Pediococcus acidilactici P48 capable of relieving constipation and diarrhea and application

Abstract

The invention belongs to the technical field of biology, and discloses a pediococcus acidilactici strain capable of relieving constipation and diarrhea, the name of the pediococcus acidilactici strain is P48, the classification name of the pediococcus acidilactici strain is pediococcus acidilactici, the preservation number of the pediococcus acidilactici strain is CGMCC No.27885, the preservation date of the pediococcus acidilactici strain is July 13, 2023, and the preservation unit of the pediococcus acidilactici strain is China General Microbiological Culture Collection Center, No. 3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing. The pediococcus acidilactici P48 can tolerate the gastrointestinal tract environment, improves the survival rate of the gastrointestinal tract, is beneficial to strain colonization, and can be used for preparing functional microbial agents, food and medicines for relieving constipation or diarrhea; the pediococcus acidilactici P48 disclosed by the invention can be used for carrying out bidirectional regulation on diarrhea and constipation at the same time; meanwhile, intestinal flora imbalance can be improved, immune system activation is promoted, inflammatory factor release is inhibited, and the gastrointestinal tract function is improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, in particular to a strain of Pediococcus acidilactici P48 capable of relieving constipation and diarrhea and application thereof. Background Art

[0002] Constipation is a common gastrointestinal disorder with a global prevalence of 12% to 19%. Its etiology is very complex and is often caused by the combined action of multiple factors. Constipated patients usually experience symptoms such as hard stools, reduced bowel movements (usually less than 3 times a week), excessive force during bowel movements, and a feeling of incomplete bowel movements. Although constipation itself is not directly life-threatening, it can cause physical and mental pain to patients and seriously affect their daily lives. In addition, constipation is prone to recurring, causing great distress to patients' lives. Over time, this can cause patients to experience more serious negative emotions, which in turn leads to worsening constipation symptoms, forming a vicious cycle.

[0003] Diarrhea refers to an increase in the frequency of bowel movements, thin stool, or stool with mucus, pus, blood, or undigested food, such as more than 3 bowel movements per day, or a total stool volume of more than 200g per day, of which the water content of the stool is more than 85%. Chronic diarrhea refers to recurrent diarrhea with a course of more than 2 months or an interval of 2 to 4 weeks. It is estimated that 5 to 10 million people die from severe diarrhea each year worldwide.

[0004] The intestinal flora plays an important role in maintaining the homeostasis and health of the host. It can interact with the body to play functions such as metabolism, immunity, and maintaining the homeostasis of the intestinal barrier. More and more evidence shows that the balance of intestinal microbiota plays a key role in the occurrence and development of constipation. For example, constipation patients often have an imbalance in intestinal flora. Compared with healthy people, constipation patients have a decrease in probiotics (Lactobacillus, Bifidobacterium, etc.) in the intestines, and an increase in pathogenic bacteria (Enterobacter, Enterococcus, etc.).

[0005] Pediococcus acidilactici can produce lactic acid, which helps regulate the pH value of the gastrointestinal tract and inhibit the growth of harmful bacteria. By competitively inhibiting pathogenic microorganisms, Pediococcus acidilactici can maintain the balance of intestinal microecology and promote the growth of beneficial bacteria. In animals, Pediococcus acidilactici can enhance the body's immune function, produce beneficial metabolites, activate the activity of acid proteases, participate in the body's metabolism, and prevent the production of harmful substances.

[0006] Through searching, it is found that the current research on commercially available lactic acid Pediococcus strains focuses on its single function, namely the physiological effect of relieving constipation. This limitation ignores the more extensive and complex mechanism of action of lactic acid Pediococcus as a probiotic in the intestinal microecology. In particular, the impact on the balance of intestinal flora and the bidirectional regulatory potential of lactic acid Pediococcus in the intestinal environment have not been fully explored and revealed. In view of the shortcomings of the prior art, the present invention aims to fill the research gap of lactic acid Pediococcus in intestinal flora regulation and bidirectional balance mechanism. By deeply analyzing the interaction between lactic acid Pediococcus and intestinal flora, the specific process and mechanism of promoting the proliferation of beneficial bacteria, inhibiting the growth of harmful bacteria, and dynamically regulating the balance of intestinal microecology are revealed, thereby providing a scientific basis and technical support for the comprehensive management of intestinal health.

[0007] Through searching, no patent publication documents related to the patent application of the present invention have been found. Summary of the invention

[0008] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a strain of Pediococcus acidilactici P48 capable of relieving constipation and diarrhea and its application.

[0009] The technical solution adopted by the present invention to solve its technical problem is:

[0010] A strain of Pediococcus acidilactici P48 that can relieve constipation and diarrhea, named: P48, classified name: Pediococcus acidilactici, preservation number: CGMCC No.27885, preservation date: July 13, 2023, preservation unit: General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC), No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0011] Furthermore, the Pediococcus acidilactici P48 has the following biological characteristics:

[0012] (1) Bacterial characteristics: Gram-positive, spherical cells, non-motile;

[0013] (2) Colony characteristics: On MRS medium, the colonies are small and white, and the growth along the agar puncture line is filamentous;

[0014] (3) Growth characteristics: At 37°C, the cells reached the late logarithmic phase after 10 h of culture in MRS medium.

