A kind of fermented lactobacillus and application thereof

Abstract

The invention discloses a fermented lactobacillus and its application. The fermented lactobacillus of the invention is classified and named as fermented mucus lactobacillus Limosilactobacillus fermentum , strain number FL16, and the deposit number is CGMCC No. 27414. The fermented Lactobacillus can be used to prepare probiotic preparations for preventing or treating porcine intestinal diseases, and can also be used to construct an in vitro probiotic-polarity-reversed porcine intestinal organoid co-culture model, which can be used to explore the interaction between bacteria and intestinal epithelium.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a fermented lactobacillus and an application thereof. Background Art

[0002] Lactic acid bacteria are widely used probiotics. Lactic acid bacteria isolated from homologous hosts are easy to exist stably and reproduce rapidly in the intestines. The intestinal microbial composition of local pig breeds in my country is rich and diverse, and has the advantages of strong disease resistance and stress resistance. Studies have shown that local pig breeds living in the north and the south have a certain proportion of lactobacilli. Among them, the lactobacilli in the intestines of southern pig breeds represented by Bama pigs and Ningxiang pigs are significantly higher than those in northern pig breeds and Duchangda commercial pigs.

[0003] Lactobacillus fermentum is a type of lactic acid bacteria widely distributed in nature. It is often isolated from fermented plant materials, dairy products, bread, naturally fermented sausages, breast milk, saliva, and human or animal feces. More and more evidence shows that Lactobacillus fermentum can both antagonize pathogens and regulate the body's immune function. Lactobacillus fermentum can inhibit the adhesion of pathogens to the intestinal mucosa and can eliminate some pathogens; it also induces mucin production and increases goblet cell density and MUC2 expression (Yu DY, Oh SH, KimIS, et al. Intestinal microbial composition changes induced by Lactobacillus plantarum GBL 16, 17 fermented feed and intestinal immune homeostasisregulation in pigs. J Anim Sci Technol . 2022;64(6):1184-1198. doi:10.5187 / jast.2022.e89).

[0004] Traditional transformed cell lines have many limitations, such as the inability to reflect the diversity of intestinal cells, and the emergence of intestinal organoid culture systems has promoted the in vitro modeling of intestinal epithelial cells. Intestinal organoids can replicate the structure and cell composition of functional native intestinal epithelium and contain all intestinal epithelial cell lines; they also have the structure of the crypt domain and its main functions, such as nutrient absorption and barrier function.

[0005] However, one challenge in using organoids is that it is difficult to access the apical or luminal surface of the epithelial cells enclosed within the organoid. Many organoid applications require access to the apical or luminal surface of the spheroid because this is the mucosal surface that typically interfaces with the external environment and has a role in absorbing nutrients, interacting with gastrointestinal microbes, and absorbing drugs or toxins.

[0006] Studies have shown that the use of EDTA to disrupt the interaction between matrix gel and basolateral β-1 integrin receptors can cause epithelial cell morphology to rearrange without changing the morphology of individual cells. After suspension culture, intestinal organoids will be everted, showing a polarity with the tip facing outward, achieving intestinal polarity reversal (Co JY, Margalef-Català M, Monack DM, Amieva MR. Controlling the polarity of human gastrointestinal organoids to investigate epithelial biology and infectious diseases. Nat Protoc . 2021;16(11):5171-5192. doi:10.1038 / s41596-021-00607-0). Summary of the invention

[0007] At present, there is no report on isolating the fermentation Lactobacillus FL16 of the present invention from healthy Tunchang pigs, nor is there any report on using the fermentation Lactobacillus FL16 of the present invention to explore its effects on intestinal epithelial homeostasis and intestinal epithelial damage repair in a polarity-reversed pig intestinal organoid model.

[0008] The present invention first provides a fermented lactobacillus, which is classified and named as fermented mucus lactobacillus Limosilactobacillus fermentum , strain number FL16, and the deposit number is CGMCC No. 27414.

[0009] The present invention further provides the use of the fermented lactobacillus in the preparation of a probiotic preparation for preventing or treating a porcine intestinal disease. Preferably, the porcine intestinal disease is intestinal epithelial damage. More preferably, the intestinal epithelial damage is caused by Escherichia coli infection.

[0010] The present invention further provides a probiotic preparation for preventing or treating pig intestinal diseases, comprising the fermented lactobacillus.

