Compound probiotics capable of relaxing bowels and improving immunity of intestinal tracts and application of compound probiotics

Abstract

The invention relates to the technical field of microorganisms, in particular to compound probiotics for relaxing bowel and improving intestinal immunity and application thereof. The compound probiotics comprise a plant lactobacillus BXM2 strain with the preservation number of CGMCC (China General Microbiological Culture Collection Center) NO.16436 and a bifidobacterium longum subsp. Longum YYS-BSM8 strain with the preservation number of CGMCC NO.28611, and the ratio of the viable count of the plant lactobacillus BXM2 to the viable count of the bifidobacterium longum subsp. Longum YYS-BSM8 is (1-10): (1-10). After the two bacteria are compounded and combined, the gastrointestinal motility efficiency of zebra fish is accelerated, constipation is relieved, enteropathogenic bacteria are inhibited, the adhesive force of intestinal cells is improved, exopolysaccharides are produced at high yield, proliferation and phagocytic activity of macrophages are promoted, oxidative damage is remarkably relieved, and synergistic interaction is achieved in the aspects of improving the intestinal motility efficiency, relieving constipation and improving body immunity; the important application value is realized.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a composite probiotic capable of moistening the intestines, promoting bowel movements and improving intestinal immunity and an application thereof. Background Art

[0002] Constipation is a common digestive system disease that is prevalent in the population, with an incidence rate of 2%-28%. The prevalence rate increases significantly with age, especially for elderly women and pregnant women, who are at high risk. Constipation can be divided into organic constipation and functional constipation. The former is caused by organic lesions such as the rectum and anus, while the latter is mainly caused by bad living habits such as bowel habits, eating habits and mental stress. The main symptoms of constipation include reduced bowel movements, dry and hard stools, and difficulty in defecation, usually accompanied by abdominal distension and pain, loss of appetite, fatigue and weakness, which seriously affect normal life. At present, the treatment of constipation mainly focuses on changing lifestyle and drug therapy, such as increasing dietary fiber intake, increasing water intake and exercise, while drug therapy mainly includes taking laxatives, such as wheat cellulose, etc. However, long-term use of such irritant laxatives may damage the intestinal mucosa, and it is easy to relapse after stopping the drug, and it cannot fundamentally solve the problem.

[0003] The intestine is not only an important organ for digestion and absorption, but also the largest immune organ in the body. The intestinal barrier plays a key role in maintaining intestinal health, and its main function is to protect the host from harmful antigens and toxic substances in the intestine. More and more studies have shown that probiotics play an important role in regulating the host's intestinal microbial barrier, chemical barrier, physical barrier and immune barrier.

[0004] Probiotics have a positive impact on intestinal function, mainly involving the regulation of intestinal microecology, the enhancement of the immune system, and the maintenance of the mucosal barrier. Probiotics can improve the body's ability to fight infection and inflammation by activating immune cells and enhancing immune responses. A strong immune system helps maintain the integrity of the intestinal barrier and reduce the invasion of pathogenic microorganisms on the intestinal mucosa, while the good adhesion ability of the strain can promote the strain's immune regulation of the host and the repair of damaged intestinal mucosa.

[0005] The exopolysaccharides produced by lactic acid bacteria are involved in the formation of biofilms and also contribute to the interaction between probiotics and other microorganisms. Related studies have shown that exopolysaccharides help antagonize pathogens, activate macrophages, promote T cell proliferation and inhibit the expression of TNF-α. In addition, exopolysaccharides can also regulate the expression of p53 and activate Caspase-3, thereby killing tumor cells. Therefore, the exopolysaccharides produced by lactic acid bacteria may be an important part of their health effects.

[0006] Therefore, how to provide a probiotic that can regulate intestinal function, improve constipation and produce extracellular polysaccharides is a technical problem that technical personnel in this field urgently need to solve. Summary of the invention

[0007] In order to solve the deficiencies of the prior art mentioned in the above background technology, the present invention provides a composite probiotic for moistening the intestines and promoting bowel movements and improving intestinal immunity, including Lactobacillus plantarum ( Lactiplantibacillus plantarum ) BXM2 strain, whose deposit number is CGMCC NO.16436, and Bifidobacterium longum subsp. longum ( Bifidobacterium longum subsp.Longum ) YYS-BSM8 strain, whose preservation number is CGMCC No.28611, and the ratio of the number of viable bacteria of Lactobacillus plantarum BXM2 and Bifidobacterium longum subspecies longum YYS-BSM8 is 1-10:1-10.

