Preparation method and application of single-cell probiotic coating

Abstract

According to the preparation method and application of the single-cell probiotic coating disclosed by the invention, the coating is used for encapsulating lactobacillus plantarum YJ7 layer by layer by using a metal-phenol network and a chitosan-casein phosphopeptide compound, so that LP-coated TP / Fe < 3 + >-coated CPC is successfully constructed. The functional coating can effectively resist invasion of severe physiological environments of gastrointestinal tracts in vivo and in vitro under the condition that normal activity of LP is not affected, and the survival rate of the functional coating in intestinal tracts is greatly improved. A mouse in-vivo safety research shows that the hydrogel has excellent biocompatibility and harmlessness. In addition, gavage experiments on mice with type 2 diabetes prove that the delivery system enhances the hypoglycemic activity of the lactobacillus plantarum YJ7. In conclusion, the oral probiotic delivery system provides a simple and efficient strategy for enhancing the survival of the probiotics in the severe intestinal environment, and is expected to provide a new view angle for beneficial effects of the probiotics in the intestinal tract.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial medicine, and specifically relates to a preparation method and application of a single-cell probiotic coating. Background Art

[0002] Lactobacillus plantarum is a type of beneficial microorganism that usually survives in the intestines to exert a variety of beneficial effects. However, oral probiotics must pass through a complex digestive tract environment, including digestive enzyme degradation, gastric acid attack, bile salt inhibition, and intestinal peristalsis, in order to exert these health effects. Previous studies by our research group found that Lactobacillus plantarum YJ7 has a significant effect on improving intestinal flora disorders and inhibiting systemic inflammation. Therefore, improving the survival rate and intestinal colonization ability of oral YJ7 is the first factor in its health effects.

[0003] In recent years, many studies have shown that probiotic delivery vehicles can effectively protect probiotics from physical and biological stresses, allowing them to maintain their biological functions in harsh environments. Traditional encapsulation techniques include spray drying, extrusion, freeze drying, gelation, and emulsification. However, each method has certain defects, such as lack of control over particle size, cumbersome preparation methods, and low in vivo release efficiency. Single-cell encapsulation technology overcomes some of the challenges faced by traditional encapsulation technology, enhances the resistance of probiotics to external environmental conditions, and improves the adhesion of probiotics.

[0004] Recently, the superior coating ability of metal-phenol networks (MPNs) on different substrates has been revealed. MPNs form stable coatings on various interfaces through the coordination of natural polyphenols and metal ions, protecting living cells from environmental stresses such as UV irradiation and freeze-drying. However, metal-phenol networks are rarely used for oral encapsulation of probiotics, probably because the protonation of hydroxyl groups in the acidic gastric environment can cause the MPN structure to collapse. This reduces the stability of MPN-encapsulated probiotics. Therefore, the present application utilizes the unique encapsulation ability of MPNs for the initial encapsulation of probiotics, and then MPNs directly adsorb chitosan-casein phosphopeptide composite layers through hydrogen bonding and electrostatic interactions to achieve efficient layer-by-layer encapsulation of probiotic single cells. Summary of the invention

[0005] In view of the problems in the prior art, the purpose of the present invention is to provide a preparation method and application of a single-cell probiotic coating to solve the shortcomings of low gastrointestinal survival efficiency, poor stability, and insufficient targeting of probiotics, and enhance the health effects of probiotics on the body, which is specifically achieved through the following technical solutions: A method for preparing a single-cell probiotic coating comprises the following steps: 1) Slowly add the metal ion solution to the probiotic solution, stir magnetically to mix, then add the polyphenol solution and stir to react. After the reaction is completed, wash twice with sterile saline to remove free iron ions and polymerized tea polyphenol solution to obtain metal-polyphenol network-coated probiotics; 2) Add chitosan-casein phosphopeptide complex solution to the metal-polyphenol network-coated probiotics, and react with magnetic stirring at room temperature. After the reaction, wash twice with sterile saline to remove the free chitosan-casein phosphopeptide complex solution to obtain LP@TP / Fe 3+ @CPC.

[0006] Furthermore, the probiotic is Lactobacillus plantarum YJ7.

