Phytobacterium plantarum for preparing diarrhea-relieving metabiotics and application of phytobacterium plantarum
By screening and preparing lactic acid bacteria postbiotics prepared from Lactobacillus plantarum Mei1101.2, the problem of animal diarrhea was solved, and intestinal health was improved and diarrhea was relieved.
Patent Information
- Application Number
- CN202510718578.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-30
- Publication Date
- 2025-10-10
AI Technical Summary
The existing technology lacks effective methods to alleviate animal diarrhea, resulting in serious losses in the livestock and poultry farming industry.
Provided is a Lactobacillus plantarum Mei1101.2 strain, which has strong antibacterial properties, the ability to produce exopolysaccharides and acid, and is used to relieve diarrhea by preparing lactic acid bacteria postbiotics.
It effectively reduces the diarrhea index and fecal water content of mice, improves intestinal structure, regulates intestinal flora, promotes the production of short-chain fatty acids, and relieves animal diarrhea.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microorganisms, and in particular to Lactobacillus plantarum for preparing a postbiotic for alleviating diarrhea and an application thereof. Background Art
[0002] Postbiotics are genetically defined, inactivated microorganisms and / or bacterial components, including or excluding their metabolites, that are beneficial to host health. Postbiotics are generally produced from microorganisms belonging to the genera Lactobacillus, Bifidobacterium, Streptococcus, Faecalibacterium, and Saccharomyces. As fourth-generation microorganisms, postbiotics play a role in enhancing intestinal barrier function, balancing intestinal flora, and modulating immunity, and have been widely used in livestock and poultry farming. The beneficial effects of postbiotics primarily depend on the various metabolites and bacterial components produced during the fermentation process. The main components of postbiotics include microbial metabolites, inactivated bacterial cells, microbial cell fractions, and cell lysates. These components exert various effects through different pathways, including regulating beneficial bacterial flora, enhancing epithelial barrier function, and modulating immunity. Organic acids, exopolysaccharides, and bacterial cells are important components of postbiotics and are key factors influencing their efficacy.
[0003] Diarrhea is a common symptom in animal husbandry that can damage animal physiological functions, reduce animal growth performance, and even cause livestock and poultry deaths, resulting in significant losses for the livestock and poultry industry. Cell-free supernatants contain a variety of products from microbial growth and metabolism, as well as active ingredients such as cell lysates and cell vesicles, and have multiple prebiotic benefits. Organic acids have a significant effect in inhibiting pathogens and alleviating diarrhea; the inhibitory effect of extracellular polysaccharides on pathogens is reflected in both inhibiting their growth and inhibiting the formation of pathogen biofilms; inactivated bacteria can compete with pathogens for attachment sites, preventing their colonization and thus reducing their damage to the body. Therefore, by screening strains suitable for preparing postbiotics and preparing them into postbiotics for alleviating animal diarrhea, we can meet the demand for postbiotics in the livestock and poultry industry and reduce losses in the livestock and poultry industry. Summary of the Invention
[0004] The present invention aims to solve at least one of the technical problems in the prior art, and provides a plant lactobacillus for preparing a postbiotic for alleviating diarrhea and its application, specifically providing a plant lactobacillus and using the plant lactobacillus to prepare a lactic acid bacteria postbiotic for alleviating animal diarrhea. The inventors have obtained a plant lactobacillus with strong antibacterial properties, high exopolysaccharide production, and good acid production and growth performance through a large number of extensive screenings. The postbiotic prepared therefrom can effectively alleviate animal diarrhea. Thus, the technical solution of the present invention is formed, and the purpose of the present invention is achieved.
[0005] The technical solutions of the present invention are as follows: In a first aspect, the present invention provides a plant lactobacillus for preparing a postbiotic for alleviating diarrhea, wherein the plant lactobacillus is plant lactobacillus ( Lactiplantibacillus plantarum ) Mei1101.2, the depository is the General Microbiology Center of China Culture Collection Administration, the deposit number is CGMCC No. 30331, the deposit date is April 15, 2024, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.
[0006] Wherein, the Lactobacillus plantarum has a nucleotide sequence as shown in SEQ ID NO.1.
[0007] In a second aspect, the present invention provides the use of the Lactobacillus plantarum and its metabolites in the preparation of products for alleviating diarrhea.
[0008] Optionally, the product is a lactic acid bacteria postbiotic.
[0009] Optionally, the product is feed or feed additive.
[0010] In a third aspect, the present invention provides a lactic acid bacteria postbiotic having the effect of alleviating diarrhea, comprising the Lactobacillus plantarum and its metabolites.
[0011] In a fourth aspect, the present invention provides a method for preparing the lactic acid bacteria postbiotics, comprising the following steps: (1) activating the Lactobacillus plantarum Mei1101.2 in a liquid culture medium; (2) The activated strain is inoculated into a liquid culture medium for fermentation and expansion to obtain a liquid fermentation liquid containing bacteria and metabolites; (3) The liquid fermentation broth was treated at 65° C. for 30 min to obtain liquid postbiotics.
