Lactobacillus johnsonii yh1136 / bs15 complex microbial inoculant, preparation method and application thereof

Abstract

The application provides a lactobacillus johnsonii YH1136 / BS15 complex microbial agent, a preparation method and application thereof, and belongs to the technical field of intestinal regulation. The complex microbial agent comprises lactobacillus johnsonii YH1136 and BS15, and can be used for preparing a product for improving insufficient gastrointestinal motility. The lactobacillus johnsonii YH1136 / BS15 complex microbial agent can significantly improve clinical symptoms and physiological indexes related to insufficient gastrointestinal motility, effectively reduce colonic inflammatory response, regulate intestinal low-grade inflammation caused by insufficient gastrointestinal motility, regulate key intestinal hormones and neurotransmitters, positively regulate key signal molecules of a brain-gut axis, improve an intestinal motility regulation network, protect intestinal barrier function, enhance the integrity of an intestinal epithelial barrier, regulate bile acid metabolism and downstream gastrointestinal motility inhibitory hormone GLP-1 expression, negatively regulate a bile acid-TGR5-GLP1 signal pathway, and improve gastrointestinal motility.

Description

Technical Field

[0001] This invention belongs to the field of intestinal regulation technology, and particularly relates to Lactobacillus johnsonii YH1136 / BS15 compound bacterial agent, its preparation method and application. Background Technology

[0002] Insufficient gastrointestinal motility is a common pathological condition of the digestive system in clinical practice and a core pathological basis for diseases such as functional constipation and functional dyspepsia. In livestock and poultry farming, it can also lead to decreased animal growth performance and certain economic losses. Its pathogenesis is complex, involving dysfunction of the enteric nervous system, damage to Cajal interstitial cells, and imbalance in the brain-gut axis. It is also closely related to multiple links such as low-grade intestinal inflammation, damage to the epithelial barrier function, and bile acid metabolism disorders. Single intervention targets are often insufficient to achieve comprehensive improvement.

[0003] While commonly used prokinetic drugs and laxatives can provide short-term symptom relief, they generally have limitations such as side effects, dependence with long-term use, or decreased efficacy. Probiotics, due to their high safety and mild effects, have become a research hotspot in the field of intestinal function regulation. However, most existing probiotic products are based on single strains, with relatively limited pathways of action, making it difficult to simultaneously cover multiple pathological processes such as motility regulation, inflammation relief, and barrier repair, resulting in limited comprehensive improvement effects. Therefore, developing compound probiotic preparations with multi-target synergistic effects has significant practical significance and application value for improving insufficient gastrointestinal motility. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a compound bacterial agent of Lactobacillus johnsonii YH1136 / BS15, its preparation method and application, which has the function of comprehensively improving gastrointestinal motility deficiency.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A compound probiotic containing *Lactobacillus johnsonii* to improve gastrointestinal motility, comprising *Lactobacillus johnsonii* (… Lactobacillus johnsonii YH1136 and Lactobacillus johnsonii ( Lactobacillus johnsonii BS15; the preservation number of Lactobacillus johnsonii YH1136 is CCTCC M 20221116; the preservation number of Lactobacillus johnsonii BS15 is CCTCC NO: M2013663.

[0006] Preferably, the viable bacteria concentration of the compound microbial agent is 1×10⁻⁶. 8 ~1×10 10 CFU / mL.

[0007] Preferably, the ratio of Lactobacillus johnsonii YH1136 to Lactobacillus johnsonii BS15 is 1:1 based on the number of live bacteria.

[0008] The present invention also provides a method for preparing the above-mentioned Lactobacillus johnsonii compound bacterial agent, comprising the following steps: taking Lactobacillus johnsonii YH1136 and Lactobacillus johnsonii BS15 bacterial strains respectively, inoculating them into liquid culture medium for rejuvenation culture; centrifuging the culture medium after rejuvenation culture to obtain bacterial sludge, resuspending and mixing them.

[0009] Preferably, the liquid culture medium is MRS broth medium; the rejuvenation culture is a static culture at 37°C for 24 hours, and a culture of 2-3 generations.

[0010] Preferably, the centrifugation speed is 4000~5000 rpm and the time is 14~16 min.

