Phytobacterium lactis for relieving constipation and preparation method thereof

Abstract

The invention discloses a lactobacillus lactis for relieving constipation and a preparation method thereof, target strains are respectively separated from a fermented bean curd sample and screened by a pectinase screening agar culture medium, and are named as lactobacillus plantarum PD-14 and lactobacillus pentosus PD-16, the lactobacillus lactis has a gene for synthesizing pectate lyase, and the lactobacillus lactis has a gene for synthesizing pectate lyase. Relates to the microbial field. The invention provides a beneficial bacterium for relieving constipation and a preparation method thereof, wherein the beneficial bacterium is capable of efficiently relieving constipation, low in cost, stable and easy to industrialize.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more particularly to a lactobacillus for relieving constipation and its preparation method. Background Technology

[0002] Constipation is a very common gastrointestinal disorder worldwide. Currently, constipation treatment mainly relies on medication, including 5-HT receptor agonists as prokinetic agents, lubiprostone and linagliptin as intestinal secretagogues, and laxatives such as senna, bisacodyl, and magnesium. However, most of these medications have toxic side effects and adverse reactions, potentially leading to drug dependence, diarrhea, and potential damage to the enteric nervous system. Therefore, exploring safer and more effective constipation relief therapies is crucial.

[0003] Gut microbiota dysbiosis is one of the important mechanisms of constipation. Compared with healthy individuals, constipated patients have a significantly reduced number of beneficial bacteria in their gut, so regulating the gut microbiota can improve constipation symptoms. Supplementing the number of beneficial bacteria in the gut can correct gut microbiota imbalance, thereby alleviating and treating constipation. Studies have found that dietary fiber, especially water-soluble dietary fiber, can promote the growth of beneficial bacteria in the gut and improve intestinal motility, thus relieving constipation symptoms. Pectin, a type of water-soluble dietary fiber, can assist intestinal peristalsis and absorb intestinal bacteria and toxins. After degradation, pectin can produce an absorbable gel that is easily transported through the intestines, possessing constipation-relieving properties. Pectin and its derivatives are potential prebiotics that can selectively increase the abundance of beneficial bacteria in the human gastrointestinal tract. However, current research results on probiotics' ability to relieve constipation are inconsistent, and clinical efficacy still needs further improvement.

[0004] Therefore, those skilled in the art are dedicated to developing a new type of safe and effective beneficial bacteria that can relieve constipation. Summary of the Invention

[0005] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a novel, effective, low-cost, safe and effective beneficial bacteria that can relieve constipation.

[0006] To achieve the above objectives, the present invention provides a lactobacillus for relieving constipation, characterized in that the lactobacillus has a gene for synthesizing pectin lyase.

[0007] In a preferred embodiment of the present invention, the *Lactobacillus plantarum* is *Lactobacillus plantarum* PD-14, with accession number GDMCC 66693. The pectin lyase gene sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. Latin name of the strain: Lactiplantibacillus plantarum PD14, classification and Latin scientific name: Lactobacillus plantarum, Lactiplantibacillus plantarumDepository: Guangdong Provincial Center for Microbial Culture Collection, Address: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, Deposit Date: July 16, 2025, Deposit Number: GDMCC 66693; In another preferred embodiment of the present invention, the *Lactobacillus* is *Lactobacillus pentosus* PD-16, with accession number GDMCC 66694, and the pectin lyase gene sequence is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4. Latin name of the strain: Lactiplantibacillus pentosus PD16, classification and Latin scientific name: Lactobacillus pentosaceus Lactiplantibacillus pentosus Depository: Guangdong Provincial Center for Microbial Culture Collection, Address: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, Deposit Date: July 16, 2025, Deposit Number: GDMCC 66694.

[0008] In another preferred embodiment of the present invention, the lactobacillus is isolated from fermented tofu.

[0009] The present invention also provides a method for preparing the above-mentioned Lactobacillus, characterized by comprising the following steps: Step 1: Isolation of Lactobacillus; Step 2: Screening of Lactobacillus that decomposes pectin.

