51%, 71.90±2.88%, and 60.98±7.62%, respectively, which were significantly higher (P<0.05) than the control group (6.72±1.25%) using WPI alone as the wall material and free *Lactobacillus paracasei* L21 (2.16±0.20%). The survival rates of probiotic microcapsules prepared using these wall materials via freeze-drying were 26.29±1.91%, 43.85±1.20%, 56.20±0.78%, 59.15±1.51%, 88.79±4.28%, 83.92±2.49%, and 69.51±3.66%, respectively, which were significantly higher (P<0.05) than those of the WPI control group (14.12±0.29%) and free *Lactobacillus paracasei* L21 (9.51±0.24%). These results indicate that protein-polysaccharide conjugates have a superior protective effect in maintaining probiotic activity. The improved survival rate of microencapsulated probiotics may be due to CP adsorption on the probiotic surface, thereby mitigating dehydration damage to the cell membrane during drying, or it may be due to the Maillard reaction products of WPIC constructing a denser network structure. The WPIC / 120 group achieved the highest survival rate, indicating that the heat treatment time of the wall material has a crucial impact on the protective effect. Both excessively short and excessively long reaction times reduce the protective effect on probiotics. Appropriately extending the heating time promotes Maillard covalent cross-linking between CP and WPI, forming a higher molecular weight complex, thereby enhancing the protection against *Lactobacillus paracasei* L21. However, excessive heating causes the degradation of Maillard products, leading to a decrease in protective performance; this trend is consistent with the variation trend of the grafting degree of the wall material. Furthermore, the microcapsules prepared by freeze-drying had a significantly higher encapsulation efficiency than those prepared by spray drying (P<0.05).

[0038] 2. Microcapsule particle size and potential determination: Take 0.1g of microcapsule sample and disperse it uniformly in 100mL of sterile water at room temperature. Stir at a constant rate for 5min using a magnetic stirrer to ensure complete dissolution of the sample. Immediately afterwards, use a laser particle size analyzer to determine the particle size distribution and zeta potential of the microcapsules in the dispersion system. Each sample is measured in triplicate.

[0039] Particle size distribution is one of the key indicators for evaluating the stability of microcapsules in solution. For example... Figure 4As shown in Figure A, different wall materials and drying methods significantly affect the particle size of the formed microcapsules. Specifically, when using WPI as the wall material, the average particle size of the spray-dried microcapsules was 681.43±4.16 nm, while the freeze-dried microcapsules had a larger particle size of 759±9.98 nm. In contrast, when using WPIC / 120 as the wall material, the particle size of the microcapsules prepared by both spray drying and freeze drying was significantly reduced, reaching 296.63±7.32 nm and 349.9±6.05 nm, respectively. This result indicates that the WPIC / 120 wall material helps to form smaller and more concentrated microcapsule structures, which may be more beneficial to improving their stability in solution. This phenomenon is mainly attributed to its better emulsification and interfacial stability, which can promote the formation of finer and more uniform microcapsules during the drying process. However, the particle size of the freeze-dried microcapsules was significantly larger than that of the spray-dried microcapsules (P<0.05). This difference is mainly due to the fact that the sublimation of ice crystals during freeze-drying forms a porous, sponge-like structure, with particles that are typically loose and large. In contrast, spray drying, through the rapid dehydration and surface shrinkage of droplets at high temperatures, can form denser and smaller particles.

[0040] Zeta potential is not only a key parameter affecting the release kinetics of microcapsules, but also an important indicator for assessing the stability of the system. Microcapsules have higher Zeta potentials ( Figure 4 (See Figure B). Further analysis showed that the zeta potential of the freeze-dried microcapsules ranged from -27.22±0.80 mV to -39.11±0.88 mV, while that of the spray-dried microcapsules ranged from -28.35±1.01 mV to -40.48±0.62 mV. Although there was a difference in zeta potential between the spray-dried and freeze-dried microcapsules, the difference was not statistically significant (P>0.05). This may be because although the processes of spray drying and freeze drying are different, neither method significantly affected the protein charge density and the resulting electrostatic repulsion.

[0041] 3. Microstructure determination (SEM): The sample powder is attached to the electron microscope disk and immediately sputtered with gold. The appearance morphology of the material is observed under a voltage of 5.0KV.

[0042] Scanning electron microscopy (SEM) was used to observe the products after spray drying and freeze drying. Figure 5 China A~ Figure 5Microcapsules encapsulated with probiotics using WPI and WPIC delivery systems were prepared by [a research team]. Microcapsules prepared by spray drying all exhibited spherical structures, but their surfaces commonly showed structural defects such as depressions, pores, and cracks. These defects were mainly attributed to particle shrinkage or WPI denaturation during the drying process. Microcapsules prepared using WPI as the wall material showed visible free probiotics attached to their surface and internal pores, indicating poor encapsulation efficiency. In contrast, microcapsules using heat-treated WPIC Maillard reaction products as the wall material, while still exhibiting some surface depressions, showed a significant reduction in depression degree and a marked decrease in the number of free probiotics on the surface with prolonged Maillard reaction time, effectively improving encapsulation efficiency.