[0015] (4) It has strong tolerance to simulated gastrointestinal fluid.

[0016] Furthermore, the Pediococcus acidilactici P48 has the function of alleviating constipation and diarrhea.

[0017] The use of the Pediococcus acidilactici P48 as described above in the preparation of common food and / or special functional food and / or health food and / or medicine for relieving constipation and / or diarrhea.

[0018] Furthermore, the food or medicine has at least one of the following effects:

[0019] Improve constipation, specifically by increasing the number and quality of feces and the water content of feces in mice;

[0020] Alternatively, constipation can be improved by repairing the colon mucosal epithelium and intestinal glands to restore the colon tissue to normal, thus providing a better colonization environment for the flora.

[0021] Alternatively, improve constipation by inhibiting the release of inflammatory factors, relieving inflammatory responses, and regulating gastrointestinal hormone levels;

[0022] Alternatively, improve constipation, specifically by regulating intestinal flora disorders, promoting the growth of beneficial bacteria, and inhibiting the growth of harmful bacteria;

[0023] Alternatively, improving diarrhea, specifically improving diarrhea loose stool rate, loose stool grade and diarrhea index.

[0024] Furthermore, the Pediococcus acidilactici P48 is a live strain, a strain metabolite or an inactivated strain;

[0025] Furthermore, the drug also contains a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier includes one or more of a filler, a diluent, a binder, a disintegrant and a lubricant.

[0026] Furthermore, the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch or dextrin; the diluent is one or more of ethanol or glycerol; the disintegrant is one or more of sodium carboxymethyl starch, sodium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone or low-substituted hydroxypropyl cellulose; the binder is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium stearyl fumarate, talc or silicon dioxide.

[0027] Use of the Pediococcus acidilactici P48 as described above in the preparation of fermented food and / or postbiotics and / or enzymes.

[0028] Furthermore, the application includes using Pediococcus acidilactici P48 as a fermentation microorganism and utilizing food raw materials for fermentation.

[0029] The advantages and positive effects achieved by the present invention are:

[0030] 1. The lactic acid Pediococcus P48 of the present invention can tolerate the gastrointestinal environment, improve the survival rate of the gastrointestinal tract, and is conducive to the colonization of the strain, and can be used to prepare functional bacterial agents, foods and medicines for relieving constipation or diarrhea; the lactic acid Pediococcus P48 of the present invention can simultaneously perform bidirectional regulation of diarrhea and constipation; at the same time, it can improve intestinal flora imbalance, promote immune system activation, inhibit the release of inflammatory factors, and improve gastrointestinal function.

[0031] 2. The toxic and side effect experiments of the Pediococcus acidilactici P48 of the present invention showed that the mice had no significant effect, no significant changes in body weight, no mortality, and no obvious pathological symptoms.

[0032] 3. The lactic acid Pediococcus P48 of the present invention can increase the number and quality of feces particles and the water content of feces of mice, repair the mucosal epithelium and intestinal glands of colon tissue, restore the colon tissue to normal, inhibit the release of inflammatory factors, and relieve inflammatory reactions.

[0033] 4. The present invention has analyzed the genome of intestinal flora and found that taking a single strain of Pediococcus acidilactici P48 can effectively change the intestinal flora structure of constipated mice, improve the intestinal environment, increase the abundance of multiple probiotics, reduce pathogenic bacteria, and effectively improve the intestinal flora structure. It can reduce the loose stool rate, loose stool grade and diarrhea index of diarrheal mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a colony morphology diagram of Pediococcus acidilactici P48 in the present invention;

[0035] Figure 2 This is a microscope picture of Pediococcus acidilactici P48 in the present invention;

[0036] Figure 3 It is a distance tree diagram of the BLAST result of the genome sequence of Pediococcus acidilactici P48 in the present invention;

[0037] Figure 4 The figures are the histological characteristics of the colon of constipated mice by Pediococcus acidilactici P48 in the present invention; wherein, (A) H&E staining (magnification 200 times); (B) AB-PAS staining (magnification 200 times);

[0038] Figure 5 The Venn diagram of the effect of Pediococcus acidilactici P48 on the intestinal flora of constipated mice;

[0039] Figure 6 It is a bar chart of the colony composition at the family level of the effect of Pediococcus acidilactici P48 on the changes in the intestinal flora of constipated mice in the present invention;

[0040] Figure 7 This is a bar graph of the genus-level colony composition of the effects of Pediococcus acidilactici P48 on the changes in the intestinal flora of constipated mice in the present invention.