[0010] In a preferred embodiment of the present invention, step 1 specifically includes: Step 1.1: Place the fermented tofu sample in a 15mL sterile centrifuge tube containing MRS liquid medium, invert and shake well, and place it in an anaerobic incubator at 37℃ for 24 hours. Step 1.2: Take the culture medium from Step 1.1 and dilute it tenfold with PBS solution. Spread the diluted solutions evenly on the surface of MRS solid culture medium. Set up two parallel groups for each concentration. Place the spread plates in an anaerobic incubator and incubate at 37°C under anaerobic conditions for 48 hours. Step 1.3: Remove the petri dish, use a disposable inoculation loop to pick up single colonies with different phenotypes and streak them on MRS solid medium, and anaerobic culture for 48 hours; Step 1.4: Repeat the steps in step 1.3 for secondary purification; Step 1.5: Pick a single colony for colony PCR to amplify the 16S rRNA gene fragment, and inoculate the same colony into MRS liquid medium and incubate under anaerobic conditions at 37°C for 24 hours. Step 1.6: Take the bacterial liquid culture from Step 1.5, mix the cultured bacterial solution with glycerol preservation solution, and the final glycerol concentration is 30%. Mix and vortex or gently blow to ensure that the glycerol evenly coats the bacterial cells, and then freeze. Step 1.7: Perform 16S rRNA gene sequencing and nucleic acid sequence alignment on the PCR products from Step 1.5 to determine the strain.

[0011] In another preferred embodiment of the present invention, step 2 specifically includes: Step 2.1: Use pectinase screening agar medium (PSAM) to preliminarily screen the pectinase activity of the isolates. Before sterilization, adjust the pH of the medium to 5.5 ± 0.5; after sterilization, pour the medium and allow it to solidify at room temperature. Step 2.2: Transfer all isolates to the culture medium from Step 2.1 and anaerobic culture at 37°C for 48 hours; Step 2.3: At the end of the culture, immerse the surface of the culture dish in 0.3% Congo red solution and let it stand at room temperature for 10 minutes. The strain that forms a transparent area around the colony is the strain obtained.

[0012] In another preferred embodiment of the present invention, the pectinase screening agar medium is composed of 1.0% citrus pectin, 0.14% ammonium sulfate, 0.6% dipotassium hydrogen phosphate, 0.20% potassium dihydrogen phosphate, 0.01% magnesium sulfate and 2.0% agar, all of which are mass / volume ratios, i.e., w / v.

[0013] Technical effect

[0014] 1. This invention identifies novel probiotics with the potential to alleviate functional constipation from fermented tofu samples collected in Pu'er City, Yunnan Province, China. These probiotics can correct intestinal flora imbalance by supplementing the number of beneficial bacteria in the gut, thereby alleviating constipation symptoms. The effects of *Lactobacillus plantarum* PD-14 and *Lactobacillus pentosus* PD-16 on constipation relief were evaluated by monitoring fecal water content, intestinal propulsion rate, and time to first black stool in mice. Bacteria were cultured anaerobically at 37°C for 48 hours on a modified culture medium with pectin as the sole carbon source. The culture dishes were then immersed in a 0.3% Congo red solution. A transparent zone formed around the colonies, indicating that the colonies could produce pectinase, and the diameter of the transparent zone was directly proportional to the relative pectinase production capacity of the bacteria. The *Lactobacillus* isolated in this invention can be used to develop novel probiotic agents to alleviate constipation symptoms; compared to drug treatment, probiotic agents are more readily accepted by the public. In this invention, *Lactobacillus plantarum* PD-14 and *Lactobacillus pentosus* PD-16 exhibit good stability and functionality. Pectinase activity of the *Lactobacillus plantarum* strains was isolated by screening using pectinase screening agar (PSAM), revealing that both strains possess excellent pectin-degrading capabilities. Whole-genome sequencing data compared with the CAZy database confirmed that *Lactobacillus plantarum* PD-14 and PD-16 contain genes encoding pectate lyase.

[0015] 2. Effective in relieving constipation: The bacterial suspensions of *Lactobacillus plantarum* PD-14 and *Lactobacillus pentosus* PD-16 in this invention significantly increased the water content of mouse feces, intestinal transit rate, and reduced the time to first black stool in a four-week animal experiment, showing the potential to relieve constipation symptoms in mice induced by loperamide.

[0016] 3. Good preservation stability of the strain: The strain of the present invention can be stored for a long time after being mixed with glycerol and stored at -80℃, thus maintaining its activity.

[0017] 4. Low cost and easy to achieve fermentation conditions: The plant lactobacillus PD-14 and pentosose lactobacillus PD-16 of the present invention can be grown in MRS medium at 37°C under anaerobic conditions. The culture conditions are relatively simple, suitable for industrial production, and the cost is low.