[0012] Optionally, the liquid culture medium in step (2) is a modified MRS medium or an MRS broth medium; The formula of the modified MRS medium specifically includes: sucrose 26.67 g / L, maltose 13.33 g / L, yeast extract powder 25 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1 g / L, initial pH 6.2, and high-pressure sterilization at 115°C for 30 min.
[0013] The formula of MRS broth medium specifically includes: peptone: 10.0 g / L, beef extract powder: 10.0 g / L, yeast powder: 5.0 g / L, glucose: 20.0 g / L, Tween 80: 1.0 g / L, dipotassium hydrogen phosphate: 2.0 g / L, sodium acetate: 5.0 g / L, ammonium citrate: 2.0 g / L, magnesium sulfate: 0.1 g / L, manganese sulfate: 0.05 g / L, initial pH 6.2, and high-pressure sterilization at 121°C for 15 min.
[0014] Optionally, in step (2), when the liquid culture medium is a modified MRS medium, the fermentation conditions include: inoculating the activated strain into the modified MRS medium at an inoculum size of 2%, with a liquid volume of 100 mL / 250 mL, a culture temperature of 33°C, and culturing at 150 rpm for 26 h; When the liquid culture medium is MRS broth medium, the fermentation conditions include: inoculating the activated strain into MRS broth at an inoculum size of 3%, culturing in a shaker at 33°C and 150 rpm for 26 h, heat-treating at 65°C for 30 min, and storing at 4°C for later use.
[0015] In a fifth aspect, the present invention provides the use of the lactic acid bacteria postbiotics in alleviating animal diarrhea.
[0016] The present invention has at least one of the following beneficial effects: 1. The present invention isolates a plant lactobacillus (Lactobacillus plantarum) from fermented rice. Lactiplantibacillus plantarum ) Mei1101.2, a strain that exhibits strong antibacterial properties and exopolysaccharide production in vitro, as well as excellent acid production and growth performance. Specifically, the fermentation broth of the present invention's Lactobacillus plantarum CGMCC No. 30331 after 24 hours of culture exhibited an inhibition zone diameter of 20.60 mm against enterotoxigenic Escherichia coli K88, an exopolysaccharide production of 2.00 g / L, and an acid production of 19.08 g / L. The fermentation entered the logarithmic phase after approximately 2 hours and the stationary phase after approximately 12 hours. 2. The lactic acid bacteria postbiotic provided by the present invention is obtained by culturing Lactobacillus plantarum Mei1101.2 in liquid culture medium and then heat-treating it. This lactic acid bacteria postbiotic can reduce the diarrhea index and fecal water content in mice, lower the level of pro-inflammatory factors in mouse serum, increase the level of anti-inflammatory factors, improve the intestinal structure and intestinal mucosal barrier damage in mice's duodenum and jejunum, regulate the intestinal flora, and promote the production of short-chain fatty acids, effectively alleviating diarrhea in animals. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0018] Figure 1 This is the growth curve of Lactobacillus plantarum Mei1101.2 in Example 4 of the present invention.
[0019] Figure 2 This is the effect of the postbiotic Mei1101.2 on diarrhea in mice in Example 6 of the present invention.
[0020] Figure 3 This is the effect of the postbiotic Mei1101.2 on the body weight and food intake of mice in Example 6 of the present invention.
[0021] Figure 4 This is the effect of the postbiotic Mei1101.2 on mouse organ indexes in Example 6 of the present invention.
[0022] Figure 5 This is the effect of the postbiotic Mei1101.2 on mouse serum inflammatory factors in Example 6 of the present invention.
[0023] Figure 6 This is the effect of the postbiotic Mei1101.2 on the duodenal morphology of mice in Example 6 of the present invention.
[0024] Figure 7 This is the effect of the postbiotic Mei1101.2 on the jejunum morphology of mice in Example 6 of the present invention.
[0025] Figure 8 This is the effect of the postbiotic Mei1101.2 on the number of duodenal goblet cells in mice in Example 6 of the present invention.
[0026] Figure 9 This is the effect of the postbiotic Mei1101.2 on the number of mouse jejunal goblet cells in Example 6 of the present invention.
[0027] Figure 10 This is an analysis of the diversity of the microbial community in the cecal contents of mice by the postbiotic Mei1101.2 in Example 6 of the present invention.
[0028] Figure 11 This is an analysis of the species composition of mouse cecal contents at the phylum and genus levels by the postbiotic Mei1101.2 in Example 6 of the present invention.
[0029] Figure 12This is an analysis of the differential species of the bacterial flora in the cecal contents of mice using the postbiotic Mei1101.2 in Example 6 of the present invention. DETAILED DESCRIPTION
[0030] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.
[0031] The MRS liquid medium used in the present invention (Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd.) is composed of the following components: 20 g / L glucose, 10 g / L peptone, 8.0 g beef extract powder, 5 g yeast extract powder, 2 g / L diammonium hydrogen citrate, 2 g / L dipotassium hydrogen phosphate, 5 g / L ammonium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, and 1 g / L Tween-80, with a pH of 5.7 ± 0.2. The preparation method is as follows: Weigh 52.24 g of this product, dissolve it in 1 L of distilled water by heating, and sterilize it by autoclaving at 121°C for 15 minutes before use.