[0043] Fine fragments often adhere to the surface of freeze-dried microcapsules, primarily due to physical structural damage caused by ice crystal sublimation during the freeze-drying process. Microcapsules prepared using WPI alone as the wall material exhibited significant surface fragmentation. However, the addition of pectin to the wall material significantly reduced the number of surface fragments and markedly improved structural integrity. Furthermore, with prolonged reaction time using Maillard reaction products as the wall material, free probiotic residues gradually disappeared from the microcapsule surface. This phenomenon may be related to the specific interaction between the active groups formed in the Maillard reaction products and the probiotic cell wall components, thereby enhancing the encapsulation and protection effect of the wall material on the bacteria.

[0044] 4. In vitro digestion stability of the microcapsules: The composition of SGF was: potassium chloride 1.12 g / L, sodium chloride 2.0 g / L, calcium chloride 0.11 g / L, potassium dihydrogen phosphate 0.4 g / L, and pepsin 0.26 g / L. After dissolution, the pH was adjusted to 2.0 with 1 mol / L hydrochloric acid. SIF contained 0.5% (w / v) pancreatin and 0.3% (w / v) bile salts, and the pH was adjusted to 7.0. Both digestion solutions were aseptically filtered through a 0.22 μm filter membrane for later use. 1 g of sample powder was mixed with 9 mL of SGF and digested in the stomach at 37 °C. Samples were taken at 0 h and 2 h, and the survival rate of *Lactobacillus paracasei* L21 was determined by plate count. After gastric digestion, 10 mL of SIF was added to the residual digestion solution, and intestinal digestion was continued at 37 °C. Plate counts were performed after 4 h of mixing.

[0045] Spray-dried and freeze-dried microcapsules prepared using WPI and WPIC as wall materials showed significant differences in the survival rate and viable count of probiotics in simulated gastrointestinal fluid (P<0.05). Figure 6 China A and Figure 6As shown in Figure B, under SGF and SIF treatments, the viable bacterial count in spray-dried microcapsules with WPI as the wall material decreased from the initial 10.66±0.08 log CFU / mL to 4.58±0.07 log CFU / mL, a reduction of 6.08 log CFU / mL, with a survival rate of 42.92±0.70%. In contrast, the viable bacterial count in freeze-dried microcapsules decreased from 10.98±0.01 log CFU / mL to 4.03±0.04 log CFU / mL, a reduction of 6.95 log CFU / mL, with a survival rate of 36.67±0.36%. Furthermore, microcapsules encapsulated with Maillard-modified WPIC / 120 wall material exhibited the strongest gastrointestinal tolerance, with a significantly higher survival rate than the WPI group microcapsules (P<0.05). After 6 hours of digestion in gastrointestinal fluid, the viable bacterial count of the WPIC / 120 microcapsules remained at 8.09±0.01 log CFU / mL in the spray-dried group and 7.19±0.01 log CFU / mL in the freeze-dried group, with survival rates of 70.74±0.09% and 62.49±0.12%, respectively. This indicates that the Maillard reaction effectively enhances the digestibility of the WPIC composite wall material. The possible mechanism is the formation of a dense interfacial structure, providing steric hindrance and reducing direct contact between digestive enzymes and probiotics. Spray-dried microcapsules demonstrate a significant advantage in improving the gastrointestinal tolerance of probiotics. Simulated gastrointestinal digestion experiments showed that the three groups of microcapsules prepared by spray drying—WPIC / 120, WPIC / 150, and WPIC / 180—all effectively maintained a viable bacterial count above 7.0 log CFU / mL, while only WPIC / 120 in the freeze-dried group achieved the same level of protection. This is attributed to the fact that spray-dried microcapsules have a typical small-diameter spherical structure with internal cavities, while freeze-dried samples exhibit a porous network structure with uneven pore size distribution. This significant morphological difference resulted in spray-dried microcapsules exhibiting a lower cell mortality rate (P<0.05) during simulated continuous gastrointestinal digestion, demonstrating superior protective effects compared to freeze-dried samples.

[0046] 5. Thermal stability of microcapsules: The microcapsules were dissolved in sterile PBS and heated in a water bath at 63°C for 10 min, 20 min and 30 min respectively. After sampling, the microcapsules were immediately placed in an ice water bath to cool to room temperature and plated to determine the thermal stability of the microcapsules.