[0018] 5. Prospects for industrial application: This invention isolates a novel probiotic strain with good potential to relieve functional constipation from fermented food samples from China, and possesses independent intellectual property rights. It provides a new solution to the problems of long-term toxic side effects and adverse reactions of drugs, as well as low public acceptance.

[0019] Food and health products: Used in the production of yogurt, beverages, health products, etc., to meet the market demand for healthy foods.

[0020] In the pharmaceutical field: for the development of probiotic preparations or adjuvants to relieve constipation symptoms.

[0021] Food industry enzyme preparations: Based on the pectin-degrading capabilities of Lactobacillus plantarum PD-14 and Lactobacillus pentosus PD-16, pectinase can be produced, which has application potential in the production of industrial enzyme preparations.

[0022] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the pectin-decomposing abilities of *Lactobacillus plantarum* PD-14 and *Lactobacillus pentosus* PD-16 according to a preferred embodiment of the present invention. Figure 2 This is a diagram showing the distribution ratio of carbohydrate enzymes in *Lactobacillus plantarum* PD-14 according to a preferred embodiment of the present invention. Figure 3 This is a diagram showing the distribution ratio of carbohydrate enzymes in *Lactobacillus pentosus* PD-16, a preferred embodiment of the present invention.

[0024] Figure 4 A is a schematic diagram of mouse weight changes according to a preferred embodiment of the present invention;

[0025] Figure 4 B is a comparison diagram of the number of mouse fecal particles in a preferred embodiment of the present invention;

[0026] Figure 4 C is a comparison chart of mouse fecal water content according to a preferred embodiment of the present invention;

[0027] Figure 4 D is a comparison chart of gastrointestinal transit rates in mice according to a preferred embodiment of the present invention;

[0028] Figure 4 E is a comparison chart of the time when mice first defecate black stool according to a preferred embodiment of the present invention.

[0029] Preservation information: Strain 1: Strain name: Lactobacillus plantarum PD-14, Strain Latin name: Lactiplantibacillus plantarum PD14, classification and Latin scientific name: Lactobacillus plantarum, Lactiplantibacillus plantarum Depository: Guangdong Provincial Center for Microbial Culture Collection, Address: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, Deposit Date: July 16, 2025, Deposit Number: GDMCC 66693; Strain 2: Strain name: Lactobacillus pentosaceus PD-16, Strain Latin name: Lactiplantibacillus pentosus PD16, classification and Latin scientific name: Lactobacillus pentosaceus Lactiplantibacillus pentosus Depository: Guangdong Provincial Center for Microbial Culture Collection, Address: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, Deposit Date: July 16, 2025, Deposit Number: GDMCC 66694. Detailed Implementation

[0030] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0031] Example 1: Isolation and Screening of Lactobacillus

[0032] 1.1 Isolation of Lactobacillus

[0033] 1) Take the fermented tofu sample from Kunming, Yunnan Province from the -80°C ultra-low temperature freezer, and place 1.0g into a 15mL sterile centrifuge tube containing 9.0mL LMRS liquid medium (the medium formula per liter is 10.0g casein digest, 10.0g beef extract, 4.0g yeast extract, 2.0g triammonium citrate, 5.0g sodium acetate, 0.2g magnesium sulfate heptahydrate, 0.05g manganese sulfate tetrahydrate, 2.0g dipotassium hydrogen phosphate, 20.0g glucose, 1.0g Tween-80 and 1000mL distilled water). Shake well by inverting and incubate anaerobically at 37°C for 24 hours.

[0034] 2) Take 1.0 mL of the above culture medium (shake well before use), add it to 9.0 mL of PBS solution, shake well, and then serially dilute from 10 mL to 10 mL. -1 Dilute to 10 -6 , absorb 10 -3 , 10 -4 , 10 -5 , 10 -6 10 μL of the diluted sample was dropped onto the surface of solid MRS medium and spread evenly using a disposable spreader. Two parallel spreads were prepared for each concentration. The plates were then anaerobically incubated at 37°C for 48 hours.

[0035] 3) Remove the culture dish, use a disposable inoculation loop to pick up single colonies with different phenotypes and streak them on MRS solid medium, and repeat the anaerobic culture for 48 hours.

[0036] 4) Repeat the above steps for secondary purification.

[0037] 5) Select a single colony for colony PCR, and add the remaining colonies to MRS liquid medium and incubate at 37°C under anaerobic conditions for 24 hours.

