Anaerobic probiotic delivery system and application thereof in enteritis treatment
Through the two-layer embedding technology, the first embedding was used with chlorogenic acid and calcium chloride, and then the second embedding was used with mucin protein, which solved the problem of Ackermans colonization in the intestine and significantly improved its efficacy in the treatment of IBDs.
Patent Information
- Application Number
- CN202411931800.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-26
- Publication Date
- 2025-04-08
AI Technical Summary
The prior art is difficult to efficiently deliver and colonize Ackermania in the intestine, limiting its efficacy in the treatment of inflammatory bowel diseases (IBDs).
Using two-layer embedding technology, the probiotics were first embedded with chlorogenic acid and calcium chloride, and then the second embedding was used with mucin protein to form a probiotic delivery system.
It significantly improves Akermans' tolerance, oxygen tolerance and antibiotic tolerance in extreme gastrointestinal environments, enhancing its colonization ability in the intestine and the efficacy of treating IBDs.
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Figure CN119770667A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a delivery system of anaerobic probiotics and application thereof in the treatment of enteritis, belonging to the technical field of microbial medicines. Background Art
[0002] Inflammatory bowel diseases (IBDs), such as Crohn's disease and ulcerative colitis, are progressive immune-mediated diseases of the large intestine. Globally, there are as many as 7 million IBDs patients each year, and the incidence is increasing year by year. IBDs patients need to take medication all year round, so the effectiveness of drug delivery has positive significance for the treatment of IBDs.
[0003] The intestinal microbiota is considered to be one of the key factors regulating host health, and bacterial therapy involving probiotics is a potential method for treating IBDs. The effectiveness of bacterial therapy largely depends on the availability of intestinal colonization after oral intervention. Most probiotics used for bacterial therapy are sensitive to the harsh environment of the intestine and have low survival rates and short retention times in the gastrointestinal tract, which limits the therapeutic effect of probiotics in the treatment of IBDs.
[0004] Akkermansia muciniphila is an important bacterium belonging to the Verrucomicrobiota discovered so far, which mainly feeds on mucus in the intestines of mammals. Changes in the abundance of Akkermansia in mammals are closely related to various diseases. Akkermansia can improve metabolic disorders in mice, enhance cancer immunotherapy or alleviate neurodegenerative diseases, and is considered to be the next generation of beneficial probiotics. The abundance of Akkermansia in the intestine is negatively correlated with the disease severity of IBD. As an anaerobic microorganism, Akkermansia is highly sensitive to oxygen. At the same time, whether the bacteria can effectively colonize in the body in monobacterial therapy is closely related to its effectiveness in disease treatment. At present, many studies have reported that the survival rate and colonization rate of probiotics in the body can be improved by surface modification. However, there are few reports on the delivery system of Akkermansia. In order to improve the colonization of Akkermansia in the body and enhance its efficacy in treating diseases, simple modifications are made to it to avoid gastrointestinal damage to Akkermansia and reduce its sensitivity to oxygen, which will greatly improve the effectiveness of Akkermansia in the treatment of IBDs. Summary of the invention
[0005] In view of the above-mentioned deficiencies of the prior art, the present invention provides a method for preparing a probiotic delivery system, comprising the following steps:
[0006] (1) First embedding: mixing probiotics with chlorogenic acid and calcium chloride in a solution to obtain embedding material 1;
[0007] (2) Second embedding: The mucus protein is mixed with the embedding material 1 described in step (1) in a solution, and a second embedding is performed to obtain a probiotic delivery system.
[0008] In one embodiment, in the solution of step (1), the concentration of chlorogenic acid is 0.15625-0.625 mg / mL, the concentration of calcium chloride is 0.5-1.5 mg / mL, and the time of the first embedding is 10-30 min.
[0009] In one embodiment, in the solution of step (2), the concentration of mucus protein is 2-8 mg / mL, and the second embedding time is 20-40 min.
[0010] In one embodiment, the probiotics include Akkermansia, and the viable count of the probiotics in the solution of step (1) is ≥ 2×10 8 CFU / mL.
[0011] The present invention also provides an anaerobic probiotic delivery system prepared by the method, wherein the probiotics are embedded with chlorogenic acid, calcium chloride and mucin.
[0012] In one embodiment, the probiotic delivery system is a probiotics encapsulation system with two layers; the inner layer encapsulation material is chlorogenic acid and calcium chloride, and the outer layer encapsulation material is mucus protein.
[0013] In one embodiment, the probiotics include but are not limited to Akkermansia.
[0014] The present invention also provides application of the method in improving the environmental tolerance of Akkermansia, characterized in that the environmental tolerance includes gastrointestinal tolerance, oxidation tolerance and antibiotic tolerance.
