Oral agarotriose preparation with function of relieving colitis

Abstract

The invention discloses an agartriose oral preparation with a function of relieving colitis, and belongs to the technical field of food biology. The invention provides a preparation method of agarotriose. Main components which mainly exert anti-inflammatory activity are analyzed through clinical evaluation. According to the invention, agarotriose is extracted by adopting an enzymolysis method to treat DSS-induced colitis mice, and the influence of main components on model mice is investigated from the aspects of fecal characters, fecal occult blood, colon pathological structures and the like. Experimental results show that compared with a model group, the agarotriose high-dose group can significantly improve colitis symptoms, relieve inflammatory reaction and promote colonic mucosa repair, so that agarotriose can be used as a good functional food for relieving colitis.

Description

Technical Field

[0001] The invention relates to an oral agarose preparation with the function of alleviating colitis, belonging to the field of food biotechnology. Background Art

[0002] Colitis is a chronic nonspecific inflammatory disease that affects the colon and terminal ileum, and its exact pathogenic mechanism has not yet been fully elucidated. Clinically, it is mainly characterized by recurrent abdominal pain, diarrhea, and mucus, pus and blood in the stool. Due to its protracted course, easy recurrence and possible induction of cancer, the quality of life of patients is significantly affected. Current clinical treatments mostly rely on salicylic acid drugs, glucocorticoids and immunosuppressants to control inflammation. Although they can achieve short-term effects, long-term use can easily cause adverse reactions such as gastric mucosal damage and endocrine disorders, and the risk of recurrence after drug withdrawal is high. Therefore, the development of natural active ingredients or functional food ingredients with significant therapeutic effects and low toxic side effects has become an important research direction to improve the current status of colitis treatment. Summary of the invention

[0003] In view of the above-mentioned deficiencies of the prior art, the present invention provides an oral agarose preparation having the function of relieving colitis, aiming to solve the technical problem in the prior art of lacking natural active ingredients with anti-inflammatory activity that can effectively relieve or assist in improving colitis and have little side effects.

[0004] The first technical solution provided by the present invention is the use of agarotriose in the preparation of a product for relieving colitis.

[0005] In certain embodiments, the product comprises a functional food or a medicine.

[0006] In certain embodiments, the drug further contains a drug carrier and / or a pharmaceutical excipient.

[0007] In certain embodiments, the drug carrier comprises a microcapsule, a microsphere, a nanoparticle and / or a liposome.

[0008] In certain embodiments, the pharmaceutical excipient comprises an excipient and / or an additive.

[0009] In certain embodiments, the excipient comprises a binder, a filler, a disintegrant and / or a lubricant.

[0010] In certain embodiments, the additional agent comprises a solubilizer, a co-solvent, a co-solvent and / or a preservative.

[0011] In certain embodiments, the drug is in the form of powder, granules, capsules, tablets, pills or oral liquid.

[0012] In some embodiments, the application includes at least one of the following effects:

[0013] (1) Alleviate weight loss in individuals with colitis;

[0014] (2) Alleviate colon shortening in individuals with colitis;

[0015] (3) Reduce colon tissue damage in individuals with colitis;

[0016] (4) Increase the number of goblet cells in individuals with colitis and improve their distribution;

[0017] (5) Up-regulate the expression levels of Claudin-1, Occludin, ZO-1 and MUC2 in the colon tissue of individuals with colitis;

[0018] (6) Inhibit the expression of pro-inflammatory factors IL-6, IL-1β and TNF-α in the colon tissue of individuals with colitis, and up-regulate the expression of anti-inflammatory factors IL-4 and IL-10.

[0019] In certain embodiments, in the product, the dose of kappa-3-sulfated galactan is 100 - 400 mg / kg.

[0020] The second technical solution provided by the present invention is a method for efficiently preparing kappa-3-sulfated galactan using α-agarase, comprising the following steps:

[0021] (1) Add agar to deionized water and mix evenly to obtain a pretreated agar solution;

[0022] (2) Add α-agarase to the pretreated agar solution for enzymatic hydrolysis;

[0023] (3) Boil the solution after enzymatic hydrolysis in step (2) to inactivate the enzyme;

[0024] (4) Centrifuge the solution after enzyme inactivation obtained in step (3), remove the precipitate to obtain the supernatant, which is the agar oligosaccharide mixture containing kappa-3-sulfated galactan.