[0041] A strain of Pediococcus acidilactici P48 that can relieve constipation and diarrhea, named: P48, classified name: Pediococcus acidilactici, preservation number: CGMCC No.27885, preservation date: July 13, 2023, preservation unit: General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC), No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0043] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0044] A strain of Pediococcus acidilactici P48 that can relieve constipation and diarrhea, named: P48, classified name: Pediococcus acidilactici, preservation number: CGMCC No.27885, preservation date: July 13, 2023, preservation unit: General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC), No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0045] Preferably, the Pediococcus acidilactici P48 has the following biological characteristics:

[0046] (1) Bacterial characteristics: Gram-positive, spherical cells, non-motile;

[0047] (2) Colony characteristics: On MRS medium, the colonies are small and white, and the growth along the agar puncture line is filamentous;

[0048] (3) Growth characteristics: At 37°C, the cells reached the late logarithmic phase after 10 h of culture in MRS medium.

[0049] (4) It has strong tolerance to simulated gastrointestinal fluid.

[0050] Preferably, the Pediococcus acidilactici P48 has the function of relieving constipation and diarrhea.

[0051] The use of the Pediococcus acidilactici P48 as described above in the preparation of common food and / or special functional food and / or health food and / or medicine for relieving constipation and / or diarrhea.

[0052] Preferably, the food or medicine has at least one of the following effects:

[0053] Improve constipation, specifically by increasing the number and quality of feces and the water content of feces in mice;

[0054] Alternatively, constipation can be improved by repairing the colon mucosal epithelium and intestinal glands to restore the colon tissue to normal, thus providing a better colonization environment for the flora.

[0055] Alternatively, improve constipation by inhibiting the release of inflammatory factors, relieving inflammatory responses, and regulating gastrointestinal hormone levels;

[0056] Alternatively, improve constipation, specifically by regulating intestinal flora disorders, promoting the growth of beneficial bacteria, and inhibiting the growth of harmful bacteria;

[0057] Alternatively, improving diarrhea, specifically improving diarrhea loose stool rate, loose stool grade and diarrhea index.

[0058] Preferably, the Pediococcus acidilactici P48 is a live strain, a strain metabolite or an inactivated strain;

[0059] Preferably, the drug further contains a pharmaceutically acceptable carrier, which includes one or more of a filler, a diluent, a binder, a disintegrant and a lubricant.

[0060] Preferably, the filler is one or more of microcrystalline cellulose, lactose, mannitol, starch or dextrin; the diluent is one or more of ethanol or glycerol; the disintegrant is one or more of sodium carboxymethyl starch, sodium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone or low-substituted hydroxypropyl cellulose; the binder is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium stearyl fumarate, talc or silicon dioxide.

[0061] Use of the Pediococcus acidilactici P48 as described above in the preparation of fermented food and / or postbiotics and / or enzymes.

[0062] Preferably, the application includes using Pediococcus acidilactici P48 as a fermentation microorganism and utilizing food raw materials for fermentation.

[0063] Specifically, the relevant preparation and detection are as follows:

[0064] Example 1: Screening of Pediococcus acidilactici P48

[0065] (I) Isolation and screening of lactic acid bacteria

[0066] (1) Take 0.1 ml of yogurt sample (taken from the home-brewed yogurt of herdsmen in Awati Township, Kashi City, Kashi Prefecture, Xinjiang Uygur Autonomous Region) and inoculate it into MRS liquid culture medium, and place it in a constant temperature incubator at 37°C for 48 hours. Use an inoculation loop to pick up a small amount of bacterial liquid and streak it into an MRS agar medium plate.

[0067] (2) After 48 hours, use an inoculation loop to pick different colonies based on their shape, size, color, etc., and then perform streak separation and purification.

[0068] (3) After Gram staining and catalase analysis, Gram-positive bacilli and catalase-negative bacteria were retained.

[0069] (4) Obtain the bacterial and colony characteristics of the final strain: Gram-positive, non-spore-forming, non-motile bacteria ( Figure 1 ); milky white, spherical ( Figure 2 ).

[0070] (II) Molecular biological identification of lactic acid bacteria

[0071] (1) Single bacterial genome extraction

[0072] (A) culturing the screened lactic acid bacteria overnight;

[0073] (B) Using the Solebro Bacterial Genomic DNA Extraction Kit, catalog number 1600, take 1 ml of the overnight bacterial suspension into a 1.5 ml centrifuge tube, centrifuge at 12000 r / min for 1 min, and aspirate the supernatant as much as possible;

[0074] (C) Add 200 μl of lysozyme (final concentration: 20 mg / ml) to the cells and treat at 37°C for 40 min.

[0075] (D) Add solution A (a mixture of 10 mmol / L Tris-HCl with a pH of 8.0 and 1 mmol / L EDTA, 0.08 g / mL SDS, and 1.5 mol / L NaCl) to the mixture obtained in step (C), and use a pipette to fully suspend the bacteria. Add 20 ul of rRNaseA (10 mg / ml) to the suspension, mix thoroughly by inverting, and let stand at room temperature for 30 min.

[0076] (E) Add 20 μl of proteinase K to the tube, mix thoroughly, and digest at 55°C for 60 min. During digestion, invert the tube several times until the sample becomes clear and viscous, indicating that the sample is completely digested.

