A probiotic and peptide combined preparation for improving gastrointestinal health

Abstract

The present application belongs to the technical field of food health care, and particularly relates to a bacteria and peptide combined preparation for improving gastrointestinal health, which is prepared from the following raw materials in parts by mass: Clostridium butyricum bacteria powder 5-15 parts, Bifidobacterium longum subsp. infantis bacteria powder 5-15 parts, wheat oligopeptide 20-40 parts, soybean peptide 15-30 parts, fructooligosaccharide 5-15 parts and sodium alginate 2-5 parts. The bacteria and peptide combined preparation DAI of the present application has a decrease of 69.8%, a barrier repair rate of 90.6%, an anti-inflammatory decrease of more than 72%, and the microcapsule embedding improves the gastrointestinal fluid survival rate from 22.5% to 73.8%, which can effectively improve the gastrointestinal health.

Description

Technical Field

[0001] This invention belongs to the field of food and health care technology, specifically relating to a combination of native bacteria and peptides for improving gastrointestinal health. Background Technology

[0002] Gastrointestinal health is fundamental to maintaining normal physiological functions. The intestinal mucosal barrier, as the first line of defense against the invasion of harmful exogenous substances, is crucial for maintaining bodily health. Impairment of the intestinal barrier function is closely related to various gastrointestinal diseases, including inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and diarrhea.

[0003] Currently, the main methods for improving gastrointestinal health include probiotic preparations and bioactive peptides. Regarding probiotics, most commercial probiotics are exogenous strains, not the dominant flora native to the human gut. Once in the gut, they are difficult to colonize long-term, and their regulatory effects quickly diminish after discontinuation. While probiotics alone can indirectly improve the intestinal environment by regulating the gut microbiota balance, they cannot provide essential nutrient substrates (such as glutamine) for damaged mucosa, resulting in insufficient mucosal repair. Orally administered probiotics are susceptible to damage from gastric acid and bile acids; naked bacterial powder has a low survival rate in simulated gastric juice, with the vast majority of live bacteria being killed in gastric acid, making it difficult to effectively reach the intestines to exert their colonization and regulatory functions. Regarding bioactive peptides, while wheat oligopeptides can provide glutamine substrates for intestinal epithelial cells, and soybean peptides have immunomodulatory activity, neither can promote the colonization and proliferation of beneficial bacteria in the intestinal mucosa, nor do they regulate the structure of the intestinal flora. Regarding the combination of probiotics and peptides, existing technologies often simply mix probiotics with active peptides. The strains used are not the dominant intestinal flora, resulting in insufficient colonization and a lack of gastric acid protection for the probiotics. Some formulations are essentially bacterial metabolites rather than a combination of bacteria and peptides, thus lacking the functions of probiotic colonization and flora regulation. Other formulations only involve the peptide's role in promoting probiotic adhesion, without addressing the effects of the combination of native flora and the repair of the gastrointestinal barrier.

[0004] In summary, the existing technology has not yet solved the following technical problems: the lack of a scientific combination of native dominant bacteria leads to insufficient colonization; the independent use of probiotics and bioactive peptides lacks synergistic design of microbial peptides; the low oral survival rate of probiotics lacks effective protective measures; and the dual functions of microbial regulation and mucosal repair have not been achieved. Summary of the Invention

[0005] The purpose of this invention is to provide a combination of bacteria and peptides that improve gastrointestinal health by native bacteria, in order to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A combination of bacteria and peptides for improving gastrointestinal health, prepared by weight of the following raw materials: 5-15 parts of Clostridium butyricum powder, 5-15 parts of Bifidobacterium longum subsp. infantis powder, 20-40 parts of wheat oligopeptides, 15-30 parts of soybean peptides, 5-15 parts of fructooligosaccharides, and 2-5 parts of sodium alginate.

[0007] Furthermore, by weight, it is prepared from the following raw materials: 10 parts of Clostridium butyricum powder, 10 parts of Bifidobacterium longum subsp. infantis powder, 30 parts of wheat oligopeptides, 20 parts of soybean peptides, 10 parts of fructooligosaccharides and 3 parts of sodium alginate.

[0008] Furthermore, the viable counts of both the Clostridium butyricum powder and the Bifidobacterium longum subsp. infantis powder are not less than 1.0 × 10⁻⁶. 10 cfu / g.

[0009] This invention also provides a method for preparing the original bacterial and peptide combination preparation for improving gastrointestinal health, specifically: Clostridium butyricum powder and Bifidobacterium longum subsp. infantis powder are mixed evenly with sodium alginate solution, and then dropped into calcium chloride solution to solidify for 20-30 minutes to form microcapsules. After collection, the microcapsules are vacuum dried to obtain the encapsulated bacterial powder; the encapsulated bacterial powder is then mixed evenly with wheat oligopeptides, soybean peptides, and fructooligosaccharides to obtain the original bacterial and peptide combination preparation for improving gastrointestinal health.

[0010] Furthermore, the sodium alginate solution has a mass concentration of 2%, and the calcium chloride solution has a mass concentration of 3%.

[0011] This invention also provides the application of the native bacteria-peptide combination preparation for improving gastrointestinal health in the preparation of products for improving gastrointestinal health.

[0012] Furthermore, the improvement of gastrointestinal health includes repairing the intestinal mucosal barrier, regulating the balance of intestinal flora, reducing intestinal inflammatory response, and enhancing intestinal immune function.

