A brown alginate oligosaccharide, its preparation method and application

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to an alginate oligosaccharide, its preparation method, and its applications. Using brown algae as raw material, the invention first removes impurities from the algae, then extracts it through subcritical acid water, followed by neutralization, decolorization, heavy metal removal, membrane ultrafiltration / nanofiltration purification, and spray drying to obtain high-purity saturated alginate oligosaccharides with a specific degree of polymerization distribution. These alginate oligosaccharides can increase butyrate production by intestinal flora and exert a laxative effect. The alginate oligosaccharide and its preparation method provided by this invention directly use brown algae as raw material, overcoming the drawbacks of traditional methods that first prepare alginate and then hydrolyze it to prepare alginate oligosaccharides, greatly shortening the process steps and improving production efficiency. The alginate oligosaccharides provided by this invention have high purity and broad application potential in the preparation of intestinal health drugs, health products, or dietary supplements.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an alginate oligosaccharide, its preparation method, and its application. Background Technology

[0002] Alginate oligosaccharides are linear oligosaccharides containing 2–25 sugar units, composed of β-D-mannuronate (M) and α-L-guluronate (G) linked by 1→4 glycosidic bonds. Studies have shown that alginate oligosaccharides possess various biological activities, including enhancing immunity, regulating gut microbiota, improving memory, promoting growth, antioxidation, and moisturizing. They have garnered significant attention and are being applied in the development of end products such as food, pharmaceuticals, cosmetics, and agricultural products. However, alginate oligosaccharides are multi-component mixtures, consisting of a group of oligosaccharide monomers with different degrees of polymerization; the degree of polymerization distribution directly affects their biological activity. Therefore, the discovery and research into preparation processes of alginate oligosaccharides with specific degree of polymerization distributions and superior efficacy for specific applications are crucial for accelerating the development of alginate oligosaccharide pharmaceuticals or functional products.

[0003] In addition, traditional methods for preparing high-purity alginate oligosaccharides involve directly using commercially available alginate as raw material and obtaining it through acid degradation, oxidative degradation, or enzymatic degradation. Acid degradation has the advantage of preserving the original structure of alginate (i.e., obtaining saturated alginate oligosaccharides) and allowing for the acquisition of oligosaccharides with different molecular weight fragments or broad polymerization degree distributions by controlling process conditions; however, it results in a high monosaccharide content in the prepared alginate oligosaccharides. Oxidative degradation is highly efficient for preparing alginate oligosaccharides, especially with hydrogen peroxide degradation combined with catalase post-treatment, which allows for relatively green production of alginate oligosaccharides; however, it results in the oxidation and destruction of some reducing end residues in the prepared alginate oligosaccharides, forming diacid structures. Enzymatic degradation can achieve the preparation of alginate oligosaccharides under mild conditions, and the resulting oligosaccharides have double bonds at the non-reducing ends, a non-original structure; however, it has the disadvantage that buffer salt systems are often used during degradation to ensure the activity of alginate lyase, leading to cumbersome desalting processes in the later stages, and the enzymatically hydrolyzed alginate oligosaccharides generally have a low degree of polymerization, mostly around 2-4 sugars.

[0004] In view of this, the present invention provides a high-purity saturated alginate oligosaccharide with a specific degree of polymerization distribution obtained directly from brown algae. This alginate oligosaccharide uses brown algae as raw material, which undergoes impurity removal treatment followed by subcritical acid extraction, neutralization, decolorization, heavy metal removal, membrane ultrafiltration / nanofiltration purification, and spray drying. It exhibits significant advantages in increasing butyrate production by intestinal flora and promoting bowel movements. The alginate oligosaccharide and its preparation method provided by the present invention directly use brown algae as raw material, overcoming the drawbacks of traditional methods that first prepare alginate and then hydrolyze it to prepare alginate oligosaccharides, greatly shortening the process steps and improving production efficiency. The alginate oligosaccharide provided by the present invention has high purity and a specific degree of polymerization distribution, exhibiting superior activity, and has broad application potential in the preparation of intestinal health drugs, health products, or dietary supplements. Summary of the Invention

[0005] The purpose of this invention is to provide an alginate oligosaccharide, its preparation method, and its application. High-purity alginate oligosaccharides with saturated structures are obtained directly from brown algae. These alginate oligosaccharides are used in the preparation of intestinal prebiotics and / or laxative drugs, health products, or dietary supplements.

