Lactic acid bacteria exopolysaccharide and application thereof in improving lamb gastrointestinal tract barrier defense function

Abstract

The invention relates to lactic acid bacteria exopolysaccharide and application thereof in improving the defense function of lamb gastrointestinal barriers. The lactobacillus exopolysaccharide is prepared from glucosamine, arabinose, galactosamine, galactose, glucose, mannose and glucuronic acid. The invention further provides application of the lactobacillus exopolysaccharide in preparation of a preparation for improving the defense function of lamb gastrointestinal barriers and a ruminant feed additive. The lactobacillus exopolysaccharide can effectively reduce gastrointestinal abnormality, edema, ulcer, erosion and inflammatory cell infiltration of the newborn lambs, greatly improve the gastrointestinal tract defense function of the newborn lambs, perfect the inherent defense mechanism of newborn lambs, enhance the intestinal tract disease resistance and health level of the newborn lambs, and improve the immunity of the newborn lambs. Passive treatment of intestinal inflammation and diseases is changed into positive intervention in advance, and great development and application prospects are achieved.

Description

Technical Field

[0001] The invention relates to lactic acid bacteria extracellular polysaccharide and application thereof in improving the gastrointestinal barrier defense function of lambs, belonging to the technical field of microbial polysaccharides and feed. Background Art

[0002] The ruminant gastrointestinal tract (GIT) protects the host from intestinal contents and pathogens while supporting the absorption and metabolism of nutrients to promote maintenance and growth. To achieve this goal, the GIT continuously senses the composition of the lumen and makes adjustments to support the maintenance of its integrity and enhance nutrient absorption. The intestinal barrier is a complex and effective defense function system that operates in a dynamic manner to maintain intestinal integrity and immune homeostasis. Gastrointestinal microorganisms play a key role in the regulation of intestinal epithelial integrity, mucosal immunity, food and drug metabolism. The intestinal flora is closely related to the physiological state of the host, especially immune function. A well-established bidirectional relationship has been established between mucosal immune function and the intestinal flora. Clinical studies have shown that intestinal barrier function can be regulated by many factors, including diet, stress, microorganisms and drugs.

[0003] Most probiotics belong to lactobacilli or bifidobacteria, which help to degrade and modify the structure of intestinal antigens, maintain the normal function of the intestinal barrier, 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 generally regarded as safe (GRAS) microorganisms. They are widely distributed in nature and are a natural component of the normal intestinal flora of humans and animals. They are often used in animal production to control the effects of pathogens such as Salmonella and Escherichia coli, which are the most common bacterial pathogens in 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 to the growth of pathogens, and their biological activity directly affects the metabolic process, dynamics, and host resistance of the intestinal flora. Current studies have shown that the above functions of lactic acid bacteria may be related to their secondary metabolites, exopolysaccharides. Lactic acid bacteria exopolysaccharide (LAB-EPS) is a carbohydrate compound secreted outside the cell wall by lactic acid bacteria during growth and metabolism. Many nutritional active substances, including polysaccharides, fatty acids, vitamins, peptides and polyphenols, have been shown to have anti-allergic and anti-inflammatory properties. These polysaccharides can regulate immune responses by directly acting on immune cells or improving microbial structure and increasing the production of SCFAs. They can also regulate intestinal microbiota by increasing bacterial diversity and SCFA-producing bacteria and reducing the number of conditional pathogens. A large number of research results have shown that LAB-EPS plays an important protective role in helping microorganisms resist adverse conditions such as dehydration, nutrient deficiency, bacteriophages, osmotic stress, antagonists and toxic substances, and has physiological activities such as lowering cholesterol, anti-tumor, antibacterial, antiviral, antioxidant and immunomodulatory. The immunomodulatory function of LAB-EPS is mainly to regulate the body's immune function by activating immune cells (such as macrophages, B lymphocytes, T lymphocytes, etc.), promoting the phagocytic ability 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 feeding systems, the chances of various stresses increase, and the incidence of gastrointestinal diseases is particularly high. In particular, the period from birth to weaning is an extremely challenging period for the GIT of young animals. The rapid changes in structure and microorganisms make young animals more susceptible to GIT diseases and disorders, which in turn affect other systems in the body. Therefore, it is of great significance in animal production systems to promote the development of the intestine of young animals and maintain its normal function and health through nutritional intervention strategies. The early development and transformation of intestinal microorganisms lay the foundation for their long-term effects on host health. Therefore, the young age of lambs is an opportune time to intervene in the development of the small intestine through nutritional strategies. Healthy animals have a balanced gastrointestinal microbiome that enables them to grow normally. However, the gastrointestinal microbiome of young ruminants is extremely unstable, especially when they are under stress, and it is very easy to become unbalanced, with a decrease in beneficial populations such as lactobacilli and bifidobacteria, and an increase in pathogens. Many problems that affect the growth performance of young animals are related to indigestion and reduced nutrient absorption caused by pathogen colonization. At present, antimicrobial growth promoters or therapeutic antibiotics are still 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 leads to more severe post-weaning syndrome and transfers antibiotic resistance to human pathogens. Therefore, the development of a green and safe alternative to antibiotics in the livestock production process is an urgent need in the industry.

[0005] In recent years, as consumers' demands have shifted to natural, residue-free, safe and healthy foods, the properties and physiological functions of LAB-EPS have received increasing attention. LAB-EPS has been commercially used in the food industry and used as a prebiotic. Compared with other types of polysaccharides, bacterial polysaccharides can be produced by fermentation, and have the advantages of a short production cycle and not being restricted by time, space and other conditions, and have broad development prospects and a huge application market. However, current research on the physiological activity of LAB-EPS is still mainly concentrated in experimental models such as humans and mice or in vitro cell culture conditions. Research and application in animal nutrition are rare. A small number of studies have been found on monogastric animals (pigs), and no reports have been found on ruminants. Summary of the invention

[0006] In view of the deficiencies of the prior art, the object of the present invention is to provide a lactic acid bacteria extracellular polysaccharide and its application in improving the gastrointestinal barrier defense function of lambs.