[0013] Compared with the prior art, the present invention has the following beneficial effects: (1) Both *Clostridium butyricum* and *Bifidobacterium longum* subsp. infantis* of this invention are native dominant flora in the human gut, exhibiting stronger intestinal colonization and adaptability compared to exogenous probiotics. Experiments showed that the DAI score of the dual-combination formulation of native bacteria in this invention decreased to 1.3 on day 14, a reduction of 69.8% compared to the model control group, comparable to the efficacy of the positive control drug mesalazine; while the DAI of the control group containing only a single native bacterium was significantly higher than that of the combined formulation of this invention, indicating a synergistic effect between the two native bacteria. This may be because butyric acid produced by *Clostridium butyricum* metabolism can directly provide energy to colonic epithelial cells and promote mucosal repair, while lowering intestinal pH and creating an acidic microenvironment conducive to the colonization of *Bifidobacterium longum* subsp. infantis; while acetic acid produced by *Bifidobacterium longum* subsp. infantis synergistically enhances intestinal barrier function.

[0014] (2) The wheat oligopeptides of this invention are rich in glutamine, which can provide energy substrates for intestinal epithelial cells and promote mucosal repair; the soybean peptides can enhance the self-aggregation ability and surface hydrophobicity of probiotics, and promote their adhesion and colonization on the intestinal mucosa. Experiments show that the DAI scores of the single peptides on day 14 were significantly higher than those of the combined formulation of this invention containing two peptides, indicating that there is a synergistic effect between wheat oligopeptides and soybean peptides. This may be because a positive feedback loop is formed between the native bacteria and the bioactive peptides: the peptides provide nutrients and adhesion promotion for the bacteria, and the bacterial metabolites such as butyric acid and acetic acid provide microenvironmental support for mucosal repair. The synergistic effect of bacteria and peptides achieves "1+1>2".

[0015] (3) The combination of bacterial peptides in this invention can significantly repair the intestinal mucosal barrier. Experiments showed that the colon length of the combined preparation of this invention was restored to 7.72 cm, and the intestinal permeability (plasma FITC-glucan) decreased to 1.48 μg / mL, which was 68.6% lower than that of the model control group; the relative expression levels of tight junction proteins Occludin, Claudin-1, and ZO-1 were restored to 0.85, 0.88, and 0.80, respectively, with recovery rates of 83.3%, 89.8%, and 76.2%; the total pathological score decreased to 1.7 points, which was 80.0% lower than that of the model control group, with the ulcer score being only 0.4 points. All of the above indicators were comparable to or better than those of the positive drug group, and were significantly better than those of each comparative group (P<0.01), indicating that the effect of the combination of bacterial peptides on barrier repair far exceeds that of the use of bacteria or peptides alone.

[0016] (4) The combination of bacterial peptides in this invention can significantly reduce intestinal inflammatory response. Experiments show that the levels of TNF-α, IL-6, and IL-1β in colon tissue of the combined preparation of this invention decreased to 33.8, 40.2, and 24.5 pg / mg prot, respectively, which were 73.7%, 74.4%, and 72.6% lower than those in the model control group, and the reduction was comparable to that in the positive control group. The levels of the three inflammatory factors in the ratio lacking any of the original bacteria were significantly higher than those in the combined preparation of this invention. This may be because Clostridium butyricum mainly reduces TNF-α by inhibiting the NF-κB pathway through butyrate, and Bifidobacterium longum subsp. infantis mainly regulates IL-6 by activating GPR43 through acetic acid, and both comprehensively inhibit the inflammatory cascade response. In addition, the levels of inflammatory factors in the ratio lacking any of the peptides were also significantly higher than those in the combined preparation of this invention. This may be because wheat oligopeptides indirectly inhibit inflammation by reducing oxidative stress through glutamine, and soybean peptides directly inhibit the release of inflammatory mediators through bioactive peptides, and the two work synergistically to achieve a more comprehensive anti-inflammatory effect.

[0017] (5) The combination of microbial peptides in this invention can effectively regulate the intestinal flora and restore its diversity and structure. Experiments show that the Chao1 index of the combined formulation of this invention recovered to 332.5, the Shannon index recovered to 4.35, and the Simpson index recovered to 0.88; at the phylum level, the relative abundance of Proteobacteria decreased to 6.5%, a decrease of 77.2% compared with the model control group, Firmicutes recovered to 50.8%, and Bacteroidetes recovered to 33.5%, and the flora structure basically returned to normal. The flora diversity and structure of each comparative group were significantly worse than those of the combined formulation of this invention, which indicates that the combined microbial peptides have a better effect on regulating the flora than using bacteria or peptides alone.

[0018] (6) The microcapsule encapsulation treatment of the present invention can significantly improve the survival rate of probiotics. Experiments show that after encapsulation with sodium alginate-calcium chloride microcapsules, the survival rate of probiotics in simulated gastric juice increased from 35.2% to 82.7% after 2 hours in simulated gastric juice and 4 hours in simulated intestinal juice. The survival rate increased from 22.5% to 73.8% after sequential treatment with simulated gastric juice and simulated intestinal juice, providing a live bacteria basis for subsequent colonization and barrier repair. Detailed Implementation

[0019] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are some embodiments of this invention, but not all embodiments.

[0020] 1. Combined formulations and their preparation methods The formula is shown in Table 1.