[0008] The present invention also provides a composite probiotic agent, the components of which include the composite probiotics for moistening the intestines and promoting bowel movements and improving intestinal immunity as described above.

[0009] In one embodiment, the viable bacteria count in the composite probiotic is ≥ 6.5×10 9 CFU / g, wherein the ratio of the viable counts of Lactobacillus plantarum BXM2 and Bifidobacterium longum subspecies longum YYS-BSM8 is 1-3:1-3.

[0010] Preferably, in the composite probiotic, the ratio of the number of viable bacteria of Lactobacillus plantarum BXM2 to that of Bifidobacterium longum subspecies longum YYS-BSM8 is 1:1.

[0011] In one embodiment, the composite probiotic further comprises a lyophilization protectant, and the lyophilization protectant comprises any one or more combinations of skimmed milk powder, maltodextrin, sodium glutamate, sucrose, lactose, trehalose, sorbitol and xylitol.

[0012] In one embodiment, the composite probiotic further comprises auxiliary additives, and the auxiliary additives comprise any one or more combinations of fructooligosaccharides, galacto-oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soy oligosaccharides, inulin, spirulina, stachyose, polydextrose, α-lactalbumin or lactoferrin.

[0013] In one embodiment, the composite probiotic further comprises excipients, which include any one or more combinations of prebiotics, fillers, acidulants, solvents, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, stabilizers, glidants, flavoring agents, preservatives, coating materials, fragrances, anti-adhesive agents, integrators, thickeners, buffers, and inclusion agents.

[0014] In one embodiment, the dosage form of the composite probiotic includes lyophilized powder, capsule, tablet or granule.

[0015] The composite probiotic provided by the present invention has at least one of the following effects: (1) Reduce oxidative damage; (2) Producing extracellular polysaccharides; (3) Resistant to gastric acid and bile salts; (4) Improve intestinal immunity; (5) Lubricates the intestines and relieves constipation.

[0016] The present invention also provides a probiotic solid beverage, the components of which include the composite probiotic agent as described above.

[0017] The present invention also provides a fermented beverage, the components of which include the composite probiotic agent as described above.

[0018] The present invention also provides a use of the composite probiotic agent as described above in the preparation of food, health products or medicines for relieving or treating constipation and improving intestinal immunity.

[0019] Compared with the prior art, the composite probiotics provided by the present invention for moistening the intestines and promoting bowel movements and improving intestinal immunity have the following beneficial effects: Compared with the use of a single BXM2 strain or a single YYS-BSM8 strain, the two bacteria have synergistic effects after compounding, and the number of live bacteria is significantly increased after mixed culture of the two. When the two bacteria are used together, the inhibitory effect on intestinal pathogens and the adhesion to intestinal cells are significantly enhanced, and the production of extracellular polysaccharides, macrophage proliferation and phagocytic activity, as well as the free radical scavenging rate are significantly improved. It also has the effect of significantly promoting gastrointestinal motility efficiency and improving constipation. It can be used in food, health products or medicines to effectively prevent or improve constipation and enhance intestinal immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The growth curves of BXM2, YYS-BSM8 and the composite bacteria of Example 1 provided by the present invention; Figure 2 The viable counts of BXM2, YYS-BSM8 and the composite bacteria of Example 1 provided by the present invention; Figure 3The size of the inhibition zone of each strain of Example 2 provided by the present invention for inhibiting intestinal pathogens; Figure 4 The number of probiotics adhered to every 100 HT-29 cells of Example 3 provided by the present invention; Figure 5 The glucose standard curve in the determination of the extracellular polysaccharide content in Example 5 provided by the present invention; Figure 6 The free radical scavenging rates of BXM2, YYS-BSM8 and the composite bacteria of Example 7 provided by the present invention; Figure 7 This is a fluorescence distribution diagram of zebrafish intestine of each strain of Example 8 provided by the present invention; Figure 8 The fluorescence intensity of zebrafish intestine of each strain in Example 8 provided by the present invention. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] The classification name of the Lactobacillus plantarum BXM2 strain involved in the following examples is Lactobacillus plantarum, Latin name: Lactiplantibacillus plantarum The depository is the General Microbiology Center of China Microbiological Culture Collection Administration Committee. The deposit date is September 6, 2018. The deposit number is CGMCC No.16436. The deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Note: This strain is also known as Lactobacillus plantarum ( Lactobacillus plantarum )BXM2, due to the changes in the new and old catalogs of bacterial species classification names, two different names appear, both referring to the same essence.

