Application of undaria pinnatifida fucoidin oligosaccharide in preparation of product for preventing / relieving intestinal inflammation induced by salmonella typhimurium

Abstract

The invention discloses application of undaria pinnatifida fucoidin oligosaccharide in preparation of a product for preventing / relieving intestinal inflammation induced by salmonella typhimurium, and belongs to the technical field of medicines. According to the application of the undaria pinnatifida fucoidin oligosaccharide in preparation of the salmonella typhimurium bacteriostatic agent, the molecular weight of the undaria pinnatifida fucoidin oligosaccharide is 4000-4500 Da, and main monosaccharides of the undaria pinnatifida fucoidin oligosaccharide are Man, Gal and Fuc. The undaria pinnatifida fucoidin oligosaccharide has a protection effect on liver and spleen organ injury, can improve the physical sign performance of the liver and spleen organ injury, relieve inflammation symptoms, effectively inhibit invasion of pathogenic bacteria, better protect colon tissues from serious damage and protect organs from pathological damage.

Description

Technical Field

[0001] The present invention relates to the use of Undaria pinnatifida fucoidan oligosaccharide in preparing a product for preventing / alleviating intestinal inflammation induced by Salmonella typhimurium, and belongs to the technical field of medicine. Background Art

[0002] Salmonella Typhimurium (ST) is a common Gram-negative facultative cytopathogen that, upon invading the human body, can cause a variety of illnesses, including typhoid fever and enteritis. Salmonellosis is one of the most important foodborne illnesses, and the number of people infected with Salmonella is increasing worldwide. Currently, the most common treatment is antibiotics. However, as Salmonella has evolved multiple drug-resistant serotypes, their resistance is increasing, making antibiotic treatment less effective or even ineffective. The overuse of antibiotics can harm the normal intestinal flora and have side effects such as toxicity. Therefore, finding effective supplements to prevent and protect against Salmonella is crucial.

[0003] Undaria pinnatifida is a kind of brown algae with medicinal and edible properties. It is rich in fucoidan (Undariapinnatifida fucoidan / UPF), which has multiple active functions such as anti-inflammatory, antioxidant, antiviral and regulating intestinal microecology. It also shows great potential in inhibiting pathogens because of its rich sulfate groups and non-toxic properties.

[0004] As shown in the applicant's previous research (application number CN202311196423.0), kelp fucoidan can inhibit the increase of liver and spleen indexes, the accumulation of Salmonella and the shortening of colon length, reduce oxidative stress damage, increase the content of short-chain fatty acids in the intestine, regulate the level of inflammation, increase the types and number of intestinal flora, and effectively alleviate typhoid enteritis caused by typhoid Salmonella infection; however, there is still the problem of poor treatment effect. Summary of the Invention

[0005] In view of the defects of the existing technology, the purpose of the present invention is to provide a use of undaria pinnatifida oligosaccharide in the preparation of a product for preventing / alleviating intestinal inflammation induced by Salmonella typhimurium. The undaria pinnatifida oligosaccharide can effectively inhibit the activity of Salmonella typhimurium and protect intestinal tissue from severe damage.

[0006] In order to achieve the above objectives, the technical solutions provided are as follows:

[0007] The present invention provides the use of Undaria pinnatifida fucoidan oligosaccharide in preparing a Salmonella typhimurium antibacterial agent.

[0008] In one embodiment, the molecular weight of the Undaria pinnatifida fucoidan is 4000-4500 Da, and the main monosaccharides are Man, Gal and Fuc.

[0009] In one embodiment, the molecular weight of the Undaria pinnatifida fucoidan is 4200Da, and the contents of neutral sugar, sulfate, and aldehyde are 64.3±1.6%, 22.6±1.3%, and 10.6±0.3%, respectively. The main monosaccharides are Man, Gal, and Fuc, with a mass ratio of 1.2:9.9:4.5.