[0032] The MRS solid culture medium (Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd.) in the present invention is composed of the following components: 20 g / L glucose, 10 g / L peptone, 8.0 g beef extract, 5 g yeast extract, 2 g / L diammonium hydrogen citrate, 2 g / L dipotassium hydrogen phosphate, 5 g / L ammonium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, 1 g / L Tween-80, 15 g / L agar, and a pH of 5.7±0.2. The preparation method is as follows: 66.2 g of the culture medium sample is weighed, dissolved in 1 L of distilled water by heating, aliquoted, and sterilized by autoclaving at 121°C for 20 min before use; If specific experimental steps or conditions are not specified in the examples, the procedures or conditions of conventional experimental steps described in the literature in the field can be followed. All raw materials and instruments used are commercially available, including but not limited to those used in the examples of this application.
[0033] Example 1 Isolation and identification of Lactobacillus plantarum Mei1101.2 1. Strain Isolation The present invention Lactobacillus plantarum ( Lactiplantibacillus plantarumMei1101.2 was isolated from fermented rice in Luocheng Zhafen. This strain was screened and obtained as follows: 25 g of sample was weighed and mixed with 225 mL of physiological saline, followed by 10-fold serial dilutions. Appropriate dilutions were plated onto MRS agar and incubated in an anaerobic chamber at 37°C for 24 hours. Individual colonies with varying morphology, size, and color were streaked onto the plate. This process was repeated multiple times until a single colony with consistent morphology appeared on the plate. This was used to obtain a pure culture of Lactobacillus plantarum. Purified individual colonies were selected and activated in MRS broth at 37°C for 24 hours. The activated solution was then mixed with 40% (vol / vol) sterile glycerol in a 1:1 ratio and stored at -80°C until ready for use.
[0034] 2. Strain screening The secondary activated Lactobacillus plantarum was inoculated into a 50 mL centrifuge tube containing 30 mL of MRS broth at a 3% inoculum volume and cultured at 37°C and 150 rpm for 24 h to obtain Lactobacillus plantarum fermentation broth. The activated ETEC K88 (enterotoxigenic Escherichia coli K88) was inoculated into the nutrient broth at a 1% inoculum volume and cultured in an air bath constant temperature shaker at 37°C and 150 rpm for 24 h. The bacterial solution concentration was adjusted to 5.0×10 5 CFU / mL, and obtain an indicator bacterial suspension for future use. Antibacterial activity was determined using the Oxford cup method. 100 μL of the indicator bacterial suspension was spread onto the surface of the nutrient agar medium. After no visible water droplets remained on the plate, the Oxford cup was evenly placed within the plate. 200 μL of Lactobacillus plantarum fermentation broth and Lactobacillus plantarum fermentation broth heat-treated at different temperatures were added, respectively. Sterilized MRS broth was used as a blank control, and the diameter of the inhibition zone was measured. Lactobacillus plantarum with an inhibition zone greater than 18.00 mm was selected. Subsequently, Lactobacillus plantarum with strong antibacterial properties was selected for secondary activation and inoculated into MRS broth to prepare Lactobacillus plantarum fermentation broth. The activated ETEC K88 strain was then inoculated into nutrient broth to prepare ETEC K88 culture. The Lactobacillus plantarum fermentation broth, Lactobacillus plantarum fermentation broth after heat treatment at different temperatures, and ETEC K88 bacterial broth were divided into 50 mL centrifuge tubes and centrifuged. The bacterial sludge was collected and washed twice with PBS, resuspended in PBS and adjusted to OD600 of 0.5±0.002, and recorded as A0 and A1, respectively. The Lactobacillus plantarum resuspension and ETEC K88 resuspension were mixed with equal volumes (3 mL) and incubated in a 37°C incubator for 24 hours. The supernatant was measured for OD600 and recorded as A2. The formula for calculating the coaggregation rate of Lactobacillus plantarum and ETEC K88 is expressed as: Coaggregation rate (%) =
[0035] By measuring the antibacterial properties and coaggregation with pathogenic bacteria of each strain, a group of strains were screened to show strong antibacterial properties after heat treatment, and good coaggregation with ETEC K88 for both live and inactivated bacteria. In this example, a total of nine strains were screened, named Mei.1101.1 to Mei.1101.9. The antibacterial properties and coaggregation with pathogenic bacteria of each strain varied, as shown in the table below. Among them, the screened Mei.1101.2 showed good antibacterial properties and coaggregation with pathogenic bacteria.