[0025] In certain embodiments, in step (1), the agar substrate concentration in the agar pretreatment is 7.5 - 15% (w / w), preferably, the agar substrate concentration in the agar pretreatment is 12.5% (w / w).

[0026] In certain embodiments, in step (1), the temperature of the agar pretreatment is 40 - 80 °C, stir at 200 - 400 rpm for 5 - 15 min; preferably, the temperature of the agar pretreatment is 60 °C, stir at 250 rpm for 10 min.

[0027] In some embodiments, in step (2), the α-agarase is α-AGA derived from Catenovulum agarivorans, and the nucleotide sequence encoding the α-agarase is as shown in SEQ ID NO.1. The degradation end products of the enzyme acting on agar include neoagarotriose and neoagaropentaose.

[0028] In some embodiments, in step (2), the addition amount of α-agarase is 90 - 150 U / g; preferably, the addition amount of α-agarase is 130 U / g.

[0029] In some embodiments, in step (2), the enzymolysis temperature is 40 - 80 °C, and the enzymolysis time is 8 - 32 h; preferably, the enzymolysis temperature is 35 °C, and the enzymolysis time is 30 h.

[0030] In some embodiments, in step (3), the enzyme is inactivated by boiling for 15 min.

[0031] In some embodiments, step (4) is to centrifuge at 10000 rpm for 10 min.

[0032] In some embodiments, it further includes step (5): after freeze-drying the product of step (4), neoagarotriose is separated and purified through a gel column.

[0033] In some embodiments, in step (5), the separation and purification is first by freeze-drying and then by purification using a Superdex 30 Increase 10 / 300 GL gel chromatography column.

[0034] The third technical solution provided by the present invention is a neoagarotriose prepared by the method described in the second technical solution.

[0035] The fourth technical solution provided by the present invention is the application of neoagarotriose in the preparation of promoters for Claudin-1, Occludin, ZO-1 and / or MUC2 expression.

[0036] The fifth technical solution provided by the present invention is an oral preparation, including neoagarotriose (80%), microcrystalline cellulose (18%), magnesium stearate (2%). After tabletting, it is coated with enteric coating and prepared into an oral preparation through granulation and drying.

[0037] Beneficial effects

[0038] The present invention provides a method for preparing kappa-tripeose, and analyzes the main components that mainly exert anti-inflammatory activity through clinical evaluation. The present invention treats DSS-induced colitis mice by extracting kappa-tripeose using an enzymatic method, and examines the effects of the main components on the model mice from aspects such as fecal traits, fecal occult blood, and colonic pathological structure. The experimental results show that compared with the model group, the high-dose kappa-tripeose group can significantly improve colitis symptoms, reduce inflammatory reactions, and promote the repair of colonic mucosa. Therefore, kappa-tripeose can be used as a functional food for relieving colitis well. Description of the Drawings

[0039] Figure 1 It is a process flow chart for the preparation of kappa-tripeose.

[0040] Figure 2 It is an ion chromatography result diagram of the product obtained in Example 2.

[0041] Figure 3 The liquid chromatography-mass spectrometry result of the product obtained in Example 3 is as shown.

[0042] Figure 4 It is a representative picture of the colon of mice in each group in Example 4.

[0043] Figure 5 It is the alcian blue staining of the colon tissue of mice in each group in Example 4.

[0044] Figure 6 It is the H&E staining diagram of the colon tissue of mice in each group in Example 4.

[0045] Figure 7 It is the immunofluorescence staining of MUC2, Claudin-1, Occludin, and ZO-1 in the colon tissue in Example 4. Detailed Embodiments

[0046] The following are descriptions of the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0047] Testing Method:

[0048] 1. Measuring the activity of Ca-AGA by DNS method:

[0049] The reducing sugar content change was determined by the 3,5-dinitrosalicylic acid (DNS) method to characterize the hydrolysis activity of Ca-AGA. An agarose substrate solution of 0.15% (w / w) was prepared with Tris-HCl buffer (20 mM, pH 8.0). Directly pipette 0.1 mL of the enzyme solution into 0.9 mL of the agarose solution, react at 35 °C for 30 min, then add 1 mL of DNS to terminate the reaction. After boiling water bath for 5 min and quickly cooling, add 2 mL of deionized water for dilution and measure the absorbance value at 540 nm. Use the inactivated enzyme as a control and draw a standard curve with galactose as the standard.