[0011] Preferably, the bacterial sludge is washed and resuspended with PBS until the viable cell concentration is 1×10⁻⁶. 8 ~1×10 10 CFU / mL.

[0012] This invention also provides the application of the above-mentioned Lactobacillus johnsonii compound bacterial agent in the preparation of products that improve gastrointestinal motility.

[0013] Preferably, the product includes pharmaceuticals, functional foods, health products, or feed additives.

[0014] Preferably, the dosage form of the product includes solution, suspension, lyophilized powder, capsule, tablet or granule.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The Lactobacillus johnsonii YH1136 / BS15 compound bacterial agent of the present invention can significantly improve clinical symptoms and physiological indicators related to insufficient gastrointestinal motility, including: increasing gastric emptying rate and accelerating the entry of gastric contents into the intestine; increasing small intestinal transit rate and promoting the propulsion of chyme in the small intestine; increasing fecal water content and effectively relieving dry and hard stools caused by constipation; shortening the whole intestinal transit time and accelerating the overall passage of intestinal contents through the digestive tract; increasing the number of fecal particles and increasing the frequency of defecation.

[0016] The Lactobacillus johnsonii YH1136 / BS15 compound bacterial agent of the present invention can also effectively reduce colonic inflammation and regulate the low-grade intestinal inflammation caused by insufficient gastrointestinal motility. Specifically, it can reduce the mRNA expression or protein level of pro-inflammatory factors such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β); increase the level of anti-inflammatory factor interleukin-10 (IL-10); and inhibit the activation of the nuclear factor κB (NF-κB) signaling pathway.

[0017] The *Lactobacillus johnsonii* YH1136 / BS15 compound probiotic of this invention can also regulate key intestinal hormones and neurotransmitters, positively regulate key signaling molecules in the "brain-gut axis," and improve the intestinal motility regulation network, including: upregulating hormones that promote gastrointestinal motility in colonic tissue, such as motilin (MTL) and gastrin (GAS); and regulating the level of the inhibitory neurotransmitter vasoactive intestinal peptide (VIP). It can also protect intestinal barrier function and enhance the integrity of the intestinal epithelial barrier, manifested by upregulating the expression of tight junction proteins in colonic tissue, such as Occludin, Claudin-1, and ZO-1.

[0018] The Lactobacillus johnsonii YH1136 / BS15 compound bacterial agent of the present invention can also regulate bile acid metabolism and the expression of downstream gastrointestinal motility inhibitory hormone GLP-1 (glucagon-like peptide-1). By negatively regulating the "bile acid-TGR5-GLP1" signaling pathway, it improves gastrointestinal motility, including: reducing the content of intestinal deoxycholic acid, reducing the expression of bile acid receptor TGR5 and hormone GLP-1, and inhibiting the expression of intestinal bile acid reabsorption receptor Slc10a2. Attached Figure Description

[0019] Figure 1 The water content of feces in mice in different drug administration groups.

[0020] Figure 2 The results of intestinal motility index detection in mice in different drug administration groups are shown in the figure. A is the whole intestinal transit time of mice, B is the number of defecation particles of mice 120 min after intervention, and C is the number of defecation particles of mice 240 min after intervention.

[0021] Figure 3 The figures show the gastric emptying rate and small intestinal propulsion rate of mice in different drug administration groups. In the figure, A represents the gastric emptying rate of mice, and B represents the small intestinal propulsion rate of mice.

[0022] Figure 4 The figures show the relative expression levels of colonic inflammatory factors mRNA in mice under different drug administration groups. In the figure, A represents the relative expression level of TNF-α mRNA, B represents the relative expression level of IL-6 mRNA, and C represents the relative expression level of IL-10 mRNA.

[0023] Figure 5 The figures show the relative expression levels of colonic bile acid signaling and key gastrointestinal regulatory molecules mRNA in mice under different drug administration groups. In the figure, A represents deoxycholic acid content, B represents the relative expression level of GLP-1 mRNA, C represents the relative expression level of TGR5 mRNA, D represents the relative expression level of FXR mRNA, and E represents the relative expression level of Slc10a2 mRNA. Detailed Implementation

[0024] This invention provides a compound probiotic agent of Lactobacillus johnsonii for improving gastrointestinal motility, comprising Lactobacillus johnsonii (… Lactobacillus johnsonii YH1136 and Lactobacillus johnsonii ( Lactobacillus johnsonii BS15.