[0038] 6) Take the above-mentioned bacterial liquid culture, mix the cultured bacterial solution with glycerol preservation solution, and the final glycerol concentration is 30%. Mix and vortex or gently blow to ensure that the glycerol evenly coats the bacterial cells. Dispense the mixed glycerol bacterial solution into sterile cryovials, label them with bacterial strain name, storage date and other information, and store the cryovials in a -80°C freezer.

[0039] 7) Sequencing of the PCR products with 16S rRNA gene was performed, and the sequencing results were analyzed by BLAST on the NCBI website to determine the strain.

[0040] 1.2 Screening of Lactobacillus species that decompose pectin

[0041] 1) Prepare pectinase screening agar medium (the formula per liter of medium is 10.0g citrus pectin, 1.4g ammonium sulfate, 6.0g dipotassium hydrogen phosphate, 2.0g potassium dihydrogen phosphate, 0.1g magnesium sulfate, 20.0g agar and about 1000mL distilled water).

[0042] 2) Pectinase activity of isolates was initially screened using pectinase screening agar (PSAM). The pH of the medium was adjusted to 5.5 ± 0.5 before sterilization, and then autoclaved at 121°C for 15 minutes. Finally, 20-25 mL of the medium was poured into sterile petri dishes and allowed to solidify at room temperature. All isolates were transferred to this medium and incubated in an anaerobic chamber at 37°C for 48 hours.

[0043] 3) At the end of the culture, immerse the petri dish in a 0.3% (w / v) Congo red solution and leave it for 10 minutes. The solution forms a clear zone around the colonies, indicating that the bacterial isolate can produce pectinase, and the diameter of the clear zone is proportional to the relative pectinase production capacity of the bacteria.

[0044] 1.3 Whole genome sequencing of Lactobacillus

[0045] The experimental procedure was performed according to the standard protocol provided by Oxford Nanopore Technologies (ONT), including sample quality testing, library construction, library quality testing, and library sequencing. The main steps are as follows: A) High-quality genomic DNA was extracted and its purity, concentration, and integrity were checked using Nanodrop, Qubit, and 0.35% agarose gel electrophoresis. B) The BluePippin fully automated nucleic acid recovery system recovers large DNA fragments; C) Library construction (SQK-LSK109 ligation kit): DNA damage repair and end repair, magnetic bead purification; adapter ligation, magnetic bead purification; Qubit library quantification; D) Sequencing.

[0046] Information analysis mainly includes the following steps: A) Raw data quality control, filtering out low-quality and excessively short reads; B) Genome assembly: The filtered reads are assembled de novo, and the assembled draft genome is corrected. C) Genome assessment, mainly including: genome kmer frequency distribution, busco assessment, checkm assessment, GC-depth, and genome coverage statistics; D) Genomic component analysis, mainly including: repetitive sequence, coding gene, non-coding RNA, prophage, gene island, CRISPR, etc. E) Functional annotations, mainly including general databases such as Nr, Uniprot, COG, KEGG, etc., as well as proprietary database annotations such as CAZyme, PHI, CARD, etc. F) Genome mapping analysis, including: genome loop maps, genome mapping tools, and genome maps.

[0047] 1.3 Technical Effects

[0048] like Figure 1 As shown, *Lactobacillus plantarum* PD-14 and *Lactobacillus pentosus* PD-16 can decompose pectin to form clear zones on pectinase screening medium.

[0049] Whole-genome sequencing of these two strains and comparison with the CAZy (Carbohydrate-active enzymesdatabase) database revealed that both *Lactobacillus plantarum* PD-14 and *Lactobacillus pentosus* PD-16 contain the pectate lyase gene, with gene sequences shown in SEQ ID NO.1 and SEQ ID NO.3, respectively, and amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO.4, respectively.

[0050] Example 2: Relieving constipation symptoms with Lactobacillus plantarum PD-14 and Lactobacillus pentosus PD-16

[0051] 1. Probiotic treatment

[0052] The *Lactobacillus plantarum* PD-14 and *Lactobacillus pentosus* PD-16 used in this invention were both stored at -80 °C. Before the experiment, the strains were removed from the freezer and thawed. First, the cultured strains were purified using the streak plate method on MRS solid plates, and then activated in MRS liquid medium. The activated bacterial solutions were serially diluted, and the CFU (Cellular Units) of the solutions were calculated using the plate count method. The concentrations were measured using a spectrophotometer at 5 × 10⁻⁶. 9 OD of CFU / mL bacterial culture 600 The bacterial culture was concentrated to 5 × 10⁻⁶. 9 After obtaining CFU / mL, centrifuge at 4000×g for 10 minutes, remove the supernatant, resuspend in physiological saline, and repeat twice. The prepared bacterial suspension is used for animal experiments.