[0010] In one embodiment, the usage amount of the Undaria pinnatifida oligosaccharide is not less than 200 mg / kg / d.

[0011] In one embodiment, the preparation of the Undaria pinnatifida oligosaccharide comprises the following steps:

[0012] (1) Dissolve the crude polysaccharide sample of Undaria pinnatifida in deionized water and perform acid hydrolysis;

[0013] (2) The solution after acid hydrolysis in step (1) was cooled to room temperature and the pH of the solution was adjusted to 7; centrifuged using a 3 kDa ultrafiltration tube, and the solution with Mw < 3 kDa after centrifugation was concentrated by rotary evaporation;

[0014] (3) dialyzing the concentrated solution in step (2), collecting the dialyzed liquid, evaporating and concentrating it, and then freeze-drying it into a solid powder to obtain Undaria pinnatifida oligosaccharide (OUPF).

[0015] In one embodiment, the acid hydrolysis in step (1) is specifically to add concentrated hydrochloric acid to the solution containing crude polysaccharide of Undaria pinnatifida to make the final concentration of hydrochloric acid in the solution reach 0.3-0.5 mol / L, and then acid hydrolyze at 90-95° C. for 10-30 min, preferably 30 min.

[0016] In one embodiment, the centrifugal separation parameters in step (2) are: 4000-8000 rpm, 10-20 min.

[0017] In one embodiment, the dialysis bag used in step (3) is a 300Da dialysis bag.

[0018] The present invention also provides the use of Undaria pinnatifida fucoidan in the preparation of a product for preventing / alleviating intestinal inflammation induced by Salmonella typhimurium.

[0019] In one embodiment, the molecular weight of the Undaria pinnatifida fucoidan is 4000-4500 Da, and the main monosaccharides are Man, Gal and Fuc.

[0020] In one embodiment, the molecular weight of the Undaria pinnatifida fucoidan is 4200Da, and the contents of neutral sugar, sulfate, and aldehyde are 64.3±1.6%, 22.6±1.3%, and 10.6±0.3%, respectively. The main monosaccharides are Man, Gal, and Fuc, with a mass ratio of 1.2:9.9:4.5.

[0021] In one embodiment, the product further contains a carrier and / or excipients, wherein the carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and adhesives commonly used in medicine.

[0022] In one embodiment, the product includes at least one of the following functions:

[0023] (1) Inhibit the number of Salmonella colonies in the liver, spleen, and colon;

[0024] (2) Reduce oxidative stress damage;

[0025] (3) Improve colon damage;

[0026] (4) regulating inflammation levels;

[0027] (5) Regulate the structure and diversity of intestinal flora.

[0028] In one embodiment, the products include medicines and health products.

[0029] In one embodiment, the product comprises a tablet, a powder, or a liquid formulation.

[0030] Beneficial effects:

[0031] The present invention provides the use of Undaria pinnatifida fucoidan oligosaccharide in treating colitis caused by Salmonella typhimurium; specifically,

[0032] (1) OUPF intake significantly increased the body weight of mice, while UPF did not significantly affect the changes in body weight;

[0033] (2) OUPF has a protective effect on liver and spleen organ damage in mice, improving their physical signs and alleviating inflammatory symptoms;

[0034] (3) By counting the number of Salmonella colonies in the liver, spleen, and colon, it was shown that OUPF can effectively inhibit the invasion of pathogenic bacteria, thereby protecting organs from pathological damage;

[0035] (4) Algal oligosaccharides can achieve antioxidant effects by scavenging free radicals and thus resist inflammation;

[0036] (5) OUPF can better protect colon tissue from severe damage than UPF;

[0037] (6)OUPF can effectively maintain the richness of the bacterial flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the molecular weight distribution diagram of Undaria pinnatifida fucoidan;

[0039] Figure 2 This is a graph showing the inhibitory effect of Undaria pinnatifida oligosaccharides with different molecular weights on Salmonella;