[0047] Probiotics are heat-sensitive, a characteristic that severely limits their stability during food processing and storage. Microencapsulation, as an effective protective measure, can significantly enhance the resistance of probiotics to heat stress. At 63℃, different heat treatment times affected the survival rate and viable count of probiotics in microcapsules, with significant differences among the treatment groups (P<0.05). Figure 7 China A Figure 7 B, Figure 7 C and Figure 7 (D). After heat treatment, the viable count of the freeze-dried free probiotics decreased sharply from 9.77±0.02 log CFU / mL to 1.89±0.01 log CFU / mL. The viable count in microcapsules prepared using WPI as the wall material and freeze-dried decreased from the initial 9.89±0.03 log CFU / mL to 3.20±0.01 log CFU / mL. In contrast, microcapsules prepared using WPIC / 120 wall material modified by the Maillard reaction exhibited the best thermal stability, with the viable count remaining at 6.58±0.05 log CFU / mL. The survival rate of probiotics in the Maillard-modified microcapsules remained above 42.03±0.19%, significantly higher than the 32.38±0.08% of the WPI control group microcapsules (P<0.05). This indicates that Maillard reaction products can effectively alleviate the damage caused by high temperatures to probiotics, possibly because WPIC forms a dense protective layer on the surface of the bacteria, delaying the transfer of heat to the interior, thereby reducing the denaturation and inactivation of Lactobacillus paracasei.

[0048] Furthermore, the *Lactobacillus paracasei* microcapsules prepared by spray drying also exhibited a protective trend consistent with the above-mentioned patterns, and their protective effect was superior to that of freeze-dried samples. Specifically, the viable count of the WPIC / 120 group of microcapsules prepared by spray drying decreased from the initial 10.62±0.02 log CFU / mL to 6.58±0.05 log CFU / mL, with a survival rate of 69.74±0.04%. In contrast, the viable count of the WPIC / 120 group of microcapsules prepared by freeze-spray drying decreased from the initial 10.56±0.02 log CFU / mL to 7.36 log CFU / mL, with a survival rate of 61.92±0.46%. This difference may be due to the higher inlet air temperature during spray drying, which causes the wall material on the droplet surface to dehydrate and form a film rapidly, enhancing the overall density and structural integrity of the microcapsules, thereby more effectively blocking the impact of thermal stress on probiotics and improving the overall protective performance of the microcapsules.

[0049] 6. Storage stability of microcapsules: To evaluate the storage stability of probiotic microcapsules obtained by spray drying and freeze drying, they were placed in environments of 4℃ and 25℃ respectively, and samples were taken every 2 weeks for plate count.

[0050] The storage stability of microcapsules prepared by spray drying and freeze drying methods was compared for 8 weeks at 4℃ and 25℃, respectively. Figure 8 China A Figure 8 B, Figure 8 C and Figure 8As shown in Figure D, the viable bacterial count in all microcapsules decreased significantly with prolonged storage time (P<0.05). Specifically, after 8 weeks of storage at 4℃, the viable bacterial count of free probiotics decreased from an initial 9.95±0.04 log CFU / mL to 5.31±0.02 log CFU / mL, a decrease of approximately 4.64 log CFU / mL. At 25℃, the decrease was even more significant, reaching only 3.91±0.02 log CFU / mL after 8 weeks, a decrease of approximately 6.04 log CFU / mL. These results indicate that storage temperature significantly affects the survival rate of probiotics in microcapsules; the higher the temperature, the more pronounced the decrease in viable bacterial count. This may be because higher temperatures accelerate cell metabolism and affect cell membrane lipid structure, leading to impaired membrane function and thus weakening the protective effect of the microcapsules. The decrease in viable bacterial count was reduced to 1.84-3.70 log CFU / mL in the freeze-drying group, while it further decreased to 1.26-3.18 log CFU / mL in the spray-drying group. These results indicate that microcapsules prepared by spray drying exhibit superior storage stability. This may be due to the more uniform and dense microcapsule structure formed by spray drying, which more effectively blocks adverse environmental factors.

[0051] 7. Determination of microcapsule permeability tolerance: A freezing point osmoremeter was used for measurement. The instrument was calibrated at three points: 0 mOsm / kg, 300 mOsm / kg, and 900 mOsm / kg. 50 μL of sample was placed in a test tube, avoiding the introduction of air bubbles. The sample was measured three times. 6%, 12%, and 18% (w / v) NaCl solutions were prepared, and 1 g of microcapsules was dissolved in 9 mL of each concentration of NaCl solution. After incubation at 37℃ for 12 h, the cell viability was measured. *Lactobacillus paracasei* L21 bacterial powder and microcapsules with WPI as the wall material were used as controls.

[0052] Significant differences in osmotic pressure were observed between different groups of microcapsules (P<0.05). Figure 9 (A) Free probiotics exhibited the lowest osmotic pressure, while WPI microcapsules increased the osmotic pressure (OP) to 330 ± 5.80 mOsm / kg. For microcapsules encapsulated with MRPs, the osmotic pressure increased and peaked in the WPIC / 120 group. Studies have shown that the osmotic pressure of honey ranges from 2.06 to 3.03 MPa. Converted according to van der Hoff's law, its OP value is approximately 1087-1224 mOsm / kg. Generally, a solution with an OP value exceeding 300 mOsm / kg is considered a hypertonic environment. High osmotic pressure induces osmotic dehydration of bacterial cells, ultimately leading to cell death. In this invention, the OP value of the WPIC / 120 spray-dried microcapsules was 770 ± 5.80 mOsm / kg, significantly higher than that of free probiotics (P < 0.05). This suggests that these microcapsules may alleviate the hypertonic stress induced by honey.