[0015] The invention also provides application of the method in preparing a product for improving colonization of Akkermansia in the intestinal tract.
[0016] The present invention also provides application of the method or the anaerobic probiotic delivery system in preparing anti-enteritis drugs.
[0017] In one embodiment, the anti-enteritis effect is achieved by enhancing the tolerance of Akkermansia to the intestinal environment, thereby improving the colonization of Akkermansia in the intestine.
[0018] In one embodiment, the drug further comprises pharmaceutically acceptable excipients, which include excipients and / or additives.
[0019] Beneficial effects:
[0020] The present invention provides an embedding method of a probiotic delivery system, wherein the probiotics are embedded in two layers, wherein the first layer embedding material is chlorogenic acid and calcium chloride, and the second layer embedding material is mucus protein. The probiotic delivery system can be used to embed Akkermansia.
[0021] The embedding method of the present invention can improve Akkermansia's tolerance to extreme gastrointestinal environments, oxygen tolerance and antibiotic tolerance: after being treated with artificial gastric juice for 2 hours, the activity of the embedded Akkermansia was 1.91 times higher than that of the uncoated Akkermansia, and the activity of the embedded Akkermansia after growing in artificial intestinal juice for 6 hours was 1.13 times that of the unencapsulated Akkermansia; after being exposed to air for 24 hours, the embedding treatment significantly reduced the inhibition of oxygen on the activity of Akkermansia by 1.24 times; and also improved the sensitivity of Akkermansia to antibiotics. The embedded Akkermansia was effectively delivered in the intestine, which alleviated the severity of dextran sulfate (DSS)-induced colitis in mice, significantly alleviated the weight loss of mice caused by DSS, delayed intestinal damage in the colon, reduced the expression of inflammatory factors, and reduced the level of reactive oxygen in DSS enteritis.
[0022] The anaerobic probiotic delivery system of the present invention has a simple preparation process, simple instruments, and a short preparation time. During the treatment process, the wall material can effectively reduce the toxic effect of air on anaerobic microorganisms, and therefore has the potential for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 The optimized data of chlorogenic acid and mucin concentrations; A The survival rate of Akkermansia in gastric juice after being treated with different concentrations of chlorogenic acid; B The growth of Akkermansia in gastric juice after being treated with different concentrations of chlorogenic acid and treated with gastric juice for 24 hours; C The survival rate of Akkermansia in gastric juice after being treated with different concentrations of mucin; D The growth of Akkermansia in gastric juice after being treated with different concentrations of mucin and treated with gastric juice for 24 hours; E The survival rate of Akkermansia in gastric juice after being treated with different proteins; F The growth of Akkermansia in gastric juice after being treated with different proteins and treated with gastric juice for 24 hours; G The survival rate of Akkermansia in gastric juice after being treated with different treatments; H The growth of Akkermansia in gastric juice after being treated with different treatments and treated with gastric juice for 24 hours;
[0024] Figure 2 Characterization and growth of Akkermansia after optimization of embedding material concentration; A particle size; B potential; C transmission electron microscopy; D growth;
[0025] Figure 3 The figure shows the tolerance of Akkermansia to the extreme environment of the gastrointestinal tract after embedding; A artificial gastric juice; B artificial intestinal juice; C various antibiotics; D hydrogen peroxide; E oxygen in the air;
[0026] Figure 4 The ability of Akkermansia to scavenge active oxygen in vitro after embedding; A DPPH; B ABTS + ; C OH - ;DO 2- ; E and F are graphs and statistical results of bacteria after active oxygen treatment;
[0027] Figure 5 The results of Akkermansia colonization in the intestine after embedding; A animal imaging shows the distribution in the intestine; B real-time fluorescence quantitative PCR;
[0028] Figure 6 Arrange and group animals for experiments;
[0029] Figure 7 The figure shows the results of Akkermansia mitochondria relieving the effects of DSS-induced enteritis on mice after embedding; A mouse weight; B mouse disease activity index (DAI); C mouse spleen index; D mouse colon length; E mouse colon length data statistics; F mouse colon pathology diagram; G mouse colon pathology statistical data;
[0030] Figure 8 The levels of inflammatory factors in mouse serum; A. IL-6 level in mouse serum; B. TNF-α level in mouse serum; C. IL-1β level in mouse serum;
[0031] Fig. 9 These are indicators related to antioxidant activity in the mouse intestine; A: malondialdehyde (MDA) level in colon tissue; B: nitric oxide (NO) level in colon tissue; C: myeloperoxide (MPO) level in colon tissue; D: reactive oxygen species (ROS) intensity in the mouse intestine in mouse colon tissue;
[0032] Fig.10 Screening results for appropriate chlorogenic acid concentration;
[0033] Fig.11 Screening results for appropriate mucin concentration. DETAILED DESCRIPTION
[0034] The technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto. Without departing from the technical concept and technical solution of the present invention, appropriate modifications and adjustments that can be implemented by ordinary technicians in the field to which the present invention belongs, and equivalent replacement methods will fall within the scope of the protection required by the present invention.