[0011] The present invention also provides an in vitro probiotic-polarity-reversed porcine intestinal organoid co-culture model, wherein the fermented lactobacillus is added to the cultured polarity-reversed porcine intestinal organoid to construct the in vitro probiotic-polarity-reversed porcine intestinal organoid co-culture model.

[0012] The fermented lactobacillus is classified and named as fermented mucus lactobacillus Limosilactobacillus fermentum , strain number FL16, and the deposit number is CGMCC No. 27414.

[0013] The present invention also provides a method for constructing the in vitro probiotic-polarity-reversed porcine intestinal organoid co-culture model, comprising the following steps:

[0014] (1) Isolation and culture of porcine intestinal organoids;

[0015] (2) constructing and culturing the porcine intestinal organoids obtained in step (1) to obtain polarity-reversed porcine intestinal organoids;

[0016] (3) Adding the fermented Lactobacillus to the polarity-reversed porcine intestinal organoids obtained in step (2), and co-culturing to obtain the in vitro probiotic-polarity-reversed porcine intestinal organoid co-culture model.

[0017] The present invention also provides the use of the in vitro probiotic-polarity-reversed pig intestinal organoid co-culture model in screening probiotic products for preventing or treating intestinal diseases.

[0018] The present invention also provides the use of the in vitro probiotic-polarity-reversed porcine intestinal organoid co-culture model in screening probiotic products that alleviate piglet weaning stress.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention isolated and identified Lactobacillus fermentum FL16 from healthy Tunchang pigs for the first time; the characteristics of this strain are: Gram staining is positive and short rod-shaped;

[0021] (2) The polarity-reversed porcine intestinal organoids constructed by the present invention can maintain complete barrier function while changing intestinal polarity; secondly, the suspended organoids with the top facing outward can be synchronously exposed to a culture medium with uniform concentration, thereby improving the effective contact between bacteria and epithelial cells.

[0022] (2) The present invention constructs an in vitro probiotic / pathogen-polarity-reversed porcine intestinal organoid co-culture model, which can be used to explore the interaction between bacteria and intestinal epithelium. Escherichia coli K88 can destroy the integrity of the intestinal epithelium and cause cellular inflammatory response; Lactobacillus fermentum FL16 can effectively adhere to the top of the intestinal epithelium, promote the proliferation and differentiation of intestinal stem cells, and maintain the integrity of the intestinal epithelium.

[0023] (3) The present invention demonstrates that Lactobacillus fermentum FL16 can activate the Wnt / β-catenin pathway to promote intestinal epithelial proliferation and differentiation, and regulate inflammatory cytokines, thereby alleviating intestinal epithelial damage caused by ETEC K88.

[0024] (4) The fermented Lactobacillus FL16 isolated by the present invention can be directly fed to animals to protect intestinal health; the bacteria is expected to be developed into a probiotic preparation for preventing or treating intestinal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is an optical microscope Gram staining observation of Lactobacillus fermentum FL16.

[0026] Figure 2 Optical microscope and laser confocal microscope observation pictures of pig intestinal organoids (Basal-out) and polarity-reversed pig intestinal organoids (Apical-out); Figure 2 a in the figure: pig intestinal organoid; Figure 2 b: Polarity-reversed pig intestinal organoid; Figure 2 c in the figure: Comparison of pig intestinal organoids and polarity-reversed pig intestinal organoids.

[0027] Figure 3 The results of the test show that Lactobacillus fermentum FL16 can promote the growth of organoids and alleviate the intestinal organoid damage caused by ETEC K88.

[0028] Figure 4 This is the result of the detection that Lactobacillus fermentum FL16 can promote the expression of Ki67 and Lgr5; Figure 4 a in the figure: Ki67 mRNA level and fluorescent protein expression; Figure 4 b: Lgr5 mRNA level and fluorescent protein expression.

[0029] Figure 5 This is the test result showing that fermented Lactobacillus FL16 can regulate the expression of inflammatory cytokines and alleviate cellular inflammatory response.

[0030] Figure 6 This is the result of the test that Lactobacillus fermentum FL16 can upregulate the expression of Wnt3a and β-catenin; Figure 6 a in the figure: Wnt3a mRNA level and fluorescent protein expression; Figure 6 b: β-catenin mRNA level and fluorescent protein expression.

[0031] Figure 7 The results of the test show that fermented Lactobacillus FL16 can increase the depth of jejunal crypts and villus height, and relieve the inflammatory response caused by Escherichia coli K88; Figure 7 a in the figure: HE staining of jejunum; Figure 7 b: Expression of inflammatory cytokines.