[0053] Because spray-dried WPIC / 120 microcapsules exhibit high encapsulation efficiency, small particle size, and excellent in vitro gastrointestinal tolerance and thermal stability, their overall performance is optimal. Therefore, WPIC / 120 spray-dried microcapsules were selected for subsequent osmotic pressure testing, with spray-dried free probiotics and WPIC / 120 microcapsules used as controls. The prepared probiotic microcapsules were placed in hypertonic sodium chloride solutions of different concentrations, and their survival rate was measured. The results are as follows: Figure 9 As shown in Figure B, when the NaCl mass fraction was 6%, the survival rate of free *Lactobacillus paracasei* powder reached 23.46±1.74%, indicating that this strain has a certain tolerance to 6.0% NaCl solution. This tolerance may stem from the large amount of soluble and neutral substances synthesized by the bacteria to cope with osmotic stress, thus mitigating the adverse effects of high-salt environment to some extent. However, as the NaCl solution mass fraction increased to 18%, the survival rate of the strains in all formulations showed a decreasing trend. Among them, the survival rate of WPIC / 120 group modified by Maillard reaction was 64.44±3.47%, significantly higher than that of free probiotics (6.65±0.34%) and WPI encapsulation alone (17.54±0.53%) (P<0.05). This indicates that WPIC / 120 probiotic microcapsules have a certain tolerance to hyperosmotic environment and can effectively reduce the damage and death of bacteria caused by adverse environment during production.

[0054] Experiment Example 2 I. Study on the targeted delivery effect of honey products prepared by encapsulating probiotic microcapsules with whey protein isolate and pectin on relieving constipation in BALB / c mice.

[0055] 1. Preparation of the solution for gavage: Free bacterial suspension: Lactobacillus paracasei L21, which had undergone two activations, was inoculated at 2% (v / v) on MRS liquid medium and incubated at 37°C for 24 h. Subsequently, the bacterial cells were collected by centrifugation at 4°C and 8000×g for 5 min, washed twice with sterile PBS buffer, and finally resuspended in PBS, adjusting the concentration to 1.0×10⁻⁶. 9 CFU / mL. Microcapsule bacterial suspension: The microcapsules prepared in Example 1 were resuspended in sterile PBS, and the viable count was adjusted to 1.0 × 10⁻⁶. 9 CFU / mL. Probiotic honey: Live bacteria count is 1.0 × 10⁻⁶ when used directly. 9 Probiotic honey containing Lactobacillus paracasei L21 at CFU / mL.

[0056] 2. Animal experiments: Seven-week-old male BALB / c mice weighing 18-22g were selected. The room temperature was 25±2℃, the humidity was 50±5%, and a 12-hour light / dark cycle was maintained. Before all experiments, the mice were fed standard mouse feed, allowed free access to water, and fasted for 24 hours.

[0057] 3. Ink preparation: Add 10g of gum arabic to 80mL of distilled water, boil, add 5g of activated carbon powder, boil again, boil 3 times, cool to room temperature, add distilled water to the total volume of 100mL, and obtain the activated carbon solution for oral gavage of mice, and store at 4℃.

[0058] 4. Induction of Constipation and Experimental Design: Based on the evaluation criteria for the modeling method of animal experiments "helping to lubricate the intestines and promote bowel movements" in the "Methods for Inspection and Evaluation of Health Foods (2023 Edition)," mice were administered loperamide hydrochloride by gavage daily (24 hours). As shown in Table 2, mice were randomly divided into 8 groups (n=8 per group): control group (D), whey protein isolate-pectin group (W), model group (M), honey group (F), mosapride group (Y), free probiotic group (L21), encapsulated probiotic group (WC), and probiotic honey group (FL). The adaptation period was 7 days, the modeling period was 7 days, and the treatment period was 14 days. During the modeling period, except for the control group, all other groups were given 0.2 mL (10 mg / kg) of loperamide. During the treatment period, except for the control group, all other groups were first given 0.2 mL (10 mg / kg) of loperamide. One hour later, the model group was administered an equal volume of sterile saline by gavage, while the other groups were administered 0.2 mL of the corresponding substance solution by gavage. During the experiment, all mice were fed standard diets and had free access to water and food. The weight of the mice was recorded every week.

[0059] Table 2 Experimental Design for Animal Models

[0060] Feces were collected from mice in each group on day 1, day 8, and day 21 of modeling to determine fecal water content and fecal SCFA levels. After day 21, all mice were fasted and deprived of water for 12 hours, then anesthetized with an intraperitoneal injection of ketamine at 100 mg / kg bw. Blood was collected from the eyeballs and placed in 1.5 mL centrifuge tubes. The tubes were incubated at 4°C for at least 30 minutes, centrifuged (4°C, 3000×g, 10 minutes), and the supernatant was carefully aspirated to obtain serum, which was then aliquoted and stored at -80°C for later use. Colon tissue was collected for histological observation, real-time quantitative PCR, and microbiome analysis.