[0025] This invention relates to Lactobacillus johnsonii ( Lactobacillus johnsonii YH1136, with accession number CCTCC M20221116, was deposited at the China Center for Type Culture Collection (CCTCC) on July 15, 2022, and disclosed in CN115869403 A-Lactobacillus johnsonii in the preparation of drugs for the treatment and / or prevention of intestinal barrier damage caused by high-altitude hypoxia (application publication date: March 31, 2023).

[0026] This invention relates to Lactobacillus johnsonii ( Lactobacillus johnsonii The accession number of BS15 is CCTCC NO: M2013663. It was deposited at the China Center for Type Culture Collection (CCTCC) on December 13, 2013, and disclosed in CN113018320 A-Lactobacillus johnsonii BS15 in the preparation of drugs for the prevention and / or treatment of intestinal damage caused by chronic fluorosis (application publication date: June 25, 2021).

[0027] In this invention, the preferred viable bacteria concentration of the compound microbial agent is 1×10⁻⁶. 8 ~1×10 10 CFU / mL, further preferably 1×10⁻⁶ 9 CFU / mL; more preferably, the ratio of viable Lactobacillus johnsonii YH1136 to Lactobacillus johnsonii BS15 is 1:1.

[0028] This invention also provides a method for preparing the above-mentioned Lactobacillus johnsonii compound inoculant, comprising the following steps: (1) Take Lactobacillus johnsonii YH1136 and Lactobacillus johnsonii BS15 respectively, and inoculate them into liquid culture medium for rejuvenation culture.

[0029] As one possible method, Lactobacillus johnsonii YH1136 and Lactobacillus johnsonii BS15 strains were preserved in 1.5 mL EP with glycerol and frozen at -80°C.

[0030] In this invention, the preferred liquid culture medium is MRS broth medium; more preferably, it is incubated at 37°C for 24 hours for rejuvenation; even more preferably, the inoculum size is 2%. Rejuvenation culture is performed for 2-3 generations.

[0031] (2) Centrifuge the culture medium after rejuvenation culture, discard the supernatant, and obtain bacterial sludge.

[0032] In this invention, the centrifugation speed is preferably 4000~5000 rpm, more preferably 4500 rpm; the centrifugation time is preferably 14~16 min, more preferably 15 min; and PBS is preferably used to mix and wash the bacterial sludge, more preferably washing 3 times.

[0033] (3) Resuspension followed by mixing.

[0034] In this invention, the cells are resuspended in PBS after washing until the viable bacterial concentration is 1×10⁻⁶. 8 ~1×10 10 The resuspension of Lactobacillus johnsonii YH1136 and Lactobacillus johnsonii BS15 was mixed in a certain proportion at CFU / mL to obtain the Lactobacillus johnsonii YH1136 / BS15 compound bacterial agent.

[0035] This invention also provides the application of Lactobacillus johnsonii YH1136 / BS15 compound bacterial agent in the preparation of products that improve gastrointestinal motility.

[0036] In this invention, preferred products include pharmaceuticals, functional foods, health products, or feed additives; more preferred dosage forms include solutions, suspensions, lyophilized powders, capsules, tablets, or granules. This invention does not impose any particular limitation on the specific preparation method of the product; any preparation process in the field is acceptable.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1 A compound bacterial agent of Lactobacillus johnsonii YH1136 / BS15 and its preparation method are as follows: (1) Rejuvenation of strains: Lactobacillus johnsonii YH1136 and Lactobacillus johnsonii BS15 strains, which were frozen and preserved in glycerol at -80℃, were inoculated into MRS broth medium that had been autoclaved at a 2% inoculation rate and cultured at 37℃ for 24 hours. The strains were then passaged for two generations under the same inoculation rate and culture conditions to complete the rejuvenation of the strains and obtain the activated culture medium of the two strains.

[0039] (2) Centrifugation to collect bacterial sludge: Transfer the activated culture medium of the two strains to sterile centrifuge tubes, set the speed to 4500 rpm, and centrifuge for 15 min; after centrifugation, discard the supernatant and collect the bacterial precipitate of the two strains respectively.