[0032] Figure 8 This is the result of fermenting Lactobacillus FL16 to reduce the diarrhea rate of piglets caused by weaning stress and increase the average daily gain (ADG) of piglets.

[0033] Fig. 9The results of the test for improving the expression of Zo-1 and Occludin proteins in the jejunal mucosa of weaned piglets by fermenting Lactobacillus FL16. DETAILED DESCRIPTION

[0034] Example 1

[0035] Isolation and identification of Lactobacillus fermentum FL16.

[0036] (1) Preliminary screening of sample sources and strains.

[0037] In the pig farm of Tunchang County, Hainan Province, 25-30 days old healthy Tunchang pigs were selected, and a sterile cotton swab was used to pick up an appropriate amount of fresh feces, which was stored in a sterile centrifuge tube. Five fecal samples were collected and microbial separation and purification was performed in the laboratory. The five fresh fecal samples were placed in a conical bottle containing sterile saline, shaken on a shaker for 5 minutes, and fully mixed to form a fecal sample stock solution. The fecal sample stock solution was diluted in a 10-fold series gradient from 10 -1 to 10 -9 , a total of 9 dilution gradients. Take 10 -5 Up to 10 -9 0.1 mL of each of the five dilution gradients of fecal suspension was spread on MRS solid culture medium, cultured anaerobically at 37°C for 48 hours, and single colonies were picked for streaking separation, and repeated 2-3 times until the colony morphology on the plate was consistent, completing bacterial purification. Single colonies on the MRS plate were selected and inoculated into MRS liquid culture medium, cultured anaerobically at 37°C for 24 hours, and a small amount of bacterial liquid was evenly spread on a glass slide. After fixation and Gram staining, the bacterial morphology was observed under a microscope.

[0038] like Figure 1 Shown is an optical microscope observation of the isolated fermentation lactobacillus, which is short rod-shaped and Gram-positive.

[0039] (2) PCR identification.

[0040] Take 200 μL of the cultured bacterial solution in a 1.5 mL centrifuge tube, centrifuge at 12000 rpm / min for 5 min, discard the supernatant to collect the bacteria, and use the TSINGKE DNA extraction kit (universal) to extract the total DNA of lactobacillus.

[0041] Table 1 16S rDNA sequencing primers

[0042]

[0043] Amplification was performed with Qingke 1×TSE101 Gold Mix, and the components of the amplification system were as follows: 1×TSE101 Gold Mix 45μL, primers (27F, 1492R) 2μL, DNA template 1μL. PCR cycle parameters were: 98℃ pre-denaturation for 2min; 98℃ denaturation for 10s, 55℃ annealing for 10s, 72℃ extension for 15s, 35 cycles; 72℃ extension for 5min.

[0044] The prepared PCR product was sent to Wuhan Qingke Biotechnology Co., Ltd. for first-generation sequencing (sequencing primers were 27F / 1492R). The sequencing results were spliced ​​and calibrated using SeqMan software. The spliced ​​sequence (sequence shown in SEQ ID No. 1) was compared with BLAST in the NCBI database. The homology of the sequence was analyzed and its species was determined. Strains with homology ≥ 99% were defined as the same species. DNA sequences of closely related species were selected, and MEGA11 was used to construct a phylogenetic tree using the Neighbor-Joining method. The strain was finally determined to be fermented Lactobacillus and was classified and named fermented mucus Lactobacillus Limosilactobacillus fermentum , strain number FL16, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), with the deposit number CGMCC No. 27414, the deposit date is May 23, 2023, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0045] Example 2

[0046] Construction of an in vitro fermentative Lactobacillus-polarity-reversed porcine intestinal organoid co-culture model.

[0047] (1) Isolation and culture of porcine intestinal organoids.

[0048] Healthy three-week-old Sanyuan piglets were purchased from Deqing Beijing Breeding Co., Ltd. After 4 hours of starvation treatment, 2 mL of propofol was injected into the neck muscle and the pigs were bled out from the armpits for killing. The crypt isolation step was performed after checking that the inguinal and mesenteric lymph nodes were not congested or swollen, and the small intestinal wall was not obviously congested or thinned.