[0077] (F) Add 200 μl of solution B (a mixture of phenol: chloroform: isoamyl alcohol = 25:24:1 by volume) to the test tube and mix thoroughly by inversion. If a white precipitate appears, place it at 75°C for 15-30 minutes and the precipitate will disappear.

[0078] (E) Add 200 μl of anhydrous ethanol to the tube and mix thoroughly. Flocculent precipitation may appear at this time, which does not affect DNA extraction. Add the solution and flocculent precipitation to the adsorption column and let it stand for 2 min.

[0079] (F) Centrifuge at 12000 rpm for 2 min, discard the waste liquid, and place the adsorption column into a collection tube;

[0080] (G) Add 600 μl of rinse solution (75% ethanol) to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste solution, place the adsorption column in a collection tube, and repeat once;

[0081] (H) Centrifuge at 12000 rpm for 2 min, and place the adsorption column in a 50°C incubator for several minutes;

[0082] (I) Place the adsorption column in a sterilized 1.5 ml centrifuge tube, drop 100 μl of the elution solution preheated in a 65°C water bath onto the center of the adsorption membrane, let stand at room temperature for 5 min, and centrifuge at 12,000 rpm for 1 min;

[0083] (J) The eluate obtained by centrifugation is added to the adsorption column, placed at room temperature for 2 minutes, and centrifuged at 12000 rpm for 2 minutes to obtain the genomic DNA of the lactic acid bacteria.

[0084] The 16S rDNA gene sequence of the Pediococcus acidilactici P48 is as follows:

[0085]

[0086] (2) 16S r DNA PCR

[0087] Bacterial 16S r DNA 50μL PCR reaction system: Taq enzyme 24μL; dd H2O: 18μL; 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), 2μL; 1492R (5'-GGTTACCTTGTTACGACTT-3'), 2μL; template: 4μL.

[0088] PCR conditions: 94°C for 5 min (i.e. step 1); 94°C for 30 s (i.e. step 2); 55°C for 30 s (i.e. step 3); 72°C for 30 s (i.e. step 4); step 2 to step 4 (30×); 72°C for 5 min.

[0089] Prepare 1% agarose gel, then mix the PCR product with 10000× loading buffer, load 2 μL, run at 120 V for 30 min, and then perform gel imaging;

[0090] The PCR products were sent to a professional sequencing company, and the sequencing results were compared with those obtained by searching and similarity comparison in Gen Bank using BLAST (see Figure 3 ), identified it as a strain of Pediococcus acidilactici, used 20% glycerol as a protective agent, added 800 μL of fresh bacterial liquid after two generations of activation into 200 μL of glycerol, repeatedly pipetted until mixed, and stored in a -80°C ultra-low temperature storage box.

[0091] Example 2: Pediococcus acidilactici P48 has good tolerance to simulated gastrointestinal fluid

[0092] In vitro simulated artificial gastric juice: After sterilizing 0.1 mol / L potassium phosphate buffer, adjust the pH to 2.5 with standard hydrochloric acid titration solution, add pepsin with a mass concentration of 10 g / L (final concentration), mix well and pass through a 0.22 μm sterile membrane to obtain in vitro simulated artificial gastric juice.

[0093] In vitro simulated artificial intestinal fluid: After sterilizing 0.1 mol / L potassium phosphate buffer, adjust the pH to 6.8 with 0.1 mol / L sodium hydroxide solution, add pancreatic enzyme with a mass concentration of 10 g / L and porcine bile salt with a mass concentration of 3 g / L (final concentration is 1L water plus 10g pancreatic enzyme and 3g porcine bile salt), mix well and pass through a 0.22 μm sterile membrane to obtain in vitro simulated artificial intestinal fluid.

[0094] Simulates gastric digestion:

[0095] Take the glycerol tube lactic acid Pediococcus P48 stored at -80℃, heat it in a 37℃ water bath until it is completely thawed, activate it for two generations, take 0.1ml and inoculate it in MRS liquid culture medium, and place it in a 37℃ constant temperature incubator for 48h. Take 3.0mL, centrifuge for 3min, collect the mycelium, place it in a 3.0mL, pH 2.5 simulated gastric juice environment, and simulate gastric juice digestion under anaerobic conditions at 37℃ and 90r / min. Take samples at 0.1, 0.2, and 0.3h, count the live bacteria using the dilution plate counting method, and calculate the survival rate.

[0096] Simulated intestinal digestion:

[0097] Take the glycerol tube lactic acid Pediococcus P48 stored at -80℃, heat it in a 37℃ water bath until it is completely thawed, activate it for two generations, take 0.1ml and inoculate it in MRS liquid culture medium, and place it in a 37℃ constant temperature incubator for 48h. Take 3.0mL of the culture medium of lactic acid Pediococcus P48 and centrifuge it for 3min, collect the mycelium, place it in a 3.0mL simulated intestinal fluid environment with a pH value of 6.8, and simulate intestinal fluid digestion under anaerobic conditions at 37℃ and 120r / min. Take samples at 0.2, 0.4, 0.6, and 0.8h, count the live bacteria using the dilution plate counting method, and calculate the survival rate.

