A lactic acid bacteria extracellular polysaccharide and its application in enhancing the gastrointestinal barrier defense function of lambs

Abstract

This invention relates to a lactic acid bacteria extracellular polysaccharide and its application in enhancing the gastrointestinal barrier function of lambs. The lactic acid bacteria extracellular polysaccharide is composed of glucosamine, arabinose, galactosamine, galactose, glucose, mannose, and glucuronic acid. This invention also provides the application of the lactic acid bacteria extracellular polysaccharide in the preparation of formulations to enhance the gastrointestinal barrier function of lambs and as a feed additive for ruminants. This lactic acid bacteria extracellular polysaccharide can effectively reduce gastrointestinal abnormalities, edema, ulcers, erosions, and inflammatory cell infiltration in newborn lambs, greatly improving their gastrointestinal defense function, perfecting their inherent defense mechanisms, enhancing their intestinal disease resistance and overall health, and shifting the treatment of intestinal inflammation and disease from passive to proactive intervention, demonstrating great potential for development and application.

Description

Technical Field

[0001] This invention relates to an extracellular polysaccharide of lactic acid bacteria and its application in improving the gastrointestinal barrier defense function of lambs, belonging to the field of microbial polysaccharide and feed technology. Background Technology

[0002] The gastrointestinal tract (GIT) of ruminants must protect the host from intestinal contents and pathogens while simultaneously supporting nutrient absorption and metabolism to promote maintenance and growth. To achieve this, the GIT continuously senses luminal components and adjusts accordingly to maintain its integrity and enhance nutrient absorption. The intestinal barrier is a complex and effective defense system that operates dynamically to maintain intestinal integrity and immune homeostasis. The gastrointestinal microbiota plays a crucial role in regulating intestinal epithelial integrity, mucosal immunity, and food and drug metabolism. The gut microbiota is closely related to the host's physiological state, particularly immune function. A strong bidirectional link has been established between mucosal immune function and the gut microbiota. Clinical studies have demonstrated that intestinal barrier function can be modulated by numerous factors, including diet, stress, microbes, and drugs.

[0003] Most probiotics belong to the Lactobacillus or Bifidobacterium families. They contribute to the degradation and structural modification of intestinal antigens, maintain normal intestinal barrier function, regulate the secretion of inflammatory mediators, and promote the development of the immune system during critical periods, such as when the immune function of young animals is immature. Lactic acid bacteria (LAB) are recognized as generally Regarded as Safe (GRAS) microorganisms, widely distributed in nature and a natural component of the normal intestinal flora in humans and animals. In animal production, they are often used to control pathogens such as Salmonella and Escherichia coli, which are the most common bacterial pathogens in animals during the first week after birth. The probiotic effects of LAB are related to their ability to colonize the gastrointestinal tract, improve microbial balance, and stimulate the immune system. Due to their growth in the intestine, they form a microbial barrier against the growth of pathogens, and their biological activity directly affects the metabolic processes, dynamics, and host resistance of the intestinal flora. Current research suggests that the above functions of lactic acid bacteria may be related to their secondary metabolites, extracellular polysaccharides. Lactic acid bacteria extracellular polysaccharides (LAB-EPS) are saccharide compounds secreted by lactic acid bacteria during their growth and metabolism. Many nutritionally active substances, including polysaccharides, fatty acids, vitamins, peptides, and polyphenols, have been shown to possess anti-allergic and anti-inflammatory properties. These polysaccharides can regulate immune responses by acting directly on immune cells or improving microbial structure and increasing the production of SCFAs. They can also regulate the gut microbiota by increasing bacterial diversity and SCFA-producing bacteria, thereby reducing the number of opportunistic pathogens. Numerous studies have shown that LAB-EPS plays a crucial protective role in helping microorganisms resist adverse conditions such as dehydration, nutrient deficiency, bacteriophages, osmotic stress, antagonists, and toxic substances, and possesses physiological activities including cholesterol reduction, antitumor, antibacterial, antiviral, antioxidant, and immunomodulatory effects. The immunomodulatory function of LAB-EPS is mainly achieved by activating immune cells (such as macrophages, B lymphocytes, T lymphocytes, etc.), promoting the phagocytic capacity of monocytes, and regulating the secretion of immune cytokines (such as complement, interleukins, etc.). Therefore, LAB-EPS can be used to prepare immune adjuvants.

[0004] In intensive animal husbandry systems, the likelihood of various stressors increases, with a particularly high incidence of gastrointestinal diseases. The period from birth to weaning is an extremely challenging time for young animals' gut microbiota (GIT), with rapid structural and microbial changes making them more susceptible to GIT diseases and disorders, which in turn affect other systems in the body. Therefore, promoting gut development and maintaining normal function and health in young animals through nutritional intervention strategies is of great importance in animal production systems. Early development and transformation of gut microbiota lay the foundation for their long-term impact on host health; thus, the early years of lambs are a favorable time for intervention in small intestinal development through nutritional strategies. Healthy animals have a balanced gut microbiota that enables normal growth. However, the gut microbiota of young ruminants is extremely unstable, especially under stress, and is prone to imbalance, with a decrease in beneficial populations such as lactobacilli and bifidobacteria, and a potential increase in pathogens. Many problems affecting the growth performance of young animals are related to indigestion and reduced nutrient absorption caused by pathogen colonization. Currently, antimicrobial growth promoters or therapeutic antibiotics remain the main means of preventing or treating gastrointestinal diseases such as diarrhea in animals. However, the use of antibiotics has led to the development of antibiotic resistance. This resistance results in more severe post-weaning syndrome and can transfer antibiotic resistance to human pathogens. Therefore, developing a green and safe antibiotic alternative for livestock production is an urgent need in the industry.

[0005] In recent years, as consumer demand has shifted towards natural, residue-free, safe, and healthy foods, the properties and physiological functions of LAB-EPS have received increasing attention. LAB-EPS is currently commercially used in the food industry as a prebiotic. Compared to other types of polysaccharides, bacterial polysaccharides can be produced through fermentation and have advantages such as short production cycles and no limitations imposed by time or space, giving them broad development prospects and a huge application market. However, current research on the physiological activity of LAB-EPS mainly focuses on experimental models such as humans and rodents or under in vitro cell culture conditions. Research and applications in animal nutrition are limited, with only a few studies on monogastric animals (pigs), and no reports on ruminants. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a lactic acid bacteria extracellular polysaccharide and its application in enhancing the gastrointestinal barrier defense function of lambs.

[0007] The technical solution of the present invention is as follows:

[0008] An extracellular polysaccharide of lactic acid bacteria, wherein the extracellular polysaccharide of lactic acid bacteria is a pyranose, composed of glucosamine, arabinose, galactosamine, galactose, glucose, mannose and glucuronic acid;

[0009] Its structural formula is shown in equation (1) below:

[0010]

[0011] According to a preferred embodiment of the present invention, the molar ratio of glucose, glucosamine and mannose in the lactic acid bacteria extracellular polysaccharide is (1.4-1.5):(1-1.1):1.