[0007] Before use, Lactobacillus plantarum YJ7 was cultured overnight in MRS medium (37°C). The culture was collected and centrifuged at 5000 rpm for 5 min at 4°C, washed twice with sterile saline, and suspended in sterile PBS.

[0008] Furthermore, in step 1), the metal ion solution is a FeCl3•6H2O solution with a concentration of 1 mg / mL.

[0009] Furthermore, the polyphenol solution in step 1) is a polymerized tea polyphenol solution with a concentration of 1 mg / mL.

[0010] Furthermore, in step 1), the volume ratio of the probiotic solution, the metal ion solution and the polyphenol solution is 1:1:3.

[0011] Furthermore, in step 1), the tea polyphenols need to be stirred and reacted at 60° C. for 3 hours before being added, and then reacted for another 30 minutes after being added.

[0012] Furthermore, in step 2), the amount of chitosan-casein phosphopeptide composite solution added is 2 mg / mL, and the stirring reaction time is 30 min.

[0013] LP@TP / Fe prepared by the above preparation method 3+ @CPC.

[0014] Furthermore, the LP@TP / Fe 3+ @Application of CPC in the preparation of drugs for lowering blood sugar.

[0015] The single-cell probiotic coating prepared by the preparation method has the advantages of good stability, high survival rate of probiotics, good environmental resistance and antibiotic resistance, and TP / Fe 3+ @CPC coating can significantly improve the gastrointestinal survival rate of probiotics in vivo, has good biosafety in vivo, and can enhance the functional activity of probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The effect of different polyphenol materials on the growth rate of LP; Figure 2 The protection of LP by different polyphenol materials; Figure 3 The effect of pH on the surface potential of the encapsulation material; Figure 4 The hydrated particle size and Zeta potential of LP and encapsulated LP are shown; Figure 5 SEM images of LP and encapsulated LP (scale bar: 1 µm); Figure 6 Transmission electron microscopy observations of LP and encapsulated LP (scale bar: 1 µm); Figure 7 Laser confocal scanning electron microscopy images of LP and encapsulated LP (scale bar: 1 µm); Figure 8 Flow cytometry images of LP and encapsulated LP; Fig. 9 It is the bacterial growth curve of LP and encapsulated LP; Fig.10 It is the bacterial viability graph of LP and encapsulated LP; Fig.11 LP@TP / Fe 3+ @CPC's in vitro resistance to gastrointestinal digestive environment (A: simulated gastric juice digestion; B: bile salt digestion); Fig.12 is the effect of temperature on the number of surviving LP and encapsulated LP; Fig.13 is the effect of antibiotics on the number of surviving LPs and encapsulated LPs; Fig.14 The effect of H2O2 on the survival number of LP and encapsulated LP; Fig.15 is the number of surviving LP and encapsulated LP in the stomach 4 h after oral administration; Fig.16 The survival numbers of LP and encapsulated LP in the intestine after oral administration (A: 4h; B: 48h; C: 96h); Fig.17 H&E staining images of mouse heart, liver, spleen, lung, and kidney (scale bar: 100 µm); Fig.18 The blood routine indicators of mice in different treatment groups (A: red blood cell number; B: mean corpuscular volume; C: hemoglobin; D: platelet number); Fig.19 LP@TP / Fe 3+@Effect of CPC on insulin resistance in T2DM mice (A: fasting blood glucose in serum; B: insulin resistance evaluation index). DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with specific embodiments to facilitate a better understanding of the present technical solution.

[0018] Example 1: Functional single cell coating probiotics LP@TP / Fe 3+ Preparation of @CPC

[0019] In all growth steps, a single LP colony was inoculated into MRS broth and grown overnight at 37 °C. Then 1 mL of the overnight culture was transferred to fresh MRS broth and incubated at 37 °C for 20 h. The culture of Lactiplantibacillus plantarum YJ7 (LP) was maintained in MRS broth containing 50% glycerol.

[0020] 1) Selection of polyphenol materials Lactobacillus plantarum YJ7 was diluted to OD600 of 0.05 in MRS, and tannic acid, proanthocyanidins or tea polyphenols were added at a final concentration of 1 mg / mL, inoculated in a 96-well plate, and cultured at 37 °C. The OD600 value was monitored every 1 h for 12 hours using a microplate reader to preliminarily evaluate whether tannic acid, proanthocyanidins or tea polyphenols had an inhibitory effect on bacterial growth.