[0035] In the following examples, chlorogenic acid (CA, Aladdin, C109404), calcium chloride (Da Mao, 3352), and mucin (Mucin, Sigma, M2378) were dissolved in sterile PBS solution to prepare a solution.
[0036] The Akkermansia involved in the following examples is Akkermansia ATTC BAA-835. Akkermansia ATTC BAA-835 was inoculated into BHI liquid culture medium containing 2.5% mucin, and cultured anaerobically at 37° C. for 48 h in a complete anaerobic culture system (10% H2, 10% CO2 and 80% N2). The culture was then centrifuged at 4000 rpm to obtain bacterial cells, and the cells were rinsed twice with 0.01 M sterile PBS for use.
[0037] All data involved in the present invention are expressed as mean ± standard error (SE). The data in the article were analyzed by SPSS23.00. One-way analysis of variance (ANOVA) was used for three groups or more, LSD test was used for multiple comparisons, and two-group comparisons were made by two-tailed unpaired Student's t test. *p<0.05, **p<0.01, ***p<0.001 were statistically significant, ns was not significant). All pictures were drawn by Origin 2019b.
[0038] Example 1 Preparation and Optimization of Anaerobic Probiotic Delivery System
[0039] Cultivation of Akkermansia: Akkermansia in the logarithmic growth phase was collected, and the collected Akkermansia was washed twice with sterile PBS, centrifuged, and the precipitate was collected.
[0040] 2×10 8 CFU / mL of Akkermansia was mixed with different final concentrations of chlorogenic acid (0.15625 mg / mL, 0.3125 mg / mL and 0.625 mg / mL) and calcium chloride with a final concentration of 1 mg / mL, mixed at 500 r / min for 20 min, then washed twice with sterile PBS, centrifuged at 5000 r / min, the precipitate was collected, and resuspended with sterile PBS to obtain a layer of Akkermansia embedded in it.
[0041] After the first layer of embedding, Akkermansia was divided into 2 × 10 8 The final concentration of CFU / mL was mixed with mucus protein at a final concentration of 2 mg / mL at 500 r / min for 30 min, washed twice with sterile PBS, centrifuged at 5000 r / min, and the precipitate was collected to obtain the embedded Akkermansia; the bacterial group without any treatment was recorded as AKK.
[0042] The effect of different concentrations of chlorogenic acid on bacterial activity was investigated by measuring the bacterial tolerance to gastric juice and the difference in growth after gastric juice treatment. 8The final concentration of CFU / mL was incubated in gastric juice (source leaves, R22155, pH = 1.3) at 37°C for 30 minutes, and then the survival rate was determined. The survival rate was determined using thiazolyl blue (MTT, McLean, T6126-5g). Take 1 mL of sample, add 100 μL of MTT to each 1 mL of sample, react at 37°C for 30 minutes, dissolve the precipitate with DMSO, and use a microplate reader to measure the absorbance at absorbance (OD) 490nm to express the survival rate. Subsequently, the above-mentioned bacterial sample treated with gastric juice was taken at a 2% (v / v) inoculation amount and cultured in BHI liquid medium containing 2.5% mucin. After anaerobic culture (10% H2, 10% CO2 and 80% N2) at 37°C for 24 hours, the absorbance was measured at OD 600nm to examine the difference in growth.
[0043] Depend on Figure 1 As shown in A, different concentrations of chlorogenic acid can improve the survival rate of Akkermansia after gastric juice treatment. When the concentration of chlorogenic acid is 0.625 mg / mL, the survival rate of bacteria treated with chlorogenic acid after gastric juice treatment is significantly higher than that of other groups (p<0.05), and compared with the bacteria in the untreated group, it is increased by 15.45%; after 24 hours of culture, the bacterial growth of the coating at this concentration is significantly higher than that of bacteria treated with other concentrations (p<0.05). Therefore, the chlorogenic acid concentration is preferably 0.625 mg / mL.
[0044] Example 2 Optimization of the preparation method of the delivery system of anaerobic probiotics
[0045] The specific implementation is the same as Example 1, except that the concentration of mucin used is 2 mg / mL, 4 mg / mL and 8 mg / mL.
[0046] 2×10 8 CFU / mL of Akkermansia was mixed with chlorogenic acid at a final concentration of 0.625 mg / mL and calcium chloride at a final concentration of 1 mg / mL, mixed at 500 r / min for 20 min, washed twice with sterile PBS, centrifuged at 5000 r / min, the precipitate was collected, and resuspended with sterile PBS; a layer of Akkermansia was obtained.