[0012] According to a preferred embodiment of the present invention, the extracellular polysaccharide of the lactic acid bacteria is derived from Lactobacillus casei WXD030, which was deposited on May 3, 2016, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.12415.

[0013] The preparation method of the above-mentioned lactic acid bacteria extracellular polysaccharide includes the following steps:

[0014] Lactic acid bacteria WXD030 was inoculated onto MRS solid medium and activated at 37°C for 20–25 h to obtain a seed culture. Then, the seed culture was inoculated into MRS liquid medium at a volume percentage of 3–6% and fermented at 37°C for 25–35 h to obtain a fermentation broth. The fermentation broth was heated to inactivate enzymes, cooled, and centrifuged to remove coagulated proteins and bacterial cells. Proteins in the supernatant were removed using 4.0% (w / v) trichloroacetic acid. After ultrafiltration and concentration, crude polysaccharide was precipitated with 75% (v / v) ethanol to obtain crude polysaccharide, and its yield was determined. The crude polysaccharide was then purified by DEAE-Sepharose Fast Flow ion exchange chromatography and Sepharose CL-6B gel chromatography to obtain lactic acid bacteria extracellular polysaccharide.

[0015] The above-mentioned lactic acid bacteria extracellular polysaccharides are used in the preparation of formulations that enhance the gastrointestinal barrier defense function of lambs.

[0016] According to a preferred embodiment of the present invention, the lactic acid bacteria extracellular polysaccharide enhances the gastrointestinal barrier defense function of lambs by strengthening the anti-inflammatory capacity of the lamb's gastrointestinal tract.

[0017] According to a preferred embodiment of the present invention, the gastrointestinal tract refers to the rumen, abomasum, duodenum, jejunum, and ileum of a lamb.

[0018] The application of the above-mentioned lactic acid bacteria extracellular polysaccharides in the preparation of ruminant animal feed additives.

[0019] More preferably, the ruminant is a cow or a sheep.

[0020] The beneficial effects of this invention are as follows:

[0021] 1. This application provides a functional lactic acid bacteria extracellular polysaccharide derived from Lactobacillus casei WXD030. The application also provides the specific components, structural formula and preparation method of the lactic acid bacteria extracellular polysaccharide, with a yield of 260 mg / L.

[0022] 2. The lactic acid bacteria extracellular polysaccharide provided by this invention can effectively reduce gastrointestinal abnormalities, edema, ulcers, erosions, and inflammatory cell infiltration in newborn lambs, greatly improve the gastrointestinal defense function of newborn lambs, improve the inherent defense mechanism of newborn lambs, enhance the intestinal disease resistance and overall health of newborn lambs, and shift the treatment of intestinal inflammation and diseases from passive to active early intervention. It can be used to prepare preparations that improve the gastrointestinal barrier defense function of lambs, as well as feed additives for ruminants, and has great development and application prospects. Attached Figure Description

[0023] Figure 1 The image shows the UV scanning results of lactic acid bacteria extracellular polysaccharide (SXJ30EPS). The horizontal axis represents the UV wavelength (nm), and the vertical axis represents the response intensity (μAu).

[0024] Figure 2 This is an infrared spectral scan of the extracellular polysaccharide of lactic acid bacteria (SXJ30EPS).

[0025] Figure 3 The molecular weight determination results are for the extracellular polysaccharide of lactic acid bacteria (SXJ30EPS).

[0026] In the figure, SXJ30EPS (black curve) represents the molecular weight distribution of polysaccharides, UV (red curve) represents the intensity of ultraviolet light signal, and dR1 (blue curve) represents the intensity of refractive signal.

[0027] Figure 4 The ion chromatogram of lactic acid bacteria extracellular polysaccharide (SXJ30EPS);

[0028] In the figure, the horizontal axis represents retention time, the vertical axis represents absorbance in units of mAU, and the numbers indicate different monosaccharides.

[0029] Figure 5 HE-stained sections of rumen tissue from lambs in different treatment groups;

[0030] In the figure, A is the rumen pathological section of the blank control group after challenge; B is the rumen pathological section of the blank control group without challenge; C is the rumen pathological section of the Astragalus polysaccharide group after challenge; D is the rumen pathological section of the Astragalus polysaccharide group without challenge; E is the rumen pathological section of the Lactobacillus extracellular polysaccharide group after challenge; and F is the rumen pathological section of the Lactobacillus extracellular polysaccharide group without challenge.

[0031] Figure 6 HE-stained sections of abomasal tissue from lambs in different treatment groups;

[0032] In the figure, A is the pathological section of the abomasum in the blank control group after challenge; B is the pathological section of the abomasum in the blank control group without challenge; C is the pathological section of the abomasum in the Astragalus polysaccharide group after challenge; D is the pathological section of the abomasum in the Astragalus polysaccharide group without challenge; E is the pathological section of the abomasum in the Lactobacillus extracellular polysaccharide group after challenge; and F is the pathological section of the abomasum in the Lactobacillus extracellular polysaccharide group without challenge.

[0033] Figure 7 HE-stained sections of duodenal tissue from lambs in different treatment groups;

[0034] In the figure, A is the duodenal pathological section of the blank control group after challenge; B is the duodenal pathological section of the blank control group without challenge; C is the duodenal pathological section of the Astragalus polysaccharide group after challenge; D is the duodenal pathological section of the Astragalus polysaccharide group without challenge; E is the duodenal pathological section of the Lactobacillus extracellular polysaccharide group after challenge; and F is the duodenal pathological section of the Lactobacillus extracellular polysaccharide group without challenge.

[0035] Figure 8 HE-stained sections of jejunal tissue from lambs in different treatment groups;

[0036] In the figure, A is the jejunal pathological section of the blank control group after challenge; B is the jejunal pathological section of the blank control group without challenge; C is the jejunal pathological section of the Astragalus polysaccharide group after challenge; D is the jejunal pathological section of the Astragalus polysaccharide group without challenge; E is the jejunal pathological section of the EPS group after challenge; and F is the jejunal pathological section of the EPS group without challenge.

[0037] Figure 9 HE staining sections of ileum tissue from different treatment groups of lambs

[0038] In the figure, A is the ileum pathological section of the blank control group after challenge; B is the ileum pathological section of the blank control group without challenge; C is the ileum pathological section of the Astragalus polysaccharide group after challenge; D is the ileum pathological section of the Astragalus polysaccharide group without challenge; E is the ileum pathological section of the EPS group after challenge; and F is the ileum pathological section of the EPS group without challenge.

[0039] Figure 10 qPCR results for the target Claudin1 in different tissues of different treatment groups;

[0040] In the diagram, A represents the duodenum, B represents the jejunum, and C represents the ileum.