[0053] 2. Animals and Experimental Design

[0054] All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University (A 2025054). Forty-two female C57BL / 6 mice (6 weeks old, weighing 18±1 g) were purchased from Lingchang Pharmaceuticals (Shanghai, China) and housed in cages with humidity ranging from 40% to 60%. Free access to water was provided. After one week of acclimatization, the mice were randomly assigned to five groups of eight mice each: a normal control group (NC), a constipation model group (CM), two experimental groups (nine mice each for PD-14 and PD-16), and a positive control group (PC). Constipation was induced in the mice by administration of loperamide (Janssen Pharmaceuticals Ltd., Shaanxi, China). Loperamide is a synthetic peripheral μ-opioid receptor agonist that attenuates colonic transport while increasing water and ion absorption. Polyethylene glycol 4000 (Beaufour Ipsen Industrie Co., Ltd., France) is a commonly used osmotic laxative for treating constipation and was selected as a positive control drug due to its established efficacy in promoting defecation. Except for the NC group mice, all mice were administered 0.2 mL of 10 mg / kg BW loperamide at 9:00 AM daily to induce constipation. One hour later, the CM group received sterile saline, the PC group received 0.2 mL of polyethylene glycol 4000 (500 mg / kg BW), and the PD-14 and PD-16 groups received 0.2 mL of 10 mg / kg BW loperamide. 9 Treatment with CFU / mL suspensions of *Lactobacillus plantarum* PD-14 and *Lactobacillus pentosus* PD-16 for four weeks.

[0055] After fasting for one night, mice were anesthetized with isoflurane, and blood samples were collected from the orbital region. The blood was allowed to coagulate at room temperature for 2 hours, then centrifuged at 3000 rpm for 10 minutes. After blood collection, the mice were euthanized by cervical dislocation, and dissected on day 29. Autopsies were then performed, and colonic tissue was extracted for analysis. Subsequently, 0.5 cm of the original colonic tissue was immersed in 4% paraformaldehyde solution (w / v). The remaining colonic tissue was divided into three portions, rapidly frozen in liquid nitrogen, and then stored at -80 °C.

[0056] 3. Assessment of defecation parameters

[0057] Mouse feces were collected weekly. After each tube-fed mouse was placed in a clean cage lined with filter paper for 5 hours, then fresh fecal samples were collected, and the quantity of feces was carefully counted. The fresh feces were collected in sterile 1.5 mL centrifuge tubes, wet-weighed, dried, and weighed again to calculate the fecal moisture content.

[0058] 4. GI transport rate

[0059] At the end of the animal experiments, after a 12-hour fast, mice were administered 0.2 mL of activated charcoal labeled with gum arabic via tube feeding. Thirty minutes later, the mice were anesthetized with isoflurane and then sacrificed. The small intestine was dissected, its total length was measured, the propulsion distance of the activated charcoal powder was determined, and the GI transport rate was calculated.

[0060] 5. The time of the first black stool

[0061] After the drug treatment ended, each mouse was given 0.2 mL of activated charcoal gum arabic-labeled solution via tube feeding one day before the end of the experiment, and the time when each mouse first showed black stool was recorded.

[0062] 6. Technical Effects

[0063] like Figure 4 As shown in AD, the body weight, fecal volume, and water content of mice in the CM group were significantly reduced, and the gastrointestinal transit rate was decreased. p < 0.01), where the data represents the SD ± mean (n = 8). Different lowercase letters indicate in p The difference was statistically significant at the < 0.05 level. Furthermore, such as... Figure 4 As shown in E, the time to first black stool defecation significantly increased ( p < 0.01 indicates that a loperamide-induced constipation model was successfully established. However, compared with the CM group mice, the PD-14 group significantly improved intestinal transit rate ( p <0.01), while the time to the first black stool was significantly longer in the PD-16 group ( p< 0.01). Furthermore, both the PD-14 and PD-16 groups significantly increased fecal water content and fecal particle number ( p < 0.01), and compared with the PC group mice, the group mice did not show significant differences in these defecation characteristics ( p > 0.05).

[0064] These results indicate that PD-14 and PD-16 can effectively relieve constipation symptoms, suggesting that taking PD-14 and PD-16 may be a potentially effective way to treat constipation.

[0065] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A type of Lactobacillus for relieving constipation, characterized in that, The lactobacillus possesses the gene for synthesizing pectin lyase.