[0050] Enzyme activity definition: Each unit of enzyme activity (U) is defined as the amount of enzyme required to generate 1 μmol of reducing sugar (calculated as galactose) per minute.

[0051] 2. Detection of neoagaro-oligosaccharides or neoagaro-pentasaccharides

[0052] Boil the sample after enzymatic hydrolysis of agar with Ca-AGA for 15 min to inactivate the enzyme, centrifuge, dilute, and filter through a 0.22 μm aqueous filter membrane, then analyze the products using a high-performance anion-exchange chromatography system (HPAEC-PAD) and an ultra-high performance liquid chromatography - electrospray ionization - quadrupole - time-of-flight mass spectrometer (UPLC-Q-TOF-MS). The conditions for HPAEC-PAD are as follows: Dionex CarboPac PA-200 anion exchange column, column temperature 35 °C, mobile phases are 100 mM NaOH, 100 mM NaOH & 500 mM NaAc, and the flow rate is 0.5 mL / min. The conditions for UPLC-Q-TOF-MS are as follows: ACQUITY UPLC BEH Amide chromatographic column, column temperature 40 °C, mobile phase A is 0.1% ammonia water - acetonitrile solution, mobile phase B is water - methanol (2:1, V / V) solution, the flow rate is 0.3 mL / min, electrospray ionization (ESI) source is used, and mass spectrometry analysis is carried out in the negative ion ionization mode.

[0053] 3. Calculation of the yield and production rate of neoagaro-oligosaccharides

[0054] The mass fraction of the agar enzymatic hydrolysis solution on a dry basis is Q, the total mass is M g, the enzyme addition amount is N g, the amount of the sampled enzymatic hydrolysis solution is m g, and the total volume after dilution and volume fixation is V mL. Dilute the sample by n times, filter through the membrane, and then perform ion chromatography detection. The concentration of the neoagaro-oligosaccharides standard is 10 μg / mL, the peak area corresponding to the standard is S1, and the total peak area corresponding to the enzymatic hydrolysis sample is S2.

[0055] The mass of neoagaro-oligosaccharides contained in m g of the sample is Y g.

[0056]

[0057] The total content of neoagaro-oligosaccharides in the agar enzymatic hydrolysis solution is Z g

[0058]

[0059] Yield of neoagaro- trisaccharide in the product (g / g agar):

[0060]

[0061] Recovery rate of neoagaro- trisaccharide in the product (%):

[0062]

[0063] In summary, it can be obtained that:

[0064]

[0065] Raw materials used in the examples:

[0066] LB liquid medium: Yeast powder (Sinopharm Group) 5 g / L, Tryptone (Shanghai Merck) 10 g / L, NaCl (Sinopharm Group) 10 g / L, pH 7.0.

[0067] LB solid medium: Yeast powder (Sinopharm Group) 5 g / L, Tryptone (Shanghai Merck) 10 g / L, NaCl (Sinopharm Group) 10 g / L, pH 7.0, 1.5% (w / v) agar (Sinopharm Group).

[0068] Fermentation liquid medium: Yeast powder (Sinopharm Group) 12 g / L, Tryptone (Shanghai Merck) 24 g / L, Sucrose (Sinopharm Group) 5 g / L, KH2PO4 (Sinopharm Group) 17 mM, K2HPO4 (Sinopharm Group) 72 mM, pH 9.0.

[0069] Plasmid pET-28a(+) and strain E. coli JM109 are both commercial plasmids and strains.

[0070] Expression of recombinant α-agarase in Example 1

[0071] The specific steps are as follows:

[0072] (1) Strain construction:

[0073] The recombinant Ca-aga gene (nucleotide sequence shown in SEQ ID NO.1) was cloned into the plasmid pET-28a(+) using the restriction endonucleases Nco I and Xho I. The obtained expression vector pET-28a(+) / Ca-aga was transformed into E. coli JM109 and spread on a plate containing kanamycin. Transformants were picked for sequencing and double digestion verification. The expression vector with correct sequencing was transformed into the competent cell E. coli BL21(DE3), and the recombinant strain E. coli BL21(DE3) / pET-28a(+)-Ca-aga was obtained by culturing.