[0021] In addition, three polyphenol compounds were explored as encapsulation coatings for probiotics to evaluate their biocompatibility. The specific operation was as follows: Lactobacillus plantarum YJ7 was cultured in MRS medium (37°C) for 20 h. 1 mL of culture was collected and centrifuged at 5000 rpm for 5 min at 4°C, washed twice with sterile saline, and then 1 mL of sterile PBS was added. Then, 1 mL of FeCl3•6H2O (1 mg / mL) was slowly added under magnetic stirring, and after mixing, 3 mL of tannic acid (1 mg / mL), proanthocyanidins (1 mg / mL) or polymerized tea polyphenol solution (1 mg / mL, tea polyphenol solution was stirred at 60°C for 3h) was added and reacted for 30 min. Then, it was washed twice in sterile saline to remove free metal ions and polyphenol solution to obtain LP@TP / Fe 3+ LP@PC / Fe 3+ and LP@TA / Fe 3+ . Equal amounts of LP, LP@TP / Fe 3+ LP@PC / Fe 3+ and LP@TA / Fe 3+(1×108 CFU) add 1 mL of SGF again, incubate for 1 h, centrifuge (5000 rpm, 5 min), discard the supernatant, add 1 mL of simulated bile salt (0.3 mg / mL) again, continue incubation for 1 h, take out 100 µL of the solution, wash with sterile saline, and evenly spread on a solid MRS agar plate, and culture at 37 ° C for 48 h before counting the bacteria.

[0022] The results are as follows Figure 1 As shown in the figure, the bacterial growth curves of LP, LP+PC and LP+TP have similar trends, and there is no significant difference in relative growth, which proves that proanthocyanidins or tea polyphenols have no significant effect on the growth of LP. However, there are significant differences in the bacterial growth curves of LP and LP+TA, indicating that tannic acid has a significant antibacterial effect on Lactobacillus plantarum.

[0023] Then LP, LP@TP / Fe 3+ LP@PC / Fe 3+ and LP@TA / Fe 3+ The survival of probiotics was assessed by incubation in simulated gastric fluid and bile salts and plate counts at designated time points. Figure 2 As shown, LP@TP / Fe 3+ LP@PC / Fe 3+ There were still a large number of surviving bacteria in the LP group, and the TP / Fe 3+ The coating has a significant protective effect on LP. 3+ The above results show that TP / Fe 3+ The coating has no effect on the cell viability and proliferation of the probiotics themselves, and plays a certain protective role. 3+ The coating may provide better survival benefits to LP.

[0024] 2) Investigation of the optimal reaction pH LP@TP / Fe with pH between 3 and 6 were prepared respectively. 3+ , CS, CPP and CPC solutions, and the Zeta potential of each sample at different pH was measured using a Malvern particle size analyzer. 3+ , CS, CPP and CPC sample solutions were exchanged for Zeta potential test dishes for Zeta potential determination, and the test was repeated 3 times.

[0025] Depend on Figure 3It can be seen that as the pH of the solution system continues to increase, the negative charge of CPP gradually increases, while the positive charge of CS gradually decreases. When the pH is around 6, CS almost reaches the isoelectric point, and the CPC complex mainly aggregates with each other by electrostatic interaction. Therefore, it is not conducive to the formation of CPC complex in a relatively alkaline environment. 3+ The CPC complex has a stable negative charge at about -20 mV, while the CPC complex has a positive charge at pH 3-5.5, which is beneficial to the LP@TP / Fe 3+ CPC composite coating is adsorbed on the surface. In addition, considering that low pH will affect the normal activity of probiotics, the optimal reaction pH is selected as 5.