[0049] Remove 8-10 cm intestinal segments from the abdomen of euthanized pigs, cut the intestines longitudinally, and scrape most of the hairs and mucus on the inner surface of the intestinal tissue with a sterile slide. Transfer the tissue to a 50 mL centrifuge tube and wash 4-5 times with 25 mL of ice-cold crypt isolation buffer (shake vigorously up and down) until the buffer is transparent. Transfer the intestinal tissue to a sterile plate, cut the tissue into 2×2 mm pieces with ophthalmic scissors, and transfer it to a 50 mL centrifuge tube. After adding 25 mL of pre-cooled crypt isolation buffer, flip the centrifuge tube on a test tube flip mixer at 4℃ 50rpm for 30 minutes. Let the fragments settle naturally and discard the supernatant. Transfer the intestinal tissue to a sterile plate and squeeze it 10 times with a round-bottom flask. Then filter the suspension with a sterile 200-mesh copper mesh sieve and collect the filtrate in the plate into a 15 mL centrifuge tube. Centrifuge at 4℃ 200×g for 5 minutes and discard the supernatant. Resuspend the crypt pellet according to Matrigel, 20-100 crypts / 50µL. Add 25µL of Matrigel-crypt mixture to the center of the pre-warmed 48-well plate. Matrigel should be applied to the center of the well so that it forms a hemispherical drop. Transfer the culture plate to a 37°C, 5% CO2 incubator. Allow Matrigel to solidify for 15 minutes and add 250µL of Crypt Growth Medium / well, changing the medium every other day.

[0050] (2) Establishment of a polarity-reversed pig intestinal organoid model.

[0051] After two passages, the porcine intestinal organoids were cultured in Matrigel for 24 h. The organoids in each well were counted under an inverted microscope to ensure that there were 200 organoids in each polarity-reversed well. If the number in one well was insufficient, multiple wells could be merged. 500 μL / well of 5 mM EDTA / PBS precooled at 4°C was added, and the suspension was transferred to a 15 mL conical centrifuge tube containing 10 mL of 5 mM EDTA / PBS by pipetting up and down 10 times. The 15 mL centrifuge tube was placed on a rocker shaker and incubated at 4°C for 1 h. Centrifuge at 200 × g, 4°C for 5 min, and the upper layer of 5 mM EDTA / PBS was carefully aspirated with a pipette. 5 mL of DMEM / F12 was added to the tube to wash the organoid pellet, and centrifuged again at 200 × g, 4°C for 5 min, and the supernatant was carefully aspirated. The obtained organoids were resuspended in the culture medium, and the pipette was gently blown up and down. After thorough mixing, 400 μL was added to each well of the ultra-low attachment culture plate (200 organoids in the original well can be divided into 3 wells for culture after processing). Cultured in a 37°C, 5% CO2 incubator, polarity reversal occurred after 1 day of suspension culture.

[0052] To reduce the residue of the top-side organoids on the tube wall during the experiment, ensure a sufficient number of experiments. All consumables (pipette tips, centrifuge tubes, etc.) that come into contact with the organoids must be rinsed with anti-adhesion rinse solution in advance, and then rinsed with DMEM / F12. Subsequent experiments can only be started after two rinses. Suspension culture in a 37°C, 5% CO2 incubator, and the culture medium is changed every 3 days. Transfer the organoids to a 15mL centrifuge tube with a pipette. Under the same conditions, multiple wells can be merged into one tube, centrifuged at 200×g, 4°C for 5min, and carefully remove the upper culture medium with a pipette. Then add the corresponding amount of freshly prepared culture medium to the tube, pipette up and down to mix, and add 400μL per well to the ultra-low adsorption culture plate. Culture in a 37°C, 5% CO2 incubator, and change the medium each time in the same way as above, and it can be cultured for 7 days.

[0053] (3) Verification of the successful establishment of the polarity reversal organoid model.

[0054] 1) Transfer the organoids from the 24-well plate to a 1.5 mL centrifuge tube, centrifuge at 300 × g for 5 min, and discard the supernatant;

[0055] 2) Add 300 µL of paraformaldehyde to the centrifuge tube for fixation and incubate overnight;

[0056] 3) Centrifuge at 300 × g for 5 min, remove the paraformaldehyde fixative with a pipette, and wash with D-PBS, repeating 3 times for 10 min each time;

[0057] 4) 0.4% Triton X-100 permeabilization for 5 minutes, D-PBS washing, repeated 3 times, 10 minutes each time;

[0058] 5) Perform immunofluorescence staining in the same test tube, resuspend the organoid pellet in diluted phalloidin staining reagent at a dilution ratio of 1:300-1:500, and incubate at room temperature for 2 hours;