[0021] Table 1. Formulation of combined preparations (unit: g)

[0022] The specific preparation method is as follows: S1. Strain culture Clostridium butyricum ( Clostridium butyricum CCTCC AB 2010154) was cultured anaerobically in RCM liquid medium at 37°C until the logarithmic growth phase, and then inoculated into fresh RCM liquid medium at a 3% (v / v) inoculum for expansion culture, and cultured anaerobically at 37°C for 20 h. Bifidobacterium longum infantis subspecies ( Bifidobacterium longum subsp. infantis CGMCC1.15639 was cultured anaerobically in BBL liquid medium at 37°C until the logarithmic growth phase, and then inoculated into fresh BBL liquid medium at a 3% (v / v) inoculum for expansion culture, and cultured anaerobically at 37°C for 22 h. The RCM liquid culture medium formula is as follows: 10 g / L peptone, 10 g / L beef extract, 3 g / L yeast extract, 1 g / L soluble starch, 5 g / L glucose, 5 g / L sodium chloride, 3 g / L sodium acetate, 0.5 g / L L-cysteine ​​hydrochloride, and pH 6.8 ± 0.2. The BBL liquid culture medium formula is as follows: peptone 15 g / L, yeast extract 2 g / L, soluble starch 0.5 g / L, L-cysteine ​​0.5 g / L, sodium chloride 5 g / L, tomato extract 5 g / L, glucose 20 g / L, Tween 1 mL, liver extract 0.3 g / L, pH 6.8 ± 0.1; S2. Preparation of bacterial powder The fermentation broths of each strain were centrifuged at 8000 r / min for 15 min at 4℃. After collecting the bacterial cells, a protective agent (10% skim milk powder, 5% trehalose, and 1% monosodium glutamate, prepared with distilled water) was added. The mixture was stirred evenly, dispensed, pre-frozen at -40°C for 4 h, and then freeze-dried under vacuum for 24 h to obtain freeze-dried bacterial powders of each strain. These powders were diluted with food-grade maltodextrin as an inert diluent until the viable count in both Clostridium butyricum and Bifidobacterium longum subsp. infantis powders was not less than 7.5 × 10⁻⁶. 10 cfu / g; S3. Microcapsule encapsulation Add the bacterial powder to a 2% sodium alginate solution and stir until homogeneous. Use a 0.5mm needle to drop the mixture into 5 times its volume of a 3% calcium chloride solution. Let it solidify for 25 minutes to form capsule particles. After collection, vacuum dry at 40°C until the moisture content is 4% to obtain the encapsulated bacterial powder. If the bacterial powder is not encapsulated, this step should be modified as follows: mix the bacterial powder with sodium alginate evenly and vacuum dry at 40°C until the moisture content is 4%.

[0023] S4. Mixing and preparation The combined preparation is made by uniformly mixing encapsulated (or unencapsulated) bacterial powder with wheat oligopeptides (Hebei Runbu Biotechnology Co., Ltd.), soybean peptides (Hebei Rencan Biotechnology Co., Ltd.), and fructooligosaccharides in a V-type mixture.

[0024] 2. The therapeutic effects of different combination formulations on DSS-induced enteritis in mice. 2.1 Experimental Objective To evaluate the therapeutic effect of the combined formulation on dextran sulfate sodium (DSS)-induced enteritis in mice.

[0025] 2.2 Laboratory Animals SPF-grade male BALB / c mice, 6-8 weeks old, weighing 20±2g. Mice were housed in an SPF-grade barrier environment at 22±2°C and 50±10% relative humidity, with a 12-hour light-dark cycle, and free access to standard pelleted feed and drinking water. Experiments began after 7 days of acclimatization.

[0026] 2.3 Test Methods 2.3.1 DSS-induced acute enteritis model Prepare a 3% (w / v) DSS solution by dissolving DSS powder in distilled water and store at 4°C. Modeling method: Administer the DSS solution as the sole drinking water to mice for 7 consecutive days. From day 8 onwards, replace the solution with normal drinking water and continue observation for another 7 days, for a total of 14 days.

[0027] 2.3.2 Experimental grouping and drug administration The animals were randomly divided into multiple groups, with 10 animals in each group. All groups received the drug via gavage at a fixed time each morning (9:00-10:00), with a gavage volume of 0.5 mL per animal. This was done once daily for 14 days, starting from day 1 of DSS modeling. All preparations and positive control drugs were prepared into a 50 mg / mL suspension with physiological saline (i.e., 0.5 mL containing 250 mg of the preparation) before use. The group assignments and administration regimens are as follows: Blank control group: Normal drinking water + daily gavage administration of 0.5 mL sterile saline Model control group: 3% DSS in drinking water + 0.5 mL of sterile saline by gavage daily. Study Group A: 3% DSS in drinking water + 0.5 mL of combined preparation A administered daily by gavage; Study Group B: 3% DSS in drinking water + 0.5 mL of combined preparation B administered daily by gavage; Study group C: 3% DSS in drinking water + 0.5 mL of combined preparation C administered daily by gavage; Study group D: 3% DSS in drinking water + 0.5 mL of combined preparation D administered daily by gavage; Study group E: 3% DSS in drinking water + 0.5 mL of combined preparation E administered daily by gavage; Positive control group: 3% DSS in drinking water + daily gavage administration of 0.5 mL mesalazine (Anjesa mesalazine enteric-coated tablets) 2.3.3 Disease Activity Index (DAI) The body weight, fecal characteristics, and fecal blood in each group of mice were observed and recorded at a fixed time each day (9:00-10:00), and scored according to the following criteria: Weight score: No decrease -- 0 points; decrease of 1-5% -- 1 point; decrease of 5-10% -- 2 points; decrease of 10-20% -- 3 points; decrease of more than 20% -- 4 points.