[0024] The classification name of the Bifidobacterium longum subspecies longum YYS-BSM8 strain involved in the following examples is Bifidobacterium longum subspecies longum, Latin name: Bifidobacterium longum subsp.Longum The depository is the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit date is October 12, 2023. The deposit number is CGMCCNo.28611. The deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0025] The formula of the MRS liquid culture medium involved in the following examples is: beef extract 10.0 g, glucose 20.0 g, tryptone 10.0 g, yeast extract powder 5.0 g, Tween 80 1.0 mL, dipotassium hydrogen phosphate 2.0 g, ammonium citrate 2.0 g, anhydrous sodium acetate 5.0 g, magnesium sulfate 0.5 g, manganese sulfate monohydrate 0.25 g, deionized water 1.0 L, pH 6.5 (1.5% agar is added to make MRS solid culture medium, and 0.05% L-cysteine ​​hydrochloride is added for culturing bifidobacteria).

[0026] The preparation method of the composite probiotics involved in the following embodiments can be prepared by conventional technical methods in the art, for example: after activating Lactobacillus plantarum BXM2 and Bifidobacterium longum subspecies YYS-BSM8 strains, they are inoculated in MRS culture medium to obtain culture solution, the culture solution is centrifuged, the bacteria are resuspended to obtain a bacterial suspension, and the two bacterial suspensions are mixed according to the ratio of viable bacteria of 1:1 to obtain composite probiotics; or further add a lyophilization protective agent for freeze drying to obtain a composite probiotic agent; a single bacterial agent can be obtained in the same way. It should be noted that the ratio of viable bacteria can be selected according to other actual ratios, for example, the ratio of viable bacteria of Lactobacillus plantarum BXM2 and Bifidobacterium longum subspecies YYS-BSM8 strains can be any one of 1:2, 1:3, 2:1, 2:3, 3:1, and 3:2.

[0027] Example 1 Synergistic symbiosis of BXM2 and YYS-BSM8 BXM2 and YYS-BSM8 strains were activated to the second generation of bacterial liquid using MRS liquid medium, and inoculated into MRS liquid medium supplemented with 0.05% L-cysteine ​​at a ratio of 1:1 at a 3% inoculation rate for mixed culture. At the same time, BXM2 and YYS-BSM8 were inoculated at a 3% inoculation rate for separate culture at 37°C for 24 h, and the growth curve was determined ( Figure 1 ), and the final live bacteria count was performed according to the plate pouring method. The results are shown in Figure 2 .

[0028] It can be seen that the mixed culture of BXM2 and YYS-BSM8 has a higher viable count than that of single culture, indicating that BXM2 and YYS-BSM8 have potential synergistic symbiosis.

[0029] Example 2 Antagonism against intestinal pathogens The punching method was used to determine the inhibitory effect of the strain on three common intestinal pathogens (Staphylococcus aureus, Escherichia coli, and Shigella sonnei). BXM2, YYS-BSM8, and a mixture of the two were cultured in MRS medium to the second generation of bacterial solution for use. The three pathogens were cultured in LB liquid medium for 24 hours, and 100 μL of each was evenly spread on the LB solid plate medium. Then, three holes were evenly punched using a puncher with a diameter of 8 mm. 200 μL of BXM2, YYS-BSM8, and composite bacterial culture solution were added to the three holes, respectively. The plate was placed at 37°C for anaerobic culture. The size of the inhibition zone (unit: mm) was measured after 36 hours. The experiment was repeated three times, and the results are shown in the table. Figure 3 It can be seen that the three bacterial cultures have good inhibitory effects on the three intestinal pathogens, among which the size of the inhibition zone of the composite bacterial culture solution is higher than that of the single bacterial culture solution, indicating that BXM2 and YYS-BSM8 also synergize in antibacterial ability.