[0040] (3) Washing and resuspending: Add sterile PBS buffer to the bacterial pellet, gently resuspend by pipetting, centrifuge at 4500 rpm for 15 min and discard the supernatant. Repeat the washing process 3 times. Finally, resuspend the bacterial cells of the two strains separately with sterile PBS buffer and adjust the viable cell concentration to 1×10⁻⁶. 9 CFU / mL.

[0041] (4) Mixing of the compound microbial agent: The adjusted concentration of *Lactobacillus johnsonii* YH1136 bacterial solution and *Lactobacillus johnsonii* BS15 bacterial solution are thoroughly mixed at a volume ratio of 1:1 to obtain the target compound microbial agent. The final viable cell concentration of this compound microbial agent is 1×10⁻⁶. 9 CFU / mL, the ratio of viable bacteria between the two strains is 1:1.

[0042] Example 2 A compound bacterial agent of Lactobacillus johnsonii YH1136 / BS15 and its preparation method are as follows: (1) Rejuvenation of strains: Lactobacillus johnsonii YH1136 and Lactobacillus johnsonii BS15 strains, which were frozen and preserved in glycerol at -80℃, were inoculated into MRS broth medium that had been autoclaved at a 2% inoculation rate and cultured at 37℃ for 24 hours. The strains were then passaged for two generations under the same inoculation rate and culture conditions to complete the rejuvenation of the strains and obtain the activated culture medium of the two strains.

[0043] (2) Centrifugation to collect bacterial sludge: Transfer the activated culture medium of the two strains to sterile centrifuge tubes, set the speed to 4000 rpm, and centrifuge for 14 min; after centrifugation, discard the supernatant and collect the bacterial precipitate of the two strains respectively.

[0044] (3) Washing and resuspending: Add sterile PBS buffer to the bacterial pellet, gently resuspend by pipetting, centrifuge at 4000 rpm for 14 min and discard the supernatant. Repeat the washing process 3 times. Finally, resuspend the bacterial cells of the two strains separately with sterile PBS buffer and adjust the viable cell concentration to 1×10⁻⁶. 8 CFU / mL.

[0045] (4) Mixing of the compound microbial agent: The adjusted concentration of *Lactobacillus johnsonii* YH1136 bacterial solution and *Lactobacillus johnsonii* BS15 bacterial solution are thoroughly mixed at a volume ratio of 1:1 to obtain the target compound microbial agent. The final viable cell concentration of this compound microbial agent is 1×10⁻⁶. 8 CFU / mL, the ratio of viable bacteria between the two strains is 1:1.

[0046] Example 3 A compound bacterial agent of Lactobacillus johnsonii YH1136 / BS15 and its preparation method are as follows: (1) Culture for strain rejuvenation: Take Lactobacillus johnsonii YH1136 and Lactobacillus johnsonii BS15 strains that were frozen and preserved in glycerol at -80℃, and inoculate them into MRS broth medium that has been autoclaved at a 2% inoculation rate. Incubate at 37℃ for 24 hours. Subculture for 3 generations under the same inoculation rate and culture conditions to complete the strain rejuvenation and obtain the activated culture medium of the two strains.

[0047] (2) Centrifugation to collect bacterial sludge: Transfer the activated culture medium of the two strains to sterile centrifuge tubes, set the speed to 5000 rpm, and centrifuge for 16 min; after centrifugation, discard the supernatant and collect the bacterial precipitate of the two strains respectively.

[0048] (3) Washing and resuspending: Add sterile PBS buffer to the bacterial pellet, gently pipette to resuspend, centrifuge at 5000 rpm for 16 min and discard the supernatant. Repeat washing 3 times. Finally, resuspend the bacterial cells of the two strains separately with sterile PBS buffer and adjust the viable cell concentration to 1×10⁻⁶. 10 CFU / mL.

[0049] (4) Mixing of compound bacterial solutions: The Lactobacillus johnsonii YH1136 bacterial solution with the adjusted concentration and Lactobacillus johnsonii BS15 bacterial solution were thoroughly mixed at a volume ratio of 1:1 to obtain a compound bacterial solution; the final viable cell concentration of this bacterial solution was 1×10⁻⁶. 10 CFU / mL, the ratio of viable bacteria between the two strains is 1:1.