[0028] Stool characteristics score: Normal - 0 points; Loose stool - 1 point; Semi-formed loose stool - 2 points; Watery diarrhea - 3 points; Stool hematochezia scoring: No bleeding - 0 points; positive occult blood - 1 point; gross hematochezia - 2 points; severe hematochezia - 3 points; Among them, the fecal occult blood test uses the guaiac resin method (o-toluidine method): take a small amount of fresh stool, add 1 drop each of 1% o-toluidine glacial acetic acid solution and 3% hydrogen peroxide solution, and a blue color within 2 minutes indicates a positive result. DAI = Weight Loss Score + Stool Characteristics Score + Rectal Blood Score; The results are shown in Table 2.

[0029] As shown in Table 2, the DAI score of the model control group increased rapidly from day 3, reaching 5.7±0.8 on day 7, peaking at 6.3±0.9 on day 10, and although it dropped somewhat on day 14 due to the discontinuation of DSS, it still remained at 4.3±0.7, indicating that 3% DSS successfully induced acute enteritis in mice.

[0030] In study group A, the DAI score was significantly lower than that of the model control group at all time points (P<0.01). On day 14, the score dropped to 1.3±0.2, which was close to the level of the blank control group, with a reduction of 69.8%. Moreover, there was no statistically significant difference between the study group A and the positive drug group (1.5±0.2). This indicates that the efficacy of the combination preparation of the present invention is comparable to that of the first-line clinical drug mesalazine.

[0031] On day 14, the DAI scores of study groups B and C were 2.3 and 2.5, respectively, both significantly higher than those of study group A (P<0.01), indicating that the absence of any of the original bacteria significantly reduced the therapeutic effect, and there was a synergistic effect between the two original bacteria. On day 14, the DAI scores of study groups D (dual bacteria + wheat oligopeptide only) and E (dual bacteria + soybean peptide only) were 2.1 and 2.2, respectively, also significantly higher than those of study group A (P<0.01), indicating a synergistic effect between wheat oligopeptide and soybean peptide, and that the effect of using either peptide alone was not as good as the combination of the two peptides. Among study groups B and E, the DAI score for the group lacking one peptide was slightly better than that for the group lacking one bacteria, indicating that the integrity of the original bacteria combination had a greater impact on the therapeutic effect.

[0032] Table 2. Effects of the combined formulation on the disease activity index in mice (x̄±s, n=10)

[0033] 2.3.4 Intestinal permeability testing and colon length measurement On day 13, after fasting for 12 hours, mice were administered FITC-glucan solution (600 mg / kg, prepared with PBS, concentration 50 mg / mL) by gavage. Four hours later, 0.5 mL of blood was collected via the orbital venous plexus, anticoagulated with heparin, and centrifuged at 3000 rpm for 15 min to separate plasma. 100 μL of plasma was collected, and fluorescence intensity was measured using a fluorescence spectrophotometer (excitation wavelength 485 nm, emission wavelength 535 nm). A standard curve was plotted using FITC-glucan standards, and the plasma FITC-glucan concentration (μg / mL) was calculated.

[0034] On day 14, after fasting for 12 hours, the mice were euthanized by cervical dislocation. The abdomen was quickly opened, and the colon was completely separated from the anus to the cecal junction. The colon was washed in ice-cold saline and the length (cm) of the colon was measured. The results are shown in Table 3.

[0035] As shown in Table 3, the colon length in the model control group was shortened to 5.13±0.52cm, which was 39.8% shorter than that in the blank control group. The plasma FITC-glucan concentration was increased to 4.72±0.68μg / mL, which was 4.5 times higher than that in the blank control group. This indicates that DSS caused significant colon shortening and intestinal barrier damage.

[0036] In the study group, the colon length of A colon recovered to 7.72±0.38cm, with a recovery rate of 90.6%, and the intestinal permeability decreased to 1.48±0.28μg / mL, which was 68.6% lower than that of the model control group. Both indicators showed no statistically significant difference from the positive drug group, indicating that the combined preparation of the present invention can significantly repair the intestinal barrier function.

[0037] In study groups B and C, colon lengths recovered to 6.35 cm and 6.18 cm, respectively, and intestinal permeability was 2.85 μg / mL and 3.02 μg / mL, respectively, both significantly worse than in study group A (P<0.01). This indicates that the synergistic effect of the native bacteria is crucial for barrier repair. This may be because *Clostridium butyricum* produces butyrate, promoting colonic epithelial proliferation, while *Bifidobacterium longum* subsp. infantis produces acetic acid, enhancing immune defense. The absence of either one would result in insufficient barrier repair. In study groups D and E, colon lengths recovered to 6.55 cm and 6.42 cm, respectively, and intestinal permeability was 2.52 μg / mL and 2.68 μg / mL, respectively, also significantly worse than in study group A (P<0.01). This may be because wheat oligopeptides directly provide energy for mucosal repair through glutamine, while soybean peptides promote probiotic adhesion and colonization to maintain the barrier for longer. The synergistic effect of these two factors achieves a closed loop of repair and maintenance. In addition, when comparing intestinal permeability indicators, group D was better than group E, indicating that the mucosal repair effect of peptides made a significant contribution to the improvement of permeability.

[0038] Table 3. Effects of the combined formulation on intestinal permeability and colon length in mice (x̄±s, n=10)

[0039] 2.3.5 Inflammatory factor detection (ELISA) Approximately 100 mg of colon tissue was taken and added to 1 mL of pre-chilled RIPA lysis buffer. The mixture was homogenized on ice and centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was collected. The protein concentration was determined by the BCA method, and the protein concentration was adjusted to 2 mg / mL. The ELISA kit was operated according to the instructions: 100 μL each of the standard and the test sample were added to a 96-well plate and incubated at 37°C for 90 min; the plate was washed 4 times, 100 μL of biotinylated antibody working solution was added, and the plate was incubated at 37°C for 60 min; the plate was washed 4 times, 100 μL of enzyme conjugate working solution was added, and the plate was incubated at 37°C for 30 min; the plate was washed 4 times, 100 μL of TMB substrate was added, and the plate was developed in the dark at 37°C for 15 min; 100 μL of stop solution was added, and the absorbance was measured at 450 nm using a microplate reader; the concentrations of TNF-α, IL-6, and IL-1β were calculated based on the standard curve and expressed as pg / mg protein. The results are shown in Table 4.