[0026] LP@TP / Fe 3+ Preparation of @CPC Lactobacillus plantarum YJ7 was cultured in MRS medium (37 °C) overnight. 1 mL of culture was collected and centrifuged at 5000 rpm for 5 min at 4 °C, washed twice with sterile saline, and suspended in 1 mL of sterile PBS. Then, 1 mL of FeCl3•6H2O solution (1 mg / mL) was slowly added under magnetic stirring, mixed, and then 3 mL of polymerized tea polyphenol solution (1 mg / mL, tea polyphenol solution was stirred at 60 °C for 3 h) was added and reacted for 30 min. Then, it was washed twice with sterile saline to remove free iron ions and polymerized tea polyphenol solution to obtain LP@TP / Fe 3+ Then the chitosan-casein phosphopeptide complex solution (2 mg / mL) prepared in advance was added to the LP@TP / Fe 3+ The reaction was stirred under magnetic stirring at room temperature for 30 min. Finally, the free chitosan-casein phosphopeptide complex solution was removed by washing twice with sterile saline to obtain LP@TP / Fe 3+ @CPC.

[0027] Example 2: LP@TP / Fe 3+ @CPC Characterization 1) Particle size and Zeta potential analysis The average particle size and Zeta potential of probiotics were measured using a Malvern particle size analyzer. 3+ @CPC、LP@TP / Fe 3+ The particle size distribution was determined with the LP sample solution, and the Zeta potential was measured using a special Zeta potential test dish. The test was repeated 3 times.

[0028] In order to verify the TP / Fe 3+The composite of LP and CPC was successfully coated on the surface of LP. The particle size and Zeta potential of LP and encapsulated LP were first measured by Malvern particle size analyzer. Figure 4 As shown, the particle size of LP is about 1504.67±10.50nm, and the particle size of LP@TP / Fe 3+ and LP@TP / Fe 3+ The particle sizes of LP@TP / Fe@CPC are approximately 1614.33±29.15 nm and 1895.33±71.34 nm, respectively. 3+ and LP@TP / Fe 3+ @CPC increased in size by 110 nm and 391 nm, respectively. 3+ and LP@TP / Fe 3+ After @CPC encapsulation, the Zeta potential also decreased from -15.33±0.44 mV to -20.06±0.54 mV and -17.89±0.43 mV. 3+ The decrease in the zeta potential indicates the presence of TP / Fe 3+ layer. 3+ Compared with the LP@TP / Fe 3+ The increase in the zeta potential of @CPC is due to the positive charge of CPC complex, which proves that CPC is successfully encapsulated in LP@TP / Fe 3+ surface layer.

[0029] 2) SEM morphology observation LP@TP / Fe 3+ @CPC、LP@TP / Fe 3+ The LP was fixed in 2.5% glutaraldehyde overnight, washed with ultrapure water, and the fixed bacteria were resuspended in water to form a dilute dispersion. 10 μL of the sample dispersion was dropped onto a glass slide, and then the sample was dehydrated in a critical point dryer, sprayed with gold, and finally observed by scanning electron microscopy.

[0030] In order to visually observe the presence of the coating, the present application used SEM to observe the microscopic morphology of the encapsulated probiotics. Figure 5 As shown, compared with the smooth surface of LP, LP@TP / Fe 3+ and LP@TP / Fe 3+ The surface of @CPC becomes wrinkled obviously, while LP@TP / Fe 3+ The wrinkling degree of @CPC is more obvious, which indicates that the coating material is successfully encapsulated on the LP surface.

[0031] 3) TEM morphology observation Take an appropriate concentration of LP@TP / Fe 3+ @CPC、LP@TP / Fe 3+ After diluting with LP sample solution, a drop was drawn onto a copper grid dedicated to transmission electron microscopy samples using a capillary, irradiated with infrared light for 5 min until dry, and then observed under a transmission electron microscope (accelerating voltage of 120 kV).

[0032] The microstructure test results of LP and encapsulated LP by TEM are as follows: Figure 6 The outer layer modification of the encapsulated probiotics can be clearly seen, which is in sharp contrast to the smooth edge of the unencapsulated LP, proving that LP@TP / Fe 3+ and LP@TP / Fe 3+ @CPC was prepared successfully.

[0033] 4) CLSM verification of coating encapsulation In order to directly observe the modification of the coating on the surface of probiotics, chitosan was labeled with FITC and tea polyphenols were labeled with rhodamine B. 3+ @CPC and LP@TP / Fe 3+ 10 μL of sample suspension was dropped onto a glass slide, and then the fluorescence labeling was directly observed by CLSM.