[0050] (5) Freeze-drying granulation: Add an equal volume of sterile 10% (w / v) skim milk powder as a freeze-drying protectant to the above compound bacterial solution, mix well, and dispense into sterile vials; after pre-freezing at -40℃ for 4 hours, transfer to a vacuum freeze dryer, dry for 24 hours, and then stopper and cap to obtain the freeze-dried powder of Lactobacillus johnsonii compound bacterial agent. The viable count of the finished product is not less than 1×10⁻⁶. 10 CFU / g.

[0051] Experimental Example 1 1. Experimental animals: 60 male C57BL / 6 mice aged 7 weeks.

[0052] 2. Model establishment: Loperamide hydrochloride was used for induction. The model group and the treatment group were administered loperamide solution (0.2 mL / animal, concentration to be adjusted according to actual conditions, such as 2 mg / kg) by gavage daily for 2 consecutive weeks.

[0053] 3. Grouping and processing: Blank control group (Group C, n=20): 0.2 mL PBS was administered by gavage in the morning and 0.2 mL PBS in the afternoon for 2 weeks.

[0054] Model group (L group, n=20): 0.2 mL loperamide solution was administered by gavage in the morning and 0.2 mL PBS was administered by gavage in the afternoon for 2 weeks.

[0055] Lactobacillus johnsonii YH1136 monoculture treatment group (Group Y, n=20): 0.2 mL of loperamide solution was administered by gavage in the morning and 0.2 mL of Lactobacillus johnsonii YH1136 suspension (live bacteria concentration 1.0 × 10⁻⁶) was administered by gavage in the afternoon daily. 9 (CFU / mL), for a total of 2 weeks.

[0056] Lactobacillus johnsonii BS15 monoculture treatment group (Group B, n=20): 0.2 mL of loperamide solution was administered by gavage in the morning and 0.2 mL of Lactobacillus johnsonii BS15 suspension (live bacteria concentration 1.0 × 10⁻⁶) was administered by gavage in the afternoon daily. 9 (CFU / mL), for a total of 2 weeks.

[0057] Treatment group with Lactobacillus johnsonii YH1136 / BS15 compound bacterial agent (YB group, n=20): 0.2 mL of loperamide solution was administered by gavage in the morning and 0.2 mL of Lactobacillus johnsonii compound bacterial suspension (live bacteria concentration 1.0 × 10⁻⁶) was administered by gavage in the afternoon daily. 9 (CFU / mL, YH1136 and BS15 mixed in equal proportions), for a total of 2 weeks.

[0058] 4. Detection indicators and results: 4.1 Results of fecal moisture content test Fecal water content directly reflects the intestines' water reabsorption function; decreased water content indicates dry, hard stools and worsening constipation. Figure 1 (There was no statistically significant difference in ns) As shown in the figure (the same below), compared with group C, the fecal water content of mice in group L was significantly reduced ( P <0.05), confirming the successful establishment of the constipation model. After intervention with YH1136 and BS15 alone, group Y ( P <0.01), Group B ( P <0.05) Fecal moisture content significantly increased compared to group L. The difference between the compound microbial agent YB group and group L was significant. P <0.001, the fecal water content was restored to a level close to that of the normal control group, and the improvement effect was significantly better than that of the two single strains. The combination of the two strains has a synergistic effect in enhancing the intestinal water retention capacity.

[0059] 4.2 Indicators related to whole intestinal transit and defecation capacity Whole intestinal transit time and the number of fecal particles at a fixed duration directly characterize the overall peristaltic emptying speed of the intestine.

[0060] Results of whole intestinal transit time are as follows Figure 2 A showed that the transport time in group L was significantly longer than that in group C. P <0.001); compared to group L, the transport time in group Y was significantly shortened ( P <0.05), the shortening effect of the YB group reached P <0.01, although Group B showed a downward trend, there was no statistically significant difference.

[0061] Short-term 120-minute stool particle count results as follows Figure 2 B showed that the stool volume in group L was significantly less than that in group C. P <0.001); Group Y ( P <0.05), YB group (P <0.01) can significantly increase the number of fecal particles, while there was no statistically significant difference in improvement in group B.