[0061] 5. Determination of mouse fecal moisture content: Mice in each group were placed individually in IVC cages lined with clean filter paper. Feces from each mouse were collected and placed into sterile EP tubes. The wet weight of the feces was recorded. After freeze-drying, the dry weight of the feces was recorded, and the fecal moisture content was calculated. The vacuum freeze-drying conditions were as follows: cooling to -40.0℃ (1.0℃ / min, 4h), heating to -30.0℃ (0.5℃ / min, 15h), heating to -10.0℃ (0.5℃ / min, 15h), heating to 10.0℃ (1.0℃ / min, 4h), and heating to 20.0℃ (1.0℃ / min, 4h). Fecal moisture content (%) = [(fecal wet weight (g) - fecal dry weight (g)] / fecal wet weight (g) × 100%.

[0062] according to Figure 10 As shown in Figure A, the weight change trend of all groups of mice showed a steady increase in weight during the loperamide gavage modeling period. After entering the treatment intervention phase (7-21 days), the weight of mice in group D increased from 21.55±0.86g to 26.60±1.42g, an increase of 22.04%; conversely, the weight of mice in group M decreased significantly, from 23.06±0.72g to 20.91±0.67g, a decrease of 9.32%. In contrast, the weight of mice in the other intervention groups increased to varying degrees, with the Y and FL groups showing the most significant increases, at 22.07% and 20.84% ​​respectively, indicating that these two intervention groups were most effective in maintaining stable weight in mice.

[0063] Constipation is often accompanied by dry, hard stools and abnormal water absorption. To evaluate the model and intervention effect, the fecal water content of mice in each group was measured. The results showed that ( Figure 10 In the B and D groups, the fecal water content was 71.08±0.57%, while in the M group it was only 38.19±1.25%, significantly lower than the other groups (P<0.05). This is consistent with the typical characteristics of slowed intestinal transit and enhanced water reabsorption in the loperamide-induced constipation model. In contrast, the fecal water content of all intervention groups was significantly improved. The water content of the FL and Y groups was 68.46±1.52% and 66.67±0.42%, respectively, with no significant difference from the D group (P>0.05). This further demonstrates that the FL group has a good effect on restoring intestinal water metabolism and relieving constipation symptoms.

[0064] 6. Determination of the first black stool in mice: Before the defecation experiment, mice were fasted for 16 hours but allowed free access to water to empty their intestines. On day 21 of the experiment, mice in each group were administered 0.20 mL of loperamide hydrochloride solution by gavage, while the control group was administered the same volume of sterile saline. 30 minutes later, the control and model groups were administered ink by gavage, while the remaining groups were administered ink solution containing the corresponding contents. The time of the first black stool in each mouse was observed and recorded, with the time of the first black stool in the last mouse of group M being the termination time.

[0065] Depend on Figure 11 It was found that there were significant differences in the time to first black feces among different groups of mice (P<0.05). Specifically, the time to first black feces in group M was 117±3.61 min, significantly longer than that in group D (50±2.65 min), confirming a significant intestinal transit delay in the constipation model mice. Compared with group M, the time to first black feces in each intervention group (Y, F, W, L21, WC, and FL groups) was shortened to varying degrees, specifically 54.67±3.01 min, 81.33±3.51 min, 86.67±4.73 min, 66.63±3.679 min, 59.33±3.21 min, and 48.67±4.04 min, respectively. Groups Y and FL showed the most significant improvement, with no significant difference in first black feces time compared to group D (P>0.05). Therefore, the results show that the FL group and the Y group can improve intestinal peristalsis function and shorten the time of first black stool in the constipation model.

[0066] 7. Small Intestinal Motility Test: Before the experiment, mice were fasted but allowed free access to water for 16 hours. On day 21, except for the control group which was administered sterile saline by gavage, all other groups were administered 0.20 mL of loperamide solution by gavage. 30 minutes later, the control and model groups were administered ink by gavage, while the other groups were administered ink solutions containing the corresponding contents. 30 minutes later, the mice were euthanized by cervical dislocation, the abdominal cavity was opened, the mesentery was separated, and the intestinal segment from the pylorus to the ileocecal junction was cut. The small intestine was gently pulled into a straight line, and the length of the segment was measured and recorded as the "total length of the small intestine." The reading at the front of the activated charcoal solution was the "activated charcoal propulsion length," and the small intestinal propulsion rate was calculated. Small intestinal propulsion rate (%) = activated charcoal propulsion length (cm) / total length of the small intestine (cm) × 100%.