[0036] strain Antibacterial properties Coaggregation with pathogenic bacteria Mei.1101.1 <![CDATA[18.74±0.86 a ]]> 55.60% Mei.1101.2 <![CDATA[20.60±1.16 a ]]> 64.00% Mei.1101.3 <![CDATA[18.96±0.75 a ]]> 65.51% Mei.1101.4 18.33 ± 1.21 a ]] 70.01% Mei.1101.5 <![CDATA[18.86±0.88 a ]]> 62.12% Mei.1101.6 <![CDATA[18.59±0.62 a ]]> 64.82% Mei.1101.7 <![CDATA[19.06±0.75 a ]]> 65.45% Mei.1101.8 <![CDATA[17.92±0.94 a ]]> 64.90% Mei.1101.9 <![CDATA[19.67±0.72 a ]]> 57.33% 3. Strain identification The strain Mei.1101.2 was extracted using a kit and amplified by PCR. The DNA was then sent to Shanghai Paisonno Biotechnology Co., Ltd. for 16S rRNA gene sequencing. The sequencing results were compared with the homologous sequences in NCBI using the BLAST tool to clarify the species relationship of the strain. The strain was confirmed to be Lactobacillus plantarum ( Lactiplantibacillus plantarum ), named Lactobacillus plantarum ( Lactiplantibacillus plantarum ) Mei1101.2. Therefore, the plant lactobacillus ( Lactiplantibacillus plantarum ) Mei1101.2 was deposited in the China General Microbiology Center (CGMCC), the deposit number is CGMCC No. 30331, the deposit date is April 15, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.
[0037] The 16S rDNA sequence of this strain is shown in SEQ ID NO.1:
[0038] Example 2 Determination of the Exopolysaccharide Production Ability of Lactobacillus plantarum Mei1101.2 Lactobacillus plantarum was streaked onto MRS solid medium and incubated at 37°C for 24 hours. A sterile inoculating loop was touched to a single colony and slowly pulled outward. The length of the threads drawn from different strains was compared to select Lactobacillus plantarum with longer threads. Lactobacillus plantarum that had been activated twice was inoculated into 250 mL of MRS liquid medium at a 3% (v / v) inoculum and incubated under anaerobic conditions at 37°C for 30 hours. The fermentation broth was boiled in a boiling water bath for 10 minutes to inactivate the exopolysaccharide-degrading enzymes. After cooling to room temperature, the broth was centrifuged at 5000 rpm at 4°C for 15 minutes, the precipitate was discarded, and the supernatant was collected. The supernatant was evaporated at 40°C to one-fifth of its original volume. 800 mg / mL trichloroacetic acid was slowly added to a final concentration of 40 mg / mL. After standing at 4°C for 8 hours, the broth was centrifuged at 10000 rpm at 4°C for 10 minutes, the precipitate was discarded, and the supernatant was collected. Anhydrous ethanol was added to the supernatant at a volume ratio of 1:4, and the mixture was incubated at 4°C overnight. The mixture was centrifuged at 10,000 rpm at 4°C for 10 minutes, and the precipitate at the bottom was collected. The precipitate was dissolved in 60°C distilled water and centrifuged at 10,000 rpm for 10 minutes. The insoluble precipitate at the bottom was discarded, and the supernatant was collected. The supernatant was placed in a dialysis bag with an 8 kDa molecular weight cutoff and dialyzed for 2 to 4 hours. The dialysate was then replaced, and the dialysate was then replaced every 6 to 8 hours for 2 days. The polysaccharide solution in the dialysis bag was collected and freeze-dried under vacuum to produce crude Lactobacillus plantarum polysaccharide.
[0039] According to the measurement, the exopolysaccharide production of the Lactobacillus plantarum Mei1101.2 of the present invention is 2.00 g / L, indicating that the strain has a good exopolysaccharide production ability.
[0040] Example 3 Determination of the Acid Production Capacity of Lactobacillus plantarum Mei1101.2 Lactobacillus plantarum, activated twice, was inoculated into MRS broth and incubated at 37°C and 150 rpm for 24 hours. The pH of the fermentation broth was measured. The total acid content of the fermentation broth was determined according to "GB 12456-2021 National Food Safety Standard - Determination of Total Acids in Foods." The fermentation broth was centrifuged, the supernatant was filtered, and the organic acid content in the fermentation broth was determined using ultra-high performance liquid chromatography.
[0041] It was determined that the pH value of the fermentation liquid of the Lactobacillus plantarum Mei1101.2 of the present invention after culturing for 24 h was 3.55, the total acid content was 19.08 g / L, and the organic acid composition was mainly lactic acid, indicating that the strain had good acid production ability.
[0042] Example 4 Growth Ability Determination of Lactobacillus plantarum Mei1101.2 1. Determination of Dry Cell Weight of Lactobacillus plantarum Mei1101.2 The dry cell weight of Lactobacillus plantarum was determined using the constant weight method. Lactobacillus plantarum Mei1101.2, activated twice, was cultured at 37°C and 150 rpm for 24 hours. A defined volume of fermentation broth was aspirated and centrifuged. The precipitate was washed and centrifuged three to four times to obtain a bacterial slurry. The slurry was then dried in a 105°C oven to a constant weight. The formula for determining dry cell weight is:
[0043] Wherein, X: dry cell weight of the strain, unit: g / L; M1: weight of the centrifuge tube and bacteria after constant weight, unit: g; M0: weight of the centrifuge tube after constant weight, unit: g; V: Volume of bacterial solution absorbed, unit: mL.