[0006] To achieve the above-mentioned objectives of the present invention, the present invention adopts the following technical solution:

[0007] An alginate oligosaccharide, comprising ≥90% of the components with the structural characteristics described in formula (I),

[0008]

[0009] In the components of the structural features described in formula (I), m and g are natural numbers, 2≤m+g≤10, m / (m+g)≥65%, where the proportion of component m+g=1 is <1%, the proportion of component m+g=2 is 13~16%, the proportion of component m+g=3 is 22~25%, the proportion of component m+g=4 is 23~26%, the proportion of component m+g=5 is 18~21%, and the proportion of component m+g≥6 is 16~19%.

[0010] This invention also provides a method for preparing and applying the above-mentioned alginate oligosaccharide, comprising the following steps:

[0011] S1: Add brown algae to an aqueous solution containing a purification aid at a material-to-liquid ratio of 1:5 to 20 (m / v), and remove impurities at 60 to 90°C for 1 to 3 hours. Filter and collect the algal residue. Repeat the above purification steps 1 to 3 times with the algal residue, and then wash the algal residue with purified water.

[0012] S2: Add the algal residue after impurity removal in step S1 to an acidic solution with a hydrogen ion concentration of 0.01-0.1 mol / L at a material-to-liquid ratio of 1:5-20 (m / v), extract at 110-140℃ for 2-5 hours, and filter to obtain the filtrate.

[0013] S3: Neutralize the filtrate extracted in step S2 with an alkaline neutralizing agent to a pH of 5.5–6.5;

[0014] S4: Add activated carbon to the solution after neutralization in step S3 at a ratio of 1:50 to 200 (m / v), decolorize at 60 to 80°C for 0.5 to 1.5 hours, and remove activated carbon by circulating filtration.

[0015] S5: The decolorized liquid from step S4 is first subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 2500-5000 Da, and the filtrate is collected. The filtrate is then subjected to nanofiltration using a nanofiltration membrane with a molecular weight cutoff of 300-800 Da, and the filtrate is collected.

[0016] S6: Spray dry or freeze dry the refined liquid from step S5 to obtain alginate oligosaccharide.

[0017] Preferably, in step S1, the aqueous solution of the impurity removal aid is either an acid solution with a hydrogen ion concentration of 0.01–0.5 mol / L or a calcium ion solution with a concentration of 0.01–0.5 mol / L.

[0018] More preferably, the acid solution is at least one of a monobasic acid such as hydrochloric acid, nitric acid, or acetic acid;

[0019] Preferably, the alkaline neutralizing agent in step S3 is at least one of the following alkaline substances containing alkali metals: sodium hydroxide, lithium hydroxide, potassium hydroxide, sodium carbonate, lithium carbonate, potassium carbonate, sodium bicarbonate, lithium bicarbonate, and potassium bicarbonate.

[0020] The present invention also provides the application of the aforementioned alginate oligosaccharide in the preparation of intestinal prebiotics and / or laxative drugs, health products or dietary supplements.

[0021] The application of the alginate oligosaccharide in specifically increasing butyric acid production by intestinal flora.

[0022] The alginate oligosaccharide is used in the preparation of drugs that increase the water content and number of stool particles in patients with constipation, increase the small intestinal propulsion rate in patients with constipation, promote intestinal peristalsis, and improve gastrointestinal transit function.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] (1) The method for preparing alginate oligosaccharides provided by the invention uses brown algae directly as raw material, which breaks through the drawback of the traditional method of first preparing alginate and then hydrolyzing it to prepare alginate oligosaccharides. It greatly shortens the process steps, improves production efficiency, is simple and easy to operate, and has broad application prospects.