[0067] By measuring small intestinal propulsion rate, the effects of different interventions on improving loperamide-induced intestinal motility dysfunction were systematically evaluated. For example... Figure 12As shown, there were significant differences in small intestinal propulsion rate among different groups of mice (P<0.05). Compared with group D, the small intestinal propulsion rate of mice in group M was significantly reduced (P<0.05). This result is consistent with the previously observed prolonged time to first-fecal blackening, jointly confirming the successful establishment of the loperamide-induced constipation model. Compared with group M, the small intestinal propulsion rate of each intervention group showed varying degrees of increase. Among them, the propulsion rate of groups Y and FL recovered most significantly, approaching the level of the control group, indicating that the intervention of these two groups had a significant promoting effect on small intestinal motility. In addition, the small intestinal propulsion rate of groups F, W, L21, and WL also showed significant improvement, but their effect was slightly less than that of groups Y and FL.

[0068] 8. Determination of SCFAs in mouse feces: After vacuum freeze-drying, 20 mg of mouse feces was taken, added to 500 µL of water and mixed. The mixture was vortexed and mixed, then 100 µL of 15% H2SO4 was added for acidification. Then, 20 µL of 4-methylvaleric acid solution (internal standard) and 280 µL of diethyl ether solution were added and homogenized for 1 min. The mixture was centrifuged at 18000 g for 15 min. The upper ether phase was dried with 250 mg of anhydrous Na2SO4 and centrifuged at 12000 g for 10 min. The supernatant was collected in a sample vial and analyzed by the instrument.

[0069] Analysis was performed using gas chromatography-mass spectrometry (GC-MS). GC conditions were as follows: Agilent DB-WAX capillary column (30 m × 0.25 mm, ID × 0.25 μm), injection volume 1 µL, injection port temperature 220 °C, helium carrier gas at a constant flow rate of 1.0 mL / min; temperature program: initial temperature 60 °C for 1 min, increased to 120 °C at 30 °C / min, then to 150 °C at 5 °C / min, and finally to 210 °C at 25 °C / min and held for 3 min. Mass spectrometry conditions were: electron ionization source, ion source temperature 230 °C, transfer line temperature 250 °C, electron energy 70 eV; selective ion monitoring (SIM) mode was used for detection.

[0070] SCFAs (Superficially Calcium Fiber Acids) are important metabolites produced by the fermentation of dietary fiber by the gut microbiota, mainly including acetic acid, propionic acid, and butyric acid, which play a crucial role in maintaining gut health and intestinal motility. Therefore, GC-MS was used to detect the levels of three SCFAs in the colonic contents of mice to evaluate the effects of different interventions on gut microbiota metabolic function. Figure 13 China A Figure 13 B, Figure 13 C and Figure 13As shown in Figure D, compared with group D, the contents of acetic acid, propionic acid, butyric acid, and total acid in group M were significantly decreased (P<0.05). Compared with group M, the contents of acetic acid, propionic acid, butyric acid, and total acid in groups Y, F, L21, WC, and FL were significantly increased (P<0.05); while the contents of acetic acid, propionic acid, and total acid in group W were significantly higher than those in group M (P<0.05), but the butyric acid content, although increased, did not differ significantly (P>0.05). Further analysis revealed that the acetic acid content in groups FL and Y recovered to similar levels as in group D (P>0.05), and the butyric acid content in group FL recovered to similar levels as in group D (P>0.05); in addition, groups FL and Y significantly increased the contents of propionic acid and total acid in the colonic contents of mice (P<0.05), but there were still some differences compared with group D (P<0.05).

[0071] 9. Determination of mouse fecal microbiota: 16S rDNA was extracted from feces using the Feces Fast DNA Spin Kit (MP Biomedical, Irvine, CA, USA) for sequencing and bioinformatics analysis, and used as a template for PCR amplification of the V3-V4 region of bacterial DNA. Primers 341F / 806R were used as forward and reverse primers, respectively. PCR products were purified using Gene CleanTurbo (MP Biomedical, Beijing, China) and quantified using the Quant-iT PicoGreen dsDNA assay kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. PCR samples were quantified and corrected to equimolar solutions, pooled to construct DNA libraries, and sequenced on the Illumina MiSeq platform according to the manufacturer's instructions.

[0072] (1) Changes at the phylum level of gut microbiota: Figure 14 As shown, the effects of different treatment groups on the phylum level of the mouse gut microbiota were analyzed. A cumulative bar chart of the relative abundance of the top 10 species at each phylum level was plotted. Figure 14 As shown, group D mice exhibited a relatively stable composition at the phylum level, with the dominant bacterial groups being Bacteroidetes (…). Bacteroidota 68.77%, Firmicutes ( Firmicutes 24.82%, Desulfobacteria ( Desulfobacterota 2.62%, Actinobacteria ( Actinobacteriota 1.78% and Proteobacteria ( ProteobacteriaThe percentage of mice with constipation induced by loperamide (Group M) was 0.46%. After establishing a constipation model, the composition of the intestinal flora in mice changed significantly. The relative abundance of Firmicutes increased to 47.75%, and Proteobacteria also increased to 5.67%. Conversely, the relative abundance of Bacteroidetes decreased to 41.03%, Actinobacteria decreased to 1.18%, and the relative abundance of Dethiobacteria was 2.48%, showing no significant difference from Group D. Compared with Group M, all intervention groups restored this intestinal disorder to some extent. Groups Y, WC, and FL showed the most significant effects, effectively inhibiting the increase in Firmicutes abundance induced by loperamide and restoring the relative abundance of Bacteroidetes to levels close to the normal levels of Group D. These results indicate that loperamide-induced constipation can lead to structural disorders at the phylum level of the intestinal flora in mice, and FL intervention can effectively alleviate this disorder and promote the return of intestinal flora composition to normal levels.