[0098] The survival rate (%) was calculated as the ratio of the number of live bacteria in the culture solution at the time of sampling to the number of live bacteria at the 0th hour, expressed in %. The experimental results are shown in Tables 1 and 2. The results show that Pediococcus acidilactici P48 has a good tolerance to artificial simulated gastrointestinal fluid.

[0099]

[0100]

[0101] Example 3: Preparation of Pediococcus acidilactici P48 bacterial powder and bacterial suspension

[0102] (1) The glycerol tube lactic acid Pediococcus acidilactici P48 stored at -80°C was inoculated into MRS liquid culture medium for activation, and cultured at 37°C for 18 hours to activate for the second generation; 5.0 g of skim milk powder was taken and diluted to 50 mL with 0.9% saline as a protective agent for preparing bacterial powder; after the strain was activated for the second generation, the previous generation of bacterial liquid was inoculated for about 10 hours, and fresh bacterial liquid was taken, centrifuged at 4°C and 4000 rpm for 15 minutes, the supernatant was removed, and the liquid was washed twice with saline, and 10% skim milk powder was added, and 10 mL of 10% skim milk powder was added to every 5 mL of fresh bacterial liquid, and the mixture was repeatedly blown and aspirated until mixed, and vacuum freeze-dried to obtain bacterial powder.

[0103] (2) The glycerol tube lactic acid Pediococcus acidilactici P48 stored at -80°C was inoculated into MRS liquid culture medium for activation and cultured at 37°C for 18 h to activate for the second generation; 5.0 g of skim milk powder was diluted to 50 mL with 0.9% saline as a protective agent for preparing bacterial powder; after the strain was activated for the second generation, the previous generation of bacterial solution was inoculated for about 10 h, and fresh bacterial solution was taken, centrifuged at 4°C and 4000 rpm for 15 min, the supernatant was removed, and the lactic acid Pediococcus acidilactici P48 was resuspended in 100 g / L skim milk solution to prepare a concentration of 4.0×10 9 CFU / mL of bacterial suspension.

[0104] Example 4: Determination of the effects of Pediococcus acidilactici P48 on defecation time, number of feces particles, feces weight and feces water content in constipated mice.

[0105] 30 SPF Balb / c mice, male, with a body weight of 18-20g, were raised in an environment with a temperature of 20-25°C, a relative humidity of (50±5)%, and a light exposure time of 12h / 12h. 30 mice were randomly divided into 3 groups, 10 in each group, set as blank control group, model control group, and experimental group. Mice in the model control group and experimental group were gavaged with loperamide hydrochloride (5mg / (kg·bw)), once in the morning and evening, for 7 consecutive days to construct a constipation model. The blank control group was gavaged with an equal amount of distilled water. After the model was successfully established, the blank control group and the model control group mice were gavaged with 0.3mL of distilled water, and the experimental group mice were gavaged with 0.3mL of the probiotic suspension prepared in Example 3, once a day, and ate normally during the test, and fed continuously for 14 days. One day before the end of the administration, the number and weight of 24h fecal pellets of each group of mice after gavage were collected, and the water content of the feces was measured. The results are shown in Table 3. It can be seen that compared with the feces of the blank group mice, the feces of the model group mice were darker and drier, and the fecal morphology of each group of mice improved to varying degrees after administration; the number of 24h fecal pellets, 24h fecal weight and fecal water content of mice were significantly reduced. Compared with the model group, the number of 24h fecal pellets, 24h fecal weight and fecal water content of the experimental group were significantly increased. In addition, the number of 24h fecal pellets in the experimental group increased significantly. The other drug-administered groups all improved to varying degrees.

[0106]

[0107] Example 5: Effect of Pediococcus acidilactici P48 on pathological changes in colon tissue of constipated mice

[0108] For Example 4, fast for 24h after 14 days of feeding. The blank control group was given an equal volume of distilled water. After 30 minutes of modeling, each group was gavaged with Chinese ink (0.1mL / 10g), and the mice were killed by cervical dislocation 20 minutes later. The small intestine was taken out by opening the abdomen, and the intestinal tube from the upper end to the pylorus and the lower end to the ileocecal part was cut off. The colon tissue of each group of mice was taken. The fixed tissue was dehydrated step by step with high concentration alcohol, transparent with xylene, embedded, sliced, dewaxed to water after slicing, HE stained, transparent with xylene, and sealed with neutral gum, and its tissue morphological changes were observed under a microscope ( Figure 4 ).

[0109] like Figure 4 As shown in the figure, the intestinal glands in the mucosal layer of the blank group were neatly arranged, with clear structures, dense, neat, smooth and complete villi, and no obvious degeneration, necrosis or shedding was observed. Compared with the blank group, the colon tissue in the model group showed mucosal epithelial necrosis and shedding; local intestinal glands in the lamina propria were degenerated and necrotic, with intestinal gland atrophy, a small amount of scattered lymphocyte infiltration in the necrotic area, and a small amount of fibrous tissue hyperplasia. Compared with the model group, the experimental group showed an improvement trend, with thickening of the colon muscle layer, intestinal gland arrangement close to normal levels, reduced damage to the submucosal layer, and improved tissue morphology compared with the model group, indicating that the probiotic group has a certain protective effect on the intestine.