[0040] Figure 3 Data graph showing the effects of Undaria pinnatifida oligosaccharides prepared in Example 1 on basic physiological and biochemical indicators in mice; (A) Experimental flow chart; (B) Weight gain; (C) Changes in mortality; (D) Liver-spleen weight ratio; (E) and (F) Colon length; *p<0.05, **p<0.01, ***p<0.001;

[0041] Figure 4 This is a statistical graph of the number of Salmonella colonies in mouse organs;

[0042] Figure 5 The data of the effect of fucoidan oligosaccharide on antioxidant indicators in mouse colon tissue; (A) CAT level; (B) SOD level; (C) MPO level; (D) MDA level;

[0043] Figure 6 The effect of Undaria pinnatifida oligosaccharide on mouse pathological tissue; (A) HE, PAS and AB staining tissue sections; (B) colon tissue scoring diagram;

[0044] Figure 7 The data of the effect of kelp oligosaccharide on the expression of inflammatory factors in mice; (A) LPS; (B) IL-1β; (C) TNF-α; (D) IL-10;

[0045] Figure 8 α-diversity analysis diagram of mouse intestinal flora; (A) ACE diagram; (B) Chao1 diagram; (C) Shannon diagram; (D) Simpson diagram;

[0046] Figure 9 β-diversity analysis diagram of mouse intestinal flora; (A) PCOA diagram; (B) species distribution diagram at the phylum level; (C) Bacteroidetes; Firmicutes, B / F data diagram. DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention. The following specific embodiments further describe the present invention.

[0048] The testing method of the present invention

[0049] 1. Determination of sulfate content

[0050] After crushing the kelp, pass it through an 80-mesh sieve. Weigh 1.002g of powder (accurate to 0.001g) on ​​a balance, add 50mL of distilled water, let it stand for 1h, heat under reflux for 4h, let it stand and cool to room temperature, centrifuge it in a centrifuge at 9000r / min for 30min, then transfer the supernatant to a 100ml volumetric flask. Wash the precipitate with a small amount of water several times, centrifuge it at 9000r / min and transfer the supernatant to the same volumetric flask. Measure 2mL of the supernatant and place it in a 50mL centrifuge tube. Add 30mL of anhydrous ethanol while stirring, shake well, and let it stand at 4℃ for 12h. After taking it out, centrifuge it at 9000r / min for 30min, discard the supernatant, and obtain an alcohol-precipitated fucose solution.

[0051] Take the alcohol-precipitated fucose solution and dry it in an oven to obtain about 50 mg of dry fucose powder. Then add 25 mL of 1 mol / L hydrochloric acid solution and heat at 100 ° C for 6 hours. After cooling, centrifuge at 8000 r / min for 20 minutes, filter, and dilute to 25 mL with HCl. Take 0.2 mL and add 3.8 mL of 3% TCA and BaCl 2- Shake 1.0 mL of gelatin solution, let it stand at room temperature for 15 minutes, and measure the absorbance A1 at a wavelength of 360 nm. Replace the barium chloride gelatin solution with 1.0 mL of gelatin solution and measure the absorbance A2. The formula is as follows:

[0052] SO4 2- Content = f × (C / 0.2) × V / M × 100%.

[0053] Where: f is the conversion factor; C is K2SO4 2- Measured concentration, mg / mL; V is the total volume of the sample hydrolyzate; W is the initial sample mass.

[0054] Determination of conversion factor: Precision absorption of K2SO4 2- Prepare 0.15mL of standard stock solution, measure the absorbance value according to the standard curve operation method, and calculate the SO4 content in the test solution by the regression equation. 2- content, the conversion factor is calculated according to f = w / cd, where w is the sulfate concentration taken, c is the measured sulfate concentration, and d is the dilution factor.

[0055] 2. Determination of molecular weight

[0056] The molecular weight was determined by high performance gel filtration chromatography (HPGFC).