[0034] After selective staining with fluorescent agents (rhodamine B for tea polyphenols and FITC for chitosan), LP@TP / Fe 3+ and LP@TP / Fe 3+ @CPC, using CLSM to visually observe the modification of the fluorescent coating on the surface of probiotics. Figure 7 As shown, in LP@TP / Fe 3+ In the group, the red fluorescence position of rhodamine B coincides with LP, indicating that LP@TP / Fe 3+ Tea polyphenols were successfully encapsulated. 3+ In the @CPC group, the red fluorescence of rhodamine B and the green fluorescence of FITC also overlap with LP, indicating that LP@TP / Fe 3+ @CPC successfully encapsulated tea polyphenols and chitosan-casein phosphopeptide complex.

[0035] 5) Flow cytometry analysis In order to further directly observe the encapsulation of the coating on the surface of probiotics, chitosan was labeled with FITC and tea polyphenols were labeled with rhodamine B. 3+ @CPC、LP@TP / Fe 3+ and LP, and finally the samples were resuspended in sterile PBS, and then the fluorescent labeling was detected by flow cytometry.

[0036] After selective staining with fluorescent agents (rhodamine B for tea polyphenols and FITC for chitosan), LP@TP / Fe 3+ and LP@TP / Fe 3+ @CPC, coating modification was analyzed by flow cytometry. Figure 8 As shown, compared with the LP group, LP@TP / Fe 3+ The red fluorescence intensity of rhodamine B and the green fluorescence intensity of FITC in the @CPC group were significantly increased. 3+ @CPC was successfully modified with tea polyphenols and chitosan-casein phosphopeptide complex.

[0037] Example 3: LP@TP / Fe 3+ @CPC growth curve investigation and bacterial activity determination 1) LP@TP / Fe 3+ @CPC growth curve investigation According to the method of the present invention, LP@TP / Fe 3+ @CPC、LP@TP / Fe 3+ and LP, LP@TP / Fe 3+ @CPC、LP@TP / Fe 3+ and LP were diluted in MRS to an OD600 of 0.05, inoculated in a 96-well plate, and cultured at 37 ° C. The OD600 value was monitored every 1 h for 12 h using a microplate reader to preliminarily evaluate whether the coating material had an inhibitory effect on bacterial growth after encapsulation.

[0038] The bacterial growth curve was used to investigate the LP@TP / Fe 3+ @CPC、LP@TP / Fe 3+ And the growth and reproduction of LP in vitro. Fig. 9 The results show that LP@TP / Fe 3+ @CPC、LP@TP / Fe 3+ The growth rate in 37°C MRS liquid medium was similar to that of unencapsulated LP, indicating that the coating material had no inhibitory effect on the activity of LP.

[0039] 2) LP@TP / Fe 3+ @CPC bacterial viability assay Cell Counting Kit-8 (CCK-8) is widely used in the determination of cell proliferation. 3+ @CPC、LP@TP / Fe 3+ and LP, LP@TP / Fe 3+@CPC、LP@TP / Fe 3+ and LP were diluted in MRS to an OD600 of 0.5. 100 μL of LP@TP / Fe 3+ @CPC、LP@TP / Fe 3+ and LP were inoculated in 96-well plates, and 10 μL of CCK-8 solution was added to each well, and the cells were cultured at 37 °C. The OD value (450 nm) was monitored every 1 h using an ELISA reader to monitor the relative bacterial activity.

[0040] After successful encapsulation of the coating, in order to determine whether the coating affects the growth and activity of LP, the cell counting kit (CCK-8) experiment was used to study the effect of the coating on LP viability. Fig.10 The results show that LP@TP / Fe 3+ @CPC、LP@TP / Fe 3+ The bacterial activity of the LP group was similar, indicating that the coating material TP / Fe 3+ @CPC has no effect on the activity of LP.

[0041] Example 4: LP@TP / Fe 3+ @CPC's tolerance to simulated in vitro harsh environment 1) LP@TP / Fe 3+ Effect of @CPC on resistance to simulated in vitro gastrointestinal digestive environment According to the present invention, LP@TP / Fe 3+ @CPC and LP@TP / Fe 3+ . Equal amounts of LP@TP / Fe 3+ @CPC、LP@TP / Fe 3+ 1 mL of simulated gastric fluid (SGF) or simulated bile salt (0.3 mg / mL) was added to LP (1×108 CFU), mixed and cultured in a 37°C incubator. 100 µL of each sample was taken at the agreed time, washed twice with sterile saline, and then diluted by gradient dilution. Finally, the bacterial suspension was spread on MRS agar plates and the number of live bacteria was counted after incubation at 37°C for 48 h.