[0047] The Akkermansia after embedding the first layer was mixed with mucin at a final concentration of 2 mg / mL, 4 mg / mL and 8 mg / mL at 500 r / min for 30 min, washed twice with sterile PBS, centrifuged at 5000 r / min, and the precipitate was collected to obtain Akkermansia embedded in two layers. The bacterial group without any treatment was recorded as AKK.
[0048] Referring to the method of Example 1, the effect of different concentrations of mucin embedding on bacterial activity was investigated by measuring the bacterial tolerance to gastric juice and the difference in growth after gastric juice treatment. The results showed that when the concentration of mucin was 2 mg / mL, the survival rate of Akkermansia in artificial gastric juice was higher and it was able to quickly resume growth ( Figure 1 C, D), therefore the preferred mucin concentration is 2 mg / mL.
[0049] Example 3 Delivery system of anaerobic probiotics and preparation method thereof
[0050] According to the chlorogenic acid concentration optimized in Example 1 and the mucin concentration optimized in Example 2, the mucus protein in the outer layer was replaced with whey protein, and the concentration of whey protein was 2 mg / mL.
[0051] 2×10 8 CFU / mL of Akkermansia was mixed with chlorogenic acid at a final concentration of 0.625 mg / mL and calcium chloride at 1 mg / mL, mixed at 500 r / min for 20 min, washed twice with sterile PBS, centrifuged at 5000 r / min, the precipitate was collected, and resuspended with sterile PBS to obtain a layer of Akkermansia embedded in it.
[0052] After the first layer of embedding, Akkermansia was divided into 2 × 10 8 The final concentration of CFU / mL was mixed with mucus protein and whey protein at a final concentration of 2 mg / mL at 500 r / min for 30 min, washed twice with sterile PBS, centrifuged at 5000 r / min, and the precipitate was collected to obtain the embedded Akkermansia, which were recorded as whey and cAKK, respectively. The bacterial group without any treatment was recorded as AKK.
[0053] Referring to the method of Example 1, the effect of different protein treatments on bacterial activity was investigated by measuring the tolerance of bacteria to gastric juice and the difference in growth after gastric juice treatment. The results showed that when the concentrations of whey protein and mucin were both 2 mg / mL, the survival rate of Akkermansia encapsulated by mucin was significantly higher than that of whey protein (p<0.05), and it could quickly resume growth under the same culture conditions ( Figure 1 E, F), so the outer layer protein is preferably mucin.
[0054] Example 4 Delivery system of anaerobic probiotics and preparation method thereof
[0055] According to the chlorogenic acid concentration optimized in Example 1 and the mucin concentration optimized in Example 2, the two are used to treat bacteria simultaneously (co-mix), or two groups of bacteria are treated in batches.
[0056] 2×10 8CFU / mL of Akkermansia was mixed with chlorogenic acid at a final concentration of 0.625 mg / mL, calcium chloride at 1 mg / mL, and mucin at 2 mg / mL, which was recorded as co-mix; the mixture was mixed in batches according to the method in Example 1 to obtain cAKK, and the bacteria without any treatment were recorded as AKK.
[0057] Referring to the method of Example 1, the effect of different treatment methods on bacterial activity was investigated by measuring the bacterial tolerance to gastric juice and the difference in growth after gastric juice treatment. The results showed that the survival rate of Akkermansia treated in batches was significantly higher than that of mixed at the same time (p<0.05), and it could quickly resume growth in culture ( Figure 1 J, H), so the processing method is preferably mixed in batches.
[0058] Example 5 Delivery system of anaerobic probiotics and preparation method thereof
[0059] According to the chlorogenic acid concentration optimized in Example 1 and the mucin concentration optimized in Example 2, the method of bacterial treatment was determined, and the following steps and related indexes were performed in sequence:
[0060] Akkermansia was mixed with chlorogenic acid at a final concentration of 0.625 mg / mL and calcium chloride at a final concentration of 1 mg / mL at 500 r / min for 20 min, washed twice with sterile PBS, and centrifuged at 5000 r / min to collect the precipitate;
[0061] The Akkermansia after embedding the first layer was mixed with mucus protein at a concentration of 2 mg / mL at 500 r / min for 30 min, washed twice with sterile PBS, centrifuged at 5000 r / min, and the precipitate was collected to obtain the embedded Akkermansia, which was recorded as cAKK, and the bacteria without any treatment were recorded as AKK.
[0062] The particle size and Zeta potential of AKK and cAKK were analyzed using a laser particle analyzer (Malvern, England), and the morphological differences between AKK and cAKK were observed using a transmission electron microscope (TEM, JEOL, Japan). CCK8 (Biyuntian, C0037) was also used to measure the activity changes of AKK and cAKK.