[0044] 2. Determination of the Growth Curve of Lactobacillus plantarum Mei1101.2 Strain growth curves were determined using a multifunctional microplate reader. Secondarily activated Lactobacillus plantarum Mei1101.2 was inoculated into MRS broth at a 3% inoculum volume. 200 μL of the test solution was pipetted into a 96-well plate. 50 μL of liquid paraffin was then added to prevent evaporation. Sterile MRS broth served as a blank control. The microplate reader was set at 37°C and a wavelength of 600 nm. The absorbance of the solution was measured every 15 minutes.
[0045] According to the absorbance measurement results, a growth curve was drawn with time as the horizontal axis and the OD600 difference between the Lactobacillus plantarum fermentation broth and the blank culture medium as the vertical axis, as shown in the following example: Figure 1 shown.
[0046] The dry cell weight of the Lactobacillus plantarum Mei1101.2 of the present invention after culturing for 24 hours is 2.46 g / L, enters the logarithmic phase at around the 2nd hour, and enters the stable phase at around the 12th hour.
[0047] Example 5 Preparation of Lactobacillus plantarum Mei1101.2 postbiotics This embodiment provides a method for preparing lactic acid bacteria postbiotics using Lactobacillus plantarum Mei1101.2: (1) The plant lactobacillus ( Lactiplantibacillus plantarum Mei1101.2 was cultured in MRS medium at 37°C for 24 h and activated twice; (2) The activated Lactobacillus plantarum Mei1101.2 was inoculated into MRS broth at a 3% inoculum size and cultured in a shaker at 33°C and 150 rpm for 26 h. After heat treatment at 65°C for 30 min, it was stored at 4°C for later use. The postbiotic was named postbiotic Mei1101.2.
[0048] Among them, the formula of MRS broth medium specifically includes: peptone: 10.0 g / L, beef extract powder: 10.0 g / L, yeast powder: 5.0 g / L, glucose: 20.0 g / L, Tween 80: 1.0 g / L, dipotassium hydrogen phosphate: 2.0 g / L, sodium acetate: 5.0 g / L, ammonium citrate: 2.0 g / L, magnesium sulfate: 0.1 g / L, manganese sulfate: 0.05 g / L, initial pH 6.2, and high-pressure sterilization at 121°C for 15 minutes.
[0049] Example 6 Application of postbiotic Mei1101.2 1. Animal experiments Eight-week-old female, specific pathogen-free (SPF) BALB / C mice (Laboratory Animal Quality Certificate No. 110324231102804174), weighing 17–19 g, were purchased from Beijing Spefoc Biotechnology Co., Ltd., Beijing [License No. SCXK (Beijing) 2019-0010]. Mice were housed in an isolated environment with a 12-h light / 12-h day / night cycle, a temperature of 25 ± 2°C, and a relative humidity of 50% ± 10%. They had free access to food and water. Mouse feed was purchased from Beijing Spefoc Biotechnology Co., Ltd.
[0050] After 7 days of adaptive feeding, BALB / C mice were randomly divided into three groups: a blank control group (NC), a model group (MC), and a Mei1101.2 group (the postbiotic Mei1101.2 described in Example 5). All mice in the different groups were fed the same diet with no dietary restrictions. The experiment lasted 10 days. The mice were first gavaged for 9 days. Each mouse in the NC and MC groups received 0.2 mL of normal saline, while each mouse in the Mei1101.2 group received 0.2 mL of the postbiotic Mei1101.2. Starting on the eighth day of the experiment, mice were challenged for two consecutive days. The NC group received an intraperitoneal injection of 0.2 mL of normal saline per mouse per day, while the MC and Mei1101.2 groups received an intraperitoneal injection of 0.2 mL of enterotoxigenic Escherichia coli K88 per mouse per day. Mice were sacrificed 2 days after the challenge.
[0051] 2. Effect of postbiotic Mei1101.2 on diarrhea in mice During the challenge period, filter paper was placed in the mouse cages to observe and record the size of diarrhea stains. The paper was changed daily. The mouse diarrhea rate was calculated according to Formula (3.1); the mouse loose stool rate was calculated according to Formula (3.2). The stains formed by fecal contamination on the filter paper were graded according to Table 1, and the mouse diarrhea index was calculated according to Formula (3.3).
[0052]
[0053] Table 1 Stool grade scores
[0054] During the challenge period, the feces of each mouse were collected and dried in an oven at 105°C to constant weight. The water content of the feces was calculated according to formula (3.4).