[0042] LP@TP / Fe 3+ @CPC conducted a culture experiment in SGF or simulated bile salt environment, using the plate colony counting method to reveal the survival status of its probiotics at various time points. The results are as follows Fig.11 As shown in A, after 1 h of SGF treatment, LP@TP / Fe 3+ The survival rate of the @CPC group was about 1.5 times that of the LP group. 3+ The LP@TP / Fe3+ There are still about 105CFU in the CPC group, which indicates that TP / Fe 3+ @CPC coating has a significant protective effect. Fig.11 The results of B in the figure showed that after 2 h of simulated bile salt treatment, a large number of LP group died, with a survival number of about 105 CFU, while LP@TP / Fe 3+ The @CPC group still had a large number of survivors, about 107 CFU, and after 6 hours, the number of survivors was still above 104 CFU, proving that LP@TP / Fe 3+ @CPC also significantly enhanced its resistance to bile salt damage. The above results show that TP / Fe 3+ @CPC coating improves LP's ability to resist the harsh environment of the gastrointestinal tract.

[0043] 2) LP@TP / Fe 3+ @CPC Storage Stability Analysis Equal amounts of LP@TP / Fe 3+ @CPC、LP@TP / Fe 3+ The storage stability of LP and LP at -20, 4 and 25 ℃ was studied. After one week, 100 μL of the solution was taken out and washed twice with sterile saline. The bacterial suspension was spread on MRS agar plates and incubated at 37 ℃ for 48 h before counting the number of live bacteria.

[0044] Considering the actual application of probiotics and the importance of temperature on the storage stability of probiotics, the LP@TP / Fe 3+ @CPC survival after storage at different temperatures (i.e. -20, 4 and 25 °C) (simulating shipping time, 7 days). Fig.12 As shown in Figure 2, at storage temperatures of 4°C and -20°C, LP@TP / Fe 3+ The survival rate of @CPC was higher than that of naked LP, with no significant difference at 25 °C. Its good environmental stress resistance greatly solved the transportation limitation of probiotics and correspondingly expanded the application scope of probiotics.

[0045] 3) LP@TP / Fe 3+ @CPC Antibiotic Resistance Analysis Equal amounts of LP@TP / Fe 3+ @CPC、LP@TP / Fe 3+ 1 mL of cephalexin solution (2 mg / mL) was added to LP (1×108 CFU) and cultured in a 37°C incubator. 100 µL of the solution was taken out at the agreed time, washed twice with sterile saline, and the bacterial suspension was spread on MRS agar plates. The number of live bacteria was counted after incubation at 37°C for 48 h.

[0046] Considering the inhibitory effect of oral antibiotics on gastrointestinal probiotics, LP@TP / Fe 3+ @CPC survival rate at different time points under antibiotic environment. Fig.13 As shown, LP@TP / Fe 3+ The resistance of LP to cephalexin was slightly improved, while LP@TP / Fe 3+ The bacterial survival level of @CPC was significantly improved. After 6 h of action, the number of survivors was about 100 times that of LP. This shows that LP@TP / Fe 3+ @CPC provides special protection for oral probiotics, preventing them from nonspecific killing by antibiotics.

[0047] 4) LP@TP / Fe 3+ @CPC resistance to H2O2 analysis Equal amounts of LP@TP / Fe 3+ @CPC、LP@TP / Fe 3+ 1 mL of H2O2 (500 µM) was added to LP (1×108 CFU) and cultured in a 37°C incubator. 100 µL of the solution was taken out at the appointed time, washed twice with sterile saline, and the bacterial suspension was spread on MRS agar plates. The number of live bacteria was counted after incubation at 37°C for 48 h.

[0048] Since tea polyphenols and casein phosphopeptides have antioxidant capacity, the LP@TP / Fe 3+ @CPC and LP@TP / Fe 3+ Resistance to H2O2. Fig.14 As shown in the figure, most of the cells in the LP group died after 1 h of incubation when 1 mL H2O2 (500 µM) was added. At 2 h of incubation, almost all of the cells in the LP group died, while the LP@TP / Fe 3+ @CPC group still had 103 CFU alive.