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

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

[0009] Its structural formula is shown in the following formula (1):

[0010]

[0011] Preferably according to 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] Preferably according to the present invention, the lactic acid bacteria exopolysaccharide is derived from lactic acid bacteria (Lactobacillus casei) WXD030, which was deposited in the General Microbiological Center of China National Committee for Microbiological Culture Collection on May 3, 2016, with the address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.12415.

[0013] The method for preparing the lactic acid bacteria extracellular polysaccharide comprises the following steps:

[0014] The lactic acid bacteria WXD030 is inoculated on an MRS solid culture medium, and activated and cultured at 37°C for 20-25 hours to obtain a seed solution; then the seed solution is inoculated into an MRS liquid culture medium at a volume percentage of 3-6%, and fermented and cultured at 37°C for 25-35 hours to obtain a fermentation solution; the fermentation solution of the strain is heated to inactivate enzymes, and then cooled and centrifuged to remove coagulated proteins and bacterial bodies, and then trichloroacetic acid with a final concentration of 4.0% (w / v) is used to remove proteins in the supernatant, and after ultrafiltration and concentration, ethanol with a final concentration of 75% (v / v) is used for precipitation to obtain crude polysaccharides, and the yield thereof is detected; the crude polysaccharides are separated and purified by a DEAE-Sepharose Fast Flow ion exchange chromatography column and a SepharoseCL-6B gel chromatography column to obtain lactic acid bacteria extracellular polysaccharides.

[0015] The application of the above lactic acid bacteria extracellular polysaccharide in the preparation of a preparation for improving the gastrointestinal barrier defense function of lambs.

[0016] Preferably according to the present invention, the lactic acid bacteria extracellular polysaccharide improves the gastrointestinal barrier defense function of lambs by enhancing the anti-inflammatory ability of the lambs' gastrointestinal tract.

[0017] Preferably according to the present invention, the gastrointestinal tract refers to the rumen, abomasum, duodenum, jejunum and ileum of lambs.

[0018] Application of the lactic acid bacteria extracellular polysaccharide in the preparation of ruminant feed additives.

[0019] Further preferably, the ruminant is a cattle or a sheep.

[0020] The beneficial effects of the present invention are:

[0021] 1. The present application provides a functional lactic acid bacteria extracellular polysaccharide, which is derived from lactic acid bacteria (Lactobacillus casei) WXD030. It also provides the specific composition, structural formula and preparation method of the lactic acid bacteria extracellular polysaccharide, and the yield reaches 260 mg / L.

[0022] 2. The lactic acid bacteria extracellular polysaccharide provided by the present 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 the newborn lambs, enhance the intestinal disease resistance and health level of the newborn lambs, and transform intestinal inflammation and diseases from passive treatment to active early intervention. It can be used to prepare preparations for improving the gastrointestinal barrier defense function of lambs, as well as ruminant feed additives, and has great development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the UV scanning result 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 the infrared spectrum scanning result of lactic acid bacteria extracellular polysaccharide (SXJ30EPS).

[0025] Figure 3 The molecular weight determination results of lactic acid bacteria exopolysaccharide (SXJ30EPS);

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

[0027] Figure 4 This is the ion chromatogram of lactic acid bacteria exopolysaccharide (SXJ30EPS);

[0028] In the figure, the abscissa represents retention time (time), the ordinate represents absorbance intensity unit (mAU), and the numbers represent different monosaccharides.

[0029] Figure 5 HE staining sections of rumen tissues of lambs in different treatment groups;

[0030] In the figure, A is the rumen pathology section of the blank control group after the virus attack; B is the rumen pathology section of the blank control group without the virus attack; C is the rumen pathology section of the astragalus polysaccharide group after the virus attack; D is the rumen pathology section of the astragalus polysaccharide group without the virus attack; E is the rumen pathology section of the lactic acid bacteria extracellular polysaccharide group after the virus attack; F is the rumen pathology section of the lactic acid bacteria extracellular polysaccharide group without the virus attack.

[0031] Figure 6 HE staining sections of abomasum tissues of lambs in different treatment groups;

[0032] In the figure, A is the true stomach pathological section of the blank control group after the virus attack; B is the true stomach pathological section of the blank control group without the virus attack; C is the true stomach pathological section of the astragalus polysaccharide group after the virus attack; D is the true stomach pathological section of the astragalus polysaccharide group without the virus attack; E is the true stomach pathological section of the lactic acid bacteria extracellular polysaccharide group after the virus attack; F is the true stomach pathological section of the lactic acid bacteria extracellular polysaccharide group without the virus attack.

[0033] Figure 7 HE staining sections of duodenal tissues of lambs in different treatment groups;

[0034] In the figure, A is a pathological section of the duodenum of the blank control group after the virus attack; B is a pathological section of the duodenum of the blank control group without the virus attack; C is a pathological section of the duodenum of the astragalus polysaccharide group after the virus attack; D is a pathological section of the duodenum of the astragalus polysaccharide group without the virus attack; E is a pathological section of the duodenum of the lactic acid bacteria extracellular polysaccharide group after the virus attack; F is a pathological section of the duodenum of the lactic acid bacteria extracellular polysaccharide group without the virus attack.

[0035] Figure 8 HE staining sections of jejunum tissues of lambs in different treatment groups;

[0036] In the figure, A is the jejunal pathological section of the blank control group after the virus attack; B is the jejunal pathological section of the blank control group without the virus attack; C is the jejunal pathological section of the APS group after the virus attack; D is the jejunal pathological section of the APS group without the virus attack; E is the jejunal pathological section of the EPS group after the virus attack; F is the jejunal pathological section of the EPS group without the virus attack.

[0037] Fig. 9 HE staining sections of ileum tissues of lambs in different treatment groups

[0038] In the figure, A is the pathological section of the ileum of the blank control group after the virus attack; B is the pathological section of the ileum of the blank control group without the virus attack; C is the pathological section of the ileum of the APS group after the virus attack; D is the pathological section of the ileum of the APS group without the virus attack; E is the pathological section of the ileum of the EPS group after the virus attack; F is the pathological section of the ileum of the EPS group without the virus attack.