[0073] (2) Changes in gut microbiota genera: The effects of different treatment groups on the gut microbiota genera levels in mice are as follows: Figure 15 As shown in the figure, the analysis of the relative abundance of the top 50 genera in the mouse gut microbiota indicates that the microbiota composition of group M mice is significantly different from that of group D. Compared with group D, group M... Muribaculum genus, Prevotell genus, Parabacteroides genus, Blautia The abundance decreased. Lactobacillus genus, Lachnoclostridium genus, Lachnospiraceae _NK4A136_group、 Escherichia-Shigella The abundance of FL increases, and intervention can restore this disorder to some extent.

[0074] (3) Species difference analysis: To further explore the dominant species among different groups, LEfSe analysis was used to compare the different species among groups D, M and FL. The results are as follows: Figure 16 China A and Figure 16 As shown in Figure B, with LDA > 4.0 and P < 0.05, a total of 35 significantly different species were detected in the three treatment groups. Among them, group M showed the highest concentration of Clostridium (c_ Clostridia ), s__ Clostridium _ sp Bacillus (c__) Bacilli ), Gammaproteobacteria (c__) Gammaproteobacteria ), family Styropodaceae (f__ Lachnospiraceae ), g__ Lachnospiraceae _NK4A136_group、g__ Lachnoclostridium Lactobacillus family (f__) Lactobacillaceae Lactobacillus (g__) Lactobacillus ), s__ Lactobacillus_prophage, Enterobacteriaceae (f__) Enterobacteriaceae ), Escherichia coli-Shigella spp. (g__ Escherichia -Shigella), s__ Escherichia _coli, anaerobic columnar bacteria (g__) Anaerocolumna The dominant bacteria were Bacteroides (c__). In group FL, Bacteroides were the dominant class. Bacteroidia S24-7 family (f__) Muribaculaceae ), g__ Muribaculum , s__ Muribaculum _intestinale、g__ Blautia , s__ Lachnospiraceae _bacterium_ and other bacteria were the dominant species. In group D, f__ Prevotellaceae g__ Prevotella , s__ Prevotella _sp, Tanneraceae (f__) Tannerellaceae ), Parabacterium spp. (g__) Parabacteroides ) and others are the dominant bacteria.

[0075] 10. Histopathological examination: Mouse colon tissue was fixed in 4% paraformaldehyde solution for more than 24 hours, then subjected to gradient dehydration and clearing, and embedded in paraffin. After preparing 4µm thick sections, hematoxylin-eosin (HE) staining was performed, and the pathological changes of the colon tissue were observed and recorded under an optical microscope, and photographs were taken and preserved.

[0076] Hematoxylin and eosin (H&E) staining allows for direct assessment of colonic mucosal morphology and structure, enabling the exploration of the repairing effect of FL on intestinal pathological damage. In constipation, the gastrointestinal tract often exhibits significant inflammatory cell infiltration, accompanied by histological changes such as villous atrophy and crypt structure destruction. Figure 17 China A~ Figure 17 As shown in Figure H, histopathological sections of mouse colon tissue revealed significant differences in colonic structure among the groups (200×). In Group D, the colonic structure was essentially normal, with neatly arranged mucosal epithelial cells, intact crypts, no submucosal edema, and no obvious inflammatory cell infiltration. In contrast, Group M showed moderate colonic structural abnormalities, characterized by swollen lymph nodes, reduced crypt numbers, and extensive neutrophil infiltration and cell necrosis (red arrows). In the intervention groups, Groups F, W, and L21 showed mild colonic structural abnormalities, with largely intact mucosal and crypt structures, no submucosal edema, but a small amount of inflammatory cell infiltration (red arrows). Groups Y, WC, and FL showed largely normal colonic structure, similar to Group D, with no significant pathological changes. Overall, Group M showed the most significant pathological damage; compared to Group M, the damage in Groups F, W, and L21 was reduced; among them, Groups WC, FL, and Y showed the most significant improvement, with the tissue structure essentially returning to normal.

[0077] 11. Measurement of mouse serum: The levels of relevant gastrointestinal regulatory peptides (SP, MTL, Ach, 5-HT) in mouse serum were measured using an ELISA kit (purchased from Beijing Andy Huatai Technology Co., Ltd.). The operation procedure was performed according to the kit instructions.