[0055]
[0056] The results of this experiment are as follows Figure 2 As shown, mice in the NC group did not develop diarrhea, with a diarrhea index of 0; mice in the MC group developed diarrhea after being challenged with ETEC K88, with feces leaving stains on the filter paper, and the diarrhea index increased. Compared with the MC group, the diarrhea index in the Mei1101.2 group was significantly lower ( p <0.05), the degree of diarrhea in mice was alleviated after intervention with postbiotic Mei1101.2. The fecal water content results showed that the fecal water content of mice in the NC group was 65.98%, while the fecal water content of mice in the MC group was significantly increased to 84.92% ( p <0.05). After the Mei1101.2 group was treated with postbiotic Mei1101.2, the fecal water content of mice decreased to 67.63%, which was significantly lower than that of the MC group ( p <0.05). This indicates that the postbiotic Mei1101.2 can alleviate the increase in fecal water content in mice caused by ETEC K88.
[0057] 3. Effects of postbiotic Mei1101.2 on body weight and food intake in mice During the experiment, mice were weighed every morning and their weights were recorded. At the beginning of the challenge, the mice's feed was weighed and their food intake was calculated. Figure 3 During the challenge period, the weight of mice in the NC group continued to increase to 102.14%, while the weight of mice in the MC group dropped sharply to 86.10%, indicating that ETEC K88 challenge not only caused diarrhea in mice, but also had a significant impact on their body weight. In the postbiotic Mei1101.2 group, ETEC K88 had a lower effect on the weight of mice. During the challenge period, the weight change rate of mice in the Mei-L12 group was only 96.96%, which was significantly higher than that in the MC group ( p<0.05), and the postbiotic Mei1101.2 could effectively alleviate the weight loss of mice induced by ETEC K88.
[0058] In this experiment, after intraperitoneal injection of ETEC K88, mice almost stopped eating, while the feeding of mice in the NC group was unaffected. Two days after intraperitoneal injection of ETEC K88, mice began to resume feeding. The average food intake of mice in each group on the second day of challenge was 3.85 g / mouse / day in the NC group, while the average food intake in the MC group decreased to 0.79 g / mouse / day. Diarrhea caused by ETEC K88 challenge resulted in decreased appetite in mice. The Mei1101.2 group averaged 2.41 g / mouse / day, which promoted feeding in mice with diarrhea.
[0059] 4. Effects of postbiotic Mei1101.2 on mouse organ indices After the mice were killed, they were dissected, the kidneys and spleen were removed, and the surrounding tissues were peeled off. The animal hair and floating blood were washed away with saline, and the surface moisture was absorbed with filter paper before weighing. The organ index was calculated according to formula (3.5).
[0060]
[0061] The results of this experiment are as follows Figure 4 Compared with the NC group, the spleen index of mice in the MC group was significantly increased ( p <0.05). The spleen, as an important immune organ, contains a large number of lymphocytes. After being stimulated by external factors, the spleen responds to the immune system. ETEC K88 may cause an inflammatory response, which in turn leads to splenomegaly. The spleen index of the Mei1101.2 group mice showed no significant difference from that of the NC group and the MC group ( p >0.05), the body maintained a sustained immune response when infected with ETEC K88. There was no significant difference in the kidney index among the mice groups ( p >0.05).
[0062] 5. Effect of postbiotic Mei1101.2 on serum inflammatory factors in mice Blood was collected from the eyeballs of mice. After standing at room temperature for 15 minutes, the blood samples were centrifuged at 3000 rpm for 10 minutes. The upper serum layer was aspirated into an EP tube and stored at −80°C. The levels of IL-10, IL-1β, and TNF-α in serum were measured using Nanjing Formase enzyme-linked immunosorbent assay (ELISA).
[0063] The results of this experiment are as follows Figure 5 Compared with the NC group, the levels of IL-1β and TNF-α in the serum of mice in the MC group were significantly increased ( p<0.05), increased by 101.36% and 158.31%, respectively, while IL-10 decreased by 33.71%. ETEC K88 challenge disrupted the secretion of pro- and anti-inflammatory factors in mice, triggering an inflammatory response and disrupting immune homeostasis. Treatment with the postbiotic Mei1101.2 reduced IL-1β and TNF-α levels, while increasing IL-10, indicating that the postbiotic Mei1101.2 can reverse the ETEC K88-induced disruption of inflammatory cytokine secretion.
[0064] 6. Effects of postbiotic Mei1101.2 on intestinal tissue morphology in mice After mouse dissection, the duodenum and jejunum were dissected, and 1-3 cm sections of intestinal tissue, free of contents, were fixed in 4% paraformaldehyde for 24 h. The tissues were removed from the fixative, rinsed with running water, dehydrated with graded ethanol solutions, cleared in xylene, and embedded in paraffin. 4 μm sections were cut, dried, and then sequentially immersed in xylene, graded ethanol solutions, and distilled water for demolding and hydration. Sections were stained with hematoxylin and eosin, dehydrated, cleared, and neutral resin was added. The sections were covered with a coverslip and air-dried. Duodenal and jejunal sections were examined under a light microscope. Villus loss was assessed under a light microscope, and villus length and crypt depth were measured using the microscope's built-in software. The ratio of villus length to crypt depth (V / C) was calculated.