[0039] Fig.10 The QPCR results of the target Claudin1 in different tissues of different treatment groups;

[0040] In the figure, A is the duodenum, B is the jejunum, and C is the ileum.

[0041] Fig.11 Western-blot grayscale value analysis of the expression results of the target Cloudin1 in different tissues of different treatment groups:

[0042] In the figure, A is the duodenum, B is the jejunum, and C is the ileum.

[0043] Fig.12 The QPCR results of target Claudin4 in different tissues of different treatment groups;

[0044] In the figure, A is the duodenum, B is the jejunum, and C is the ileum.

[0045] Fig.13 Western-blot grayscale value analysis of the expression results of the target Cloudin4 in different tissues of different treatment groups:

[0046] In the figure, A is the duodenum, B is the jejunum, and C is the ileum.

[0047] Fig.14 The QPCR results of the target Occludin in different tissues of each experimental group;

[0048] In the figure, A is the duodenum, B is the jejunum, and C is the ileum.

[0049] Fig.15 Western-blot grayscale analysis of the expression results of the target Occludin in different tissues of different treatment groups:

[0050] In the figure, A is the duodenum, B is the jejunum, and C is the ileum. DETAILED DESCRIPTION

[0051] The specific implementation process of the present invention is further described in detail below in conjunction with embodiments. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0052] Unless otherwise specified, the experimental techniques and scientific terms used in the following examples have the same meanings as those generally understood by ordinary technicians. The experimental consumables and reagents involved are available through general commercial channels unless otherwise specified.

[0053] The lactic acid bacteria (Lactobacillus casei) WXD030 described in the embodiment was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration Committee on May 3, 2016, with the address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 12415. The strain has been disclosed in Chinese patent document CN109182186A-A lactic acid bacteria extracellular polysaccharide and immune adjuvant, and is an existing strain.

[0054] The lambs described in the examples are owned by the Comprehensive Experimental Demonstration Base of the Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences, and the animal program complies with the animal ethics and experimental regulations of the Research Department of the Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences.

[0055] Example 1. Preparation of lactic acid bacteria exopolysaccharide

[0056] A method for preparing lactic acid bacteria extracellular polysaccharide comprises the following steps:

[0057] (1) Lactobacillus casei WXD030 was inoculated into MRS solid culture medium and activated and cultured in an anaerobic incubator at 37°C for 24 hours; 4% (v / v) inoculation amount was inoculated into 50 mL of MRS liquid culture medium and fermented and cultured in an anaerobic incubator at 37°C for 30 hours; the fermented bacterial liquid was centrifuged at 8000 rpm and 4°C for 15 minutes, and the bottom sediment was discarded; trichloroacetic acid was slowly added to the supernatant after centrifugation to adjust its concentration to 40 mg / mL; after standing at 4°C for 12 hours, the supernatant was centrifuged at 8000 rpm and 4°C Centrifuge for 15 minutes, discard the bottom precipitate, and collect the supernatant; add anhydrous ethanol to 80% of the total volume of the supernatant, and precipitate at 4°C for 24 hours; after centrifugation at 4°C for 15 minutes, collect all the precipitates at the bottom; dissolve the precipitates in 60°C distilled water, centrifuge at 10,000rpm for 15 minutes, and discard the insoluble precipitates at the bottom; load the supernatant into a dialysis bag with a molecular weight cutoff of 8k to 12kDa, and the dialysate should not exceed two-thirds of the dialysis bag; replace the dialysate every 8 hours, and dialyze continuously for 3 days. Collect all the solutions in the dialysis bag, and vacuum freeze-dry to obtain crude extracellular polysaccharides of lactic acid bacteria;

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

[0059] The DEAE-Sepharose Fast Flow filler well preserved in the laboratory was loaded into the Column XK 26 / 40 chromatographic column (height: 40cm, inner diameter: 26mm), and the column loading was stopped when the filler was 5cm away from the top of the column; ultrapure water and Tris-HCl solution (55mM, pH 7.8) were used to balance the column at a flow rate of 2mL / min, and eluted for 2 to 3 column volumes; 100mg of crude lactic acid bacteria extracellular polysaccharide was weighed and fully dissolved in 10mL of Tris-HCl, filtered through a 0.22μm filter membrane, loaded, and after standing for 30min, elution began; Tris-HCl eluent and 1M The eluent of NaCl was eluted in sequence at a flow rate of 2 mL / min, and 6 mL was collected in each tube to obtain the crude extracellular polysaccharide of lactic acid bacteria purified by DEAE column; the polysaccharide content in the eluent was determined by phenol-sulfuric acid method, the absorbance value was detected at 490 nm, and the elution curve was drawn; according to the drawn elution curve, the eluent was collected and loaded into a dialysis bag with a molecular weight cutoff of 8k~12k Da, and the dialysate loaded should not exceed two-thirds of the dialysis bag. The dialysate was replaced every 8 hours, and the dialysis was continued for 3 days, and the polysaccharide was 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 filler was loaded into a chromatography column of Column XK 16 / 100 (height: 100 cm, inner diameter: 16 mm), and the filling was stopped when the filler was 10 cm away from the top of the chromatography column; ultrapure water and Tris-HCl solution (55 mM, pH 7.8) were used to balance the column at a flow rate of 2 mL / min, and 2 to 3 column volumes were eluted; 50 mg of crude lactic acid bacteria extracellular polysaccharide sample purified by DEAE column was weighed, fully dissolved in 5 mL Tris-HCl, filtered through a 0.22 μm filter membrane, and loaded; Tris-HCl eluent was used as the mobile phase for elution at a flow rate of 2 mL / min, and 6 mL was collected in each tube; the polysaccharide content was determined by the phenol-sulfuric acid method, and an elution curve was drawn; and after freeze-drying, lactic acid bacteria extracellular polysaccharide was obtained, which was named SXJ30EPS.