[0025] (2) The preparation method of alginate oligosaccharide provided by the present invention not only removes non-target sugar components, water-soluble small organic molecules, inorganic salt components, etc. in the impurity removal process of raw materials, but also effectively removes pigments in brown algae. The algae no longer need to adopt the formaldehyde color fixing process used when extracting alginate, so that the prepared alginate oligosaccharide has no formaldehyde residue problem.

[0026] (3) The preparation method of alginate oligosaccharide provided by the present invention effectively and selectively removes monosaccharide components through membrane treatment in the refining process. The resulting product is alginate oligosaccharide with high purity and special degree of polymerization distribution. Its purity is ≥90% and monosaccharide content is ≤1%, which can be applied to the fields of medicine and food.

[0027] (4) The alginate oligosaccharide obtained by the method provided by the present invention has superior efficacy in terms of intestinal prebiotics and / or intestinal lubricating bioactivity. Attached Figure Description

[0028] Figure 1 The diagram shows the monosaccharide composition analysis of alginate oligosaccharides in Example 1-(1);

[0029] Figure 2 Infrared spectrum of alginate oligosaccharides in Example 1-(1);

[0030] Figure 3 The 1H NMR spectrum of alginate oligosaccharides in Example 1-(1) is shown below.

[0031] Figure 4 Example 1-(1) Degree of polymerization analysis of alginate oligosaccharides;

[0032] Figure 5 This is a comparative analysis diagram of the effects of alginate oligosaccharides in Example 1-(1) and comparative examples on acetic acid production by human intestinal flora;

[0033] Figure 6 The diagram shows a comparative analysis of the effects of alginate oligosaccharides in Example 1-(1) and comparative examples on propionic acid production by human intestinal flora.

[0034] Figure 7 This is a comparative analysis diagram of the effects of alginate oligosaccharides in Example 1-(1) and comparative examples on butyric acid production by human intestinal flora;

[0035] Figure 8 The graph shows a comparative analysis of the number of fecal particles in mice using alginate oligosaccharides from Example 1-(1) and the comparative example.

[0036] Figure 9 Figure 1 shows the comparative analysis of the time of first black stool in mice in Example 1-(1) and the comparative example;

[0037] Figure 10 The graph shows the comparative analysis of the effects of alginate oligosaccharides in Example 1-(1) and comparative examples on the water content of mouse feces.

[0038] Figure 11 The graph shows the comparative analysis of alginate oligosaccharide in Example 1-(1) and the small intestinal propulsion rate in mice compared with those in comparative examples.

[0039] Figure 12 This is a comparative analysis of the gastrointestinal transit rate of alginate oligosaccharides in Examples 1-(1) and comparative examples in mice;

[0040] Figure 13 This is a comparative analysis of the amount of alginate oligosaccharide in Example 1-(1) and the amount of gastric residue in mice compared with the comparative example. Detailed Implementation Plan

[0041] The embodiments of the present invention will be described in detail below with reference to specific examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0042] Example 1: Preparation of alginate oligosaccharides