[0059] 6) Centrifuge at 300 × g for 5 min, carefully remove phalloidin, and wash with PBS three times, 10 min each time;

[0060] 7) Resuspend the organoid pellet in DAPI and incubate at room temperature for 10 minutes;

[0061] 8) Centrifuge at 300 × g for 5 min, carefully remove DAPI, and wash with PBS three times, 10 min each time;

[0062] 9) Resuspend the precipitate in anti-fluorescence quenching sealing medium, drop it in the center of the slide, apply silicone grease on the four corners of the cover glass, cover it with a cover glass to form a three-dimensional space, and use a laser confocal microscope to image. Figure 2As shown in Figure 2, organoid morphology changes after suspension culture. Organoids with the basal side facing outward have clear lumens, while suspended organoids have visible edges of columnar epithelial cells and often no lumens. Confocal immunofluorescence imaging of F-actin shows that the epithelial apex has successfully turned outward.

[0063] (4) In vitro co-culture of probiotic / pathogen-polarity-reversed porcine intestinal organoids.

[0064] Transfer the fermented Lactobacillus FL16 bacterial suspension to a 15 mL centrifuge tube, centrifuge at 1200 × g for 10 min, and carefully remove the supernatant to obtain the bacterial precipitate. 6 Calculate the required amount of fermented Lactobacillus by CFU / well, add DMEM / F12 without antibiotics, mix evenly with a pipette up and down, and form a culture medium containing fermented Lactobacillus FL16 for standby use. Collect the suspended cultured organoids into a 15mL centrifuge tube, centrifuge at 200×g, 4℃ for 5min, and carefully remove the upper culture medium with a pipette. Add the corresponding amount of the above-mentioned bacterial culture medium to the tube, mix evenly by pipetting up and down, and add 400μL per well to the ultra-low adsorption culture plate. Co-culture in a 37℃, 5% CO2 incubator.

[0065] Transfer the E. coli K88 bacterial suspension to a 15 mL centrifuge tube, centrifuge at 1200 × g for 10 min, and carefully remove the supernatant to obtain the bacterial precipitate. 6 Calculate the amount of fermented lactobacillus required by CFU / well, add DMEM / F12 without antibiotics, mix evenly with a pipette up and down, and form a culture medium containing Escherichia coli K88 for standby use. Collect the suspended organoids into a 15mL centrifuge tube, centrifuge at 200×g, 4℃ for 5min, and carefully remove the upper culture medium with a pipette. Add the corresponding amount of the above-mentioned bacterial culture medium to the tube, mix evenly by pipetting up and down, and add 400μL per well to the ultra-low adsorption culture plate. Co-culture in a 37℃, 5% CO2 incubator.

[0066] Example 3

[0067] Detecting the effect of Lactobacillus fermentum FL16 on polarity-reversed porcine intestinal organoids.

[0068] The experimental group was divided into 4 groups, the control group ( Figure 3 Con group), Lactobacillus fermentum FL16 group ( Figure 3 FL16 group), ETECK88 (Escherichia coli K88) infection group ( Figure 3 ETEC K88 group), Lactobacillus fermentum FL16 repair group after damage caused by ETEC K88 ( Figure 3 The Lactobacillus fermentum FL16 group was prepared by adding 10 6CFU Lactobacillus fermentum FL16 was co-cultured for 24 h; the ETEC K88 infection group was added to each well of the polarity reversal organoid model constructed in Example 2. 6 CFU ETEC K88, co-cultured for 24 h; the Lactobacillus fermentum FL16 repair group was added to each well of the polarity reversal organoid model constructed in Example 2. 6 CFU ETEC K88 was added after 1 h of injury 6 CFU fermented Lactobacillus FL16, continued for 24 hours; the control group was the polarity reversal organoid model constructed in Example 2, cultured in normal medium without any treatment. The FITC-dextran permeability test was used to observe the changes in cell permeability; the expression of inflammatory cytokines was measured to observe the inflammatory response of cells; the proliferation of intestinal organoids was observed by Lgr5 and Ki67 immunofluorescence staining, and the expression of Wnt3a and β-catenin was determined by fluorescence quantification and immunofluorescence staining.

[0069] (1) FITC-dextran permeability test was used to observe changes in cell permeability.