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

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

[0064] The characterization process of the structure of the lactic acid bacteria extracellular polysaccharide (SXJ30EPS) prepared by the present invention is as follows: the pure lactic acid bacteria extracellular polysaccharide component is determined by ultraviolet scanning to determine whether there are impurities, that is, 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 ultraviolet and infrared spectral scanning results; then, an 18-angle laser light scattering instrument is used to determine the molecular weight of the lactic acid bacteria extracellular polysaccharide, and an ion chromatography is used to determine the monosaccharide composition of the lactic acid bacteria extracellular polysaccharide.

[0065] 1. The analysis was performed using an ultraviolet-visible 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, the supernatant was collected, and deionized water was used as a control. The ultraviolet spectrophotometer was used to scan in the range of 190 to 600 nm. The results of the ultraviolet scanning were as follows: Figure 1 shown.

[0066] Depend on Figure 1 It can be seen that the polysaccharide only has a response absorption peak at 206nm; there is no absorption peak at 260nm and 280nm, indicating that EPS does not contain two types of impurities, nucleic acid and protein.

[0067] 2. An infrared spectrophotometer (Shimadzu Corporation, Tokyo, Japan) was used to analyze the characteristic absorption peaks of SXJ30EPS by infrared spectroscopy. Specifically, 1.5 mg of dry SXJ30EPS sample and 150 mg of potassium bromide powder were mixed and pressed into a tablet with a thickness of about 0.1 mm. The sample was infrared scanned. The infrared scanning results were as follows: Figure 2 shown.

[0068] Depend on Figure 2 It can be seen that the SXJ30EPS is 3408.32cm -1 There is a strong absorption peak at 2939.41cm, which is the OH bond stretching vibration absorption peak of the hydroxyl group of sugar compounds; -1 The absorption peak at 1652.93cm is the stretching vibration absorption peak of the CH bond; -1 Nearby is the C=O asymmetric stretching vibration peak; at 1411.84 cm -1 The peak at 1226.68cm is the angle-variable vibration peak of the CH bond. -1 The peak at 1053.10 cm is the angle-variable vibration peak of C=O; -1 The strong absorption peak at 808.15 cm is the COC characteristic absorption peak of pyranose. -1 The peak at the bottom is the CH angle vibration of the pyranose terminal group. That is, SXJ30EPS has the characteristic absorption peak of polysaccharides and has an α-type pyranose ring configuration.

[0069] Combining the UV and IR spectral scanning results, it can be seen that SXJ30EPS is a sugar and the sample does not contain other impurities.

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

[0071] The chromatographic conditions are as follows: chromatographic column: TSK Gel G4000PWxl; mobile phase: 0.1M NaCl solution; flow rate: 0.5mL / min; injection volume: 200μL. Weigh SXJ30EPS and dissolve it in 0.1M NaCl solution to make a solution with a concentration of 1mg / mL, and load it. Use Astra data analysis software to calculate the molecular weight of the sample.

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

[0073]

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

[0075] 4. Ion chromatography was used to determine the monosaccharide composition of SXJ30EPS. Chromatographic conditions: Chromatographic column: Analytical column: CarboPac TM PA203×150mm Analytical; Eluent: 250mM NaOH and 1M NaAC; Flow rate: 0.5mL / min; Injection volume: 10μL; Column temperature: 35℃; Detector: Pulsed amperometric detector, gold electrode. Gradient elution conditions: A is water, B is 250mM NaOH, and 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. Determination of monosaccharide composition: Weigh 10 mg of the sample into a hydrolysis bottle, add 4 mL of 4M trifluoroacetic acid, fill with N2 for 1 min to exhaust the air in the tube, tighten the screw cap, hydrolyze at 120℃ for 2 h, filter with a 0.22μm filter membrane, and inject according to the conditions described in 3.2.6.1. Determine the monosaccharide composition of the cleavage product by comparing the retention time RT. The results are as follows: Figure 4 shown.

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

[0077] That is, SXJ30EPS is composed of seven monosaccharides, namely, glucosamine, arabinose, galactosamine, galactose, glucose, mannose and glucuronic acid, among which the molar ratio of glucose:glucosamine:mannose, the main components of SXJ30EPS, is approximately 1.4:1.1:1.

[0078] Example 3. Obtaining samples from the gastrointestinal tract (rumen, duodenum, jejunum, ileum) of lambs

[0079] Twenty-four healthy newborn lambs were selected, half male and half female. The 24 lambs were divided into three groups according to the principle of weight, gender, and age, namely, blank control group, lactic acid bacteria extracellular polysaccharide (EPS) group and astragalus polysaccharide (APS) group. The experimental lambs were raised in separate pens. Among them, the lactic acid bacteria extracellular polysaccharide group was added with lactic acid bacteria extracellular polysaccharide at a dose of 1g / head / day, the astragalus polysaccharide group was added with a dose of 1g / head / day, and the blank control group was added with normal saline at a dose of 1g / head / day. The experiment set a 3-day transition period to allow the experimental lambs to become familiar with the experimental separate pen feeding mode to avoid stress. On the fourth day of the experiment, different treatment groups were given oral administration of different solutes. The different treatment solutes were dissolved in 100ml sterilized milk replacer powder and fed for 49 consecutive days. During the lamb experiment, the feeding procedures were carried out according to the original procedures of the ranch. On the 43rd and 44th days of feeding, 4 experimental lambs were randomly selected from each group and 10 9 The Escherichia coli solution with a dose of CFU / head was administered orally for challenge treatment, and on the 49th day of the experiment, 4 lambs were randomly selected from each group (2 challenged and 2 not challenged) and slaughtered to collect gastrointestinal tract samples (rumen, abomasum, duodenum, jejunum, and ileum). These samples corresponded to the blank control group (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).

[0080] One sample was preserved in 10% phosphate buffered formalin for tissue morphology analysis, and the other was preserved in liquid nitrogen for related gene and protein expression determination.