[0110] Example 6: Effect of Pediococcus acidilactici P48 on GAS (gastrin), MTL (gastrointestinal hormone), IL-6 (interleukin 6), and IFN-γ (inflammatory cytokine gamma interferon) in the colon tissue of constipated mice. For Example 5, an appropriate amount of colon tissue was taken, weighed, and added with 10-fold phosphate buffer solution (PBS), homogenized, and heated at 4°C and 4000 r / min. -1 Centrifuge for 10 min (centrifugal radius 5.5 cm, the same below) and set aside. GAS, MTL, IFN-γ, and IL-6 detection kits were purchased from Solebao, and the GAS, MTL, IL-6, and IFN-γ contents in each sample were determined according to the corresponding methods of each kit.

[0111] As shown in Table 4, compared with the blank group, the levels of GAS and MTL in the model group were significantly reduced, and the levels of IL-6 and IFN-γ were significantly increased. Compared with the model group, the levels of GAS and MTL in the experimental group were significantly increased to varying degrees, the level of IL-6 was significantly reduced, and the level of IFN-γ had a decreasing trend.

[0112]

[0113] Example 7: Effect of Pediococcus acidilactici P48 on intestinal flora of constipated mice

[0114] Thirty male SPF Balb / c mice, weighing 18-20g, were raised in an environment with a temperature of 20-25℃, a relative humidity of (50±5)%, and a light exposure time of 12h / 12h. 40 mice were randomly divided into 4 groups, with 10 mice in each group, namely, the blank group (NC), the model group (MC), the experimental group (Pediococcus acidilactici P48, 4 billion CFU / g, viable bacteria count.) (YS), and the mixed bacteria positive group (YX) (Pediococcus acidilactici: Bifidobacterium lactis: Bifidobacterium bifidum: Lactobacillus acidophilus: Lactobacillus paracasei mixed bacteria, with a mass ratio of 1:1:1:1:1, which is a commercial product, 4 billion CFU / g, viable bacteria count.). Mice in the model control group, the experimental group, and the mixed bacteria positive group were gavaged with loperamide hydrochloride (5mg / (kg·bw)), once in the morning and evening, for 7 consecutive days to construct a constipation model. The blank control group was gavaged with an equal amount of distilled water. After successful modeling, the blank control group and the model control group mice were gavaged with 0.3 mL of distilled water, and the experimental group and the mixed bacteria positive group mice were gavaged with 0.3 mL of the probiotic suspension prepared in Example 3, once a day, and ate normally during the test, and fed continuously for 14 days, and fasted for 24 hours after 14 days. 1 hour after the end of administration, the feces were collected and frozen at -80°C for sequencing research. The Solebao bacterial genomic DNA extraction kit, item number 1600, was purchased to extract mouse fecal DNA, stored in a -20°C refrigerator, and transferred to Shanghai Meiji Biotechnology Co., Ltd. for sequencing. The formal test PCR reaction parameters are: (a) 95°C denaturation for 3min; (b) cycle number (95°C denaturation for 30s; annealing temperature for 30s; 72°C extension for 45s); (c) 72°C extension for 10min, 10°C insulation. PCR products were detected using 2% agarose gel electrophoresis. PCR products were purified using Vazyme VAHTSTM DNAClean Beads, quantified using Quant-iT PicoGreen dsDNA Assay Kit, and sequenced using the Illlumina NovaSeq platform. Linear discriminant analysis (LDA) was used for differential bacterial genus analysis, with LDA score>4 as the screening condition.

[0115] 1. Analysis of sample species composition: Venn diagrams can be used to count the number of common and unique species (such as ASV) in different groups, and can more intuitively show the uniqueness and overlap of species (such as ASV) composition of colon content samples. The number of OTUs in four groups of mice (blank group, model group, positive control group, and lactic acid Pediococcus group) was analyzed, and a Venn diagram was drawn, see Figure 5. The figure shows that there are unique ASVs in common among different samples, and 148 ASVs overlap between the blank group, model group, positive control group, and Pediococcus acidilactici group. There are 1196, 695, 782, and 713 unique ASVs in the blank group, model group, positive control group, and Pediococcus acidilactici group, respectively. The results showed that the number of ASVs in the intestinal flora increased in the positive control group and Pediococcus acidilactici group after administration, and the number of ASVs in the Pediococcus acidilactici group and the positive control group were higher than those in the model group, and more similar to the blank group.