[0074] The Ca-aga gene sequence is as follows (SEQ ID NO.1):

[0075]

[0076] (2) Streak the plate:

[0077] Use an inoculation loop to dip into the recombinant strain E. coli BL21(DE3) / pET-28a(+)-Ca-aga bacterial solution and streak it on an LB solid medium containing 100 μg / mL kanamycin, and incubate it in a 37°C constant temperature incubator for 12 h;

[0078] (3) Activation:

[0079] Pick a single colony and transfer it to an LB medium containing 100 μg / mL kanamycin, and shake flask culture it at 37°C and 200 rpm for 8 - 12 h to prepare a seed solution;

[0080] (4) Fermentation:

[0081] Transfer the seed solution obtained in step (3) to a 50 mL fermentation medium containing 100 μg / mL kanamycin at an inoculation amount of 4% (v / v). When the OD 600 value reaches 0.6, add isopropyl-β-D-thiogalactoside (IPTG, final concentration 0.01 mM), and shake flask culture it at 25°C and 200 rpm for 20 h; Collect the fermentation broth, centrifuge it at 4°C and 10000 rpm for 20 min to obtain the cells, resuspend the cells by adding an equal volume of lysis buffer (20 mM Tris-HCl, pH 8.0), and treat it with an ultrasonic cell disruptor for 15 min.

[0082] Centrifuge at low temperature and high speed to take the supernatant, that is, obtain the crude intracellular enzyme solution, and the Ca-AGA enzyme activity measured by the DNS method is 80.79 U / mL.

[0083] Example 2: Method for preparing kistose

[0084] The specific steps are as follows:

[0085] (1) Weigh 25 g of agar, add 200 mL of ionized water, stir it at 60°C and 350 rpm for 10 min, and mix evenly to obtain an agar gel with a concentration of 12.5% (w / w);

[0086] (2) After the temperature of the water bath is reduced to 35°C, add 130 U / g of the Ca-AGA crude enzyme solution, enzymatically hydrolyze it for 30 h, and then boil it for 15 min to inactivate the enzyme;

[0087] (3) Place the enzymatic hydrolysis solution obtained in step (2) in a centrifuge and centrifuge it for 10 min, take the supernatant, and remove the precipitate.

[0088] (4) Take 1 g of the enzymatic hydrolysate obtained in step (3) and make up the volume to 10 mL. After diluting 2000 times, filter it through a 0.22 μm aqueous filter membrane for HPAEC-PAD detection. The results are as Figure 1 shown, and its main product is neoagaro-oligosaccharide.

[0089] (5) The product obtained in step (4) is first freeze-dried and then purified using a Superdex 30 Increase 10 / 300GL gel chromatography column to obtain neoagaro-oligosaccharide with a purity of 98.3%.

[0090] (6) Calculate the yield and recovery rate of neoagaro-oligosaccharide.

[0091] The results show that: the yield of neoagaro-oligosaccharide is 0.3638 g / g agar, and the recovery rate of neoagaro-oligosaccharide is 36.38%.

[0092] Example 3: Isolation and Characterization of Neoagaro-oligosaccharide

[0093] Centrifuge the reaction system obtained in step (3) of Example 2 to take the supernatant, freeze-dry, concentrate and weigh it. After re-dissolving, use a Superdex 30 Increase 10 / 300GL chromatographic column to separate the sample by size exclusion chromatography (SEC). Elute with Milli-Q water at a flow rate of 0.1 mL / min, collect in fractions, and analyze by HPAEC-PAD and UPLC-Q-TOF-MS to verify the purity of neoagaro-oligosaccharide.

[0094] The ion chromatography results of the obtained product are as Figure 2 shown. The purified neoagaro-oligosaccharide has no other miscellaneous sugars, and the purity is high and can reach 98.3%. Patent CN108715841A provides an immobilized enzyme producing 3,6-anhydro-L-galactose for degrading agarose to prepare neoagaro-oligosaccharide. The purity of the neoagaro-oligosaccharide obtained after purification is higher than 96.0%. It can be seen that: the method for preparing neoagaro-oligosaccharide in the present invention is superior to the existing preparation techniques.