[0030] Example 3 Intestinal cell adhesion HT-29 cells were activated and cultured in DMEM medium containing 10% heat-inactivated fetal bovine serum and 1% double antibodies (streptomycin and penicillin). After culturing to the logarithmic phase, the cells were counted and resuspended in DMEM medium containing 10% heat-inactivated fetal bovine serum and 1% double antibodies to a concentration of 1×10 5 The cells were inoculated into a 6-well plate (2 mL) with an acid-washed sterilized coverslip, cultured for 12 h, and washed three times with sterile PBS. The experimental strain was cultured at 37°C for 18 h, and the Lactobacillus cells were harvested by centrifugation at 8000×g, 4°C for 5 min, and washed twice with sterile PBS. The cells were resuspended in DMEM medium to 5×10 7 CFU / mL, and inoculated into 6-well plates (2 mL), incubated at 37°C for 2 h, discarded the culture medium, and washed the cells 3 times with sterile PBS. Add 2 mL of methanol to each well and fix at room temperature for 1 h. After fixation, discard the methanol and stain with crystal violet solution. Examine the coverslips in the 6-well plates under a microscope and count the number of bacteria adhered per 100 HT-29 cells ( Figure 4 ).

[0031] The results showed that the average number of BXM2 adhered to every 100 HT-29 cells was 61, the average number of YYS-BSM8 adhered to every 100 HT-29 cells was 55, and the average number of composite bacteria adhered to every 100 HT-29 cells was 67, indicating that each strain has good intestinal cell adhesion, and the adhesion effect of composite bacteria is significantly higher than that of single strains.

[0032] Example 4 Heat, acid and bile resistance of bacterial agents 1. Heat resistance BXM2, YYS-BSM8 and their composite were inoculated into MRS liquid medium at 2% (w / v) and cultured at 40°C and 50°C for 2 h, respectively. The plate count method was used to count the viable bacteria (LogCFU / mL). The results are shown in Table 1.

[0033] Table 1 Study on heat resistance

[0034] As shown in Table 1, there was no significant difference in the survival rate of BXM2, YYS-BSM8 and the composite bacteria after being cultured at 40℃ and 50℃ for 2 h.

[0035] 2. Acid and bile salt resistance BXM2, YYS-BSM8 and their composite were inoculated at 2% (w / v) at pH 4.0, pH 3.0, pH 2.0 and 0.2% bile salt concentration at 37°C for 2 h and then the plate count method was used to count the viable bacteria (Log CFU / mL). The results are shown in Table 2.

[0036] Table 2 Study on acid resistance and bile salt resistance

[0037] As can be seen from Table 2, under the condition of pH 2.0, each strain still had a sufficient number of live bacteria to pass through the gastric environment after 2 h, and had good bile salt tolerance at a bile salt concentration of 0.2%.

[0038] 3. Tolerance in artificial gastric juice and artificial intestinal juice A certain amount of pepsin was dissolved in a PBS buffer solution at pH 3.0 and its final concentration was adjusted to 3.0 g / L to obtain simulated artificial gastric juice. A certain amount of trypsin was dissolved in a PBS buffer solution at pH 8.0 and its final concentration was adjusted to 1.0 g / L to obtain simulated artificial intestinal juice. Both artificial gastric juice and artificial intestinal juice were filtered with a 0.22 μm filter membrane under sterile conditions.

[0039] BXM2, YYS-BSM8 and their composite were inoculated into artificial gastric fluid at a 2% (w / v) inoculation rate and cultured at 37°C for 2 h. Then, the fermentation broth in artificial gastric fluid was inoculated into artificial intestinal fluid at a 10% inoculation rate and cultured at 37°C for 2 h. The plate count method was used to count the viable bacteria (Log CFU / mL). The results are shown in Table 3.

[0040] Table 3 Tolerance of bacterial agents in artificial gastric juice and artificial intestinal juice

[0041] As can be seen from Table 3, after passing through artificial gastric juice and then artificial intestinal juice, BXM2, YYS-BSM8 and the composite bacteria were well tolerated and could successfully colonize in the intestine and exert their efficacy.

[0042] Example 5 Production of exopolysaccharides 1.1 Reagent preparation Concentrated sulfuric acid solution: Accurately measure 82 mL of concentrated sulfuric acid, add it to 18 mL of water, and set aside after cooling.

[0043] Anthrone solution: Accurately weigh 0.2 g of anthrone and add 100 mL of 82% concentrated sulfuric acid solution to dissolve it.

[0044] 1.2 EPS content determination Anthrone colorimetry was used to quantify EPS production. Prepare glucose standard sample solutions of 10, 20, 40, 60, 80, and 100 μg / mL. Pipette 1 mL of standard sample of different concentrations and add it to a 10 mL stoppered colorimetric tube with pure water as a control. Add 4 mL of anthrone solution, shake well, and cool in an ice water bath. Move to a boiling water bath and heat for 10 min, cool with ice water, and place at room temperature away from light for 10 min. Measure the absorbance at a wavelength of 620 nm and draw a standard curve ( Figure 5 ).