[0043] (1) Preparation Example 1

[0044] Antarctic seaweed (Durvillaea antarctica (Chamisso) Hariot) was pulverized and passed through a 20-mesh sieve. 1 kg of the seaweed powder was added to 10 L of 0.15 mol / L calcium chloride solution and extracted at 70 °C for 2.5 h. After extraction, the liquid was filtered, and the resulting residue was extracted twice more. After impurity removal, the residue was rinsed with purified water to remove residual calcium chloride. The resulting residue was then added to 15 L of 0.03 mol / L hydrochloric acid solution and extracted at 120 °C for 3 h. After extraction, the liquid was filtered. The filtrate was adjusted to pH 6 with sodium hydroxide solution, and then 100 g of activated carbon was added for decolorization at 70 °C for 1 h. The filtrate was then filtered. The resulting liquid was ultrafiltered using a 5000 Da ultrafiltration membrane, and the filtrate was collected. Purified water was repeatedly added for ultrafiltration until the filtrate showed no sugar content. The filtrates were then combined. The resulting filtrate was treated with a 500 Da nanofiltration membrane, with purified water repeatedly added for nanofiltration until no sugar content was detected in the filtrate. The retentate was then collected. The retentate was directly spray-dried using a spray dryer with an inlet temperature set at 180℃ and an outlet temperature set at 80℃. The alginate oligosaccharide was obtained after spray drying.

[0045] (2) Preparation Example 2

[0046] Antarctic seaweed (Durvillaea antarctica (Chamisso) Hariot) was pulverized and passed through a 20-mesh sieve. 1 kg of the algal powder was added to 10 L of 0.1 mol / L hydrochloric acid solution and extracted at 50 °C for 2.5 h. After extraction, the solution was filtered, and the resulting algal residue was subjected to the same steps three times. After impurity removal, the residue was rinsed with purified water until the pH reached 3–4 to remove most of the residual hydrochloric acid. The resulting residue was again added to 10 L of 0.04 mol / L hydrochloric acid solution and extracted at 130 °C for 2 h. After extraction, the solution was filtered. The filtrate was adjusted to pH 5.5 with sodium hydroxide solution, and then 80 g of activated carbon was added. The solution was decolorized at 65 °C for 0.5 h, and then filtered. The resulting solution was ultrafiltered using a 5000 Da ultrafiltration membrane, and the filtrate was collected. Purified water was repeatedly added for ultrafiltration until the filtrate showed no sugar content. The filtrates were then combined. The resulting filtrate was treated with a 500 Da nanofiltration membrane, with purified water repeatedly added for nanofiltration until no sugar content was detected in the filtrate. The retentate was then collected. The retentate was directly spray-dried using a spray dryer with an inlet temperature set at 180℃ and an outlet temperature set at 80℃. The alginate oligosaccharide was obtained after spray drying.

[0047] (3) Preparation Example 3

[0048] Pulverize kelp (Laminaria japonica) and pass it through a 2-mesh sieve. Take 1 kg of kelp powder and add it to 15 L of 0.4 mol / L calcium chloride solution, extract at 80℃ for 1.5 h. After extraction, filter the solution, and repeat the above steps 3 times with the resulting kelp residue. After impurity removal, rinse the kelp residue with an appropriate amount of purified water to remove residual calcium chloride. Add the resulting kelp residue to 17 L of 0.05 mol / L hydrochloric acid solution again and extract at 125℃ for 4 h. After extraction, filter the solution. Adjust the pH of the filtrate to 5.5 with sodium hydroxide solution, then add 120 g of activated carbon and decolorize at 70℃ for 0.5 h, then filter the solution. The resulting solution is ultrafiltered using a 2500 Da ultrafiltration membrane, and the filtrate is collected. Repeatedly add purified water for ultrafiltration until the filtrate shows no sugar content, then combine the filtrates. The resulting filtrate was treated with a 300 Da nanofiltration membrane, with purified water repeatedly added for nanofiltration until no sugar content was detected in the filtrate. The retentate was then collected. The retentate was directly spray-dried using a spray dryer with an inlet temperature set at 180℃ and an outlet temperature set at 80℃. The alginate oligosaccharide was obtained after spray drying.