[0062] The results of the cumulative number of fecal particles over a long period of 240 minutes are as follows: Figure 2 C showed that the number of particles in group L was significantly lower than that in group C. P <0.01); the levels in groups Y, B, and YB were all significantly higher than those in group L ( P <0.05), with the YB group showing a significantly higher number of bowel movements than the B group, indicating a more pronounced effect in promoting defecation. These indicators collectively demonstrate that a single bacterial strain can mildly improve intestinal peristalsis, while a combination of bacterial strains can synergistically enhance the overall propulsion and emptying capacity of the intestines.

[0063] 4.3 Results of gastric emptying rate and small intestinal propulsion rate tests Gastric emptying rate and small intestinal propulsion rate were used to evaluate the gastric motility and small intestinal peristalsis of the upper gastrointestinal tract, respectively.

[0064] Gastric emptying rate results as follows Figure 3 A showed that the gastric emptying rate in group L was significantly lower than that in group C. P <0.05); After single-bacterial intervention, groups Y and B only showed a rebound trend, with no statistical difference compared to group L. The compound bacterial agent YB group could significantly improve the gastric emptying rate ( P <0.01), returning to the normal control level.

[0065] Small intestine propulsion rate results are as follows: Figure 3 B showed that the small intestinal propulsion capacity of group L was significantly lower than that of group C. P <0.01); Single-strain intervention in groups Y and B showed no significant improvement, while only the compound bacterial agent YB group significantly increased the small intestinal propulsion rate ( P <0.05), effectively alleviating small intestinal peristalsis inhibition. Intergroup comparisons showed no significant statistical differences between individual strains or between the single strain and the blank control group; only the dual-strain combination preparation could simultaneously and significantly repair damaged gastric motility and small intestinal propulsion function.

[0066] 4.4 Results of mRNA expression detection of inflammatory factors in colon tissue Colon tissues from mice in groups C, L, and YB were selected, and the relative transcriptional levels of pro-inflammatory factors TNF-α and IL-6 and anti-inflammatory factor IL-10 were detected by qPCR to explore the regulatory effect of the compound bacterial strain on constipation accompanied by low-grade intestinal inflammation.

[0067] The results are as follows Figure 4 The results showed that, compared with group C, the relative expression level of TNF-α mRNA in group L showed an increasing trend, but there was no statistically significant difference. After intervention with the compound bacterial agent, the relative expression level of TNF-α mRNA in group YB returned to normal levels, and there were no significant differences between any two groups. Compared with group C, the relative expression level of IL-6 mRNA in group L was significantly increased (P <0.01), confirming that the constipation model was accompanied by significant intestinal inflammation activation; after intervention with compound bacterial agent, the relative expression level of IL-6 mRNA in the YB group decreased significantly, showing a significant difference compared with the L group ( P <0.05), which can effectively inhibit overactivated pro-inflammatory signaling pathways. The relative expression level of IL-10 mRNA in group L was significantly lower than that in group C ( P <0.05), indicating an imbalance in intestinal anti-inflammatory homeostasis; after intervention with compound bacterial agents, the relative expression level of IL-10 mRNA in the YB group significantly increased ( P <0.01), to rebuild the intestinal anti-inflammatory immune balance.

[0068] 4.5 Results of detection of bile acid signaling and mRNA expression of key molecules in gastrointestinal regulation To elucidate the core molecular pathways by which the compound bacterial strain improves constipation, the mRNA expression levels of bile acid membrane receptor TGR5, nuclear receptor FXR (farnesol X receptor, NR1H4), bile acid reabsorption transporter Slc10a2, and gastrointestinal hormone GLP-1 in colonic tissue were detected by qPCR. The colonic bile acid content in each group was detected by bile acid targeted metabolomics to explore the regulatory role of the bacterial strain on the bile acid metabolism-intestinal motility signaling axis.

[0069] like Figure 5 As shown in A, compared with group C, the colonic deoxycholic acid level in group L was significantly upregulated ( P <0.05). After intervention with compound microbial agents, the deoxycholic acid content in the YB group decreased significantly, and the difference between the YB group and the L group was significant ( P <0.05), returning to levels close to the blank control group. There was no statistically significant difference between group C and group YB, indicating that the compound strain can reverse the abnormal increase of deoxycholic acid in constipation and remodel gastrointestinal bile acid metabolism.