[0063] Depend on Figure 2 As shown in Figure 1, the particle size and zeta potential of Akkermansia after layer-by-layer coating were analyzed using a laser particle analyzer. Figure 2 A, the particle sizes of AKK and cAKK were 1543.67±22.48nm and 1684.33±33.95nm, respectively. The particle size of cAKK increased after treatment (p<0.05). The zeta potentials of AKK and cAKK were -6.7±0.4 and -5.5±0.1, respectively. The zeta potential decreased significantly ( Figure 2 B). Transmission electron microscopy results show that there is a transparent coating on the surface of AKK ( Figure 2 C). Effects of CCK8 assay modification on AKK activity ( Figure 2 D), bacteria were co-incubated with WST-8 reagent, and then the dehydrogenase in the live bacteria reduced WST-8 to formazan through the action of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate. The amount of formazan generated is proportional to the average number of live bacteria. By measuring the amount of formazan generated, the proliferation of bacteria can be evaluated. The results showed that the treatment of chlorogenic acid and mucin had no effect on the activity of bacteria. In addition, it was found that the growth of cAKK was significantly higher than that of AKK, which may be that the addition of chlorogenic acid and mucin provided nutrition for the bacteria.
[0064] Example 6 Analysis of cAKK's gastrointestinal, antibiotic and oxygen tolerance in extreme environments
[0065] The bacteria were treated according to the embedding method of Example 5 and recorded as cAKK; those without any treatment were recorded as AKK. 8 CFU / mL of AKK and cAKK were incubated with artificial gastric fluid SGF (source leaves, R22155, pH=1.3), artificial intestinal fluid SIF (source leaves, R22156, pH=6.8), different types and concentrations of antibiotics, different concentrations of hydrogen peroxide and oxygen (21%) to investigate the changes in bacterial activity and growth after treatment.
[0066] The incubation time of AKK and cAKK in different environmental factors is as follows: artificial gastric juice: 0.5, 1, 1.5 and 2h; artificial intestinal juice: 1, 2 and 4h; oxygen: 8, 12 and 24h, and the bacterial activity of the two was determined using thiazolyl blue (MTT) at each time point. The antibiotics used in this example include: ampicillin (AMP, 2.5mg / mL), streptomycin (SM, 2.5mg / mL), gentamicin (GEN, 5.0mg / mL), carbenicillin (CAR, 5.0mg / mL) and kanamycin (KAN, 5.0mg / mL); the exposed hydrogen peroxide concentrations are 5, 10, 20, 40, 80mM, respectively. AKK or cAKK is co-incubated with antibiotics and hydrogen peroxide at 37°C, and its absorbance change is measured at OD 600nm after 24h.
[0067] like Figure 3 As shown in A, compared with AKK, the OD values of the cAKK group increased by 11.76%, 7.92%, 32.13%, and 91.31% after incubation in SGF for 0.5, 1, 1.5, and 2 hours, respectively, with significant differences (p<0.05), indicating that this treatment can protect bacteria from the effects of SGF. Figure 3As shown in B, the results show that after the bacteria were incubated in SIF for 1, 2 and 4 hours, the absorbance of cAKK increased by 38.39%, 59.15% and 13.35% respectively compared with AKK. This result shows that the coating can protect the bacteria and the outer layer modified mucin provides nutrients for AKK, promoting its growth in SIF.
[0068] Antibiotics are one of the factors that affect the growth and abundance of intestinal microorganisms. Exploring the effect of antibiotics on bacteria is one of the indicators for exploring the effectiveness of the treatment. AKK and cAKK were incubated with various antibiotics for 24 hours, and the growth of the two groups of bacteria was measured. The OD value of Akkermansia that had not been incubated with antibiotics for the same period of time was used as a reference to examine the changes in the OD value of bacteria after antibiotic incubation. Figure 3 As shown in C, after incubation with different antibiotics for 24 h, the bacteria in all treatment groups were inhibited by the antibiotics, but the growth of cAKK was higher than that of AKK (p<0.05).
[0069] In order to further analyze the tolerance of bacteria to the treatment, the bacteria were treated with different concentrations of hydrogen peroxide to explore their survival. The OD value of Akkermansia that had not been treated with hydrogen peroxide for the same period of time was used as a reference (counted as 100%), and the OD value after hydrogen peroxide treatment was detected to calculate the relative survival rate. Figure 3 D shows that 5mM hydrogen peroxide reduces the activity of bacteria, with the relative survival rate of the AKK group being 56.62% and the relative survival rate of the cAKK group being 131.44%. This shows that the tolerance of the treated bacteria to hydrogen peroxide has increased. When the hydrogen peroxide concentration is higher than 10mM, the activity of the untreated AKK drops below 20%, while the activity of the treated bacterial cAKK remains at 88.53%; as the hydrogen peroxide concentration increases to 40mM, the cAKK survival rate remains at 22.47%, while the untreated AKK activity is only 13.21%, indicating that the tolerance of cAKK to hydrogen peroxide is much higher than that of AKK ( Figure 3 D).