[0045] The experimental strain was cultured at 37°C for 18 h, then expanded and centrifuged at 4°C at 12,000×g for 10 min. The supernatant was taken, 80% (v / v) trichloroacetic acid was added to a final concentration of 4%, and the mixture was allowed to stand at 4°C for 30 min, centrifuged at 8,000×g for 10 min, and the protein precipitate was discarded. The obtained supernatant was mixed with anhydrous ethanol at a ratio of 1:3, allowed to stand at 4°C for 12 h, centrifuged at 8,000×g for 10 min, and the polysaccharide precipitate was taken. 2 mL of pure water was taken to re-dissolve the polysaccharide precipitate, and the EPS content was determined according to the standard curve method. The EPS content is expressed as mg / mL.

[0046] Table 4 EPS production of each strain

[0047] The results showed that the polysaccharide production of BXM2 strain was about 1.14±0.18 mg / mL, the polysaccharide production of YYS-BSM8 was about 2.09±0.25 mg / mL, and the polysaccharide production of the composite bacteria was about 2.61±0.33 mg / mL. In addition, compared with the single bacteria, the composite bacteria had a higher polysaccharide production ( P <0.01).

[0048] Example 6 Effects of strains on the proliferation and phagocytic ability of macrophages RAW264.7 Phagocytosis is an important defense mechanism of vertebrates against pathogen invasion, and enhanced phagocytosis is a prominent feature of macrophage activation.

[0049] (1) CCK-8 assay to determine the effect of strains on RAW264.7 cell proliferation RAW264.7 cells were inoculated into DMEM medium containing 10% heat-inactivated fetal bovine serum (BI) and 1% double antibodies (streptomycin and penicillin) for activation and passage. After culturing to the logarithmic phase, the cells were counted and resuspended in DMEM medium containing 10% heat-inactivated fetal bovine serum (BI) and 1% double antibodies (streptomycin and penicillin) to a cell concentration of 1×10 5 Inoculate 100 μL of RAW264.7 cells in a 96-well cell culture plate, and add new DMEM medium to replace the original culture medium after the cells adhere to the wall.

[0050] The experiment set up a blank group (DMEM culture medium) and an experimental group (BXM2, YYS-BSM8 and mixed bacteria, the concentration of the bacterial solution was 10 8 CFU / mL), with 3 replicates per group. 37°C, 5% CO 2 After the culture was completed, the old culture medium was removed and 100 μL of DMEM culture medium containing 10% CCK-8 was added to each well. The well was protected from light and placed in a CO 2 The cells were cultured in the incubator for 30 min. The OD value of each well was detected at 450 nm using an enzyme-labeled instrument to calculate the relative cell proliferation rate.

[0051] Relative cell proliferation rate%=OD value of experimental group / OD value of blank group.

[0052] (2) The neutral red method was used to determine the effect of the strain on the phagocytic ability of RAW264.7 cells.

[0053] RAW264.7 cells were inoculated into DMEM medium containing 10% heat-inactivated fetal bovine serum (BI) and 1% double antibodies (streptomycin and penicillin) for activation and passage. After culturing to the logarithmic phase, the cells were counted and resuspended in DMEM medium containing 10% heat-inactivated fetal bovine serum (BI) and 1% double antibodies (streptomycin and penicillin) to a cell concentration of 1×10 5 100 μL RAW264.7 cells were inoculated into a 96-well cell culture plate, and new DMEM medium was added to replace the original culture medium after the cells adhered to the wall. The experimental grouping was the same as in Example 6 (1), with 3 replicates per group. 37°C, 5% CO 2 The cells were cultured in an incubator for 24 h. The supernatant culture medium in the wells was discarded, and then 0.1% neutral red-PBS solution was added under light-proof conditions and placed at 37°C and 5% CO2 Incubate in the incubator for 1 hour; after the incubation, discard the supernatant and wash three times with PBS at room temperature. Then add lysis solution (acetic acid: anhydrous ethanol = 1:1) to each well and keep it in the dark. After the cells are completely lysed, use an enzyme reader to measure the absorbance value of each well at a wavelength of 540nm.

[0054] Cell phagocytosis index = OD value of experimental group / OD value of blank group.