[0049] Example 2: Comparative Sample Preparation Example

[0050] (1) Comparative Example 1

[0051] Antarctic seaweed (Durvillaea antarctica (Chamisso) Hariot) was pulverized and passed through a 20-mesh sieve. 1 kg of the algal powder was added to 10 L of 0.15 mol / L calcium chloride solution and extracted at 70 °C for 2.5 h. After extraction, the solution was filtered, and the algal residue was extracted twice more. After impurity removal, the residue was rinsed with purified water to remove residual calcium chloride. The resulting residue was then added to 15 L of 0.3 mol / L sodium carbonate solution and extracted at 90 °C for 3 h. After extraction, the solution was filtered. The filtrate was adjusted to pH 6 with hydrochloric acid solution, and then 100 g of activated carbon was added. The solution was decolorized at 70 °C for 1 h, and then filtered. The resulting solution was ultrafiltered using a 5000 Da ultrafiltration membrane, and the retentate was collected. The retentate was directly spray-dried using a spray dryer with an inlet temperature of 180 °C and an outlet temperature of 80 °C. The sample for Comparative Example 1 was obtained after spray drying.

[0052] (2) Comparative Example 2

[0053] Antarctic seaweed (Durvillaea antarctica (Chamisso) Hariot) was pulverized and passed through a 20-mesh sieve. 1 kg of the algal powder was added to 10 L of 0.15 mol / L calcium chloride solution and extracted at 70 °C for 2.5 h. After extraction, the solution was filtered, and the algal residue was extracted twice more. After impurity removal, the residue was rinsed with purified water to remove residual calcium chloride. The resulting residue was then added to 15 L of 0.1 mol / L hydrochloric acid solution and extracted at 100 °C for 3 h. After extraction, the solution was filtered. The filtrate was adjusted to pH 6 with sodium hydroxide solution, and then 100 g of activated carbon was added. The solution was decolorized at 70 °C for 1 h, and then filtered. The resulting solution was ultrafiltered using a 1000 Da ultrafiltration membrane, and the retentate was collected. The retentate was directly spray-dried using a spray dryer with an inlet temperature of 180 °C and an outlet temperature of 80 °C. The sample for Comparative Example 2 was obtained after spray drying.

[0054] (3) Comparative Example 3

[0055] Antarctic seaweed (Durvillaea antarctica (Chamisso) Hariot) was pulverized and passed through a 20-mesh sieve. 1 kg of the seaweed powder was added to 10 L of 0.15 mol / L calcium chloride solution and extracted at 70 °C for 2.5 h. After extraction, the liquid was filtered, and the resulting residue was extracted twice more. After impurity removal, the residue was rinsed with purified water to remove residual calcium chloride. The resulting residue was then added to 15 L of 0.04 mol / L hydrochloric acid solution and extracted at 120 °C for 3 h. After extraction, the liquid was filtered. The filtrate was adjusted to pH 6 with sodium hydroxide solution, and then 100 g of activated carbon was added for decolorization at 70 °C for 1 h. The filtrate was then filtered. The resulting liquid was ultrafiltered using a 1000 Da ultrafiltration membrane, and the filtrate was collected. Purified water was repeatedly added for ultrafiltration until the filtrate showed no sugar content. The filtrates were then combined. The filtrate was treated with a 150 Da nanofiltration membrane, and purified water was repeatedly added for nanofiltration until no sugar content was detected in the filtrate. The retentate was then collected. The retentate was directly spray-dried using a spray dryer with an inlet temperature set at 180℃ and an outlet temperature set at 80℃. The sample of Comparative Example 3 was obtained after spray drying.