[0040] As shown in Table 4, the levels of the three pro-inflammatory factors TNF-α, IL-6 and IL-1β in the model control group were 128.6, 156.8 and 89.5 pg / mg prot, respectively, which were 5.9 times, 6.0 times and 4.9 times higher than those in the blank control group, indicating that DSS induced a strong intestinal inflammatory response.

[0041] In study group A, the levels of the three pro-inflammatory factors decreased to 33.8, 40.2, and 24.5 pg / mg prot, respectively, representing reductions of 73.7%, 74.4%, and 72.6% compared to the model control group. These reductions were comparable to or even better than those in the positive control group, indicating that the combined formulation of this invention has a significant anti-inflammatory effect. In contrast, study group B (Clostridium butyricum only + dipeptide) showed levels of 72.5, 88.6, and 48.2 pg / mg prot, respectively, while study group C (Bifidobacterium longum subsp. infantis only + dipeptide) showed levels of 78.3, 95.2, and 52.6 pg / mg prot, respectively, all significantly higher than study group A (P<0.01). This suggests that the anti-inflammatory capacity of a single native bacterium is limited. This may be because Clostridium butyricum primarily inhibits the NF-κB pathway by butyrate to reduce TNF-α, while Bifidobacterium longum subsp. infantis primarily regulates IL-6 by acetic acid activating GPR43; the synergistic effect of these two factors is necessary to comprehensively inhibit the inflammatory cascade response. The levels of the three factors in study group D were 62.8, 76.5, and 42.3 pg / mg prot, respectively, while those in study group E were 68.5, 82.3, and 45.8 pg / mg prot, respectively. Both were significantly higher than those in study group A (P<0.01), indicating the necessity of synergistic anti-inflammatory effects of the dipeptides. This may be because wheat oligopeptides indirectly inhibit inflammation by reducing oxidative stress through glutamine, while soybean peptides directly inhibit the release of inflammatory mediators through bioactive peptides; the synergistic effect of both achieves a more comprehensive anti-inflammatory effect.

[0042] Table 4. Effects of the combined formulation on the levels of inflammatory factors in mouse colon tissue (x̄±s, n=10)

[0043] 2.3.6 Tight junction protein detection (Western blot) Take approximately 80 mg of colon tissue, add 800 μL of pre-cooled RIPA lysis buffer (containing PMSF), homogenize on ice, centrifuge at 12000 rpm for 15 min at 4°C, and collect the supernatant. Determine the protein concentration using the BCA method, adjust the protein concentration to 4 μg / μL with 5×SDS loading buffer, and boil at 100°C for 5 min to denature the protein.

[0044] (1) SDS-PAGE electrophoresis: Prepare 10% separating gel and 5% stacking gel, load 30 μg protein per well, electrophore at 80V constant voltage for 30 min, after entering the separating gel, switch to 120V constant voltage electrophoresis until bromophenol blue reaches the bottom; (2) Transfer membrane: Use wet transfer method, transfer membrane at 300mA constant current for 90 min to transfer protein to PVDF membrane; (3) Blocking: Block with 5% skim milk powder / TBST at room temperature for 2 h; (4) Primary antibody incubation: Add rabbit anti-occludin (1:1000) and rabbit anti-claudin-1 (1:1000) respectively. Rabbit anti-ZO-1 (1:1000) and rabbit anti-β-actin (1:5000) were incubated overnight at 4°C; (5) Washing: the membrane was washed 3 times with TBST for 10 min each time; (6) Secondary antibody incubation: HRP-labeled goat anti-rabbit IgG (1:5000) was added and incubated at room temperature for 1 h; (7) Washing: the membrane was washed 3 times with TBST for 10 min each time; (8) ECL chemiluminescence color development and gel imaging system were used to acquire images; (9) The gray values ​​of the bands were analyzed using ImageJ software, and the relative expression level of the target protein was calculated with β-actin as an internal reference. The results are shown in Table 5.

[0045] Table 5. Effects of the combined formulation on tight junction protein expression in mouse colon tissue (x̄±s, n=10)

[0046] As shown in Table 5, the relative expression levels of Occludin, Claudin-1, and ZO-1 in the model control group decreased to 0.28, 0.32, and 0.25, respectively, which were 72.5%, 67.3%, and 76.2% lower than those in the blank control group, indicating that DSS severely disrupted the expression of intestinal tight junction proteins.