[0070] 1) Transfer the organoids from the 24-well plate to a 1.5 mL centrifuge tube, centrifuge at 300 × g for 5 min, and discard the supernatant;

[0071] 2) Resuspend the organoid pellet in 4kDa FITC-dextran solution and let stand for 5 minutes at room temperature;

[0072] 3) Aspirate most of the 4kDa FITC-dextran solution, leaving 10μL at the bottom of the centrifuge tube, drop it on the center of the slide, cover it with a coverslip, and use a laser confocal microscope to image. Figure 3 As shown, infection of organoids with ETEC K88 resulted in impaired barrier integrity, allowing FITC-dextran to enter the interior of the spheres. Organoids in the control group completely excluded FITC-dextran, while those in the K88+FL16 group did have FITC-dextran enter, but this was significantly better than in the ETEC K88 infection group.

[0073] (2) Lgr5 and Ki67 immunofluorescence staining was used to observe the proliferation of intestinal organoids.

[0074] 1) Transfer the organoids from the 24-well plate to a 1.5 mL centrifuge tube, centrifuge at 300 × g for 5 min, and discard the supernatant;

[0075] 2) Add 300 µL of paraformaldehyde to the centrifuge tube for fixation overnight;

[0076] 3) Centrifuge at 300 × g for 5 min, remove the paraformaldehyde fixative with a pipette, and wash with D-PBS, repeating 3 times for 10 min each time;

[0077] 4) 0.4% Triton X-100 permeabilization for 5 minutes, D-PBS washing, repeated 3 times, 10 minutes each time;

[0078] 5) Perform immunofluorescence staining in the same test tube, resuspend the organoid pellet in diluted Lgr5 and Ki67 antibodies (Servicebio, China) at a dilution ratio of 1:100-1:1000, and incubate at room temperature for 4 hours;

[0079] 6) Centrifuge at 300 × g for 5 min, carefully remove the primary antibody, and wash with PBS three times, 10 min each time;

[0080] 7) Resuspend the organoid pellet in the secondary antibody and incubate at room temperature for 2 hours;

[0081] 8) Centrifuge at 300 × g for 5 min, carefully remove the secondary antibody, and wash with PBS three times, 10 min each time;

[0082] 9) Resuspend the precipitate in anti-fluorescence quenching sealing medium, drop it in the center of the slide, apply silicone grease on the four corners of the cover glass, cover it with a cover glass to form a three-dimensional space, and use a laser confocal microscope to image. Figure 4 As shown, Lactobacillus fermentum FL16 was able to significantly increase the number of Lgr5 and Ki67 positive cells.

[0083] (3) Expression of inflammatory cytokines.

[0084] Intestinal organoids were collected, and 1 mL of Trizol was added to each tube for RNA extraction. The reverse transcription kit was used to reverse transcribe and synthesize cDNA. The mRNA levels of individual genes were detected by real-time fluorescence quantitative PCR, and the values ​​were obtained based on the cycle threshold (CT) and internal reference comparative analysis. The genes specifically included: TNF-α, IL-6, IL-1β, IFN-γ, IL-10 , TGF-β .like Figure 5 As shown in Figure 2, the expression of pro-inflammatory cytokines in the K88 group was significantly increased, while the expression of anti-inflammatory cytokines ( IL-10 and TGF-β ) mRNA level expression was significantly decreased; K88+FL16 group significantly decreased pro-inflammatory cytokines ( TNF-α, IL-6, IL-1β and IFN-γ ) mRNA level, while inhibiting inflammatory cytokines ( IL-10 and TGF-β ) mRNA level expression was significantly increased, and the cellular inflammatory response caused by Escherichia coli was alleviated.

[0085] (4) Fluorescence quantification and immunofluorescence staining were used to determine the expression of Wnt3a and β-catenin.

[0086] 1) Real-time fluorescence quantitative PCR technology

[0087] Intestinal organoids were collected, and 1 mL of Trizol was added to each tube for RNA extraction, and reverse transcription was used to synthesize cDNA. Real-time fluorescence quantitative PCR was used to detect the mRNA level of individual genes, and the values ​​were obtained by comparative analysis based on the cycle threshold (CT) and the internal reference.