[0057] Chromatographic conditions: Column: YMC Pack-Diol 200 column (300 mm × 8.0 mm, S-5 μm, 20 nm); Mobile phase: distilled water; Flow rate: 0.8 mL min -1 ; Column temperature: 35℃; Detector: differential refractive index detector; Injection volume: 20μL.

[0058] Standard curve drawing: Glucan T-10, T-40, T-70, T-500, T-2000 and glucose were used as standard substances and dissolved in mobile phase to prepare 2 mg·mL -1 The solution is injected and the retention time of the elution peak is recorded. The molecular weight Mw and its elution volume V on the gel column e , distribution coefficient K av The following relationship exists:

[0059] V e =a-blgM w

[0060] K av =K1-K2lgM w

[0061] K av =(V e -V0) / (V t -V0)

[0062] Where: a, b, K1, K2 are constants; V0 is the elution volume of T-2000; V t The elution volume of glucose is expressed in terms of retention time.

[0063] K av lg M w Perform linear regression to obtain the linear regression equation. The sample is loaded onto the column according to the above conditions to obtain V e , according to its K av The molecular weight of the sample was calculated from the standard curve.

[0064] 3. Determination of monosaccharide composition

[0065] (1) Preparation of monosaccharide standards

[0066] 2 mg of each sample was hydrolyzed with 2 mL of 2M TFA at 121°C for 2 hours. The dried hydrolyzate was dissolved with 2.0 mL of 0.5 mol / L Na2CO3 and reduced with NaBH4 at room temperature for 1.5 hours. Excess NaBH4 was decomposed with acetic acid. The solution was mixed with Dowex 50WX8 (H', 200-400 mesh) and eluted with 6 ml of water. The eluate was dried on a rotary evaporator at 45°C. 0.1% (v / v) HCl-methanol was added to the borate in the mixture and evaporated to dryness repeatedly. The dried hydrolyzate was then stored at 85°C for 2 hours to convert the aldonic acid into aldonic lactone. The reaction product was then dissolved in 2 ml of pyridine and 2 ml of acetic anhydride and incubated at 100°C for 30 minutes.

[0067] (2) Sample preparation

[0068] Dissolve OUPF in water to a 10 mg / mL solution. Transfer 1 mL of this solution to a 5 mL hydrolysis tube. Add 0.3 mL of trifluoroacetic acid dropwise to the solution and hydrolyze in a 105°C oven for 3 hours. Remove, cool, freeze-concentrate, and spin-dry. Add 1.0 mL of chromatography-grade methanol and repeat three times. Dissolve the sample in 1 mL of 1% acetic acid and extract with 1 mL of CHCl₃. Repeat three times. Pass the sample through a 0.22 μm organic filter into a brown vial and analyze by liquid chromatography.

[0069] Sources of materials involved in the present invention:

[0070] Undaria pinnatifida fucoidan was provided by Qingdao Mingyue Seaweed Group Co., Ltd.

[0071] Culture and preparation of strains:

[0072] Preparation of LB medium: Take 1 g of tryptone, 0.5 g of yeast extract, 1 g of sodium chloride, and 1.5 g of agar (for solid culture medium) in a conical flask, shake and dissolve in 100 mL of deionized water, seal with parafilm, and sterilize under high pressure at 121°C for 15 min. Add 100 μg / mL of LB medium under sterile conditions, pour the solid culture medium into a plate and seal it. Store the culture medium at 4°C until use.

[0073] Strain Culture: Streak Salmonella Typhimurium ATCC 14028 frozen at -80°C onto LB agar containing 100 μg / mL streptomycin. After incubation at 37°C, single colonies were selected and placed into LB liquid medium containing 100 μg / mL streptomycin. Culture on a shaker at 160 rpm at 37°C for approximately 16 hours. Activate for two generations before use.