[0081] Example 5: Effects of lactic acid bacteria exopolysaccharides on the physiological structure of lamb gastrointestinal tissues

[0082] Paraffin embedding sectioning and HE staining were used to observe the changes in the physiological structure of the gastrointestinal tract (rumen, abomasum, duodenum, jejunum, ileum) samples of the blank control group, lactic acid bacteria exopolysaccharide group and astragalus polysaccharide group obtained in Example 4. The specific process is as follows:

[0083] 1. Tissue embedding

[0084] (1) Ethanol dehydration: The tissue was dehydrated in different concentrations of ethanol solution: 75%, 85%, 95%, 100%, 100%, and each level of ethanol for 40 min (Note: Bony and calcified tissues need to be immersed in decalcification solution for 24 h, and then the ethanol dehydration step can be performed after the tissue softens);

[0085] (2) Transparency: The tissue was immersed in three xylene tanks in sequence, each tank for 30 min;

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

[0087] (4) Embedding: Pour liquid paraffin into the mold box, and then place the wax-soaked tissue block flat on the bottom, with the cut surface facing downward. After the paraffin solidifies, remove the embedding frame. After it has completely cooled and hardened, trim the wax block. Keep an appropriate amount of paraffin on the outside of the tissue to facilitate slicing.

[0088] 2. Slice production

[0089] Fix the pre-cooled wax block on the paraffin slicer so that the cut surface of the wax block is parallel to the knife edge. The inclination of the knife is usually 15 degrees. Turn the rotary propeller to adjust the slice thickness to 3μm and cut into slices with uniform thickness; hold the brush in your left hand and rotate the slicer handle with your right hand. After the slice is brought out, gently lift it up with the brush, and then use tweezers to gently tweeze the wax slice, and put it into the slide exhibition box with the front side. The water temperature is about 40℃, and after it is flattened, pick up the slice; attach the slice. Hold one end of the glass slide in your left hand and put it vertically into the water to attach the slice. Use tweezers in your right hand to help push it. Attach it to two-thirds of the slide. After the slice is attached, place it in the air to dry slightly, bake it on a 60℃ slide oven for 1h, and then bake it in an oven for 2h.

[0090] 3. Dewax paraffin sections to water

[0091] Place the paraffin sections in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 10 min, anhydrous ethanol II in turn.

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

[0093] 4. HE staining

[0094] The paraffin sections were stained with hematoxylin for 5-10 minutes, rinsed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, then blued with saturated aqueous solution of lithium carbonate for 1 minute, rinsed with running water for a few seconds, stained with eosin solution for a few seconds, and rinsed with running water.

[0095] 5. Dehydration and sealing

[0096] The paraffin sections were sequentially placed 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 make them transparent. The sections were taken out of xylene and sealed with neutral gum.

[0097] 6. Interpretation of results

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

[0099] HE staining sections of rumen tissue of the blank control group (blank challenge and blank control), lactic acid bacteria exopolysaccharide group (EPS challenge and EPS control), and astragalus polysaccharide group (APS challenge and APS control) after and without challenge in this example are shown in Figure 2. Figure 5 shown.

[0100] Depend on Figure 5 It can be seen that Figure 5 A After the attack, the overall structure of the rumen tissue in the blank control group was basically normal, the mucosal layer was basically normal, the mucosa was covered with keratinized squamous epithelium, the papillae were arranged neatly, there were no abnormalities in the submucosal layer and muscular layer, and no obvious inflammatory cell infiltration was found in the tissue. Figure 5 B. The overall structure of the rumen tissue in the blank control group without infection was basically normal, with mild abnormalities in the mucosal layer. The mucosa was covered with keratinized squamous epithelium, focal mucosal epithelial ulcers accompanied by inflammatory cell infiltration, the papillae became shorter and arranged neatly, and no abnormalities were found in the submucosal layer and muscular layer. Figure 5 After the attack, the overall structure of the rumen tissue in the APS group was basically normal, the mucosal layer was basically normal, the mucosa was covered with keratinized squamous epithelium, the papillae became shorter and arranged neatly, there were no abnormalities in the submucosal layer and muscular layer, and no obvious inflammatory cell infiltration was found in the tissue. Figure 5 In the non-challenged APS group, the overall structure of the rumen tissue was basically normal, the mucosal layer was basically normal, the mucosa was covered with keratinized squamous epithelium, the papillae were flat, there were no abnormalities in the submucosal layer and muscular layer, and no obvious inflammatory cell infiltration was found in the tissue. Figure 5 After E infection, the overall structure of the rumen tissue in the EPS group was basically normal, the mucosal layer was basically normal, the mucosa was covered with keratinized squamous epithelium, the papillae were flat, there were no abnormalities in the submucosal layer and muscular layer, and no obvious inflammatory cell infiltration was found in the tissue. Figure 5 In the F non-infected EPS group, the overall structure of the rumen tissue was basically normal, the mucosal layer was basically normal, the mucosa was covered with keratinized squamous epithelium, the papillae were arranged regularly, there were no abnormalities in the submucosal layer and muscular layer, and no obvious inflammatory cell infiltration was found in the tissue.

[0101] HE staining sections of abomasum tissue of the blank control group (blank challenge and blank control), lactic acid bacteria exopolysaccharide group (EPS challenge and EPS control), and astragalus polysaccharide group (APS challenge and APS control) after and without challenge in this example are shown in Figure 2. Figure 6 shown.