[0116] 2. Community composition analysis

[0117] At the phylum level, Bacteroidetes, Firmicutes, Campilobacterota and Actinobacteria were the main species, among which the relative abundance of Bacteroidetes and Firmicutes reached more than 90%. Compared with the blank, the proportion of Firmicutes in the model group decreased, and the proportion of Bacteroidetes increased; compared with the model group, the proportion of Firmicutes and Bacteroidetes in the lactic acid Pediococcus group was adjusted back. At the genus level, norank_f__Muribaculaceae, Lactobacillus, Bacillus, Alistipes, Alloprevotella, Bacteroides, Helicobacter, etc. were mainly present in each group. Compared with the blank group, the proportions of Lactobacillus, Bacillus, Bacteroides, and Plevofloxacin in the lactic acid Pediococcus group decreased, and the proportion of Helicobacter increased; compared with the model group, the proportions of Lactobacillus, Bacillus, and Helicobacter in the lactic acid Pediococcus group decreased. Figure 6 . At the genus level, the abundance of Pseudomonas in the blank group was higher than that in the model group, and the abundance of Helicobacter and Bacillus in the model group was higher than that in the blank group; the abundance of Lactobacillus in the Pediococcus acidilactici group was higher than that in the model group; the abundance of Bacillus in the mixed bacteria positive group was higher than that in the model group. Based on the above results, it is speculated that the treatment of model mice with Pediococcus acidilactici is related to the decrease in the abundance of Helicobacter and Bacillus, and the increase in the abundance of Lactobacillus and g_unclassified_c_Bacilli. Figure 7 .

[0118] Example 8: Effect of Pediococcus acidilactici P48 on intestinal flora of diarrheal mice

[0119] 40 SPF Balb / c mice, male, with a body weight of 18-20g, were raised in an environment with a temperature of 20-25℃, a relative humidity of (50±5)%, and a light exposure time of 12h / 12h. They were randomly divided into 4 groups, 10 mice in each group, set as blank control group, model group, probiotic experimental group and positive control group (Imodium). Mice in the blank control group were given a solvent (a 3.2% Tween-80 aqueous solution) of 10mL / kg, mice in the model group were given a solvent (a 3.2% Tween-80 aqueous solution) of 10mL / kg, mice in the probiotic experimental group were given 1.0g / kg probiotic powder (the solvent was a 3.2% Tween-80 aqueous solution), and mice in the positive control group were given Imodium 10mg / kg (the solvent was a 3.2% Tween-80 solution), each group once / d for 7 days. On the evening of the 7th day of administration, fasting but not water was allowed. 0.5h after administration on the 8th day, the model group, probiotic experimental group and positive control group mice were gavaged with 0.5mL castor oil to establish the model, and the control group mice were gavaged with an equal amount of saline. After modeling, the mice were fed individually in a single cage, and filter paper was placed in the cage to observe the diarrhea of ​​the mice. The filter paper was changed every hour and observed continuously for 4h. The filter paper was collected to record the total defecation, normal stool, soft stool, watery stool, and loose stool grades of the mice, and the diarrhea score and diarrhea index were calculated. After 4h, the mice were eyeballed for blood collection. The collected blood samples were left to stand for more than 2h, centrifuged at 1500r / min for 10min, and the upper serum was taken for ELISA detection.

[0120] 1. Calculation of mouse diarrhea score and diarrhea index

[0121] Diarrhea score: 0 for normal stool; 2 for semi-solid stool; 3 for watery stool. Diarrhea index = sum of loose stool levels / number of loose stools. Loose stool rate (%) = sum of loose stool levels / total number of defecations of mice × 100%. Loose stool level: diameter <1.0 cm is level 1; 1.0-1.9 cm is level 2; 2.0-3.0 cm is level 3; >3.0 cm is level 4. If the stain is a regular circle, the diameter is measured. If the stain is irregular, the diameter of the longest and shortest points is measured, and the two numbers are added and divided by 2. As shown in Table 5, the diarrhea score and diarrhea index of mice in the model group were significantly higher than those in the control group, indicating that the castor oil model was successfully established. The diarrhea score and diarrhea index of mice in the positive control group were significantly lower than those in the model group, and the diarrhea score and diarrhea index of mice in the probiotic experimental group were significantly lower than those in the model group. At the same time, the diarrhea index of the probiotic group was slightly lower than that of the Imodium group. This shows that Pediococcus acidilactici has a significant preventive and therapeutic effect on the castor oil diarrhea model.

[0122]

[0123] 2. Detection of inflammatory cytokines in serum

[0124] Solebao IL-1β (interleukin 1β), IL-6, IL-10 (interleukin 10), and TNF-α (tumor necrosis factor α) ELISA kits were purchased, and the collected serum was operated according to the ELISA kit instructions: add the sample and incubate at 37°C for 1 hour; after aspiration, add detection solution A and incubate for 1 hour, wash the plate: add detection solution B and incubate for 30 minutes, wash the plate; add TMB substrate and incubate for 10 to 20 minutes; after adding the stop solution, immediately use an enzyme reader to read at 450nm to determine the levels of IL-1β, IL-6, IL-10, and TNF-α cytokines.

[0125] As shown in Table 6, compared with the control group, there were no significant differences in the levels of IL-1β, IL-6 and TNF-α in the serum of the model group mice, and the IL-10 content was significantly increased. Compared with the model group, the concentrations of IL-1β, IL-6 and IL-10 in the serum of the mice in the probiotic experimental group were significantly or extremely significantly reduced. It is speculated that lactic acid Pediococcus can reduce the inflammatory response in mice by downregulating the expression of IL-1β, IL-6 and IL-10, and play a certain anti-diarrhea effect.