[0070] During the onset of the disease, the oxygen level in the intestinal cavity of IBD patients increases, which may inhibit the growth of AKK, an absolute anaerobic bacterium, and on the contrary promote the growth of facultative anaerobic bacteria, such as Enterobacteriaceae, thereby affecting the oxygen utilization and metabolite production in the intestine, leading to an imbalance in the intestinal flora. As a typical anaerobic microorganism, improving AKK's ability to resist oxygen will help it survive in an aerobic environment and its subsequent application. AKK and cAKK were placed in an oxygen-containing environment (21%). Figure 3E), after standing for 8 hours, the relative survival rate of the treated bacteria cAKK was the same as the initial survival rate, and the survival rate of the untreated bacteria AKK decreased significantly (p<0.05); as time went on, the activity of all bacteria decreased significantly (p<0.05), and compared with the AKK group, at 8, 12 and 24 hours, the relative survival rate of the cAKK group increased by 16.99%, 12.94% and 23.83%, respectively, indicating that the embedding method of the present invention can effectively improve the tolerance of Akkermansia to oxygen.
[0071] Example 7 Antioxidant capacity of cAKK
[0072] A prominent feature of inflammatory bowel disease (IBD) is the overproduction of reactive oxygen species (ROS). This excessive ROS not only disrupts the balance of intestinal microbial communities, but also easily causes damage to the intestinal barrier function and aggravates the intestinal inflammatory response. In addition, the accumulation of ROS plays a key role in promoting the progression of inflammatory bowel disease. This example explores the scavenging effect of AKK and cAKK on reactive oxygen-related free radicals, including DPPH, ABTS + OH - and O2 - .
[0073] The Akkermansia prepared according to the method described in Example 5 is denoted as cAKK, and the untreated bacteria is denoted as AKK. Figure 4 The results showed that compared with AKK, cAKK has obvious ability to scavenge reactive oxygen free radicals, including DPPH, ABTS + OH - and O 2- , and in a dose-dependent manner ( Figure 4 AD). For example, when the viable count is 5×10 8 CFU / mL, cAKK to DPPH, ABTS + OH - and O 2- The clearance rates were 1.25, 1.01, 1.35 and 1.44 times that of AKK, respectively.
[0074] At the same time, the level of reactive oxygen species after AKK and cAKK were measured after hydrogen peroxide treatment. The method for measuring reactive oxygen species was as follows: ROS was measured using 2,7-dichlorofluorescein (DCFH-DA, Sigma, USA). 8CFU / mL of AKK and cAKK were incubated with H2O2 aqueous solution at 37°C for 60 min, and washed twice with 0.01 M PBS to remove residual H2O2. Subsequently, 1 mL of AKK solution and cAKK solution were incubated with 5 μM DCFH-DA at 37°C for 30 min, and then washed twice with 0.01 M PBS to remove residual DCFH-DA. The fluorescence intensity was evaluated by a fluorescence microplate reader (Tecan, Switzerland), and representative fluorescence images were taken with a fluorescence microscope (Nikon, Japan) at ×10 magnification. Figure 4 As shown in E and F, compared with bacteria not incubated with hydrogen peroxide, the production of AKK reactive oxygen species in untreated bacteria increased by 37.74%, while the production of cAKK reactive oxygen species in treated bacteria increased by only 11.39%, a decrease of 3.31 times ( Figure 4 E, F). It can be seen that after AKK is treated by the present invention, the ability of the strain to scavenge free radicals is significantly enhanced. In addition, the treated strain significantly inhibits the effect of oxides on bacteria, and this treatment shows its potential application value in reducing oxidative stress and maintaining microbial balance.
[0075] Example 8 Colonization of cAKK in the Intestinal Tract
[0076] Specific Pathogen Free (SPF) C57B / 6J mice, 6-8 weeks old, weighing 16-18 g, were housed in Individually Ventilate Cages (IVC) with a 12-hour light-dark cycle and an ambient temperature of 22-23°C. The mice received irradiated feed and autoclaved water. After one week of adaptation, the mice were divided into three groups: control group, AKK group, and cAKK group, with 3 mice in each group. The control group was gavaged with sterile PBS; the AKK and cAKK groups received 2×10 8 CFU of AKK or cAKK, 24 hours after gavage, fresh mouse feces were collected and the expression of AKK bacteria was analyzed using real-time fluorescence quantitative method. The mice were euthanized, the intestinal tract was removed, and the distribution of Akkermansia in the intestine was analyzed using MIIS XFP-BIX living imaging system.