[0095] The liquid mass spectrometry results of the product obtained in Example 2 are as Figure 3 shown. The component analysis is as follows: neoagaro-oligosaccharide [M-H] - . The double-charged ion (m / z 242.0021) in the mass spectrometry appears at 1 / 2 of the mass of the single-charged ion (m / z 485.0080) with the same mass unit, and the two peaks are the same substance. The mass spectrometry analysis results further illustrate that high-purity neoagaro-oligosaccharide is obtained after separation and purification.

[0096] Example 4: Application of Neoagaro-oligosaccharide in Alleviating DSS-induced Colitis in Mice

[0097] (1) Use the neoagaro-oligosaccharide prepared in Example 3 at different doses as raw materials for animal experiments:

[0098] Fifty 6-week-old male C57 BL / 6 mice (20 - 22 kg) purchased from Cavens, Changzhou were evenly divided into 5 groups, with 10 mice in each group, namely the control group, the model group, the low-dose fucoidan group (100 mg / kg), the medium-dose fucoidan group (200 mg / kg), and the high-dose fucoidan group (400 mg / kg). All mice were fed adaptively for 7 days. The intervention period started from the 1st day. The blank control group was intragastrically administered PBS phosphate buffer every day; the model group was intragastrically administered PBS phosphate buffer in the first seven days and 3.0% dextran sulfate sodium (DSS) in the next seven days for modeling; the low-dose fucoidan group (100 mg / kg) was intragastrically administered low-dose fucoidan in the first seven days and first intragastrically administered 3.0% dextran sulfate sodium (DSS) and then low-dose fucoidan 1 h later in the next seven days; the medium-dose fucoidan group (200 mg / kg) was intragastrically administered medium-dose fucoidan in the first seven days and first intragastrically administered 3.0% dextran sulfate sodium (DSS) and then medium-dose fucoidan 1 h later in the next seven days; the high-dose fucoidan group (400 mg / kg) was intragastrically administered high-dose fucoidan in the first seven days and first intragastrically administered 3.0% dextran sulfate sodium (DSS) and then high-dose fucoidan 1 h later in the next seven days. The body weights of the mice were weighed every 2 days, and relevant indicators were measured. The mice were sacrificed on the 7th day after modeling (the 14th day of the intervention period), and the distal part of the colon of the mice was taken, fixed with 4% neutral formaldehyde, embedded in paraffin, and then tissue sections were made. A part of the sections was used for HE staining and analyzed under a light microscope.

[0099] (2) Experimental method:

[0100] During the experiment, the body weight of the mice was measured every other day, and the change curve was plotted. The daily body weight change rate was calculated. At the same time, fecal samples were collected for trait evaluation and occult blood detection. After the mice were sacrificed at the end of the experiment, the colon length was measured. The colon tissues were routinely paraffin-embedded. After preparing sections, hematoxylin-eosin (H&E) staining (Jiangsu KeyGen Biotech Co., Ltd.) and Alcian blue staining (Jiangsu KeyGen Biotech Co., Ltd.) were performed. In the hematoxylin-eosin staining method, the hematoxylin stain solution is alkaline, mainly staining the chromatin in the nucleus and ribosomes in the cytoplasm purple-blue. Eosin is an acidic dye, mainly staining the components in the cytoplasm and extracellular matrix red. Alcian blue is a cationic copper phthalocyanine dye. Under acidic conditions (pH 2.5 - 3.0), it specifically binds to sulfated or carboxylated acidic mucopolysaccharides (such as chondroitin sulfate, hyaluronic acid) in mucus through electrostatic interaction, forming a blue precipitate. Goblet cells are stained blue due to their rich acidic mucus components, and the nucleus can be shown in a contrasting color through counterstaining (such as nuclear fast red). The morphology of the colon tissue, infiltration of inflammatory cells, and ulcer conditions were observed by H&E staining, and the mucosal thickness was measured. The number and distribution of goblet cells were observed by Alcian blue staining. Immunofluorescence staining is a localization technique based on the specific binding of antigen-antibody. The expression and distribution of target proteins in tissues or cells are shown by fluorescence labeling. The expression and distribution of tight junction proteins (Claudin-1, Occludin, ZO-1) and MUC2 in colon tissues were detected by immunofluorescence staining (Nanjing Jiancheng Bioengineering Institute), and fluorescence intensity quantitative analysis was performed. Enzyme-linked immunosorbent assay (ELISA) is a highly sensitive immunological detection technique based on the specific binding of antigen-antibody. The concentration of target proteins (such as cytokines) is quantified by the enzyme-catalyzed substrate chromogenic reaction. The levels of IL4, IL-10, IL-6, TNF-α, and IL-1β in mouse intestinal tissues were detected by enzyme-linked immunosorbent assay (ELISA) (Wuhan Cloud-Clone Corp.).