[0041] Figure 11 Western blot analysis of the expression levels of the target Cloudin1 in different tissues of different treatment groups:

[0042] In the diagram, A represents the duodenum, B represents the jejunum, and C represents the ileum.

[0043] Figure 12 qPCR results for the target Claudin4 in different tissues of different treatment groups;

[0044] In the diagram, A represents the duodenum, B represents the jejunum, and C represents the ileum.

[0045] Figure 13 Western blot analysis of the expression levels of the target Cloudin4 in different tissues of different treatment groups:

[0046] In the diagram, A represents the duodenum, B represents the jejunum, and C represents the ileum.

[0047] Figure 14 The results of qPCR for the target Occludin in different tissues of each experimental group;

[0048] In the diagram, A represents the duodenum, B represents the jejunum, and C represents the ileum.

[0049] Figure 15 Western blot analysis of Occludin expression levels in different tissues from different treatment groups:

[0050] In the diagram, A represents the duodenum, B represents the jejunum, and C represents the ileum. Detailed Implementation

[0051] The specific implementation process of the present invention will be further described in detail below with reference to the embodiments. The described embodiments are only some embodiments of the present invention, and not all embodiments.

[0052] Unless otherwise specified, the experimental techniques and scientific terms used in the following examples have the same meanings as commonly understood by a person skilled in the art. Unless otherwise noted, all experimental consumables and reagents are commercially available.

[0053] The lactic acid bacteria (Lactobacillus casei) WXD030 described in this example was deposited on May 3, 2016, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.12415. This strain has been disclosed in Chinese patent document CN109182186A – A Lactic Acid Bacterial Extracellular Polysaccharide and Immunoadjuvant, and is an existing strain.

[0054] The lambs mentioned in this embodiment belong to the Comprehensive Experimental Demonstration Base of the Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences, and the animal program complies with the regulations of the Research Department of the Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences regarding animal ethics and experiments.

[0055] Example 1: Preparation of extracellular polysaccharides from lactic acid bacteria

[0056] A method for preparing extracellular polysaccharides from lactic acid bacteria includes the following steps:

[0057] (1) Lactobacillus casei WXD030 was inoculated into MRS solid medium and activated in an anaerobic incubator at 37℃ for 24 h; 4% (v / v) inoculation was added to 50 mL of MRS liquid medium and fermented in an anaerobic incubator at 37℃ for 30 h; the fermented bacterial solution was centrifuged at 8000 rpm and 4℃ for 15 min, and the bottom precipitate was discarded; trichloroacetic acid was slowly added to the supernatant after centrifugation to adjust its concentration to 40 mg / mL; after standing at 4℃ for 12 h, it was centrifuged at 8000 rpm and 4℃. Centrifuge for 15 min, discard the bottom precipitate, and collect the supernatant. Add anhydrous ethanol to the supernatant to 80% of its total volume, and precipitate at 4°C for 24 h. Centrifuge at 4°C for 15 min, and collect all the bottom precipitate. Dissolve the precipitate in distilled water at 60°C, centrifuge at 10,000 rpm for 15 min, and discard the water-insoluble precipitate at the bottom. Transfer the supernatant to a dialysis bag with a molecular weight cutoff of 8k-12kDa, ensuring the dialysis solution does not exceed two-thirds of the bag's capacity. Replace the dialysis solution every 8 h, and perform dialysis continuously for 3 days. Collect all the solution in the dialysis bag and freeze-dry under vacuum to obtain crude extracellular polysaccharide from lactic acid bacteria.

[0058] (2) Purification of crude extracellular polysaccharides from lactic acid bacteria using DEAE-Sepharose Fast Flow ion exchange column

[0059] Pack the well-preserved DEAE-Sepharose Fast Flow packing material into a Column XK 26 / 40 column (height: 40 cm, inner diameter: 26 mm). Stop packing when the packing material is 5 cm from the top of the column. Equilibrate the column sequentially with ultrapure water and Tris-HCl solution (55 mM, pH 7.8) at a flow rate of 2 mL / min, eluting for 2–3 column volumes. Weigh 100 mg of crude lactic acid bacteria extracellular polysaccharide and dissolve it thoroughly in 10 mL of Tris-HCl. Filter the solution through a 0.22 μm filter membrane, load the sample, and allow it to stand for 30 min before elution. Add the Tris-HCl eluent and 1 M... Elution was performed sequentially with NaCl eluent at a flow rate of 2 mL / min, collecting 6 mL from each tube to obtain crude extracellular polysaccharides of lactic acid bacteria purified by DEAE column chromatography. The polysaccharide content in the eluent was determined by the phenol-sulfuric acid method, with absorbance measured at 490 nm to plot the elution curve. Based on the plotted elution curve, the eluent was collected and placed into a dialysis bag with a molecular weight cutoff of 8k–12kDa, ensuring the bag was not filled more than two-thirds full. The dialysis solution was replaced every 8 hours, and dialysis was performed continuously for 3 days. The polysaccharides were then recovered by vacuum freeze-drying.

[0060] (3) Purification of crude extracellular polysaccharides from lactic acid bacteria using Sepharose CL-6B molecular sieve

[0061] Sepharose CL-6B packing material was loaded into a Column XK 16 / 100 chromatography column (height: 100 cm, inner diameter: 16 mm). Packing was stopped when the packing material was 10 cm from the top of the column. The column was equilibrated with ultrapure water and Tris-HCl solution (55 mM, pH 7.8) at a flow rate of 2 mL / min, eluting for 2-3 column volumes. 50 mg of crude lactic acid bacteria extracellular polysaccharide sample purified by DEAE column was weighed, dissolved thoroughly in 5 mL of Tris-HCl, filtered through a 0.22 μm filter membrane, and loaded onto the column. The mobile phase was eluted with Tris-HCl elution buffer at a flow rate of 2 mL / min, and 6 mL was collected from each tube. The polysaccharide content was determined by the phenol-sulfuric acid method, and an elution curve was plotted. After lyophilization, the lactic acid bacteria extracellular polysaccharide was obtained and named SXJ30EPS.

[0062] The extracellular polysaccharide yield of lactic acid bacteria in this example was determined to be 260 mg / L by the sulfuric acid phenol method.

[0063] Example 2: Structural characterization of lactic acid bacteria extracellular polysaccharides

[0064] The characterization process of the structure of the lactic acid bacteria extracellular polysaccharide (SXJ30EPS) prepared in this invention is as follows: the pure lactic acid bacteria extracellular polysaccharide component is determined by ultraviolet scanning to determine whether there are impurities, i.e. whether there are proteins and nucleic acids; the characteristic absorption peaks of the lactic acid bacteria extracellular polysaccharide are analyzed by infrared spectroscopy, and the lactic acid bacteria extracellular polysaccharide is identified by combining the results of ultraviolet and infrared spectroscopy; then, the molecular weight of the lactic acid bacteria extracellular polysaccharide is determined by 18-angle laser light scattering instrument, and the monosaccharide composition of the lactic acid bacteria extracellular polysaccharide is determined by ion chromatography.