[0102] Depend on Figure 6 It can be seen that Figure 6 A After the attack, the overall structure of the abomasum tissue of the blank control group was basically normal, the mucosal layer was basically normal, the mucosal glands were arranged regularly and neatly, there were no abnormalities in the submucosal layer and muscular layer, and no obvious inflammatory cell infiltration was found in the tissue. Figure 6 B. The overall structure of the true stomach tissue of the blank control group without virus infection was basically normal, with mild abnormalities in the mucosal layer, focal mucosal epithelial exfoliation accompanied by inflammatory cell infiltration, regular and neat arrangement of mucosal glands, no abnormalities in the submucosal layer and muscular layer, and no obvious inflammatory cell infiltration in the tissue. Figure 6After the APS challenge, the overall structure of the true stomach tissue in the APS group was basically normal, with a slight abnormality in the mucosal layer, sparse and loose arrangement of focal mucosal glands, no abnormality in the submucosal layer and muscular layer, and no obvious inflammatory cell infiltration in the tissue. Figure 6 D. The overall structure of the true stomach tissue in the non-challenged APS group was basically normal, the mucosal layer was basically normal, the mucosal glands were arranged regularly and neatly, the submucosal layer was slightly edematous, the muscular layer was normal, and no obvious inflammatory cell infiltration was found in the tissue. Figure 6 After E infection, the overall structure of the true stomach tissue in the EPS group was basically normal, the mucosal layer was basically normal, the mucosal glands were arranged irregularly with intestinal metaplasia, there were no abnormalities in the submucosa and muscular layer, and no obvious inflammatory cell infiltration was observed in the tissue. Figure 6 In the EPS group not challenged with poison, the overall structure of the true stomach tissue was basically normal, the mucosal layer was basically normal, the mucosal glands were arranged regularly and neatly, there were no abnormalities in the submucosal layer and muscular layer, and no obvious inflammatory cell infiltration was found in the tissue.

[0103] HE staining sections of duodenal tissue of the blank control group (blank challenge and blank control), lactic acid bacteria exopolysaccharide group (EPS challenge and EPS control), and astragalus polysaccharide group (APS challenge and APS control) after and without challenge in this example are shown in Figure 2. Figure 7 shown.

[0104] Depend on Figure 7 It can be seen that Figure 7 A After the attack, the overall structure of the duodenal tissue in the blank control group was slightly abnormal, the mucosal layer was slightly abnormal, the villi were slightly eroded with inflammatory cell infiltration, and no abnormalities were found in the submucosal layer and muscular layer. Figure 7 B. The overall structure of the duodenal tissue in the blank control group without infection was slightly abnormal, the mucosal layer was slightly abnormal, villous erosion was accompanied by inflammatory cell infiltration, and no abnormality was found in the submucosal layer and muscular layer. Figure 7 After the APS attack, the overall structure of the duodenal tissue in the APS group was basically normal, the mucosal layer was basically normal without erosion or shedding, duodenal glands were visible in the submucosal layer, and no abnormalities were found in the muscular layer. Figure 7 D. The overall structure of the duodenal tissue in the non-challenged APS group was slightly abnormal, the mucosal layer was slightly abnormal, the villi were flat and fell off, and no abnormalities were found in the submucosal layer and muscular layer. Figure 7 After E infection, the overall structure of the duodenal tissue in the EPS group was basically normal, with mild abnormalities in the mucosal layer, flat villi and shedding, duodenal glands visible in the submucosal layer, and no abnormalities in the muscular layer. Figure 7 The overall structure of the duodenal tissue in the EPS group F was basically normal, with mild abnormalities in the mucosal layer, flat villi with erosion, duodenal glands visible in the submucosal layer, and no abnormalities in the muscular layer.

[0105] The HE staining sections of the jejunal tissue of the blank control group (blank challenge and blank control), the lactic acid bacteria exopolysaccharide group (EPS challenge and EPS control), and the astragalus polysaccharide group (APS challenge and APS control) after and without challenge in this example are shown in Figure 2. Figure 8 shown.

[0106] Depend on Figure 8 It can be seen that Figure 8 A After the attack, the overall structure of the jejunal tissue in the blank control group was basically normal, the mucosal layer was basically normal, the mucosal glands were arranged regularly and neatly, small adenomas were visible inside, the submucosal layer was slightly edematous, the muscular layer was normal, and no obvious inflammatory cell infiltration was found in the tissue. Figure 8 B. The overall structure of the jejunal tissue in the blank control group without virus infection was basically normal, the mucosal layer 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 submucosal layer and muscular layer. Figure 8 After the APS attack, the overall structure of the jejunum tissue in the APS group was basically normal, the mucosal layer 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 submucosal layer and muscular layer. Figure 8 D. The overall structure of the jejunum tissue in the non-challenged APS group was basically normal, the mucosal layer 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 submucosal layer and muscular layer. Figure 8 After E infection, the overall structure of the jejunal tissue in the EPS group was basically normal, the mucosal layer was abnormal, the mucosal glands were arranged regularly and neatly, some villi were eroded, a small amount of inflammatory cell infiltration was visible, and no abnormalities were found in the submucosal layer and muscular layer. Figure 8 In the EPS group not challenged with virus, the overall structure of the jejunum was basically normal, the mucosal layer was abnormal, the mucosal glands were arranged regularly and neatly, some villi were eroded, a small amount of inflammatory cell infiltration was visible, and no abnormalities were found in the submucosal layer and muscular layer.

[0107] HE staining sections of ileum tissue of the blank control group (blank challenge and blank control), lactic acid bacteria exopolysaccharide group (EPS challenge and EPS control), and astragalus polysaccharide group (APS challenge and APS control) after and without challenge in this example are shown in Figure 2. Fig. 9 shown.

[0108] Depend on Fig. 9 It can be seen that Fig. 9 A After the virus attack, the overall structure of the ileum tissue in the blank control group was basically normal, the mucosal layer was basically normal, the mucosal glands were arranged regularly and neatly, a large number of lymphocytes were seen gathering in the submucosal layer, and no abnormalities were found in the muscular layer. Fig. 9 B. The overall structure of the ileum tissue in the blank control group without virus infection was basically normal, the mucosal layer was basically normal, the mucosal glands were arranged regularly and neatly, a large number of lymphocytes were seen gathering in the submucosal layer, and no abnormalities were found in the muscular layer. Fig. 9After C challenge, the overall structure of the ileum tissue in the APS group was basically normal, the mucosal layer was basically normal, the mucosal glands were arranged regularly and neatly with inflammatory cell infiltration, a large number of lymphocytes were seen gathering in the submucosal layer, and no abnormalities were found in the muscular layer. Fig. 9 D. The overall structure of the ileum tissue in the non-challenged APS group was basically normal, the mucosal layer was basically normal, the mucosal glands were arranged regularly and neatly, the villi were flat and shed, a large number of lymphocytes were seen in the submucosal layer, and no abnormalities were found in the muscular layer. Fig. 9 After E infection, the overall structure of the ileum tissue in the EPS group was basically normal, the mucosal layer was basically normal, the mucosal glands were arranged regularly and neatly, a small amount of mucosal epithelium was shed, a large number of lymphocytes were seen gathering in the submucosal layer, and no abnormalities were found in the muscular layer. Fig. 9 In the EPS group not challenged with poison, the overall structure of the ileum tissue was basically normal, the mucosal layer was basically normal, the mucosal glands were arranged regularly and neatly, a small amount of mucosal epithelium was shed, a large number of lymphocytes were seen gathering in the submucosal layer, and no abnormalities were found in the muscular layer.