[0055] Table 5 Effects of various strains on the proliferation and phagocytic ability of macrophage RAW264.7 cells

[0056] The results are shown in Table 5. Each strain can promote the proliferation and phagocytic activity of RAW264.7 cells and effectively play an immunomodulatory role. The proliferation and phagocytic activity of the composite bacteria on RAW264.7 cells are higher than that of the single strain, indicating that BXM2 and BSM8 have a synergistic effect ( P <0.01).

[0057] Example 7 Antioxidant Effect 5.1 DPPH free radical scavenging rate The strain was activated to the second generation, the cells were collected by centrifugation, washed twice with 1× PBS, and resuspended to 10 9 CFU / mL, ultrasonically disrupted at 300w, ultrasonically for 5s every 5s, for 10 min, centrifuged, removed the precipitate, and kept 800 uL of the supernatant, added 1 mL of DPPH (containing 0.2 mmol / L anhydrous ethanol) in a brown centrifuge tube, reacted at room temperature in the dark for 30 min, centrifuged at 6000 rpm for 10 min, kept the supernatant, and measured the sample absorbance at 517 nm (denoted as A). For the control, distilled water was used instead of the sample (denoted as B).

[0058] DPPH free radical scavenging rate = (BA) / B×100% 5.2 Hydroxyl radical scavenging rate Take 1 mL of 2.5 mmol / L 1,10-phenanthroline, 1 mL of PBS buffer with a pH of 7.4, and 1 mL of 2.5 mmol / L FeSO 4 Mix well, add 1 mL of the sample to be tested, mix well, and then add 1 mL of 20 mmol / L H 2 O 2 , mix well and place in a 37℃ constant temperature water bath for 1.5 h, and measure the absorbance A at a wavelength of 536 nm; replace 1 mL of H 2 O 2 Measure its absorbance B; use 1mL of distilled water to replace 1mL of sample and measure its absorbance C.

[0059] Hydroxyl free radical scavenging capacity = (AC) / (BC)×100% The results are as follows Figure 6 As shown, the composite bacteria have a higher free radical scavenging rate than single bacteria ( P <0.01).

[0060] Example 8 Zebrafish bowel-moistening and laxative efficacy test The anatomical structure and tissue cell morphology of the zebrafish digestive tract are similar to those of the human digestive tract, with an epithelial layer, a lamina propria, and a muscle layer. When zebrafish develop to 5 days (5 dpf), spontaneous rhythmic muscle contraction activity appears in their digestive tract. After feeding a specific living fluorescent marker dye, Nile red, the amount of Nile red in the zebrafish intestine (fluorescence intensity) can be clearly observed. If the sample has a laxative effect, the intestinal peristalsis will be accelerated, the residual Nile red in the intestine will be reduced, and the fluorescence intensity will be significantly weakened.

[0061] The zebrafish in this experiment were provided by Huante Biotechnology. Wild-type AB strain zebrafish at 5 dpf after fertilization were randomly selected and placed in a culture dish. Aluminum sulfate was given in water to establish a zebrafish constipation model. At the same time, Nile red was given in water as a fluorescent indicator of intestinal contents. After feeding at 28℃ for 3 h, the model zebrafish were randomly assigned to a 6-well plate, and 30 zebrafish were treated in each well (experimental group). Samples (10 9 CFU / mL), positive control domperidone 50.0 µg / mL concentration, and model control group were set up at the same time, with a volume of 3 mL per well. After 4.5 h of treatment at 28℃, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope and saved. The fluorescence intensity of the zebrafish intestine was analyzed using Image J image processing software. The results of statistical analysis of this index were used to evaluate the efficacy of the samples in promoting intestinal peristalsis and improving constipation in zebrafish. "**" indicates that the experimental group shown is significantly higher than the model control group (t test). P The difference was significant at the <0.01 level. The results showed that ( Figure 7 , Figure 8 ), compared with the model control group, the intestinal fluorescence intensity of zebrafish fed with compound bacteria was significantly weaker than that of zebrafish fed with single bacteria, indicating that the compound bacteria has significant effects on moisturizing the intestine and promoting bowel movements and improving constipation.

[0062] Example 9 Application of composite bacterial agent in the preparation of solid beverage A probiotic solid beverage is obtained by mixing 10% of Lactobacillus plantarum BXM2 powder, 10% of Bifidobacterium longum subspecies longum YYS-BSM8, 10% of erythritol, 10% of skimmed milk powder, 19% of hawthorn powder, 11% of prune powder, 10% of maltodextrin and 20% of oligofructose (wherein the percentages are by mass ratios).