[0088] Table 2 Primer sequences for real-time fluorescence quantitative PCR

[0089]

[0090] 2) Immunofluorescence staining

[0091] Transfer the organoids from the 24-well plate to a 1.5 mL centrifuge tube, centrifuge at 300 × g for 5 min, and discard the supernatant; add 300 µL of paraformaldehyde to the centrifuge tube for fixation overnight; centrifuge at 300 × g for 5 min, gently remove the paraformaldehyde fixative with a pipette, and wash with D-PBS, repeat 3 times, 10 min each time; 0.4% Triton X-100 permeabilization for 5 minutes, D-PBS washing, repeat 3 times, 10 minutes each time; perform immunofluorescence staining in the same test tube, resuspend the organoid pellet in the diluted antibodies Wnt3a and β-catenin, dilution ratio 1:100-1:1000, incubate at room temperature for 4 hours; 300×g, centrifuge for 5 minutes, carefully remove the primary antibody, wash 3 times with PBS, 10 minutes each time; resuspend the organoid pellet in the secondary antibody, incubate at room temperature for 2 hours; 300×g, centrifuge for 5 minutes, carefully remove the secondary antibody, wash 3 times with PBS, 10 minutes each time; resuspend the pellet in anti-fluorescence quenching sealing agent, drop it in the center of the slide, apply silicone grease on the four corners of the coverslip, cover it with a coverslip to form a three-dimensional space, and use a laser confocal microscope for imaging. Figure 6 As shown, Lactobacillus fermentum FL16 significantly upregulated the expression of Wnt3a and β-catenin, and the Wnt / β-catenin signaling pathway was activated.

[0092] Example 4

[0093] To detect the effect of Lactobacillus fermentum FL16 on small intestinal damage in piglets.

[0094] Twelve litters of "Du × Chang × Da" three-way crossbred piglets of the same birth age and parity were randomly divided into 4 groups (3 litters in each group, 10-12 piglets in each litter): control group (Con group), Lactobacillus fermentum FL16 group (FL16 group), Escherichia coli K88 group (K88 group) and Lactobacillus fermentum FL16 repair group after Escherichia coli damage (K88+FL16 group). On the 1st to 3rd day of the experiment, the K88 group and the K88+FL16 group were gavaged with 2 mL of ETEC K88 suspension (1×10 9CFU / mL) once; on the 4th to 15th day of the experiment, the K88+FL16 group was gavaged with 1.5 mL of fermented Lactobacillus FL16 suspension (1×10 9 CFU / mL) once, and the K88 group was gavaged with the same amount of sterile PBS buffer every day. The Con group was gavaged with the same amount of PBS buffer during the experiment. Figure 7 The results showed that feeding piglets with Lactobacillus fermentum FL16 increased the crypt depth and villus height of the jejunum and alleviated the inflammatory response caused by Escherichia coli K88.

[0095] Example 5

[0096] To detect the effect of Lactobacillus fermentum FL16 on alleviating weaning stress in piglets.

[0097] Nine litters of newborn piglets of the "Du×Chang×Da" hybrid with the same birth age and parity were randomly divided into three groups (three litters in each group, 10-12 piglets in each litter): 1) lactation group (S group); 2) weaning group (W group); 3) fermented Lactobacillus FL16 group (FL16 group). The piglets in the FL16 group were gavaged with 1.5 mL of fermented Lactobacillus FL16 suspension (1×10 9 CFU / mL) once, and the piglets in the lactation group and the weaning group were gavage-fed with the same amount of sterile PBS buffer. The piglets in the weaning group and the fermented Lactobacillus FL16 group were fed with piglet feed after weaning at 21 days of age, and the piglets in the lactation group continued to be lactated until 28 days of age, and the experiment ended. During the experiment, the piglets had free access to food and water, and none of them received antibiotic treatment. The diarrhea of ​​the piglets was observed and recorded, and the piglets were weighed on an empty stomach at the beginning and end of the experiment. Figure 8 The results show that fermented Lactobacillus FL16 can significantly reduce the diarrhea rate of piglets after weaning, relieve diarrhea in piglets caused by weaning stress, and increase daily weight gain. Fig. 9 As shown in the figure, compared with the weaning group, fermented Lactobacillus FL16 can significantly increase the expression of tight junction proteins Zo-1 and Occludin in the jejunal mucosa, and alleviate the jejunal mucosal epithelial damage caused by weaning stress.

Claims

1. Application of fermented lactobacillus in the preparation of probiotic products for alleviating diarrhea and jejunal mucosal epithelial damage caused by weaning stress in piglets, The fermented lactobacillus is classified and named as fermented mucus lactobacillus Limosilactobacillus fermentum , strain number FL16, and the deposit number is CGMCC No. 27414.

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