[0074] Preparation of strains: The activated bacterial solution was centrifuged at 6000 rpm for 10 min at 4°C to collect bacterial sludge, which was then resuspended in sterile PBS and washed twice. The OD was adjusted. 600nm =0.5 (bacterial concentration is about 10 8 CFU / mL), and then diluted with sterile PBS to a bacterial concentration of 10 5 CFU / mL was used for subsequent experiments.

[0075] Example 1

[0076] The preparation of Undaria pinnatifida oligosaccharide comprises the following steps:

[0077] (1) The crude polysaccharide powder sample of Undaria pinnatifida was dissolved in deionized water at a concentration of 5 mg / mL and stirred until completely dissolved; 12 mol / L concentrated hydrochloric acid was added to make the final concentration of hydrochloric acid in the solution 0.3 mol / L, and then hydrolyzed at 90°C for 30 min;

[0078] (2) The solution after hydrolysis in step (1) was cooled to room temperature and the pH of the solution was adjusted to 7 with NaOH; the solution was centrifuged at 4000 rpm for 20 min using a 3 kDa ultrafiltration tube, and the solution with Mw < 3 kDa after centrifugation was concentrated by rotary evaporation;

[0079] (3) The concentrated solution in step (2) was dialyzed using a 300Da dialysis bag with running water, using tap water for the first two days and deionized water for the third day; the dialyzed liquid was collected, evaporated and concentrated, and then freeze-dried into a solid powder to obtain Undaria pinnatifida oligosaccharide powder (OUPF powder).

[0080] Example 2

[0081] The only difference from Example 1 is that the acid hydrolysis time in step (1) is controlled to 5 min, 10 min, and 20 min, respectively; other parameters and conditions are the same as those in Example 1.

[0082] Result Analysis

[0083] 1. The molecular weight of undaria pinnatifida oligosaccharides obtained at different acid hydrolysis times is shown in Table 1:

[0084] Table 1. Molecular weight of Undaria pinnatifida oligosaccharides

[0085]

[0086] In the first 20 minutes of hydrolysis, the proportion of OUPF with this structure is relatively small, up to 50%, while after 30 minutes of acid hydrolysis, the yield of OUPF with this structure in the product can reach 80%.

[0087] 2. Composition of Undaria pinnatifida oligosaccharides

[0088] Figure 1 As shown, the molecular weight of the Undaria pinnatifida fucoido-oligosaccharide (OUPF) prepared in Example 1 is 4200 Da, and the contents of neutral sugar, sulfate, and aldehyde are 64.3±1.6%, 22.6±1.3%, and 10.6±0.3%, respectively. The main monosaccharides are mannose (Man), galactose (Gal), and fucose (Fuc), and the mass ratio is 1.2:9.9:4.5.

[0089] 3. The Undaria pinnatifida oligosaccharides obtained in Examples 1 and 2 were used to determine the inhibitory effect on Salmonella

[0090] (1) Salmonella activation

[0091] Preparation of LB medium: Take 1 g of tryptone, 0.5 g of yeast extract, 1 g of sodium chloride, and 1.5 g of agar (for solid culture medium) in a conical flask, shake and dissolve in 100 mL of deionized water, seal with parafilm, and sterilize under high pressure at 121°C for 15 min. Add 100 μg / mL of streptomycin to the culture medium under sterile conditions, pour the solid culture medium into a plate and seal it. Store the culture medium at 4°C until use.

[0092] Strain culture: Streak Salmonella Typhimurium ATCC 14028 frozen at -80°C onto LB agar containing 100 μg / mL streptomycin. After incubation at 37°C, single colonies were selected and placed into LB liquid medium containing 100 μg / mL streptomycin. Culture on a shaker at 160 rpm at 37°C for approximately 16 hours. Activate for two generations before use.

[0093] Preparation of strains: The activated bacterial solution was centrifuged at 6000 rpm for 10 min at 4°C to collect bacterial sludge, which was then resuspended in sterile PBS and washed twice. The OD was adjusted. 600nm =0.5 (bacterial concentration is about 10 8 CFU / mL) were used for subsequent experiments.