[0056] Example 3: Analysis Methods and Results

[0057] (1) Monosaccharide composition analysis

[0058] After a suitable amount of the test sample was completely hydrolyzed into monosaccharides with trifluoroacetic acid, it was derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP); then liquid chromatography was performed under the following chromatographic conditions: C18 column (4.6×250mm); mobile phase: acetonitrile:0.1mol / L PBS solution (pH=7.0)=17:83 (v / v); flow rate: 0.8mL / min; detector: UV detector (254nm); column temperature: 30℃. Taking the alginate oligosaccharide prepared in Example 1-(1) as an example, the monosaccharide composition was analyzed, and the results are shown in the appendix. Figure 1 As shown. Among them, peak 1 (P1) is guluronic acid (GluA), peak 2 (P2) is mannuronic acid (ManA), and these two peaks are the main monosaccharide components of alginate oligosaccharides; peak 3 (P3) is other monosaccharide components in brown algae. The content of mannuronic acid was calculated according to the relative peak area, P2 / (P1+P2) was 79.07%; the content of alginate oligosaccharides was calculated according to the monosaccharide peak area as (P1+P1) / (P1+P2+P3)=94.03%. Using the same method, the content of alginate oligosaccharides prepared in Example 1-(2) was 95.11%, and the content of alginate oligosaccharides prepared in Example 1-(3) was 92.78%. The above results prove that the method provided by the present invention can directly extract high-purity alginate oligosaccharides from brown algae.

[0059] (2) Structural characterization

[0060] The alginate oligosaccharides prepared in Example 1-(1) were characterized by infrared spectroscopy and proton nuclear magnetic resonance spectroscopy. The structures were consistent with the spectral characteristics of high-purity alginate oligosaccharides, as shown in the attached figure. Figure 2 and attached Figure 3 As shown in the figure. The above results further demonstrate that the method provided by the present invention can directly extract high-purity alginate oligosaccharides from brown algae.

[0061] (3) Polymerization degree composition analysis

[0062] High-performance gel permeation chromatography (HPLC) was used to analyze the degree of polymerization under the following chromatographic conditions: Superdex 30 column, 0.1 mol / L ammonium bicarbonate mobile phase, flow rate of 0.3 mL / min, column temperature of 30℃, and detection time of 110 min. (See attached image) Figure 4 This is an example of degree of polymerization analysis of the alginate oligosaccharides prepared in Example 1-(1). The degree of polymerization of the examples and comparative examples was analyzed using the above method, and the results are shown in the table below. The above results demonstrate that the method provided by this invention can control the degree of polymerization and corresponding content of the obtained alginate oligosaccharides within the expected range.

[0063]

[0064]

[0065]

[0066] Example 3 Activity Evaluation Example

[0067] (1) Prebiotic effect of the test sample on intestinal flora

[0068] VI. Medium preparation: tryptone 3g / L, peptone 3g / L, yeast extract 3g / L, mucin 0.5g / L, bile salt No. 3 0.4g / L, L-cysteine ​​hydrochloride 0.8g / L, heme 0.05g / L, Tween 80 1mL / L, sodium chloride 4.5g / L, potassium chloride 2.5g / L, magnesium chloride 4.5g / L, calcium chloride 0.2g / L, potassium dihydrogen phosphate 0.4g / L, trace elements 2mL / L. The solvent is distilled water, and the pH value is 6.4-6.5. The medium is poured into anaerobic vials and sterilized by purging with nitrogen.

[0069] The final concentrations of the trace elements are as follows: MgSO4·7H2O 3.0 g / L, CaCl2·2H2O 0.1 g / L, MnCl2·4H2O 0.32 g / L, FeSO4·7H2O 0.1 g / L, CoSO4·7H2O 0.18 g / L, ZnSO4·7H2O 0.18 g / L, CuSO4·5H2O 0.01 g / L, and NiCl2·6H2O 0.092 g / L.

[0070] Sodium alginate and alginate oligosaccharide were added to VI medium at a concentration of 4 g / L. The pH of the medium was adjusted to 6.5 before autoclaving.

[0071] Pretreatment of feces: Fresh feces from volunteers were prepared into a 20% (wt / vol) suspension with 0.1M PBS, thoroughly mixed, and then filtered through a 2mm diameter metal sieve to remove large particles, resulting in a fecal suspension solution.

[0072] Inoculation and Culture: 1 mL of fecal suspension was inoculated into 9 mL of VI medium containing comparative examples 2-(1-3) and alginate oligosaccharide as a carbon source. The medium was sealed in Hungate tubes and cultured in an anaerobic chamber at 37°C. After fermentation for 48 h, preliminary enrichment culture was performed.