[0101] (3) Experimental results:

[0102] As Figures 4 to 7As shown, the experiment explored the protective effect of A3 on DSS-induced colitis in mice. Three dose groups of 100 mg / kg, 200 mg / kg, and 400 mg / kg were set up. The experimental results fully confirmed the protective effect of A3: in terms of body weight change, the control group showed a stable growth trend, while the body weight of the DSS model group decreased significantly (p<0.001), and each dose of A3 intervention group could effectively relieve this symptom (p<0.05); the evaluation results of the disease activity index (DAI) showed that compared with the normal control group, the DAI score of the DSS model group increased significantly (p<0.001), manifested as abnormal fecal traits, increased defecation frequency, and severe occult blood phenomenon. After intervention with different doses of A3, the DAI score decreased significantly compared with the model group (p<0.05), and showed a clear dose-effect relationship; measurement of colon length found that DSS caused the colon to shorten to 4.93±0.17 cm, while the A3 intervention group, especially the high-dose group (6.90±0.23 cm) and the medium-dose group (6.88±0.21 cm), significantly reversed this pathological change, and the effect of the low-dose group was weaker (5.94±0.23 cm). HE staining results showed that the colon tissue structure of the control group was intact and the glandular arrangement was regular; the DSS model group showed mucosal erosion, ulcers, massive infiltration of inflammatory cells, and destruction of the crypt structure; the degree of colon tissue damage in each dose of A3 intervention group was significantly reduced, and the improvement effect of the high- and medium-dose groups was better than that of the low-dose group. Alcian blue staining analysis showed that the density of goblet cells in the colon mucosa of the DSS group was significantly reduced and unevenly distributed (p<0.001); while each A3 intervention group could effectively increase the number of goblet cells and improve their distribution, showing a dose-dependent effect, indicating that A3 could effectively repair the mucus layer barrier function; immunofluorescence staining revealed that DSS caused the expression of tight junction proteins such as Claudin-1, Occludin, and ZO-1 to weaken or even disappear, while A3 treatment successfully restored the expression and distribution of these proteins; MUC2 staining also showed that A3 could effectively reverse the down-regulation of mucin expression caused by DSS.

[0103] Table 1. Comparison of anti-inflammatory factors in colon tissues of mice in each group (unit: pg / mL protein; data are expressed as mean ± standard error of the mean (SEM) (n = 10))

[0104]

[0105] Table 2. Comparison of pro-inflammatory factors in mice in each group (unit: pg / mL protein; data are expressed as mean ± standard error of the mean (SEM) (n = 10))

[0106]

[0107] As shown in Tables 1 and 2, all doses of A3 could significantly inhibit the release of pro-inflammatory factors IL-6, IL-1β and TNF-α, while up-regulating the expression of anti-inflammatory factors IL-4 and IL-10. The effects were most significant in the medium and high dose groups. These results comprehensively indicated that A3 had multi-target protective effects on DSS-induced colitis by improving clinical symptoms, repairing intestinal barrier function and regulating the balance of inflammatory factors, and showed an obvious dose-dependence.

[0108] Example 5: An oral preparation

[0109] An enteric coating, the enteric coating comprising 200 mg of kappa-carrageenan, 37.5 mg of microcrystalline cellulose, 7.5 mg of hypromellose, and 5 mg of magnesium stearate. The freeze-dried kappa-carrageenan was crushed and passed through an 80-100 mesh sieve, and the moisture was controlled ≤5% to avoid sticking during tabletting. The AOS and microcrystalline cellulose were mixed in equal increments and stirred in a V-type mixer for 15-20 minutes. An adhesive solution (HPMC dissolved in pure water) was added for wet granulation, and the granules were passed through a 16-mesh sieve. Fluidized bed drying was carried out at 50-60 °C until the moisture ≤3%, and after sizing, magnesium stearate was added and mixed for 5 minutes. Tabletting was carried out, and finally coating was performed to obtain the kappa-carrageenan oral preparation. The prepared oral preparation had the efficacy of relieving colitis in mice.