[0094] (2) Determination of antibacterial effect

[0095] The acid-hydrolyzed fucoidan oligosaccharides from kelp at different times were dissolved in Salmonella colorimetric medium (HB7007-1, Qingdao Haibo Biotechnology Co., Ltd.) at 1% (mg / mL), and the bacterial solution was gradiently diluted to 10 -7 Then, 100 μL of each gradient was applied to the medium containing fucose and placed in a constant temperature incubator at 37°C for 48 hours. The plates with 30-300 colonies were selected for counting. The results were as follows: Figure 2 As shown in the figure, it can be seen that the number of Salmonella in the culture medium with the addition of fucoidan oligosaccharide hydrolyzed for 30 minutes is the least, and it has a significant inhibitory effect on Salmonella.

[0096] Example 3 Construction of animal model

[0097] The mice were fed freely in an SPF-grade experimental animal room for one week, with alternating light and dark periods of 12 h, an ambient temperature of 23 ± 2 °C, and a relative humidity of 50 ± 5%. The experimental procedures were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals of Dalian Polytechnic University.

[0098] After one week of adaptation, the mice were randomly divided into 4 groups (n=10 / group): blank group (CN group), model group (ST group), Undaria pinnatifida crude polysaccharide group (UPF group), and Undaria pinnatifida oligosaccharide group (OUPF group);

[0099] All mice were fed with normal maintenance feed, and mice in the CN and ST groups drank sterile water freely. Mice in the UPF and OUPF groups drank a solution containing fucoidan (200 mg / kg / d) and a solution containing fucoidan oligosaccharides prepared in Example 1 (200 mg / kg / d), respectively.

[0100] During the experiment, mice in the CN group were given a continuous oral gavage of 0.2 mL of a mixture of gentamicin sulfate and cefradine (23.3 mg / kg / d) once a day for 5 consecutive days. Then, mice in the CN group were given a continuous oral gavage of metronidazole (30 μg / kg / d) for 5 consecutive days. In the following week, mice in the CN group were given a continuous oral gavage of Salmonella typhimurium (10 5 CFU / mL). Successful modeling was considered when symptoms such as sudden weight loss, lethargy, and occult blood in the stool were observed. The experimental period lasted approximately 5 weeks. The weight of each mouse was recorded during the experiment, and feces were collected for high-throughput sequencing analysis approximately one week before the end of the experiment. After the mice were sacrificed, the weight of their liver and spleen and the length of their colon were measured. The contents of the cecum were collected for the determination of short-chain fatty acids. Part of the liver was removed and the number of Salmonella colonies contained was recorded. A section of the colon was immersed in tissue fixative for staining analysis. The remaining organs were quickly frozen in liquid nitrogen and stored in a -80°C freezer until further use.

[0101] Result Analysis

[0102] 1. Effects of Undaria pinnatifida oligosaccharide prepared in Example 1 on basic physiological and biochemical indicators of mice

[0103] The results showed that the body weight of mice in the ST group decreased significantly compared with the control group (CN group), while the body weight of mice in the OUPF group was similar to that of the CN group, with no significant change. The body weight change trends of the UPF and ST groups were more similar, indicating that the intake of OUPF can significantly increase the body weight of mice, while UPF has no significant effect on the change in body weight. Figure 3B). Mice with DSS colitis often show splenomegaly. The ST group had the highest liver and spleen index, while the OUPF and UPF groups had lower indexes. The OUPF group showed a significant decrease, indicating that OUPF has a protective effect on liver and spleen damage in mice ( Figure 3 C and D). The colon length of mice in the OUPF group was significantly restored ( Figure 3 E and F). In summary, OUPF can improve the physical signs and alleviate the inflammatory symptoms of mice.