[0065] 1. Analysis was performed using a UV-Vis spectrophotometer (UV-1800 Shimadzu, Tokyo, Japan). Specifically, the purified SXJ30EPS sample was prepared into a solution with a concentration of 1.0 mg / mL, centrifuged at 1000 rpm for 10 min, and the supernatant was collected. Deionized water was used as a control. The UV spectrophotometer was used to scan the sample in the range of 190–600 nm. The UV scanning results are shown below. Figure 1 As shown.

[0066] Depend on Figure 1 It can be seen that polysaccharides only showed a response absorption peak at 206 nm, which is characteristic of polysaccharides; there were no absorption peaks at 260 nm and 280 nm, indicating that EPS does not contain either nucleic acid or protein impurities.

[0067] 2. The characteristic absorption peaks of SXJ30EPS were analyzed using an infrared spectrophotometer (Shimadzu Corporation, Tokyo, Japan). Specifically, 1.5 mg of dried SXJ30EPS sample and 150 mg of potassium bromide powder were mixed and compressed into a tablet approximately 0.1 mm thick. The sample was then scanned using infrared spectroscopy. The infrared scanning results are shown below. Figure 2 As shown.

[0068] Depend on Figure 2 As can be seen from this, the SXJ30 EPS is 3408.32cm. -1 There is a strong absorption peak at 2939.41 cm⁻¹, which is the absorption peak of the stretching vibration of the OH bond of the hydroxyl group in carbohydrate compounds. -1 The absorption peak appearing at 1652.93 cm⁻¹ is the absorption peak of the stretching vibration of the CH bond. -1 Nearby is the C=O asymmetric stretching vibration peak; at 1411.84 cm⁻¹ -1 The peak appearing at this point is a change-angle vibration peak of the CH bond; it appears at 1226.68 cm⁻¹. -1 The peak at 1053.10 cm⁻¹ represents the variable-angle vibration peak of C=O. -1 The strong absorption peak appearing at 808.15 cm⁻¹ is a characteristic absorption peak of COC in pyranose; -1 The peak at the pyranose terminal group (CH-C) is a variable-angle vibration. This indicates that SXJ30EPS exhibits characteristic absorption peaks of polysaccharides and possesses an α-pyranose ring configuration.

[0069] Based on the combined results of ultraviolet and infrared spectroscopy, it can be seen that SXJ30EPS is a sugar and the sample does not contain any other impurities.

[0070] 3. The molecular weight of SXJ30EPS was determined using a multi-angle laser detector (Wyatt Technology DAWN EOS, 18-angle). The results are as follows: Figure 3 As shown in Table 1.

[0071] The chromatographic conditions were as follows: column: TSK Gel G4000PWxl; mobile phase: 0.1M NaCl solution; flow rate: 0.5 mL / min; injection volume: 200 μL. SXJ30EPS was weighed and dissolved in 0.1M NaCl solution to prepare a 1 mg / mL solution, which was then loaded onto the sample. The molecular weight of the sample was calculated using Astra data analysis software.

[0072] Table 1. Results of weight-average and number-average molecular weight determination of SXJ30EPS

[0073]

[0074] Depend on Figure 3As shown in Table 1, the molecular weight of SXJ30EPS is relatively small, at 3.737 × 10⁻⁶. 4 Da.

[0075] 4. Ion chromatography was used to determine the monosaccharide composition of SXJ30EPS. Chromatographic conditions: Column: CarboPac TM PA203×150mm Analytical; Eluent: 250mM NaOH and 1M NaAC; Flow rate: 0.5mL / min; Injection volume: 10μL; Column temperature: 35℃; Detector: Pulse amperometric detector, gold electrode. Gradient elution conditions: A is water, B is 250mM NaOH, C is 1M NaAC. 0-20min, 94% A, 6% B, 0% C; 20-20.1min, 89% A, 6% B, 5% C; 20.1-35min, 74% A, 6% B, 20% C; 35.1-45min, 20% A, 80% B; 45.1-55min, 94% A, 6% B. Monosaccharide composition determination: Weigh 10 mg of the sample into a hydrolysis flask, add 4 mL of 4M trifluoroacetic acid, purge the air from the tube with N2 for 1 min, tighten the screw cap, and hydrolyze at 120℃ for 2 h. After filtration through a 0.22 μm filter membrane, inject the sample according to the conditions described in 3.2.6.1. The monosaccharide composition of the lysis products was determined based on the comparative retention time RT. The results are as follows: Figure 4 As shown.

[0076] Depend on Figure 4 It can be seen that the main monosaccharide composition of SXJ30EPS is as follows: glucosamine (T=5.68) accounts for 34.89% of the total peak area; glucose (T=9.53) accounts for 27.95% of the total peak area; and mannose (T=12.01) accounts for 17.97% of the total peak area. In addition, there are small amounts of arabinose (T=6.65) accounting for 0.98% of the total peak area; galactosamine (T=7.32) accounting for 8.05% of the total peak area; galactose (T=8.33) accounting for 3.68% of the total peak area; and glucuronic acid (T=27.73) accounting for 6.48% of the total peak area. The molar ratio of each major component, glucose:glucosamine:mannose, is approximately 1.4:1.1:1.

[0077] SXJ30EPS is composed of seven monosaccharides, including glucosamine, arabinose, galactosamine, galactose, glucose, mannose, and glucuronic acid. The molar ratio of glucose, glucosamine, and mannose in SXJ30EPS is approximately 1.4:1.1:1.

[0078] Example 3: Obtaining samples of lamb's gastrointestinal tract (rumen, duodenum, jejunum, ileum)

[0079] Twenty-four healthy newborn lambs, half male and half female, were selected and paired according to weight, sex, and age. They were divided into three groups: a control group, a lactic acid bacteria extracellular polysaccharide (EPS) group, and an astragalus polysaccharide (APS) group. The lambs were housed in separate pens. The EPS group received 1 g / lamb / day of EPS, the APS group received 1 g / lamb / day of APS, and the control group received 1 g / lamb / day of physiological saline. A three-day transition period was included to allow the lambs to become accustomed to the pen-based housing system and avoid stress. On day 4, different solutes were administered orally to the different treatment groups. The different solutes were dissolved in 100 ml of sterilized milk replacer and fed to the lambs. This feeding continued for 49 days, and the lambs' feeding program followed the farm's existing procedures. On days 43 and 44, four lambs from each group were randomly selected to receive 10 ml of APS. 9 CFU / lamb solution was administered orally to challenge the lambs. On day 49 of the experiment, four lambs from each group were randomly selected (two challenged and two not challenged) and slaughtered to collect gastrointestinal (rumen, abomasum, duodenum, jejunum, and ileum) samples. These samples corresponded to the blank control group (blank challenge and blank control), the lactic acid bacteria extracellular polysaccharide group (EPS challenge and EPS control), and the astragalus polysaccharide group (APS challenge and APS control).