[0065] HE staining results Figure 6 、 Figure 7 As shown in the figure, the villi in the duodenum and jejunum of mice in the NC group were intact and neatly arranged, with no villus shedding. However, the duodenum and jejunum in the MC group showed significant structural damage, with atrophy and shortening of the villi, severe villus breakage, and severe intestinal epithelial cell shedding, along with a large number of inflammatory cell infiltrations in the intestinal mucosa. In the Mei1101.2 group, the villi were relatively neatly arranged, with less breakage and shedding, and the villus morphology was essentially restored to normal, with no inflammatory infiltration, alleviating the intestinal damage caused by ETEC K88 in mice.
[0066] Table 2 Ratio of villus height to crypt depth in mouse intestine Group Duodenal V / C Jejunum V / C NC group <![CDATA[6.51±0.38 a ]]> <![CDATA[4.84±0.18 a ]]> MC Group <![CDATA[3.75±0.28 b ]]> 2.91 ± 0.22 b ]] Mei1101.2 group <![CDATA[5.84±0.16 a ]]> <![CDATA[4.16±0.57 a ]]> The ratio of villus length to crypt depth (V / C) in the duodenum and jejunum of mice is shown in Table 2. The V / C value reflects the strength of intestinal absorption function to a certain extent. A decrease in the ratio indicates that the intestinal absorption function decreases and the secretion function increases, thus causing diarrhea. The V / C value of the MC group was significantly lower than that of the NC group ( p <0.05), after intervention with postbiotic Mei1101.2, V / C was significantly increased compared with the MC group ( p<0.05), indicating that the postbiotic Mei1101.2 can alleviate animal diarrhea by improving the shortening of villus height and deepening of crypt depth in the duodenum and jejunum caused by ETEC K88.
[0067] 7. Effects of postbiotic Mei1101.2 on the intestinal mucosal barrier in mice Duodenal and jejunal sections were collected and sequentially stained with PAS, dehydrated, and transparentized. Neutral resin was added, and coverslips were placed. The sections were then air-dried and sealed. Goblet cell distribution was observed under a light microscope, and goblet cells in the duodenum and jejunum were counted using the microscope's built-in software.
[0068] The results of this experiment are as follows Figure 8 、 Figure 9 Compared with healthy mice, ETEC K88 invasion leads to a decrease in the number of goblet cells in the duodenum and jejunum of mice and a lighter coloration. The postbiotic Mei1101.2 can inhibit ETEC K88 damage to duodenal and jejunal goblet cells, thereby improving intestinal mucosal barrier function. 8. Effects of postbiotic Mei1101.2 on short-chain fatty acids in mouse colon A gradient mixture of acetic acid (C2), propionic acid (C3), isobutyric acid (iC4), butyric acid (C4), isovaleric acid (iC5), and valeric acid (C5) standards was prepared and filtered through a 0.22 mm filter. The chromatographic peak area corresponding to each concentration of the mixed standard was obtained to generate a standard curve. Fifty mg of mouse colonic feces was added to a 2 mL grinding tube containing 520 μL of saturated sodium chloride solution. The pH of the solution was adjusted to pH 2-3 with 6 mol / L hydrochloric acid, and a 2-ethylbutyric acid internal standard solution was added to a final concentration of 1 mmol / L. The mixture was ground using a cryo-grinder and centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was collected and filtered through a 0.22 μm filter. The peak areas of the corresponding SCFA peaks were measured by gas chromatography. The SCFA concentrations in the supernatant were calculated and converted to their contents in the fecal samples.
[0069] Table 3 SCFAs content in mouse colon contents
[0070] The results of this experiment are shown in Table 3. Compared with the NC group, the MC group had significantly lower levels of acetic acid, butyric acid, and total acid ( p <0.05), propionic acid content also decreased. ETEC K88 reduced the content of SCFAs in the colon of mice. The acetic acid and total acid contents in the Mei1101.2 group were significantly higher than those in the MC group ( p<0.05), indicating that the postbiotic Mei1101.2 intervention increased the SCFAs content in the mouse colon to a certain extent.
[0071] 9. Effects of postbiotic Mei1101.2 on intestinal flora in mice The cecal content samples of each mouse were collected in sterile and enzyme-free cryopreservation tubes, and the species composition of the intestinal flora of each group of mice was analyzed by 16S rRNA gene sequencing.
[0072] The results of the diversity analysis of the microbial community in the cecal contents of mice by the postbiotic Mei1101.2 in this experiment are as follows: Figure 10 Compared with the NC group, the Chao 1 index and observed_species index of the intestinal microbiota of mice in the MC group were significantly increased, and the richness and uniformity of the intestinal microbiota of mice were improved after ETEC K88 infection. After intervention with the postbiotic Mei1101.2, the Chao 1 index and observed_species index of the intestinal microbiota of mice were further increased, and the intestinal microbiota of mice had higher richness and uniformity. PCoA analysis based on the unweighted Unifrac distance algorithm was used to analyze the β-diversity of the intestinal microbiota of mice in different groups. The microbial structure of mice in different groups could be clearly distinguished, and the microbial flora of mice in different groups had undergone significant changes.