[0077] like Figure 5 As shown in A, the fluorescence of treated AKK in the gastrointestinal tract after 24 hours was higher than that of untreated AKK. In addition, since it is difficult to obtain the accurate number of AKK in the intestine using traditional coated plates, the abundance of AKK in feces after oral gavage was analyzed by RT-PCR, and it was found that the colonization of treated AKK in the intestine was higher than that of untreated AKK ( Figure 5 B)
[0078] Example 9 Application of AKK-coated probiotic delivery system in DSS-induced colitis
[0079] Specific Pathogen Free (SPF) C57B / 6J mice, 6-8 weeks old, weighing 16-18 g, were housed in Individually Ventilate Cages (IVC) with a 12-hour light-dark cycle and an ambient temperature of 22-23°C. The mice received irradiated feed and autoclaved water. After one week of adaptation, the mice were divided into four groups and received 200 μL of PBS (Control group), PBS (DSS group), 2×10 8 CFU AKK (AKK group) or 2×10 8 CFU cAKK (cAKK group) for 10 days; then the mice in the DSS group, AKK group or cAKK group received 3.5% (v / v) DSS with free drinking water for 5 days. On the fifth day, the body weight and disease activity index (DAI) of the mice were recorded. Figure 6 shown.
[0080] Disease Activity Index (DAI): weight score (0 point, no change; 1 point, weight loss of 0-2.5%; 2 points, weight loss of 2.5-5%; 3 points, weight loss of 5-10%; 4 points, weight loss of 10%), stool consistency (0 point, normal; 2 points, slightly loose stools; 3 points, loose stools; 4 points, diarrhea) and blood in stool (0 point, none; 2 points, slight blood; 3 points, bleeding; 4 points, continuous bleeding).
[0081] The mice were then euthanized, and serum, colon, and spleen were collected for subsequent experiments.
[0082] The spleen index was calculated as follows: spleen index = spleen weight (g) on the day after mouse euthanasia / body weight (g) on the day × 100%.
[0083] The inflammatory factors in serum, including interleukin 6 (IL-6, Shanghai ELISA, mL098430), interleukin 10 (IL-1β, Shanghai ELISA, mL098416), and tumor necrosis factor α (TNF-α, Shanghai ELISA, mL002095), were determined according to the instructions of the reagent manufacturers.
[0084] The colon pathology scoring method of mice is as follows: 4% paraformaldehyde-fixed colon tissue sections were stained with hematoxylin and eosin, and the colon histopathological characteristics were scanned under a microscope (Nikon, Japan). The histopathological scores are as follows: the degree of submucosal edema (from low to high, 0-4 points), the degree of inflammatory cell infiltration (from low to high, 0-4 points), the severity of inflammation (from low to high, 0-4 points), and the degree of crypt damage (from low to high, 0-4 points).
[0085] The oxidative substances in the colon of mice, including malondialdehyde (Nanjing Jiancheng, A003-1-2), nitric oxide (Nanjing Jiancheng, A013-2-1) and myeloperoxidase (Nanjing Jiancheng, A044-1-1), were determined according to the instructions of the reagent manufacturers.
[0086] The method for measuring ROS in mouse colon was as follows: dihydroethidium (DHE, MCE, USA) was used to measure ROS levels in colon. Briefly, colon tissue was incubated with 20 μM DHE for 30 min and then washed twice with 0.01 M PBS to remove residual DHE. ROS levels were observed under a fluorescence microscope (Nikon, Japan) at ×10 magnification.
[0087] Compared with the control group, the average weight loss of mice treated with DSS was 1.62 g, that of the AKK group was 1.44 g, and that of the cAKK group was 0.7 g, indicating that cAKK treatment can significantly alleviate the weight loss of mice, and the degree of relief is better than that of AKK without coating modification ( Figure 7 A).
[0088] Five days after the intestinal inflammation model was established, the intestinal tissues of the mice were scored for DAI. Figure 7 As shown in B, the index of the DSS group was 6.29, the index of the AKK group was 5.14, and the index of the cAKK group was 3.86. Oral administration of AKK could significantly reduce the DAI index of mice, and the index of the cAKK group was significantly lower than that of the AKK group and DSS group (p<0.05), indicating that cAKK can better alleviate the disease severity of mice.
[0089] The mice in the DSS group all showed swollen spleens. Compared with the control group, the spleen index of the DSS group increased by 44.12%, the spleen index of the AKK group increased by 28.96%, and the spleen index of the cAKK group increased by 21.23%. Compared with the DSS group, the spleen index of the AKK group and the cAKK group decreased by 1.53 times and 2.08 times, respectively ( Figure 7 C).