[0104] 2. Effects of Undaria pinnatifida oligosaccharides on the number of bacterial colonies in mouse organs and tissues

[0105] The number of Salmonella colonies in the liver, spleen, and colon was used to judge the degree of organ damage. The results showed that the number of colonies in the liver, spleen, and colon of the ST and UPF groups was significantly higher than that of the OUPF group ( Figure 4 ), indicating that OUPF can effectively inhibit the invasion of pathogenic bacteria, thereby protecting organs and reducing pathological damage.

[0106] 3. Effects of Undaria pinnatifida oligosaccharides on antioxidant indicators in mice

[0107] Fucoidan oligosaccharides can achieve antioxidant effects by scavenging free radicals and thus resist inflammation. Figure 5 The results showed that the CAT and T-SOD levels, which were reduced in the model group mice, were significantly restored in the OUPF group. The elevated MDA and MPO levels were significantly reduced in the OUPF group. These changes in the levels of different enzymes demonstrate the effective inhibitory effect of OUPF on excessive oxidative stress. These antioxidant indices did not change significantly in the UPF group mice, indicating that OUPF has a stronger antioxidant capacity than UPF.

[0108] 4. Effects of Undaria pinnatifida oligosaccharides on colon tissue pathology in mice

[0109] Observe the pathological colon sections of mice in each group by HE staining ( Figure 6 A) In the Salmonella-infected group, mice showed extensive inflammatory cell infiltration, elongated and damaged crypts, and severe epithelial loss. In contrast, mice in the OUPF group showed significantly reduced inflammatory cell infiltration, crypt depths restored to a morphology similar to that of the normal group, and significantly improved epithelial cell damage. Colon sections from mice in the UPF group were almost indistinguishable from those in the ST group. This demonstrates that OUPF significantly reduces intestinal tissue damage caused by ST, effectively preventing pathogenic bacteria from colonizing the intestine.

[0110] PAS staining observations showed that compared with the CN group, the goblet cells of the ST group were severely damaged and the glycoprotein content was greatly reduced. The degree of colon lesions in the mice that ingested UPF was almost not restored. However, the degree of colon tissue damage in the OUPF group was significantly improved, the goblet cell morphology returned to normal, the damage was reduced, and the glycoprotein density was significantly increased, indicating that OUPF avoided the increase in intestinal permeability caused by ST invading the intestine to ingest glycoproteins.

[0111] According to the colon tissue scoring table, the ST group had the highest score, which means the pathological degree was the highest. The scores of the UPF and OUPF groups decreased, but the OUPF group decreased significantly, which means the pathological degree was significantly alleviated ( Figure 6 B) OUPF better protected colonic tissue from severe damage than UPF, restoring it to a near-healthy state. It also reduced the permeability of the intestinal mucus layer, preventing pathogen colonization. Hematoxylin and eosin (HE) and post-transcriptional succinate (PAS)-stained sections showed that polysaccharides from Lachnum sp. reduced excessive inflammation in mice with colitis and restored intestinal barrier integrity, demonstrating similar effects to OUPF of the present invention.

[0112] 5. Effects of Undaria pinnatifida oligosaccharides on inflammatory factor levels in mice

[0113] In the ST group of mice, the levels of lipopolysaccharide (LPS) and pro-inflammatory factors (IL-1β and TNF-α) increased significantly, while the levels of anti-inflammatory factors decreased significantly, indicating that inflammation occurred in the body. OUPF and UPF can regulate their levels, and OUPF significantly inhibited the degree of inflammation compared to UPF ( Figure 7 ), UPF has little to no significant effect on inflammatory responses. When Salmonella invades the host, it binds to intestinal epithelial cells and releases toxins, triggering an immune response and the secretion of various inflammatory cytokines and mediators. LPS is an endotoxin derived from Gram-negative bacteria. Excessive LPS activation of macrophages can cause fatal endotoxin shock in mice, triggering an inflammatory response.