[0080] One sample was preserved in 10% phosphate-buffered formalin for histological analysis. Another sample was preserved in liquid nitrogen for the determination of related gene and protein expression.

[0081] Example 5: Effects of Lactic Acid Bacteria Extracellular Polysaccharides on the Physiological Structure of Lamb Gastrointestinal Tissue

[0082] The changes in the physiological structure of gastrointestinal (rumen, abomasum, duodenum, jejunum, ileum) tissues obtained in Example 4 were observed using paraffin embedding sectioning and HE staining. The specific process is as follows:

[0083] 1. Tissue embedding

[0084] (1) Ethanol dehydration: The tissue was dehydrated stepwise with different concentrations of ethanol solution: 75%, 85%, 95%, 100%, 100%, each ethanol solution was used for 40 minutes (Note: Bone and calcified tissues need to be soaked in decalcification solution for 24 hours to soften the tissue before the ethanol dehydration step is performed).

[0085] (2) Transparency: The tissue was sequentially immersed in three xylene solutions for 30 minutes each;

[0086] (3) Wax immersion: The tissue was immersed in three paraffin baths in sequence, for 1 hour in the first bath, 1.5 hours in the second bath, and 2 hours in the third bath;

[0087] (4) Embedding: Pour liquid paraffin into the mold box, then place the tissue block soaked in paraffin flat at the bottom, making sure the cut surface is facing down. After the paraffin solidifies, remove the embedding frame. After it has completely cooled and hardened, trim the paraffin block. Leave an appropriate amount of paraffin around the tissue for sectioning.

[0088] 2. Slicing

[0089] Fix the pre-cooled wax block onto the paraffin microtome, ensuring the cut surface of the wax block is parallel to the blade. The blade's inclination is typically 15 degrees. Rotate the rotary feeder to adjust the section thickness to 3 μm, cutting into uniformly thick sections. Hold a brush in your left hand and rotate the microtome handle with your right hand. Once the section is pulled out, gently lift it with the brush and then lightly tweezers it, placing it face up into the slide box at approximately 40°C. After it has spread out evenly, retrieve the slide. For attaching sections, hold one end of the glass slide in your left hand and vertically immerse it in water to attach the section. Use tweezers in your right hand to assist in pushing the section, attaching it to two-thirds of the way up the slide. After attaching the section, allow it to air dry slightly, then bake it in a 60°C slide oven for 1 hour, followed by baking it in an oven for 2 hours.

[0090] 3. Dewaxing paraffin sections to water

[0091] Paraffin sections were sequentially immersed in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 10 min, and anhydrous ethanol II.

[0092] Gradient dewaxing: 10 min - 95% alcohol 10 min - 85% alcohol 10 min.

[0093] 4. HE staining

[0094] Stain paraffin sections with hematoxylin for 5-10 minutes, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, then blue with saturated lithium carbonate solution for 1 minute, rinse with running water for a few seconds, stain with eosin solution for a few seconds, and rinse with running water.

[0095] 5. Dehydration sealing sheet

[0096] Paraffin sections were sequentially immersed in 75% ethanol for 2 min, 85% ethanol for 2 min, anhydrous ethanol for 2 min, anhydrous ethanol for 2 min, and xylene for 2 min to clear. The sections were then removed from the xylene and mounted with neutral resin.

[0097] 6. Result Interpretation

[0098] The cell nucleus is blue, and the cytoplasm is red.

[0099] HE-stained sections of rumen tissue from the blank control group (blank challenge and blank control), the lactic acid bacteria extracellular polysaccharide group (EPS challenge and EPS control), and the astragalus polysaccharide group (APS challenge and APS control) after and without challenge are shown in the figure below. Figure 5 As shown.

[0100] Depend on Figure 5 It can be seen that, Figure 5 In the blank control group after challenge, the overall structure of the rumen tissue was basically normal, the mucosa was basically normal, the mucosa was covered with keratotic squamous epithelium, the papillae were neatly arranged, no abnormalities were found in the submucosa and muscle layer, and no obvious inflammatory cell infiltration was found in the tissue. Figure 5 B. The untreated blank control group had a basically normal overall rumen tissue structure, with mild abnormalities in the mucosa. The mucosa was covered with keratotic squamous epithelium, with focal mucosal epithelial ulcers accompanied by inflammatory cell infiltration. The papillae were shorter and arranged neatly. No abnormalities were found in the submucosa and muscularis propria. Figure 5 After C-type challenge, the overall structure of the rumen tissue in the Astragalus polysaccharide group was basically normal, the mucosa was basically normal, the mucosa was covered with keratotic squamous epithelium, the papillae were shorter and neatly arranged, no abnormalities were found in the submucosa and muscle layer, and no obvious inflammatory cell infiltration was found in the tissue. Figure 5 The overall structure of the rumen tissue in the untreated Astragalus polysaccharide group (D) was basically normal, the mucosa was basically normal, the mucosa was covered with keratotic squamous epithelium, the papillae were flat, and no abnormalities were found in the submucosa and muscle layer. No obvious inflammatory cell infiltration was found in the tissue. Figure 5 After E challenge, the overall structure of the rumen tissue in the EPS group was basically normal, the mucosa was basically normal, the mucosa was covered with keratotic squamous epithelium, the papillae were flat, and no abnormalities were found in the submucosa and muscularis propria. No obvious inflammatory cell infiltration was found in the tissue. Figure 5 The overall structure of the rumen tissue in the unchallenged EPS group was basically normal, the mucosa was basically normal, the mucosa was covered with keratinized squamous epithelium, the papillae were arranged regularly, no abnormalities were seen in the submucosa and muscularis propria, and no obvious inflammatory cell infiltration was seen in the tissue.

[0101] HE-stained sections of abomasal tissue from the blank control group (blank challenge and blank control), the lactic acid bacteria extracellular polysaccharide group (EPS challenge and EPS control), and the astragalus polysaccharide group (APS challenge and APS control) after and without challenge are shown in the figure below. Figure 6 As shown.