[0073] The results of the species composition analysis of mouse cecal contents at the phylum level (A) and genus level (B) in this experiment are shown in Figure 2. Figure 11 As shown. Compared with the NC group, the composition of the intestinal microbial community of mice in the MC group changed significantly. ETEC K88 challenge increased the relative abundance of Bacteroidetes and Proteobacteria in the mouse intestine, and reduced the relative abundance of Firmicutes and Actinobacteria. After intervention with the postbiotic Mei1101.2, the relative abundance of Firmicutes and Bacteroidetes increased, while the relative abundance of Proteobacteria and Actinobacteria decreased. Figure 12 As shown, LEfSe was used to analyze the differential marker species of each group of mice compared with the MC group. The NC group and Mei-L12 group had different Lactobacillus species from the MC group. Lactobacillus This is consistent with the relative abundance changes of microbial communities at the genus level mentioned above, indicating that the postbiotic Mei1101.2 upregulated Lactobacillus levels, and improved diarrhea in mice by regulating intestinal flora.
[0074] Example 7 Preparation of Improved Postbiotic Mei1101.2 for Alleviating Diarrhea This embodiment provides a method for preparing a lactic acid bacteria postbiotic with the effect of alleviating diarrhea using Lactobacillus plantarum Mei1101.2: (1) Lactobacillus plantarum ( Lactiplantibacillus plantarum ) Mei1101.2 was activated twice in liquid culture medium; the activation method was the same as in Example 5.
[0075] (2) The activated Lactobacillus plantarum Mei1101.2 was inoculated into the modified MRS medium at a 2% inoculum volume of 100 mL / 250 mL, the culture temperature was 33°C, and the culture was carried out at 150 rpm for 26 h to obtain the postbiotic, which was named the improved postbiotic Mei1101.2.
[0076] The modified MRS medium formula is: sucrose 26.67 g / L, maltose 13.33 g / L, yeast extract 25 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 801 g / L, initial pH 6.2, and autoclave at 115°C for 30 min.
[0077] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.
Claims
1. A Lactobacillus plantarum for preparing a postbiotic for alleviating diarrhea, characterized in that: The plant lactobacillus is plant lactobacillus ( Lactiplantibacillus plantarum ) Mei1101.2, the depository is the General Microbiology Center of China Culture Collection Administration, the deposit number is CGMCC No. 30331, the deposit date is April 15, 2024, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.
2. The plant lactobacillus according to claim 1, wherein The Lactobacillus plantarum has a nucleotide sequence as shown in SEQ ID NO.
1.
3. Use of the plant lactobacillus and its metabolites according to claim 1 in preparing products for alleviating diarrhea.
4. The use according to claim 3, characterized in that The product is a lactic acid bacteria postbiotic.
5. The use according to claim 3, characterized in that The product is feed or feed additive.
6. A lactic acid bacteria postbiotic having the effect of alleviating diarrhea, characterized in that: The invention comprises the plant lactobacillus and its metabolites according to claim 1 or 2.
7. The method for preparing lactic acid bacteria postbiotics according to claim 6, wherein: The following steps are involved: (1) activating the Lactobacillus plantarum in a liquid culture medium; (2) The activated strain is inoculated into a liquid culture medium for fermentation and expansion to obtain a liquid fermentation liquid containing bacteria and metabolites; (3) The liquid fermentation broth was treated at 65° C. for 30 min to obtain liquid postbiotics.
8. The preparation method according to claim 7, characterized in that The liquid culture medium in step (2) is a modified MRS culture medium or an MRS broth culture medium; The modified MRS medium formula specifically includes: sucrose 26.67 g / L, maltose 13.33 g / L, yeast extract 25 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1 g / L, initial pH 6.2, and autoclave sterilization at 115°C for 30 min. The formula of MRS broth medium specifically includes: peptone: 10.0 g / L, beef extract powder: 10.0 g / L, yeast powder: 5.0 g / L, glucose: 20.0 g / L, Tween 80: 1.0 g / L, dipotassium hydrogen phosphate: 2.0 g / L, sodium acetate: 5.0 g / L, ammonium citrate: 2.0 g / L, magnesium sulfate: 0.1 g / L, manganese sulfate: 0.05 g / L, initial pH 6.2, and high-pressure sterilization at 121°C for 15 min.
9. The preparation method according to claim 8, characterized in that In step (2), when the liquid culture medium is a modified MRS medium, the fermentation conditions include: inoculating the activated strain into the modified MRS medium at an inoculum size of 2%, the liquid volume is 100 mL / 250 mL, the culture temperature is 33°C, and the culture is carried out at 150 rpm for 26 h; When the liquid culture medium is MRS broth medium, the fermentation conditions include: inoculating the activated strain into MRS broth at an inoculum size of 3%, culturing in a shaker at 33°C and 150 rpm for 26 h, heat-treating at 65°C for 30 min, and storing at 4°C for later use.
10. Use of the lactic acid bacteria postbiotics according to claim 6 in alleviating diarrhea in animals.