[0126]

[0127] Comparative Example 1

[0128] The specific implementation method is the same as Example 5, except that the lactic acid Pediococcus P48 is replaced by lactic acid Pediococcus BNCC137601 (commercially available strain of Suzhou Beina Chuanglian Biotechnology Research Institute), and the feces particle number, feces mass and feces water content of constipated mice are measured. The results show that the feces particle number of the constipated mice in the lactic acid Pediococcus BNCC137601 group is 47.58±1.30, the feces mass is 1.55±0.22g, and the feces water content is 38.48±0.55%. It can be seen that lactic acid Pediococcus P48 has a significant effect on the small intestinal propulsion rate of constipated mice. Compared with lactic acid Pediococcus BNCC137601, lactic acid Pediococcus P48 relieves constipation and promotes the peristalsis of the small intestine, which is significantly better than lactic acid Pediococcus BNCC137601.

[0129] Comparative Example 2

[0130] The specific implementation method is the same as Example 8, except that Pediococcus acidilactici P48 is replaced by Pediococcus acidilactici BNCC137601 (a commercially available strain from Suzhou Beina Chuanglian Biotechnology Research Institute), and the diarrhea score and diarrhea index of diarrheal mice are measured. The results show that the diarrhea score and diarrhea index of Pediococcus acidilactici BNCC137601 for diarrheal mice are 15.28±5.71 and 1.38±0.02, respectively. Compared with the diarrhea model group (55.66±12.71, 1.81±0.42), Pediococcus acidilactici BNCC137601 has no effect on alleviating the loose stool rate, loose stool grade and diarrhea index of diarrheal mice.

[0131] The bidirectional regulation process of the lactic acid bacteria in the intestine is a dynamic equilibrium process. It can not only flexibly adjust its own metabolic strategy and growth pattern according to changes in the intestinal environment, but also maintain the stability and harmony of the intestinal microecology through interaction with other intestinal microorganisms.

[0132] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A strain of Pediococcus acidilactici P48 capable of relieving constipation and diarrhea, characterized in that: Its name is P48, its classification name is Pediococcus acidilactici, its preservation number is CGMCC No.27885, its preservation date is July 13, 2023, and its preservation unit is General Microbiology Center of China Culture Collection Administration, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

2. The Pediococcus acidilactici P48 according to claim 1, characterized in that: The lactic acid Pediococcus P48 has the following biological characteristics: (1) Bacterial characteristics: Gram-positive, spherical cells, non-motile; (2) Colony characteristics: On MRS medium, the colonies are small and white, and the growth along the agar puncture line is filamentous; (3) Growth characteristics: At 37°C, the cells reached the late logarithmic phase after 10 h of culture in MRS medium. (4) It has strong tolerance to simulated gastrointestinal fluid.

3. The Pediococcus acidilactici P48 according to claim 1 or 2, characterized in that: The Pediococcus acidilactici P48 has the function of alleviating constipation and diarrhea.

4. Use of Pediococcus acidilactici P48 according to any one of claims 1 to 3 in the preparation of a medicament for relieving constipation and diarrhea.

5. The use according to claim 4, characterized in that: The drug has at least one of the following effects: Improve constipation, specifically by increasing the number and quality of feces and the water content of feces in mice; Alternatively, constipation can be improved by repairing the colon mucosal epithelium and intestinal glands to restore the colon tissue to normal, thus providing a better colonization environment for the flora. Alternatively, improve constipation by inhibiting the release of inflammatory factors, relieving inflammatory responses, and regulating gastrointestinal hormone levels; Alternatively, improve constipation, specifically by regulating intestinal flora disorders, promoting the growth of beneficial bacteria, and inhibiting the growth of harmful bacteria; Alternatively, improving diarrhea, specifically improving the diarrhea loose stool rate, loose stool grade and diarrhea index.

6. The use according to claim 4, characterized in that: The Pediococcus acidilactici P48 is a live strain.

7. The use according to any one of claims 4 to 6, characterized in that: The drug further contains a pharmaceutically acceptable carrier, which includes one or more of a filler, a diluent, a binder, a disintegrant and a lubricant.

8. The use according to claim 7, characterized in that: The filler is one or more of microcrystalline cellulose, lactose, mannitol, starch or dextrin; the diluent is one or more of ethanol or glycerol; the disintegrant is one or more of sodium carboxymethyl starch, sodium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone or low-substituted hydroxypropyl cellulose; the binder is one or more of starch paste, syrup, maltose, refined honey or liquid glucose; the lubricant is one or more of magnesium stearate, sodium stearyl fumarate, talc or silicon dioxide.

9. Use of Pediococcus acidilactici P48 according to any one of claims 1 to 3 in the preparation of fermented food and / or postbiotics and / or enzymes.

10. The use according to claim 9, characterized in that: The application includes using Pediococcus acidilactici P48 as a fermentation microorganism and utilizing food raw materials for fermentation.

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