[0070] GLP-1 is a key hormone that inhibits gastrointestinal motility. Results are as follows: Figure 5 B showed that, compared with group C, the relative expression level of colonic GLP-1 mRNA in group L was significantly upregulated ( P <0.001). After intervention with compound bacterial agent, GLP-1 expression in group YB decreased significantly, and the difference between group YB and group L was significant. P <0.001, returning to levels close to the blank control group. There was no statistically significant difference between group C and group YB, indicating that the compound strain can reverse the compensatory abnormal increase of GLP-1 in constipation and reshape gastrointestinal hormone homeostasis.

[0071] Further examination of the signaling genes TGR5 and FXR that promote GLP-1 expression yielded the following results: Figure 5 CD showed that, compared with group C, the relative expression level of colonic TGR5 mRNA in group L was significantly increased (P <0.01). After intervention with the compound microbial agent, the relative expression level of TGR5 mRNA in the YB group was significantly downregulated, and the difference between the YB group and the L group was <0.01. P <0.01. There was no statistically significant difference between group C and group YB. The relative expression level of colonic FXR mRNA in group L was significantly higher than that in group C. P <0.001). After intervention with the compound bacterial agent, the relative expression level of FXR mRNA in the YB group decreased significantly, and the difference between the YB group and the L group was extremely significant ( P <0.001). There was no statistically significant difference between group C and group YB. TGR5 and FXR are both bile acid agonist receptors, and Slc10a2 mediates ileal bile acid reabsorption. Results are as follows. Figure 5 E showed that, compared with group C, the relative expression level of Slc10a2 mRNA in group L was significantly increased ( P <0.01). After intervention with the compound bacterial agent, the relative expression level of Slc10a2 mRNA in the YB group was significantly reduced, and the difference between the YB group and the L group was significant. P <0.01. There was no statistically significant difference between group C and group YB, indicating that the compound strain significantly improved bile acid metabolism disorder.

[0072] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound probiotic preparation of *Lactobacillus johnsonii* to improve gastrointestinal motility deficiency, characterized in that, Including Lactobacillus johnsonii ( Lactobacillus johnsonii YH1136 and Lactobacillus johnsonii ( Lactobacillus johnsonii BS15; the preservation number of Lactobacillus johnsonii YH1136 is CCTCC M 20221116; the preservation number of Lactobacillus johnsonii BS15 is CCTCC NO: M2013663.

2. The Lactobacillus johnsonii compound bacterial agent according to claim 1, characterized in that, The viable bacteria concentration of the compound microbial agent is 1×10⁻⁶. 8 ~1×10 10 CFU / mL.

3. The Lactobacillus johnsonii compound bacterial agent according to claim 1, characterized in that, The ratio of Lactobacillus johnsonii YH1136 to Lactobacillus johnsonii BS15 is 1:1 based on the number of live bacteria.

4. The method for preparing the Lactobacillus johnsonii complex bacterial agent according to any one of claims 1 to 3, characterized in that, Includes the following steps: Lactobacillus johnsonii YH1136 and Lactobacillus johnsonii BS15 were respectively inoculated into liquid culture medium for rejuvenation culture; the culture medium after rejuvenation culture was centrifuged to obtain bacterial sludge, which was then resuspended and mixed.

5. The preparation method according to claim 4, characterized in that, The liquid culture medium is MRS broth medium; the rejuvenation culture is a static culture at 37℃ for 24 hours, and a culture of 2-3 generations.

6. The preparation method according to claim 4, characterized in that, The centrifugation speed is 4000~5000 rpm, and the time is 14~16 min.

7. The preparation method according to claim 4, characterized in that, The bacterial sludge was washed and resuspended with PBS until the viable cell concentration reached 1×10⁻⁶. 8 ~1×10 10 CFU / mL.

8. The use of the Lactobacillus johnsonii complex bacterial agent according to any one of claims 1 to 3 in the preparation of products that improve gastrointestinal motility.

9. The application according to claim 8, characterized in that, The products include pharmaceuticals, functional foods, health products, or feed additives.

10. The application according to claim 8, characterized in that, The dosage forms of the product include solutions, suspensions, lyophilized powders, capsules, tablets, or granules.

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