[0114] 6. Regulatory effect of Undaria pinnatifida oligosaccharide on intestinal flora of mice

[0115] Figure 8 A and B are box plots of ACE and Chao1 index. The OUPF group is similar to the CN group, and the UPF group is similar to the ST group. Salmonella invasion will reduce the richness of species, while OUPF can effectively maintain the richness of the flora. The Shannon index and Simpson index represent the structure and species diversity of the intestinal flora ( Figure 8 C and D), OUPF intake restored the reduction in microbial diversity caused by Salmonella invasion. Changes in the structure, richness, and diversity of the intestinal microbiota can reflect changes in the overall microecological environment.

[0116] Figure 9 A is the principal coordinate analysis PCoA diagram based on the Bray-Curtis differences between samples. It can be seen from the figure that the samples of the ST group and the UPF group are relatively clustered, indicating that the colony structures of the two groups are relatively similar. Figure 9 B is a bar graph of the abundance of different bacterial colonies at the phylum level. The largest phyla in the intestine are Bacteroidetes and Firmicutes, which are the two main bacteria that make up the intestine. Figure 9 Figure C shows the changes in the abundance of these two bacteria among different groups, as well as the changes in their ratio (B / F). Compared with the control group, the abundance of Bacteroidetes increased in both the ST and UPF groups, while the abundance of Firmicutes decreased. The changes in the microbiome of ST mice were more significant, and OUPF significantly adjusted it to a level close to that of healthy mice.

[0117] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. The application of Undaria pinnatifida oligosaccharide in the preparation of a Salmonella typhimurium antibacterial agent, characterized in that: The molecular weight of the Undaria pinnatifida fucoidan is 4000-4500 Da, and the main monosaccharides are Man, Gal and Fuc.

2. The use according to claim 1, characterized in that The molecular weight of the undaria pinnatifida fucoidan is 4200Da, and the contents of neutral sugar, sulfate and aldehyde are 64.3±1.6%, 22.6±1.3% and 10.6±0.3% respectively. The main monosaccharides are Man, Gal and Fuc, and the mass ratio is 1.2:9.9:4.

5.

3. The use according to claim 1, characterized in that The preparation of the Undaria pinnatifida oligosaccharide comprises the following steps: (1) Dissolve the crude polysaccharide sample of Undaria pinnatifida in deionized water and perform acid hydrolysis; (2) The solution after acid hydrolysis in step (1) was cooled to room temperature and the pH of the solution was adjusted to 7; centrifuged using a 3 kDa ultrafiltration tube, and the solution with Mw < 3 kDa after centrifugation was concentrated by rotary evaporation; (3) dialyzing the concentrated solution in step (2), collecting the dialyzed liquid, evaporating and concentrating it, and then freeze-drying it into a solid powder to obtain the Undaria pinnatifida oligosaccharide OUPF.

4. The use according to claim 3, characterized in that The acid hydrolysis in step (1) is specifically to add concentrated hydrochloric acid to the solution containing the crude polysaccharide of Undaria pinnatifida to make the final concentration of hydrochloric acid in the solution reach 0.3-0.5 mol / L, and then acid hydrolyze at 90-95° C. for 10-30 min.

5. The use according to claim 3, characterized in that (3) The dialysis bag used in the dialysis is a 300Da dialysis bag.

6. Application of Undaria pinnatifida fucoidan in the preparation of a product for preventing / alleviating intestinal inflammation induced by Salmonella typhimurium, characterized in that: The molecular weight of the Undaria pinnatifida fucoidan is 4000-4500 Da, and the main monosaccharides are Man, Gal and Fuc.

7. The use according to claim 6, characterized in that The product further contains a carrier and / or excipients.

8. The use according to claim 7, characterized in that The carrier includes one or more of fillers, binders, wetting agents, disintegrants, lubricants, and adhesives commonly used in medicine.

9. The use according to claim 6, characterized in that The products include medicines and health products.

10. The use according to claim 6, characterized in that The products include tablets, powders or liquid preparations.

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