[0109] The summary of HE staining section analysis of gastrointestinal tract (rumen, abomasum, duodenum, jejunum, ileum) tissue samples of lamb blank control group, lactic acid bacteria extracellular polysaccharide group and astragalus polysaccharide group is shown in Table 2.

[0110] Table 2

[0111]

[0112] Depend on Figures 5 to 9 As shown in Table 2, the lactic acid bacteria extracellular polysaccharide (SXJ30EPS) provided by the present invention can effectively resist inflammation of the gastrointestinal tract (rumen, true stomach, duodenum, jejunum, and ileum) of lambs caused by Escherichia coli, and has the function of improving the gastrointestinal defense function of newborn lambs, improving the inherent defense mechanism of the newborn lambs, and enhancing the intestinal disease resistance and health level of the newborn lambs.

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

[0114] 1. Real-time fluorescence quantitative PCR analysis

[0115] (1) Sample processing: 100 mg of gastrointestinal tract (rumen, true stomach, duodenum, jejunum, ileum) sample tissues of the blank control group, lactic acid bacteria extracellular polysaccharide group and astragalus polysaccharide group obtained in Example 4 were respectively taken and cut into small pieces as much as possible before grinding. 1 ml of TRIzol reagent was added and the mixture was quickly and fully homogenized using a high-throughput tissue grinder (6 m / s, grinding for 20 s, standing for 15 s, grinding for 2 cycles).

[0116] (2) RNA extraction: After adding TRIzol, the sample was reacted at room temperature for 10 minutes; 200 μl of chloroform was added, and the sample was shaken vigorously for 15 seconds. Do not use a vortex shaker, and the sample was placed at room temperature for 10 minutes; centrifuged at 4℃ and 12000rpm for 15 minutes. After centrifugation, the solution was divided into three layers, from bottom to top: phenol layer, middle white protein layer and clear water layer, and RNA was present in the water layer; 400 μL of the upper aqueous phase was taken into a new centrifuge tube, and an equal volume of isopropanol (precooled at 4℃) was added, and the sample was placed at -20℃ for 15 minutes; centrifuged at 4℃ and 12000rpm for 10 minutes; the supernatant was discarded, and 1 mL of 75% ethanol (precooled at 4℃) was added to wash the RNA precipitate; the sample was gently turned upside down and washed thoroughly. Centrifuged at 4℃ and 12000rpm for 5 minutes; the supernatant was discarded, and the alcohol precipitation was repeated once; the supernatant was discarded, and the residual liquid was carefully sucked clean with a pipette, and dried. Add 50 μL DEPC water and blow gently with a gun to fully dissolve it; RNA purity and concentration detection: NanoDrop 2000 determines the OD value and concentration of the extracted RNA;

[0117] RNA integrity detection: 1.5% agarose gel electrophoresis was used to identify the integrity of RNA, and RNA samples were stored in a -80°C refrigerator for a long time.

[0118] (3) In vitro reverse transcription

[0119] Add the following reagents to the RNase / DNase-free PCR tube according to the reverse transcription system, mix gently on ice, centrifuge, and then use Oligo (dT) 18 or gene-specific primers, incubate at 42 ° C for 60 minutes, and then heat at 70 ° C for 5 minutes to terminate the reaction and obtain cDNA. After dilution, cDNA can be directly used for PCR reaction or stored at -20 ° C for short-term storage, and stored for long-term storage in a -80 ° C refrigerator.

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

[0121] (4) PCR amplification

[0122] The diluted cDNA (about 100 ng / μl) was used as a PCR reaction template and PCR amplification was performed using primers F / R to obtain a 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, a total of 20μL.

[0124] PCR amplification program: pre-denaturation, 94℃30s; denaturation, 94℃5sec; annealing, 60℃30sec; extension, 95℃15sec, 40 cycles; melting curve 60℃1min, heating 0.3℃ every 15s, 95℃15sec; termination extension, 72℃8min; finally keeping warm at 4℃.

[0125] (5) Data analysis

[0126] ABI7500 software was used to read the Ct value of each PCR reaction. 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 subtracted from the ΔCt of the experimental group to obtain ΔΔCt. 2-ΔΔCt was used to calculate the expression change of the target gene in the experimental group relative to the control group.

[0127] 2. Western-blot analysis

[0128] (1) Sample processing: 100 mg of gastrointestinal tract (rumen, abomasum, duodenum, jejunum, ileum) sample tissues of the blank control group, lactic acid bacteria extracellular polysaccharide group and astragalus polysaccharide group obtained in Example 4 were respectively taken, 1 mL of RIPA lysis buffer (10 μL of PMSF was added to each 1 mL of lysis buffer), homogenized using a tissue grinder, lysed at 4°C for 30 min, and then centrifuged at 4°C, 12000 rpm for 10 min, and the supernatant was taken; the supernatant was subjected to BCA protein quantification and stored at -80°C for a long time.

[0129] (2) BCA protein quantification: Use the Biotech (P0011) kit and follow the instructions of the kit.

[0130] (3) Denaturation of protein samples: Based on the BCA protein quantification results, approximately 80 μg of total protein was added to an appropriate amount of 5× protein loading buffer, mixed well, and denatured in boiling water for 10 min. The sample was then loaded after brief centrifugation.

[0131] (4) SDS-PAGE electrophoresis: Prepare fresh electrophoresis buffer and run the electrophoresis at 140 V until the loading point just runs out of the separation gel.