[0047] In study group A, the expression levels of the three tight junction proteins recovered to 0.85, 0.88, and 0.80, respectively, with recovery rates of 83.3%, 89.8%, and 76.2%, comparable to or slightly better than the positive drug group, indicating that the combined formulation of this invention can effectively promote the restoration of tight junction protein expression. In contrast, the expression levels of the three proteins in study groups B and C only recovered to 0.50 / 0.48 (Occludin), 0.53 / 0.51 (Claudin-1), and 0.46 / 0.43 (ZO-1), respectively, all significantly lower than in study group A (P<0.01), suggesting that the synergistic effect of the native bacteria is indispensable for tight junction repair. This may be because butyrate produced by Clostridium butyricum inhibits and upregulates ZO-1 transcription via HDAC, while metabolites of Bifidobacterium longum subsp. infantileum promote the expression of Occludin and Claudin-1 via the TLR-MyD88 pathway. These two bacteria act on different targets of tight junctions, and their synergy enables comprehensive repair. Furthermore, the expression levels of the three proteins in study groups D and E recovered to 0.55 / 0.52 (Occludin), 0.58 / 0.55 (Claudin-1), and 0.50 / 0.48 (ZO-1), respectively, which were also significantly lower than those in study group A (P<0.01), indicating that the synergistic effect of the two peptides is equally important for tight junction repair. This may be because wheat oligopeptides provide glutamine substrates for epithelial cell proliferation, while soybean peptides promote probiotic colonization to continuously maintain tight junctions; the absence of either one results in incomplete repair. In addition, the recovery of ZO-1 was more difficult than that of Occludin and Claudin-1 in all groups, which may be because ZO-1, as a transmembrane junction backbone protein, requires a more complete microenvironment for synergistic microbial peptide repair.

[0048] 2.3.7 Gut microbiota analysis (16S rRNA high-throughput sequencing) After the mice were sacrificed on day 14, approximately 200 mg of cecal contents were aseptically collected from 6 mice, which were then flash-frozen in liquid nitrogen and stored at -80°C.

[0049] Qiagen DNA extraction kit was used. Primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') were used to amplify the V3-V4 region of the 16S rRNA gene. The PCR reaction mixture (25 μL) consisted of 10 ng template DNA, 12.5 μL 2×Taq Master Mix, and 0.5 μM each of forward and reverse primers. PCR program: 95°C for 3 min; 95°C for 30 s, 55°C for 30 s, 72°C for 45 s, for a total of 30 cycles; 72°C for 10 min; PCR products were detected by 2% agarose gel electrophoresis, and purified by gel extraction; PCR products from each sample were mixed in equimolar ratio and subjected to paired-end sequencing (2×300 bp) on the Illumina MiSeq platform; the original sequences were quality controlled and chimeras removed using the QIIME2 platform, clustered into OTUs with 97% similarity, and species were annotated based on the Silva database. Alpha diversity was assessed by calculating the Chao1 index, Shannon index, and Simpson index; phylum-level species composition analysis was performed using the R language vegan package, and the results are shown in Tables 6 and 7.

[0050] As shown in Table 6, the Chao1 index, Shannon index, and Simpson index in the model control group decreased by 47.4%, 44.7%, and 29.3% respectively compared with the blank control group, indicating that DSS caused a serious loss of gut microbiota diversity.

[0051] In the study group, the Chao1 index recovered to 332.5 (recovery rate 94.3%), the Shannon index to 4.35 (recovery rate 89.7%), and the Simpson index to 0.88 (recovery rate 95.7%). All three indicators were significantly better than the model control group (P<0.01), and there was no statistically significant difference compared to the positive drug group, indicating that the combined formulation of this invention can effectively restore intestinal flora diversity. However, the Chao1 index in the study group (BC, 258.3) and in the study group C (265.8) were significantly lower than in the study group A (P<0.01), indicating that the synergistic effect of the native bacteria is crucial for the restoration of flora diversity. This may be because butyric acid produced by Clostridium butyricum lowers the intestinal pH, creating an acidic environment that promotes the proliferation of various anaerobic bacteria; and Bifidobacterium longum subsp. infantis inhibits pathogenic bacteria through occupancy effects and nutrient competition. Only through the synergy of both can the flora's ecological niche be fully restored. The Chao1 index of study group D (285.6) and study group E (278.2) were significantly lower than those of study group A (P<0.05), indicating that the synergistic effect of the two peptides also contributed to the restoration of the gut microbiota. This may be because wheat oligopeptides provide nitrogen and carbon sources for gut symbiotic bacteria, promoting their proliferation, while soybean peptides enhance the colonization of probiotics, enabling them to continuously exert their role in gut microbiota regulation. The two complement each other to achieve a more comprehensive gut microbiota reconstruction. In addition, study group D was slightly better than study group B (3.42, 0.76) in the Shannon index (3.82) and Simpson index (0.82), with statistically significant differences (P<0.05). This indicates that the peptide combination has a more significant effect on improving gut microbiota homogeneity than the bacterial combination. This may be related to the fact that wheat oligopeptides provide nutrition for multiple symbiotic bacteria (rather than just native bacteria).

[0052] Table 6. Effects of the combined formulation on the Alpha diversity index of mouse gut microbiota (x̄±s, n=6)

[0053] As shown in Table 7, the relative abundance of Proteobacteria in the model control group increased to 28.5%, which was 7.9 times higher than that in the blank control group. Firmicutes decreased to 38.5% and Bacteroidetes decreased to 22.6%, showing typical characteristics of dysbiosis—the expansion of pathogenic Proteobacteria and the reduction of beneficial bacteria.