[0049] Example 5: LP@TP / Fe 3+ @CPC survival rate in healthy mice 1) Experimental animal feeding and grouping Male C57BL / 6 mice aged 6 to 8 weeks were placed in an animal room and fed with a normal diet for 7 days to adapt to the experimental environment. After the adaptation period, the mice were randomly divided into 3 groups, and the oral gavage dose was set at 108 CFU / mouse / day. Group 1: using normal feed and gavage of LP; Group 2: using normal feed and gavage of LP@TP / Fe 3+ ; Group 3: using normal feed and gavage of LP@TP / Fe 3+@CPC. The mice were housed in nine cages, with five mice in each cage. The temperature was controlled at 24 °C and the air humidity was controlled at 50%. This experiment was approved by the Ethics Committee of Zhejiang University of Technology, number MGS20240530060.

[0050] 2) Sample collection and processing The mice in the three groups were killed by cervical dislocation at the predetermined time points (4 h, 48 h, and 96 h) after oral gavage. The stomach, small intestine, colon, and cecum tissues of the mice were collected and placed in 1 mL of sterile PBS, and the tissues were fully ground using a homogenizer at 4°C. 100 μL of the tissue suspension was taken out and then diluted by gradient dilution. Finally, the bacterial suspension was spread on MRS agar plates containing kanamycin sulfate and the number of live bacteria was counted after incubation at 37°C for 48 h.

[0051] 3) Oral administration of LP@TP / Fe 3+ Intestinal survival rate of CPC In order to determine the LP@TP / Fe 3+ @CPC can improve the survival of LP in vivo. The survival number of unencapsulated and encapsulated LP in the gastrointestinal tract (stomach, intestine, cecum and colon) was detected at different time points after oral administration of 108 CFU samples. Fig.15 As shown, LP@TP / Fe 3+ The colony counts in the stomach of the @CPC group were significantly higher than those of the LP group and the LP@TP / Fe 3+ group, compared with LP group and LP@TP / Fe 3+ The levels of leukemia in the control group were about 12 and 11 times higher, respectively. Fig.16 It showed that LP@TP / Fe 3+ @CPC survived in large numbers in the small intestine, colon, and cecum tissues, and was significantly higher in the small intestine than the LP group and LP@TP / Fe 3+ The levels of LP@TP / Fe in the colon were 13 and 20 times higher than those in the LP group and LP@TP / Fe 3+ The cecum was about 7 times higher than that of the LP group and LP@TP / Fe 3+ group were about 20 times and 8 times higher, respectively. In addition, even when the time was increased to 48 and 96 h, the 3+ Compared with the LP@TP / Fe 3+ Similar results were obtained in the @CPC group in the intestine, colon and cecum of mice, with significant increases in number. 3 + The stability and survival of CPC in the overall gastrointestinal environment were significantly enhanced. These findings strongly confirm that coating encapsulation has a significant effect on improving the survival of LP in the mouse digestive tract.

[0052] Example 6: LP@TP / Fe 3+ Safety evaluation of @CPC in healthy mice Male C57BL / 6 mice aged 6 to 8 weeks were placed in an animal room and fed with a normal diet for 7 days to adapt to the experimental environment. After the adaptation period, the mice were randomly divided into 4 groups, and the oral gavage dose was set at 108 CFU / mouse / day. Group 1: using normal feed and oral gavage of PBS; Group 2: using normal feed and oral gavage of LP; Group 3: using normal feed and oral gavage of LP@TP / Fe 3+ ; Group 4: using normal feed and gavage of LP@TP / Fe 3+ @CPC. The mice were housed in four cages, with five mice in each cage. The temperature was controlled at 24 °C and the air humidity was controlled at 50%. This experiment was approved by the Ethics Committee of Zhejiang University of Technology, number MGS20240530060.

[0053] Evaluation of LP@TP / Fe in mouse models 3+ @CPC biosafety. Fig.17 As shown in the results, hematoxylin and eosin (H&E) staining analysis of the main organs (heart, liver, spleen, lung, and kidney) of mice treated with PBS, encapsulated, or unencapsulated LP showed no histological damage or inflammatory cell infiltration after three oral gavages. 3+ @CPC has good biological safety and no side effects in the body.