[0132] (5) Transfer: After electrophoresis, take out the gel and place it in the prepared 1× transfer solution for 20 minutes; cut the PVDF membrane and thin filter paper to appropriate size, activate the PVDF membrane with methanol, and then place the PVDF membrane and thin filter paper in the transfer solution for 20 minutes; place them in the order of negative electrode-thin filter paper-gel-PVDF membrane-thin filter paper-positive electrode. Transfer the membrane at 200mA constant current for 1 hour.

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

[0134] (7) ECL color development: Prepare an appropriate amount of ECL working solution according to the instructions of the ECL color development kit; drain the liquid on the PVDF membrane, evenly drop the prepared ECL working solution on the membrane to cover the entire membrane, incubate at room temperature for 2 min, then remove the excess color development solution, place the membrane in the fully automatic chemiluminescence image analysis system for imaging, and take pictures; use the gray value software Gelpro32 to analyze the gray value of the bands.

[0135] The QPCR results of the target Claudin1 in the duodenum, jejunum, and ileum tissue samples of the blank control group, lactic acid bacteria exopolysaccharide group, and astragalus polysaccharide group after and without the challenge are shown in the following table. Fig.10 As shown, A is the duodenum, B is the jejunum, and C is the ileum.

[0136] Western-blot grayscale value analysis of the expression of the target Cloudin1 in the duodenum, jejunum, and ileum tissue samples of the blank control group (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) after and without challenge Fig.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 increased compared with the blank control group after challenge.

[0138] The QPCR results of the target Claudin4 in the duodenum, jejunum, and ileum sample tissues of the blank control group (blank challenge and blank control), lactic acid bacteria exopolysaccharide group (EPS challenge and EPS control), and astragalus polysaccharide group (APS challenge and APS control) after and without challenge in this example are as follows: Fig.12 As shown, A is the duodenum, B is the jejunum, and C is the ileum.

[0139] Western-blot grayscale value analysis of the expression of the target Cloudin4 in the duodenum, jejunum, and ileum tissue samples of the blank control group (blank challenge and blank control), lactic acid bacteria exopolysaccharide group (EPS challenge and EPS control), and astragalus polysaccharide group (APS challenge and APS control) after and without challenge Fig.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 grayscale value of each group in the challenge group showed an increasing trend compared with the control group.

[0141] The QPCR results of the target Occludin in the duodenum, jejunum, and ileum sample tissues of the blank control group (blank challenge and blank control), lactic acid bacteria exopolysaccharide group (EPS challenge and EPS control), and astragalus polysaccharide group (APS challenge and APS control) after and without challenge in this example are as follows: Fig.14 As shown, A is the duodenum, B is the jejunum, and C is the ileum.

[0142] Western-blot grayscale value analysis of the target Occludin expression in the duodenum, jejunum, and ileum tissue samples of the blank control group (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) after and without challenge Fig.15 As shown, A is the duodenum, B is the jejunum, and C is the ileum.

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

[0144] Depend on Figures 10 to 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 lactic acid bacteria extracellular polysaccharide group after challenge was significantly upregulated in the duodenum, jejunum and ileum, 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 the present invention can effectively improve the gastrointestinal defense function of newborn lambs, improve the inherent defense mechanism of the newborn lambs, enhance the intestinal disease resistance and health level of the newborn lambs, and transform intestinal inflammation and diseases from passive treatment to active early intervention. It can be used to prepare preparations for improving the gastrointestinal barrier defense function of lambs, as well as ruminant feed additives, and has great development and application prospects.

Claims

1. A lactic acid bacteria exopolysaccharide, characterized in that: The lactic acid bacteria extracellular polysaccharide is pyranose, which is composed of glucosamine, arabinose, galactosamine, galactose, glucose, mannose and glucuronic acid; Its structural formula is shown in the following formula (1):

2. The lactic acid bacteria exopolysaccharide according to claim 1, characterized in that The lactic acid bacteria extracellular polysaccharide is derived from lactic acid bacteria (Lactobacillus casei) WXD030, which was deposited in the General Microbiological Center of China National Committee for the Preservation of Microorganisms on May 3, 2016, with the address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNO.12415.

3. The lactic acid bacteria exopolysaccharide according to 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.

4. The method for preparing lactic acid bacteria exopolysaccharide according to claim 1, characterized in that: The steps include: The lactic acid bacteria WXD030 is inoculated on an MRS solid culture medium, and activated and cultured at 37°C for 20-25 hours to obtain a seed liquid; then the seed liquid is inoculated into an MRS liquid culture medium at a volume percentage of 3-6%, and fermented and cultured at 37°C for 25-35 hours to obtain a fermentation liquid; the fermentation liquid of the strain is heated to inactivate enzymes, and then cooled and centrifuged to remove coagulated proteins and bacterial bodies, and then trichloroacetic acid with a final concentration of 4.0% (w / v) is used to remove proteins in the supernatant, and after ultrafiltration and concentration, ethanol with a final concentration of 75% (v / v) is used for precipitation to obtain a crude polysaccharide; the crude polysaccharide is separated and purified by a DEAE-Sepharose Fast Flow ion exchange chromatography column and a SepharoseCL-6B gel chromatography column to obtain a lactic acid bacteria extracellular polysaccharide.

5. Use of the lactic acid bacteria extracellular polysaccharide according to claim 1 in the preparation of a preparation for improving the gastrointestinal barrier defense function of lambs.

6. The use according to claim 5, characterized in that The lactic acid bacteria extracellular polysaccharide improves the gastrointestinal barrier defense function of the lambs by enhancing the anti-inflammatory ability of the lambs' gastrointestinal tract.

7. The use according to claim 5, characterized in that The gastrointestinal tract refers to the rumen, true stomach, duodenum, jejunum and ileum of the lamb.

8. Use of the lactic acid bacteria extracellular polysaccharide according to claim 1 in the preparation of a ruminant feed additive.

9. The use according to claim 8, characterized in that The ruminant is a cow or a sheep.

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