[0054] In study group A, the abundance of Proteobacteria decreased to 6.5%, a 77.2% reduction compared to the model control group, approaching the level of the blank control group; Firmicutes recovered to 50.8%, and Bacteroidetes recovered to 33.5%, indicating that the phylum-level structure of the bacterial community had basically returned to normal, demonstrating the significant effect of the combined formulation of this invention on the restoration of bacterial community structure. In contrast, the abundance of Proteobacteria in study group B remained at 16.5%, and in study group C at 14.8%, both significantly higher than in study group A (P<0.01), indicating that the native bacteria combination has a synergistic effect in inhibiting the proliferation of pathogenic bacteria. This may be because butyric acid production by Clostridium butyricum directly inhibits the proliferation of facultative anaerobes such as Escherichia coli in Proteobacteria, while Bifidobacterium longum subsp. infantis inhibits pathogenic bacteria colonization through competitive adhesion sites and acid production; the synergy of both is necessary to effectively reduce the abundance of Proteobacteria. Furthermore, the abundance of Proteobacteria in study group D (10.5%) and study group E (11.2%) was also significantly higher than in study group A (P<0.05), indicating that the synergistic effect of the dipeptide is equally important in inhibiting pathogenic bacteria. This may be because wheat oligopeptides promote the proliferation of beneficial bacteria (such as Lactobacillus) in Firmicutes, thereby competitively inhibiting Proteobacteria; soybean peptides enhance the adhesion and colonization of native bacteria, forming a stable biofilm barrier to prevent the invasion of pathogenic bacteria. The synergistic effect of both is the most effective way to inhibit Proteobacteria expansion. Secondly, there was no significant difference in the abundance of Actinobacteria among the groups, indicating that DSS has a relatively small impact on Actinobacteria. The microbial regulation effect of the combined formulation of this invention is mainly reflected in restoring the Firmicutes / Bacteroidetes balance and inhibiting Proteobacteria expansion.

[0055] Table 7. Effects of the combined formulation on the relative abundance of gut microbiota at the phylum level in mice (x̄±s, n=6)

[0056] 2.3.8 Histopathological examination (HE staining) Colon tissue approximately 1 cm long was taken from 1-2 cm from the anus, fixed in 4% paraformaldehyde for 24 h, routinely dehydrated, embedded in paraffin, serially sectioned at 5 μm, stained with hematoxylin and eosin (HE), mounted with neutral resin, and observed and photographed under a microscope.

[0057] The case evaluation criteria are as follows: Inflammation infiltration score: No infiltration - 0 points; slight infiltration of the mucosal layer - 1 point; significant infiltration of the mucosal layer - 2 points; infiltration extending to the submucosa - 3 points; infiltration reaching the muscular layer - 4 points.

[0058] Crypt destruction score: Normal -- 0 points; 1 / 3 of the crypt base damaged -- 1 point; 2 / 3 of the crypt base damaged -- 2 points; Only the mucosal surface epithelium intact -- 3 points; The entire crypt and epithelium are destroyed -- 4 points.

[0059] Ulcer scoring: No ulcer - 0 points; Mucosal erosion - 1 point; Ulcer area <10% - 2 points; Ulcer area 10-25% - 3 points; Ulcer area >25% - 4 points.

[0060] The total pathological score is calculated as follows: Inflammation infiltration score + crypt destruction score + ulcer score.

[0061] The results are shown in Table 8.

[0062] Table 8. Effects of the combined formulation on the histopathological score of mouse colon tissue (x̄±s, n=10)

[0063] As shown in Table 8, the total pathological score of the model control group was 8.5±1.2 points, including 3.2 points for inflammatory infiltration, 2.8 points for crypt destruction, and 2.5 points for ulceration, indicating that DSS caused severe damage to the entire colon tissue.

[0064] The total pathological score in study group A decreased to 1.7, a reduction of 80.0% compared to the model control group, and the difference was statistically significant compared to the positive drug group (study group A was even lower), indicating that the combined preparation of this invention is superior to the first-line clinical drug mesalazine in terms of tissue pathological repair. HE staining and microscopic observation showed that the colonic mucosal epithelium in study group A was basically intact, with regular crypt structures and only a small amount of inflammatory cell infiltration. In contrast, the total pathological scores of study groups B and C were significantly higher than those of study group A (P<0.01), and microscopic examination revealed incomplete mucosal epithelium, partial loss of crypts, and significant inflammatory cell infiltration, indicating that a single native bacterium cannot achieve sufficient tissue repair. Butyric acid from Clostridium butyricum promotes epithelial proliferation and repairs crypts and ulcers, while Bifidobacterium longum subsp. infantis regulates immunity and reduces inflammatory infiltration; these two mechanisms must work synergistically. The total pathological scores of study groups D and E were also significantly higher than those of study group A (P<0.01), and microscopic examination revealed irregular crypt structures and mild erosion on the mucosal surface, indicating that a single peptide cannot simultaneously meet the dual needs of tissue repair and colonization maintenance. This may be because wheat oligopeptides promote crypt regeneration by providing energy, while soybean peptides maintain the bacterial membrane barrier and prevent ulcer recurrence by promoting adhesion; the complementary effects of these two components achieve optimal tissue repair. Furthermore, study group A scored only 0.4 on the ulcer score, lower than those with inflammatory infiltration and crypt destruction, indicating that the combined formulation of this invention is particularly effective in promoting ulcer healing.

[0065] 3. Effect of combined formulation on the survival rate of probiotics in simulated gastrointestinal fluid The combined formulation is the same as that of research group A. The only difference in the preparation method of the combined formulation is whether or not the bacterial powder is encapsulated.

[0066] 3.2 Preparation of simulated gastric juice Dissolve 2.0g of NaCl in 80mL of distilled water, adjust the pH to 2.0 with hydrochloric acid, dissolve 3.2g of pepsin, and bring the volume to 1000mL with distilled water.

[0067] 3.3 Preparation of Simulated Intestinal Fluid Dissolve 6.8g of KH2PO4 in 500mL of distilled water, adjust the pH to 6.8 with NaOH, dissolve 10.0g of trypsin, and bring the volume to 1000mL with distilled water.