[0102] Depend on Figure 6 It can be seen that, Figure 6 In the blank control group after challenge, the overall structure of the abomasal tissue was basically normal, the mucosa was basically normal, the mucosal glands were arranged regularly and neatly, no abnormalities were found in the submucosa and muscular layer, and no obvious inflammatory cell infiltration was found in the tissue. Figure 6 B. The untreated blank control group had a basically normal overall structure of abomasal tissue, with mild abnormalities in the mucosa, focal mucosal epithelial shedding accompanied by inflammatory cell infiltration, regular and orderly arrangement of mucosal glands, no abnormalities in the submucosa and muscularis propria, and no obvious inflammatory cell infiltration in the tissue. Figure 6After C-type challenge, the overall structure of the abomasal tissue in the Astragalus polysaccharide group was basically normal, with mild abnormalities in the mucosal layer, sparse and loose arrangement of local mucosal glands, no abnormalities in the submucosa and muscular layer, and no obvious inflammatory cell infiltration in the tissue. Figure 6 In the untreated Astragalus polysaccharide group (D), the overall structure of the abomasal tissue was basically normal, the mucosa was basically normal, the mucosal glands were arranged regularly and neatly, the submucosa showed mild edema, the muscle layer showed no abnormalities, and no obvious inflammatory cell infiltration was observed in the tissue. Figure 6 After E challenge, the overall structure of the abomasal tissue in the EPS group was basically normal, the mucosa was basically normal, the mucosal glands were irregularly arranged with intestinal metaplasia, no abnormalities were found in the submucosa and muscularis, and no obvious inflammatory cell infiltration was found in the tissue. Figure 6 In the unchallenged EPS group, the overall structure of the abomasal tissue was basically normal, the mucosa was basically normal, the mucosal glands were arranged regularly and neatly, no abnormalities were found in the submucosa and muscular layer, and no obvious inflammatory cell infiltration was found in the tissue.

[0103] In this embodiment, HE-stained sections of duodenal tissue from the blank control group (blank challenge and blank control), the lactic acid bacteria extracellular polysaccharide group (EPS challenge and EPS control), and the astragalus polysaccharide group (APS challenge and APS control) after and without challenge are shown below. Figure 7 As shown.

[0104] Depend on Figure 7 It can be seen that, Figure 7 In the blank control group after viral challenge, the overall structure of the duodenal tissue was slightly abnormal, the mucosa was slightly abnormal, the villi were slightly eroded with inflammatory cell infiltration, and no abnormalities were found in the submucosa and muscle layer. Figure 7 B. In the untreated control group, the overall structure of the duodenum was slightly abnormal, the mucosa was slightly abnormal, the villi were eroded with inflammatory cell infiltration, and no abnormalities were found in the submucosa and muscle layer. Figure 7 After the C-type challenge, the overall structure of the duodenal tissue in the Astragalus polysaccharide group was basically normal, the mucosa was basically normal, and no erosion or shedding was observed. Duodenal glands were visible in the submucosa, and no abnormalities were observed in the muscle layer. Figure 7 In the untreated Astragalus polysaccharide group (D), the overall structure of the duodenal tissue was slightly abnormal, the mucosa was slightly abnormal, the villi were flattened and shed, and no abnormalities were found in the submucosa and muscle layer. Figure 7 After E challenge, the overall structure of the duodenum in the EPS group was basically normal, with mild abnormalities in the mucosa, flattened villi with shedding, duodenal glands visible in the submucosa, and no abnormalities in the muscle layer. Figure 7 In the untreated EPS group, the overall structure of the duodenal tissue was basically normal, with mild abnormalities in the mucosa, flattened villi with erosion, duodenal glands visible in the submucosa, and no abnormalities observed in the muscle layer.

[0105] In this embodiment, HE-stained sections of jejunal tissue from the blank control group (blank challenge and blank control), the lactic acid bacteria extracellular polysaccharide group (EPS challenge and EPS control), and the astragalus polysaccharide group (APS challenge and APS control) after and without challenge are shown below. Figure 8 As shown.

[0106] Depend on Figure 8 It can be seen that, Figure 8 After viral challenge, the jejunal tissue of the blank control group was basically normal in overall structure, the mucosa was basically normal, the mucosal glands were arranged regularly and neatly, small adenomas were visible in them, the submucosa was slightly edematous, the muscle layer was not abnormal, and no obvious inflammatory cell infiltration was seen in the tissue. Figure 8 B. The unchallenged blank control group had a basically normal overall jejunal tissue structure, a basically normal mucosa, and regularly arranged mucosal glands. A small amount of inflammatory cell infiltration was visible, and no abnormalities were found in the submucosa and muscular layer. Figure 8 After C-type viral challenge, the overall structure of the jejunal tissue in the Astragalus polysaccharide group was basically normal, the mucosa was basically normal, the mucosal glands were arranged regularly and neatly, a small amount of inflammatory cell infiltration was visible, and no abnormalities were found in the submucosa and muscle layer. Figure 8 The overall structure of the jejunal tissue in the untreated Astragalus polysaccharide group (D) was basically normal, the mucosa was basically normal, the mucosal glands were arranged regularly and neatly, a small amount of inflammatory cell infiltration was visible, and no abnormalities were found in the submucosa and muscle layer. Figure 8 After E challenge, the overall structure of the jejunal tissue in the EPS group was basically normal, but the mucosal layer was abnormal. The mucosal glands were arranged regularly and neatly, some villi were eroded, and a small amount of inflammatory cell infiltration was visible. No abnormalities were found in the submucosa and muscular layer. Figure 8 In the unchallenged EPS group, the overall structure of the jejunal tissue was basically normal, but the mucosal layer was abnormal. The mucosal glands were arranged regularly and neatly, some villi were eroded, and a small amount of inflammatory cell infiltration was visible. No abnormalities were found in the submucosa and muscular layer.

[0107] In this embodiment, HE-stained sections of ileum tissue from the blank control group (blank challenge and blank control), the lactic acid bacteria extracellular polysaccharide group (EPS challenge and EPS control), and the astragalus polysaccharide group (APS challenge and APS control) after and without challenge are shown below. Figure 9 As shown.

[0108] Depend on Figure 9 It can be seen that, Figure 9 After viral challenge, the ileum tissue of the blank control group was basically normal in general, the mucosa was basically normal, the mucosal glands were arranged regularly and neatly, a large number of lymphocytes were observed in the submucosa, and no abnormalities were found in the muscle layer. Figure 9 B. The unchallenged blank control group had a basically normal overall ileal tissue structure, a basically normal mucosa, regular and orderly arrangement of mucosal glands, a large number of lymphocytes were observed in the submucosa, and no abnormalities were found in the muscle layer. Figure 9After the C-type challenge, the overall structure of the ileum tissue in the Astragalus polysaccharide group was basically normal, the mucosa was basically normal, the mucosal glands were arranged regularly and neatly with inflammatory cell infiltration, a large number of lymphocytes were observed in the submucosa, and no abnormalities were found in the muscle layer. Figure 9 In the untreated Astragalus polysaccharide group (D), the overall structure of the ileum tissue was basically normal, the mucosa was basically normal, the mucosal glands were arranged regularly and neatly, the villi were flat and shed, a large number of lymphocytes were observed in the submucosa, and no abnormalities were found in the muscle layer. Figure 9 After E challenge, the overall structure of the ileum tissue in the EPS group was basically normal, the mucosa was basically normal, the mucosal glands were arranged regularly and neatly, a small amount of mucosal epithelium was sloughed off, a large number of lymphocytes were observed to be aggregated in the submucosa, and no abnormalities were found in the muscle layer. Figure 9 The overall structure of the ileum tissue in the unchallenged EPS group was basically normal, the mucosa was basically normal, the mucosal glands were arranged regularly and neatly, a small amount of mucosal epithelial shedding was observed, a large number of lymphocytes were seen in the submucosa, and no abnormalities were found in the muscle layer.