2. The *Lactobacillus* as described in claim 1, characterized in that, The Lactobacillus plantarum is Lactobacillus plantarum PD-14, with accession number GDMCC 66693. The pectin lyase gene sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

3. The *Lactobacillus* as described in claim 1, characterized in that, The Lactobacillus is Lactobacillus pentosus PD-16, with accession number GDMCC 66694. The pectin lyase gene sequence is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.

4.

4. The *Lactobacillus* as described in claim 1, characterized in that, The lactobacillus was isolated from fermented tofu.

5. A method for preparing *Lactobacillus lactis* as described in any one of claims 1-4, characterized in that, Includes the following steps: Step 1: Isolation of Lactobacillus; Step 2: Screening of Lactobacillus that decomposes pectin.

6. The preparation method according to claim 5, characterized in that, Step 1 specifically includes: Step 1.1: Place the fermented tofu sample in a 15mL sterile centrifuge tube containing MRS liquid medium, invert and shake well, and place it in an anaerobic incubator at 37℃ for 24 hours. Step 1.2: Take the culture medium from Step 1.1 and dilute it tenfold with PBS solution. Spread the diluted solutions evenly on the surface of MRS solid culture medium. Set up two parallel groups for each concentration. Place the spread plates in an anaerobic incubator and incubate at 37°C under anaerobic conditions for 48 hours. Step 1.3: Remove the petri dish, use a disposable inoculation loop to pick up single colonies with different phenotypes and streak them on MRS solid medium, and anaerobic culture for 48 hours; Step 1.4: Repeat the steps in step 1.3 for secondary purification; Step 1.5: Pick a single colony for colony PCR to amplify the 16S rRNA gene fragment, and inoculate the same colony into MRS liquid medium and incubate under anaerobic conditions at 37°C for 24 hours. Step 1.6: Take the bacterial liquid culture from Step 1.5, mix the cultured bacterial solution with glycerol preservation solution, and the final glycerol concentration is 30%. Mix and vortex or gently blow to ensure that the glycerol evenly coats the bacterial cells, and then freeze. Step 1.7: Perform 16S rRNA gene sequencing and nucleic acid sequence alignment on the PCR products from Step 1.5 to determine the strain.

7. The preparation method according to claim 6, characterized in that, The formulation of the MRS liquid culture medium per liter is as follows: 10.0g casein digest, 10.0g beef extract powder, 4.0g yeast extract powder, 2.0g triammonium citrate, 5.0g sodium acetate, 0.2g magnesium sulfate heptahydrate, 0.05g manganese sulfate tetrahydrate, 2.0g dipotassium hydrogen phosphate, 20.0g glucose, 1.0g Tween-80 and 1000mL distilled water.

8. The preparation method according to claim 5, characterized in that, Step 2 specifically includes: Step 2.1: Use pectinase screening agar medium (PSAM) to preliminarily screen the pectinase activity of the isolates. Before sterilization, adjust the pH of the medium to 5.5 ± 0.5; after sterilization, pour the medium and allow it to solidify at room temperature. Step 2.2: Transfer all isolates to the culture medium from Step 2.1 and anaerobic culture at 37°C for 48 hours; Step 2.3: At the end of the culture, immerse the surface of the culture dish in 0.3% Congo red solution and let it stand at room temperature for 10 minutes. The strain that forms a transparent area around the colony is the target strain.

9. The preparation method according to claim 8, characterized in that, The pectinase screening agar medium consists of 1.0% citrus pectin, 0.14% ammonium sulfate, 0.6% dipotassium hydrogen phosphate, 0.20% potassium dihydrogen phosphate, 0.01% magnesium sulfate, and 2.0% agar. All percentages are mass / volume ratios, i.e., w / v.

10. The preparation method according to claim 7, characterized in that, This also includes the treatment of *Lactobacillus lactis*. The prepared *Lactobacillus lactis* strain was removed from the refrigerator and thawed. First, the strain was purified using the streak plate method on MRS solid plates. Then, it was activated in MRS liquid medium. The activated bacterial solution was serially diluted, and the CFU (Cost Per Quantity) of the bacterial solution was calculated using the plate count method. The concentration at 5 × 10⁻⁶ CFU / mL was measured using a spectrophotometer. 9 OD of CFU / mL bacterial culture 600 The bacterial culture was concentrated to 5 × 10⁻⁶. 9 After reaching CFU / mL, centrifuge at 4000×g for 10 minutes, remove the supernatant, resuspend in physiological saline, repeat twice, and then use for later use.

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