[0063] Example 10 Application of composite bacterial agent in fermented beverages 1.0 kg of water, 0.3 kg of concentrated prune juice and 0.2 kg of concentrated pitaya juice were mixed, inoculated with 0.5 g of Lactobacillus plantarum BXM2 powder and 0.5 g of Bifidobacterium longum subsp. longum YYS-BSM8 powder, and fermented at 30°C for 48 h. The pH value at the fermentation end point was 3.78, thereby obtaining a fermented beverage.

[0064] It should be noted that: (1) Definition: The term "food" as used herein is broad and includes food and drink for humans. In certain embodiments, the food product is suitable for and designed for human consumption. The application can be used to prepare solid preparations such as powders, tablets, gels, and also dispersed in liquids to prepare liquid preparations, including but not limited to the embodiment schemes.

[0065] (2) Relevant prior art means or prior art terms involved in this application: "OD" is the abbreviation of optical density, also known as absorbance. When light passes through the object, the energy difference before and after is the energy absorbed by the object. At a specific wavelength, there is a quantitative relationship between the concentration of the same object and the absorbed energy. This quantitative relationship can be used to determine the concentration of the object. 405 " is the optical density value measured when the wavelength is set to 405nm. It is a standard indicator for tracking the density of microorganisms in liquid cultures and is usually used to indicate bacterial cell density. Among them, the "OD" value determination method is a prior art, and its principles and methods will not be repeated here.

[0066] Although more terms such as plant lactobacillus, long bifidobacterium longum subspecies longum, composite bacterial agent, probiotics and MRS culture medium are used herein, the possibility of using other terms is not excluded. These terms are used only to more conveniently describe and explain the essence of the present invention; interpreting them as any additional limitation is contrary to the spirit of the present invention.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composite probiotic for moistening the intestines and promoting bowel movements and improving intestinal immunity, characterized in that: Includes Lactobacillus plantarum ( Lactiplantibacillus plantarum ) BXM2 strain, whose deposit number is CGMCC NO.16436, and Bifidobacterium longum subsp. longum ( Bifidobacterium longum subsp.Longum ) YYS-BSM8 strain, whose preservation number is CGMCC No.28611, and the ratio of the number of viable bacteria of Lactobacillus plantarum BXM2 and Bifidobacterium longum subspecies longum YYS-BSM8 is 1-10:1-10.

2. A composite probiotic, characterized in that: The components thereof include the composite probiotics for moistening the intestines, promoting bowel movements and improving intestinal immunity as claimed in claim 1.

3. The composite probiotic according to claim 2, characterized in that: The number of viable bacteria in the composite probiotic is ≥ 6.5×10 9 CFU / g, wherein the ratio of the viable counts of Lactobacillus plantarum BXM2 and Bifidobacterium longum subspecies longum YYS-BSM8 is 1-3:1-3.

4. The composite probiotic according to claim 2, characterized in that: The composite probiotics further include a freeze-drying protective agent, which includes any one or more combinations of skimmed milk powder, maltodextrin, sodium glutamate, sucrose, lactose, trehalose, sorbitol and xylitol.

5. The composite probiotic according to claim 2, characterized in that: The composite probiotic also includes auxiliary additives, which include any one or more combinations of fructooligosaccharides, galacto-oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, spirulina, stachyose, polydextrose, α-lactalbumin or lactoferrin.

6. The composite probiotic according to claim 2, characterized in that: The composite probiotics also include excipients, which include any one or more combinations of prebiotics, fillers, acidulants, solvents, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, lubricants, wetting agents, stabilizers, glidants, flavoring agents, preservatives, coating materials, fragrances, anti-adhesive agents, integrators, thickeners, buffers and inclusion agents.

7. The composite probiotic according to claim 2, characterized in that: The dosage form of the composite probiotic includes freeze-dried powder, capsule, tablet or granule.

8. A probiotic solid beverage, characterized in that: Its components include the composite probiotic agent as claimed in claim 2.

9. A fermented beverage, characterized in that: Its components include the composite probiotic agent as claimed in claim 2.

10. Use of the composite probiotic according to claim 2 in the preparation of food, health products or medicines for relieving or treating constipation and improving intestinal immunity.

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