[0109] Table 2 summarizes the HE staining analysis of gastrointestinal (rumen, abomasum, duodenum, jejunum, ileum) tissue samples from the lamb blank control group, lactic acid bacteria extracellular polysaccharide group, and astragalus polysaccharide group.

[0110] Table 2

[0111]

[0112] Depend on Figures 5-9 As shown in Table 2, the lactic acid bacteria extracellular polysaccharide (SXJ30EPS) provided by this invention can effectively resist inflammation of the lamb's gastrointestinal tract (rumen, abomasum, duodenum, jejunum, and ileum) caused by Escherichia coli. It can improve the gastrointestinal defense function of newborn lambs, improve the inherent defense mechanism of newborn lambs, and enhance the intestinal disease resistance and overall health of newborn lambs.

[0113] Example 6: Effects of lactic acid bacteria extracellular polysaccharides on the expression of tight junction-related genes and proteins in lamb gastrointestinal tissue.

[0114] 1. Real-time quantitative PCR analysis

[0115] (1) Sample processing: Take 100mg of gastrointestinal (rumen, abomasum, duodenum, jejunum, ileum) tissue samples from the blank control group, lactic acid bacteria extracellular polysaccharide group and astragalus polysaccharide group obtained in Example 4 respectively. Before grinding, cut the tissue into small pieces as much as possible, add 1ml TRIzol reagent, and use a high-throughput tissue homogenizer to quickly and thoroughly homogenize the tissue (6m / s, grind for 20s, stand for 15s, grind for 2 cycles).

[0116] (2) RNA extraction: After adding TRIzol, react the sample at room temperature for 10 min; add 200 μl of chloroform, shake vigorously for 15 s (do not use a vortex mixer), and incubate at room temperature for 10 min; centrifuge at 12000 rpm for 15 min at 4℃. After centrifugation, the solution separates into three layers, from bottom to top: phenol-form layer, middle white protein layer, and clear aqueous layer, with RNA present in the aqueous layer; take 400 μL of the upper aqueous phase into a new centrifuge tube, add an equal volume of isopropanol (pre-cooled at 4℃), and incubate at -20℃ for 15 min; centrifuge at 12000 rpm for 10 min at 4℃; discard the supernatant, add 1 mL of 75% ethanol (pre-cooled at 4℃) to wash the RNA precipitate; gently invert and wash thoroughly. Centrifuge at 12000 rpm for 5 min at 4℃; discard the supernatant, repeat the ethanol precipitation washing once more; discard the supernatant, carefully aspirate the remaining liquid with a pipette, and air dry. Add to 50 μL of DEPC water and gently blow with a pipette to dissolve completely; RNA purity and concentration detection: NanoDrop 2000 was used to determine the OD value and concentration of the extracted RNA;

[0117] RNA integrity testing: RNA integrity was assessed by 1.5% agarose gel electrophoresis. RNA samples were stored at -80°C for extended periods.

[0118] (3) In vitro reverse transcription

[0119] Add the following reagents to an RNase / DNase-free PCR tube according to the following reverse transcription system. Gently mix on ice, centrifuge, and then incubate at 42°C for 60 min using Oligo(dT)18 or gene-specific primers. Terminate the reaction by heating at 70°C for 5 min to obtain cDNA. The diluted cDNA can be used directly for PCR reactions or stored at -20°C for short-term storage. For long-term storage, store at -80°C.

[0120] Reverse transcription system: 4 μL 5×Reaction Buffer, 1 μL reverse transcription primer, 5 μg RNA, 1 μL Ribolock RNase inhibitor (20 U / μL), 1 μL Revertaid M-MuLV reverse transcriptase (200 U / μL), 2 μL 10 mM dNTP Mix, total volume 9 μL.

[0121] (4) PCR amplification

[0122] The diluted cDNA (approximately 100 ng / μl) was used as a template for the PCR reaction, and PCR amplification was performed using primers F / R to obtain the PCR product.

[0123] PCR amplification system: cDNA 1μL, primer F (10μM) 0.5μL, primer R (10μM) 0.5μL, ROX ReferenceDye (50×) 0.5μL, SYBR mix 10μL, RNase / DNase-free ddH2O 8.6μL, total 20μL.

[0124] PCR amplification program: pre-denaturation, 94℃ for 30s; denaturation, 94℃ for 5s; annealing, 60℃ for 30s; extension, 95℃ for 15s, 40 cycles; melting curve, 60℃ for 1min, increasing the temperature by 0.3℃ every 15s, 95℃ for 15s; termination extension, 72℃ for 8min; final incubation at 4℃.

[0125] (5) Data Analysis

[0126] The Ct values ​​of each PCR reaction were read using ABI 7500 software. The Ct value of the target gene was subtracted from the Ct value of the internal reference gene to obtain ΔCt. The mean ΔCt of the control group was obtained by subtracting the mean ΔCt of the experimental group from the mean ΔCt of the control group. The change in target gene expression in the experimental group relative to the control group was calculated using 2-ΔΔCt.

[0127] 2. Western blot analysis

[0128] (1) Sample processing: Take 100 mg of gastrointestinal (rumen, abomasum, duodenum, jejunum, ileum) tissue samples from the blank control group, lactic acid bacteria extracellular polysaccharide group and astragalus polysaccharide group obtained in Example 4, add 1 mL of RIPA lysis buffer (10 μL of PMSF should be added to each 1 mL of lysis buffer), homogenize using a tissue homogenizer, lyse at 4℃ for 30 min, then centrifuge at 4℃ and 12000 rpm for 10 min, and collect the supernatant; after BCA protein quantification, store at -80℃ for a long time.

[0129] (2) BCA protein quantification: The Beyotime (P0011) kit was used, and the specific operation was performed according to the kit instructions.

[0130] (3) Denaturation of protein samples: Based on the BCA protein quantification results, take about 80 μg of total protein, add an appropriate amount of 5× protein loading buffer, mix well, denature in boiling water for 10 min, and then load the sample after a short centrifugation.

[0131] (4) SDS-PAGE electrophoresis: Prepare fresh electrophoresis buffer and use 140V electrophoresis until the loading just runs out of the separating gel and then stop electrophoresis.

[0132] (5) Transfer: After electrophoresis, remove the gel and equilibrate it in the prepared 1× transfer buffer for 20 min; cut the PVDF membrane and thin filter paper to an appropriate size, activate the PVDF membrane with methanol, and then equilibrate the PVDF membrane and thin filter paper in the transfer buffer for 20 min; place them in the following order: negative electrode - thin filter paper - gel - PVDF membrane - thin filter paper - positive electrode. Transfer at a constant current of 200 mA for 1 h.