[0068] 3.4 Experimental Methods Take 1.0 g of each of the embedded and unembedded bacterial powders and add them to 100 mL of simulated gastric fluid. Incubate at 37°C with shaking at 100 rpm for 2 h. After 2 h of treatment with simulated gastric fluid, take 5 mL of each suspension. For the unembedded bacterial powder group, dilute it serially with 0.85% NaCl solution and directly spread it on the corresponding selective culture medium plates for counting. Calculate the survival rate of the simulated gastric fluid. For the embedded bacterial powder group, add 10 mL of 55 mmol / L sodium citrate solution to dissolve the microcapsule wall, incubate at 37°C for 30 min to completely disintegrate the microcapsules, and then dilute it serially, spread it on plates for counting. Calculate the survival rate of the simulated gastric fluid. After centrifuging the remaining simulated gastric fluid suspensions from each group at 4000 r / min for 10 min, the precipitate was collected and transferred to 100 mL of simulated intestinal fluid. The suspensions were then treated with shaking at 100 r / min in a 37°C constant temperature water bath for 4 h. 5 mL of the suspensions from each group were taken. For the unencapsulated bacterial powder group, the suspensions were directly serially diluted and plated for counting. For the encapsulated bacterial powder group, 10 mL of 55 mmol / L sodium citrate solution was added to dissolve the microcapsule walls. The microcapsules were then completely disintegrated in a 37°C water bath for 30 min. The suspensions were then serially diluted and plated for counting. The overall survival rate of the simulated gastrointestinal fluid sequential treatment was calculated, and the results are shown in Table 9.

[0069] Survival rate (%) = Number of viable bacteria after treatment / Initial number of viable bacteria × 100%.

[0070] Table 9. Effect of encapsulated and unencapsulated bacterial powders on the survival rate in simulated gastrointestinal fluid (x̄±s, n=3)

[0071] As shown in Table 9, the survival rate of unencapsulated bacterial powder after 2 hours of treatment in simulated gastric fluid was only 35.2%. After sequential treatment with simulated gastric fluid for 2 hours and simulated intestinal fluid for 4 hours, the survival rate further decreased to 22.5%. This indicates that the survival rate of naked bacterial powder in the gastrointestinal environment is extremely low. The vast majority of live bacteria are killed in gastric acid and cannot effectively reach the intestine to exert colonization and regulatory functions.

[0072] After encapsulation with sodium alginate microcapsules, the 2-hour survival rate in simulated gastric fluid increased to 82.7%, a 47.5 percentage point increase compared to the unencapsulated group; the survival rate after sequential treatment with simulated gastrointestinal fluid was 73.8%, with both differences being highly statistically significant (P<0.01). This may be because sodium alginate and calcium chloride form an alginate-calcium gel network, encapsulating the bacteria within the microcapsules. The gel wall then acts as a physical barrier against gastric acid and pepsin, delaying the absorption of H2S. +Diffusion and contact between enzymes and bacteria; after entering the intestinal fluid, calcium alginate is gradually degraded under the action of phosphate and pancreatic enzymes, achieving slow release of bacteria and ensuring that live bacteria reach the intestine.

[0073] The survival rate data shows that microcapsule encapsulation significantly increased the number of probiotics that effectively reached the intestines, providing a viable bacterial basis for subsequent colonization, acid production, and barrier repair. This also demonstrates the necessity and effectiveness of the microcapsule encapsulation process of this invention.

[0074] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A combination preparation of native bacteria and peptides for improving gastrointestinal health, characterized in that, It is prepared from the following raw materials by weight: 5-15 parts of Clostridium butyricum powder, 5-15 parts of Bifidobacterium longum subsp. infantis powder, 20-40 parts of wheat oligopeptides, 15-30 parts of soybean peptides, 5-15 parts of fructooligosaccharides and 2-5 parts of sodium alginate.

2. The combined bacterial and peptide preparation for improving gastrointestinal health according to claim 1, characterized in that, By weight, it is prepared from the following raw materials: 10 parts of Clostridium butyricum powder, 10 parts of Bifidobacterium longum subsp. infantis powder, 30 parts of wheat oligopeptides, 20 parts of soybean peptides, 10 parts of fructooligosaccharides and 3 parts of sodium alginate.

3. The combined bacterial and peptide preparation for improving gastrointestinal health according to claim 1, characterized in that, The viable counts of both Clostridium butyricum powder and Bifidobacterium longum subsp. infantis powder are not less than 1.0 × 10⁻⁶. 10 cfu / g.

4. A method for preparing a combined preparation of native bacteria and peptides for improving gastrointestinal health, as described in any one of claims 1-3, characterized in that, Specifically, the mixture of Clostridium butyricum powder and Bifidobacterium longum subsp. infantis powder is mixed evenly with sodium alginate solution, and then added dropwise into calcium chloride solution to solidify for 20-30 minutes to form microcapsules. After collection, the microcapsules are vacuum dried to obtain the encapsulated bacterial powder. The encapsulated bacterial powder is then mixed evenly with wheat oligopeptides, soybean peptides, and fructooligosaccharides to obtain the original bacterial and peptide combination preparation for improving gastrointestinal health.

5. The preparation method according to claim 4, characterized in that, The sodium alginate solution has a mass concentration of 2%, and the calcium chloride solution has a mass concentration of 3%.

6. The use of the native bacteria-peptide combination preparation according to any one of claims 1-3 in the preparation of products for improving gastrointestinal health.

7. The application according to claim 6, characterized in that, The improvement of gastrointestinal health includes repairing the intestinal mucosal barrier, regulating the balance of intestinal flora, reducing intestinal inflammation, and enhancing intestinal immune function.

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