[0078] Among the gastrointestinal regulatory peptides closely related to constipation, excitatory regulatory substances mainly include motilin (MTL), acetylcholine (Ach), substance P (SP), and serotonin (5-HT), which play a synergistic role in maintaining normal intestinal motility. Figure 18 China A Figure 18 B, Figure 18 C and Figure 18 As shown in Figure D, compared with group D, the serum levels of 5-HT, Ach, SP, and MTL in group M mice were significantly decreased (P<0.05), indicating that intestinal motility was significantly inhibited under constipation. After different interventions, each treatment group showed varying degrees of improvement in serum levels of gastrointestinal regulatory peptides. Specifically, compared with group M, groups Y, F, W, L21, WC, and FL significantly increased the expression levels of 5-HT, Ach, SP, and MTL (P<0.05); among them, groups Y and FL showed the most significant recovery effects, especially SP and MTL levels, which recovered to levels similar to those in group D (P>0.05); the other intervention groups (group F, W, L21, and WC) also showed significant upregulation effects. These results indicate that loperamide-induced constipation can significantly reduce the serum levels of gastrointestinal regulatory peptides in mice, while interventions with FL and Y can significantly promote their expression recovery, thereby improving intestinal motility.

[0079] 12. Real-time quantitative PCR: Total RNA was extracted from colon tissue using a total RNA extraction kit (Bioss Antibodies). 500 ng of total RNA was reverse transcribed into cDNA using SuperScript II reverse transcriptase. The cDNA was then used to prepare the qPCR reaction system according to the instructions of the ChamQ Universal SYBR qPCR Master Mix kit. Primers were synthesized by Jilin Kumei Biotechnology Co., Ltd.

[0080] The results are as follows Figure 19 China A~ Figure 19 As shown in Figure H, compared to group D, group M... 5HT 4 R , 5HT 3 R , TPH1 and GPR43 Gene expression levels were significantly reduced (P<0.05), while SERT , AQP 4 , AQP 8 and MAOA Gene expression levels were significantly increased (P<0.05); compared with group M, each intervention group showed varying degrees of improvement: in increasing... 5-HT 4 R Regarding expression, the levels were significantly increased in groups Y, F, W, L21, WC, and FL (P<0.05); groups Y, WC, and FL showed significant improvement. 5- HT 3 R , TPH1 and GPR43 The expression level was [high] (P<0.05); while downregulation [was observed]. SERT , AQP 4 and AQP 8 In terms of expression, groups Y, F, W, L21, WC, and FL all showed significant effects (P<0.05); Regarding... MAOA Gene expression was significantly reduced in groups Y, L21, WC, and FL (P<0.05), while groups F and W showed no significant effect (P>0.05). Overall, groups Y and FL showed the most significant improvement.

[0081] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing whey protein isolate-pectin encapsulated probiotic microcapsules, characterized in that, Includes the following steps: 1) After dissolving pectin in a water bath, mix it with whey protein isolate and hydrate it to obtain a whey protein isolate-pectin mixture; 2) Adjust the pH of the whey protein isolate-pectin mixture obtained in step 1) to 8.8~9.2, and heat in a water bath to obtain the whey protein isolate-pectin complex; 3) Lactobacillus paracasei ( Lacticaseibacillus paracasei L21 was inoculated into the culture medium, cultured, centrifuged to collect the precipitate, and resuspended in the whey protein isolate-pectin complex obtained in step 2). The mixture was then spray-dried to obtain whey protein isolate-pectin encapsulated probiotic microcapsules.

2. The preparation method according to claim 1, characterized in that, The water bath dissolution temperature in step 1) is 35~45℃.

3. The preparation method according to claim 1, characterized in that, The mass ratio of pectin to whey protein isolate in step 1) is 1:1 to 4.

4. The preparation method according to claim 1, characterized in that, The hydration temperature in step 1) is 4°C, and the hydration time is 10~14h.

5. The preparation method according to claim 1, characterized in that, The water bath heating temperature in step 2) is 70~90℃, and the water bath heating time is 90~150min.

6. The preparation method according to claim 1, characterized in that, The inoculation amount in step 3) is 1.5~2.5% (v / v), the culture temperature in step 3) is 37℃, the culture time is 20~28h, the centrifugation temperature in step 3) is 4℃, the centrifugation speed is 7000~9000g, and the centrifugation time is 3~7min.

7. The preparation method according to claim 1, characterized in that, In step 3), the inlet temperature of the spray dryer is 110~130℃, the outlet temperature is 60~80℃, the feed rate is 400~600mL / h, and the air flow rate is 4~6m³ / h. 3 / h.

8. The whey protein isolate-pectin-encapsulated probiotic microcapsules prepared by any one of claims 1 to 7.

9. The application of the whey protein isolate-pectin encapsulated probiotic microcapsules as described in claim 8 in the preparation of honey products for relieving constipation.

10. The application according to claim 9, characterized in that, Specifically, whey protein isolate-pectin encapsulated probiotic microcapsules are added to honey and treated in a water bath at 25~35℃ for 10~30 minutes to obtain a honey product that relieves constipation.

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