[0133] (6) Blocking and antibody incubation: Blocking was performed using 1×TBST to prepare a 5% skim milk powder solution; according to the antibody instructions (1:1000), 6 μL of primary antibody was added and incubated overnight at 4°C on a shaker. Then, the membrane was washed 3 times with 1×TBST for 10 min each time. The washed membrane was placed in diluted secondary antibody (1:3000) and incubated on a shaker at room temperature for 1 h. Then, the membrane was washed 3 times with 1×TBST for 10 min each time.

[0134] (7) ECL color development: Prepare an appropriate amount of ECL working solution according to the ECL color development kit instructions; drain the liquid on the PVDF membrane, and evenly drop the prepared ECL working solution onto the membrane to cover the entire membrane. Incubate at room temperature for 2 minutes, then remove the excess color development solution, place the membrane into a fully automated chemiluminescence image analysis system for imaging and photography; use the grayscale software Gelpro32 to perform grayscale analysis on the bands.

[0135] In this embodiment, the qPCR results of the target Claudin1 in duodenal, jejunal, and ileal tissue samples from the blank control group (after and without infection), the lactic acid bacteria extracellular polysaccharide group, and the astragalus polysaccharide group are as follows: Figure 10 As shown, A is the duodenum, B is the jejunum, and C is the ileum.

[0136] Western blot analysis of Cloudin1 expression levels in duodenal, jejunal, and ileal tissue samples after and without viral challenge (blank challenge and blank control), lactic acid bacteria extracellular polysaccharide group (EPS challenge and EPS control), and astragalus polysaccharide group (APS challenge and APS control) is shown in the figure. Figure 11 As shown, A is the duodenum, B is the jejunum, and C is the ileum.

[0137] Depend on Figures 10-11 It can be seen that in the duodenum, jejunum, and ileum, the expression level of Claudin1 protein in the lactic acid bacteria extracellular polysaccharide group after challenge was significantly higher than that in the blank control group after challenge.

[0138] In this embodiment, the qPCR results of the target Claudin4 in duodenal, jejunal, and ileal tissue samples from the blank control group (blank challenge and blank control), the lactic acid bacteria extracellular polysaccharide group (EPS challenge and EPS control), and the astragalus polysaccharide group (APS challenge and APS control) after challenge and without challenge are as follows: Figure 12 As shown, A is the duodenum, B is the jejunum, and C is the ileum.

[0139] Western blot analysis of Cloudin4 expression levels in duodenal, jejunal, and ileal tissue samples after and without viral challenge (blank challenge and blank control), lactic acid bacteria extracellular polysaccharide group (EPS challenge and EPS control), and astragalus polysaccharide group (APS challenge and APS control) is shown in the figure. Figure 13 As shown, A is the duodenum, B is the jejunum, and C is the ileum.

[0140] Depend on Figures 12-13 It can be seen that in the duodenum, there was no significant difference in the QPCR results of Cloudin4 among the groups, while the protein gray value of each challenge group showed an increasing trend compared with the control group.

[0141] In this embodiment, the qPCR results of the target Occludin in duodenal, jejunal, and ileal tissue samples from the blank control group (blank challenge and blank control), the lactic acid bacteria extracellular polysaccharide group (EPS challenge and EPS control), and the astragalus polysaccharide group (APS challenge and APS control) after challenge and without challenge are as follows: Figure 14 As shown, A is the duodenum, B is the jejunum, and C is the ileum.

[0142] Western blot analysis of Occludin expression levels in duodenal, jejunal, and ileal tissue samples after and without viral challenge (blank challenge and blank control), lactic acid bacteria extracellular polysaccharide group (EPS challenge and EPS control), and astragalus polysaccharide group (APS challenge and APS control) is shown in the figure. Figure 15 As shown, A is the duodenum, B is the jejunum, and C is the ileum.

[0143] Depend on Figures 14-15 It was found that in the jejunum and ileum, the expression level of Occludin protein in the lactic acid bacteria extracellular polysaccharide group after challenge was significantly higher than that in the blank control group after challenge, while there was no significant difference in the duodenum.

[0144] Depend on Figures 10-15 It can be seen that, compared with the blank control group after challenge, the expression of tight junction molecule-related protein Claudin1 in the exopolysaccharide group after challenge was significantly upregulated in the duodenum, jejunum and ileum, while Claudin4 was significantly upregulated only in the jejunum and ileum, and Occludin was significantly upregulated only in the ileum and jejunum.

[0145] In summary, the lactic acid bacteria extracellular polysaccharide (SXJ30EPS) provided by this invention can effectively improve the gastrointestinal defense function of newborn lambs, improve the inherent defense mechanism of newborn lambs, enhance the intestinal disease resistance and overall health of newborn lambs, and shift the treatment of intestinal inflammation and diseases from passive to active early intervention. It can be used to prepare preparations that improve the gastrointestinal barrier defense function of lambs, as well as feed additives for ruminants, and has great development and application prospects.

Claims

1. The application of lactic acid bacteria extracellular polysaccharides in the preparation of formulations that enhance the gastrointestinal barrier defense function of lambs, characterized in that, The extracellular polysaccharide of lactic acid bacteria is derived from lactic acid bacteria. Lactobacillus casei WXD030 is a pyranose composed of glucosamine, arabinose, galactosamine, galactose, glucose, mannose, and glucuronic acid. Its structural formula is shown in formula (1) below: Equation (1); The gastrointestinal tract refers to the duodenum, jejunum, and ileum of a lamb; The method for preparing the lactic acid bacteria extracellular polysaccharide includes the following steps: lactic acid bacteria Lactobacillus casei WXD030 was inoculated onto MRS solid medium and activated at 37°C for 20-25 hours to obtain a seed culture. Then, the seed culture was inoculated into MRS liquid medium at a volume percentage of 3-6% and fermented at 37°C for 25-35 hours to obtain a fermentation broth. The fermentation broth was heated to inactivate enzymes, cooled, and centrifuged to remove coagulated proteins and bacterial cells. Then, the supernatant was purified with 4.0% trichloroacetic acid to remove proteins. After ultrafiltration and concentration, the broth was precipitated with 75% ethanol to obtain crude polysaccharide. The crude polysaccharide was then purified by DEAE-Sepharose Fast Flow ion exchange chromatography and Sepharose CL-6B gel chromatography to obtain lactic acid bacteria extracellular polysaccharide.

2. The application as described in claim 1, characterized in that, The lactic acid bacteria Lactobacillus casei WXD030 was deposited on May 3, 2016, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.12415.

3. The application as described in claim 1, characterized in that, The molar ratio of glucose, glucosamine, and mannose in the lactic acid bacteria extracellular polysaccharide is (1.4~1.5):(1~1.1):1.

Images