[0110] Example 6

[0111] When the substrate concentration was changed to 10%, and other conditions or parameters were the same as in Example 2, the yield of kappa-carrageenan was 27.76%; when the substrate concentration was changed to 15%, and other conditions or parameters were the same as in Example 2, the yield of kappa-carrageenan was 35.59%.

[0112] Example 7

[0113] When the enzymatic hydrolysis time was changed to 24 h, and other conditions or parameters were the same as in Example 2, the yield of kappa-carrageenan was 31.80%; when the enzymatic hydrolysis time was changed to 32 h, and other conditions or parameters were the same as in Example 2, the yield of kappa-carrageenan was 33.02%.

[0114] Example 8

[0115] When the enzyme addition amount was changed to 120 U / g, and other conditions or parameters were the same as in Example 2, the yield of kappa-carrageenan was 33.86%; when the enzyme addition amount was changed to 140 U / g, and other conditions or parameters were the same as in Example 2, the yield of kappa-carrageenan was 24.77%.

[0116] Example 9

[0117] When the agar pretreatment temperature was changed to 50 °C, and other conditions or parameters were the same as in Example 2, the yield of kappa-carrageenan was 33.86%; when the agar pretreatment temperature was changed to 70 °C, and other conditions or parameters were the same as in Example 2, the yield of kappa-carrageenan was 28.96%.

[0118] Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Use of D - galactose trisaccharide in the preparation of a medicament for relieving colitis.

2. The application according to claim 1, wherein The said use includes at least one of the following effects: (1) Relieving weight loss in individuals with colitis; (2) Relieving colon shortening in individuals with colitis; (3) Alleviating colon tissue damage in individuals with colitis; (4) Increasing the number of goblet cells and improving their distribution in individuals with colitis; (5) Up - regulating the expression levels of Claudin - 1, Occludin, ZO - 1 and MUC2 in the colon tissue of individuals with colitis; (6) Inhibiting the expression of pro - inflammatory factors IL - 6, IL - 1β and TNF - α in the colon tissue of individuals with colitis, and up - regulating the expression of anti - inflammatory factors IL - 4 and IL - 10.

3. The application according to claim 1, characterized in that, In the said medicament, the dosage of D - galactose trisaccharide is 100 - 400 mg / kg.

4. A method for preparing neoagaro-oligosaccharide using α-agarase, characterized in that, It includes the following steps: (1) Add agar into water and mix evenly to obtain an agar pretreatment solution; (2) Add α - agarase into the agar pretreatment solution for enzymatic hydrolysis; (3) Boil the solution after enzymatic hydrolysis in step (2) to inactivate the enzyme; (4) Centrifuge the solution after enzyme inactivation obtained in step (3), remove the precipitate to obtain the supernatant, which is an agar oligosaccharide mixture containing D - galactose trisaccharide.

5. The method according to claim 4, characterized in that In step (1), the agar substrate concentration in the agar pretreatment is 7.5 - 15% (w / w); the temperature of the agar pretreatment is 40 - 80°C, and stir at 200 - 400 rpm for 5 - 15 min.

6. The method according to claim 4, characterized in that, In step (2), the said α - agarase is α - AGA derived from Catenovulum agarivorans, and the nucleotide sequence encoding the α - agarase is as shown in SEQ ID NO.1; The addition amount of α - agarase is 90 - 150 U / g, the enzymatic hydrolysis temperature is 40 - 80°C, and the enzymatic hydrolysis time is 8 - 32 h.

7. The method according to claim 4, characterized in that It further includes step (5): Freeze - dry the solution obtained in step (4) and then separate and purify D - galactose trisaccharide through a gel column.

8. The method according to claim 7, wherein In step (5), the said separation and purification is to first conduct freeze - drying and then use a Superdex 30 Increase 10 / 300GL gel chromatography column for separation and purification.

9. An oral preparation of kistose, characterized in that, The said oral preparation is prepared by mixing 80% of D - galactose trisaccharide prepared by any one of the methods in claims 4 - 8, 18% of microcrystalline cellulose, and 2% of magnesium stearate, tabletting, coating with enteric - coating, and granulating and drying.

10. Use of D - galactose trisaccharide in the preparation of a promoter for the expression of Claudin - 1, Occludin, ZO - 1 and / or MUC2.

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