[0054] Fig.18 It showed that after three oral administrations of probiotics, LP@TP / Fe 3+ @CPC group, LP@TP / Fe 3+ The results of blood routine indicators (erythrocytes, hemoglobin, and platelets) in the LP group and the PBS group were similar and there was no significant difference, indicating that LP@TP / Fe 3+ @CPC has good biological safety and no side effects in the body.

[0055] Example 7: LP@TP / Fe 3+ Effects of CPC on fasting blood glucose and insulin resistance in type 2 diabetic mice After one week of adaptive feeding, 5-week-old male C57BL / 6J mice were fed a high-fat diet (60% fat energy supply) except for the normal feed group. From the 4th week, unencapsulated probiotics, encapsulated probiotics and saline were given by gavage, and the gavage dose was set at 108 CFU / mouse / day. Starting from the 6th week, STZ solution was injected intraperitoneally at a dose of 50 mg / kg according to the weight of the animals for 5 consecutive days. After modeling type 2 diabetic mice (T2DM), unencapsulated probiotics, encapsulated probiotics and saline were used for daily gavage intervention for 4 weeks, and fasting blood glucose was measured at the end of the feeding experiment.

[0056] Fasting blood glucose (FBG) and insulin (FI) in mouse serum were detected according to the operating procedures of the commercial kit.

[0057] The calculation formula of insulin resistance evaluation index (HOMA-IR) is:

[0058] Note: Fasting insulin (FI, mU / L) and fasting blood glucose (FBG, mmol / L) Depend on Fig.19 It can be seen that compared with the NCD group, the fasting blood glucose and insulin resistance index (HOMA-IR) of the T2DM group mice were significantly higher (p<0.05), which indicates that the T2DM group mice have developed significant insulin resistance. 3+ @CPC、LP@TP / Fe 3+ , LP significantly reduced hyperglycemia and HOMA-IR in T2DM mice (p<0.05). 3+ @Fasting blood glucose and HOMA-IR in CPC and NCD groups were significantly lower (p<0.05). 3+ @CPC coating can significantly enhance the effect of LP in improving insulin resistance and hyperglycemia in type 2 diabetic mice.

Claims

1. A method for preparing a single-cell probiotic coating, characterized in that: The following steps are involved: 1) slowly adding the metal ion solution to the probiotic solution, stirring the mixture by magnetic stirring, then adding the polyphenol solution, stirring the mixture to react, and obtaining the metal-polyphenol network-coated probiotics; 2) Add chitosan-casein phosphopeptide complex solution to the metal-polyphenol network-coated probiotics and react with magnetic stirring at room temperature to obtain LP@TP / Fe 3+ @CPC.

2. The method for preparing a single-cell probiotic coating according to claim 1, characterized in that: The probiotic is Lactobacillus plantarum YJ7.

3. The method for preparing a single-cell probiotic coating according to claim 1, characterized in that: In step 1), the metal ion solution is FeCl3•6H2O solution with a concentration of 1 mg / mL.

4. The method for preparing a single-cell probiotic coating according to claim 1, characterized in that: The polyphenol solution in step 1) is a polymerized tea polyphenol solution with a concentration of 1 mg / mL.

5. The method for preparing a single-cell probiotic coating according to claim 1, characterized in that: In step 1), the volume ratio of the probiotic solution, the metal ion solution and the polyphenol solution is 1:1:

3.

6. The method for preparing a single-cell probiotic coating according to claim 1, characterized in that: In step 1), the reaction needs to be stirred at 60°C for 3 hours before the addition of tea polyphenols, and then reacted for another 30 minutes after the addition.

7. The method for preparing a single-cell probiotic coating according to claim 1, characterized in that: In step 2), the amount of chitosan-casein phosphopeptide complex solution added was 2 mg / mL, and the stirring reaction time was 30 min.

8. LP@TP / Fe prepared by the method described in any one of claims 1 to 7 3+ @CPC.

9. LP@TP / Fe according to claim 8 3+ @Application of CPC in the preparation of drugs for lowering blood sugar.

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