[0090] The colon lengths of mice in different groups were measured. The results showed that compared with the control group, the colon length of mice in the DSS group was 6.2 cm, while the colon lengths of mice in the AKK and cAKK groups were 7.13 cm and 7.67 cm, respectively. Figure 7 D, E), were significantly higher than those in the DSS group (p<0.05). Meanwhile, the colon length of the treated cAKK mice was significantly higher than that in the AKK group (p<0.05).
[0091] Colonic histopathology in the DSS group showed severe damage to the epithelial crypts and more inflammatory cell infiltration ( Figure 7 F). Histological scores of the colon of each group of mice Figure 7 As shown in G, the score of the DSS group was significantly higher than that of the other two groups (p<0.05). It is worth noting that compared with the DSS group, the colon pathology scores of the AKK group and the cAKK group were reduced by 0.30 times and 0.46 times, respectively; cAKK had a significantly better effect than AKK in improving the pathological damage of the mouse colon. The above results show that AKK can effectively alleviate the colon tissue inflammation caused by DSS and treatment can enhance the therapeutic effect of AKK.
[0092] Figure 8 The level of inflammatory factors in mouse serum. The coated cAKK can significantly reduce the level of inflammation in mice, such as reducing the expression of IL-6 and TNF-α. The expression level of IL-1β in the cAKK group was significantly lower than that in the AKK group (p<0.05), which better alleviated the inflammation induced by DSS ( Figure 8 AC).
[0093] Fig. 9 is the level of reactive oxygen species in the mouse intestine. Fig. 9 As shown in A and B, the AKK and cAKK treatment groups were able to significantly reduce the levels of intestinal endogenous oxides and related enzymes (such as MDA, NO, and MPO). The cAKK treatment had a significantly higher effect on reducing the DSS marker MPO than the AKK treatment ( Fig. 9 C); At the same time, the ROS in the tissue was stained, and the fluorescence intensity of ROS in the cAKK group was weaker, lower than that in the DSS group and the AKK group ( Fig. 9 D).
[0094] Comparative Example 1
[0095] Specific implementation method Referring to Example 1, the difference is that the concentration of chlorogenic acid is changed to 1.25 mg / mL, 2.5 mg / mL, 5 mg / mL and 10 mg / mL. Fig.10 As shown, when the concentration of chlorogenic acid is greater than 0.625 mg / mL, it will inhibit the growth of bacteria.
[0096] Comparative Example 2
[0097] Specific implementation method Referring to Example 1, the difference is that the concentration of mucin is changed to 1 mg / mL or 16 mg / mL. The results are as follows Fig.11 As shown, when the concentration of mucin was 1 mg / mL, the growth of bacteria was inhibited; while when the concentration of mucin was 16 mg / mL, the embedded bacteria were too viscous and difficult to wash.
[0098] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.
Claims
1. A method for preparing a probiotic delivery system, characterized in that: The following steps are involved: (1) First embedding: mixing probiotics with chlorogenic acid and calcium chloride in a solution to obtain embedding material 1; (2) Second embedding: The mucus protein is mixed with the embedding material 1 described in step (1) in a solution, and a second embedding is performed to obtain a probiotic delivery system.
2. The method according to claim 1, characterized in that In the solution of step (1), the concentration of chlorogenic acid is 0.15625-0.625 mg / mL, and the concentration of calcium chloride is 0.5-1.5 mg / mL; mixing is performed for 10-30 minutes.
3. The method according to claim 2, characterized in that In the solution of step (2), the concentration of mucus protein is 2-8 mg / mL, and the embedding time is 20-40 minutes.
4. The method according to claim 3, characterized in that The probiotics include Akkermansia. In the solution of step (1), the number of viable probiotics is ≥ 2×10 8 CFU / mL.
5. A probiotic delivery system prepared by the method according to any one of claims 1 to 4.
6. The probiotic delivery system according to claim 5, characterized in that The probiotic delivery system is a system that embeds probiotics in two layers; the inner layer embedding material is chlorogenic acid and calcium chloride, and the outer layer embedding material is mucus protein.
7. The probiotic delivery system according to claim 6, characterized in that The probiotics include but are not limited to Akkermansia.
8. Use of the method according to any one of claims 1 to 4 in improving the environmental tolerance of Akkermansia, characterized in that: The environmental resistance includes gastrointestinal resistance, oxidative resistance and / or antibiotic resistance.
9. Use of the method according to any one of claims 1 to 4 in preparing a product for improving Akkermansia colonization in the intestine.
10. Use of the method according to any one of claims 1 to 4 or the probiotic delivery system according to any one of claims 5 to 7 in the preparation of an anti-enteritis drug.