[0073] Short-chain fatty acid analysis: Fermentation medium broth was collected and centrifuged at 12000 rpm for 10 min to remove bacteria and insoluble substances. The concentration of short-chain fatty acids (SCFAs) in the medium was analyzed using an Agilent 1260 high-performance liquid chromatograph. The sample volume was 20 μL, the column was an Aminex HPX-87H Exclusion Column, the mobile phase was 5 mM H2SO4, the flow rate was 0.6 mL / min, isocratic elution was performed, and the detection was performed with a UV detector at 210 nm.

[0074] Results Analysis

[0075] Promoting the production of short-chain fatty acids such as propionic acid and butyric acid by gut microbiota, especially butyric acid, is an important indicator for evaluating the effectiveness of prebiotics. Figures 5-7 This is a comparative analysis of the effects of alginate oligosaccharides prepared in Example 1-(1) and comparative examples in Examples 2-(1-3) on the production of acetic acid, propionic acid, and butyric acid by human intestinal flora. The alginate oligosaccharides prepared in Example 1 differ in degree of polymerization from those prepared in Examples 2-(1-3). (See attached diagram...) Figures 5-7 As can be seen, compared with the comparative example, the alginate oligosaccharide prepared in Example 1 significantly increased the butyric acid content produced by intestinal flora, increased the propionic acid content to a certain extent, and significantly reduced the acetic acid content. Literature reports that butyric acid in the intestine is synthesized from acetic acid. The above results indicate that the alginate oligosaccharide prepared in Example 1 can promote the conversion of acetic acid to butyric acid in the intestine. In summary, the alginate oligosaccharide provided by this patent has superior efficacy as an intestinal prebiotic within its specified degree of polymerization distribution range.

[0076] (2) The therapeutic effect of the test sample on mice with constipation induced by loperamide hydrochloride model

[0077] Sixty SPF-grade male BALB / c mice, weighing 21±2g, were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. They were randomly divided into six groups: normal control group, model control group, comparative group 1, comparative group 2, alginate oligosaccharide group, and comparative group 3. The weight and food intake of the mice were recorded daily.

[0078] Solvent preparation: Loperamide hydrochloride 10 mg / kg / d was used as the modeling agent, and the gavage dose of comparative examples 1, 2, 3 and alginate oligosaccharide was 166.65 mg / kg / d.

[0079] Model establishment: Throughout the experiment, except for the normal group, mice were administered 0.2 mL of loperamide hydrochloride by gavage daily. After 5 days of gavage, the morphology of the mice's feces was observed, and it was found that the mice had obvious constipation. After administering loperamide hydrochloride by gavage, Comparative Example 1, Comparative Example 2, alginate oligosaccharide and Comparative Example 3 were administered by gavage at 5-hour intervals for 16 days.

[0080] Time of first black feces and number of feces: On day 15, all mice were fasted for 12 hours but allowed free water. They were then administered activated charcoal / gum arabic solution by gavage. The time of first black feces excreted by each mouse within 6 hours after gavage was observed and recorded. The feces of the mice were collected and placed in 5mL centrifuge tubes. The number of feces was also recorded.

[0081] Fecal moisture content determination: The collected mouse feces were weighed and the wet weight was recorded. The feces were placed in an oven for 72 hours and the dry weight was recorded. The fecal moisture content was calculated using the following formula:

[0082] Fecal moisture content (%) = (Fecal wet weight - Fecal dry weight) / Fecal wet weight × 100

[0083] Small intestinal propulsion rate determination: On day 16, all mice were fasted for 12 hours but allowed free access to water. They were then administered activated charcoal / gum arabic solution by gavage. After 30 minutes of precise timing, the mice were sacrificed. The total length of the small intestine from the pylorus to the ileocecal junction, as well as the length propelled by the activated charcoal solution, were measured. The propulsion rate was calculated using the following formula:

[0084] Small intestine propulsion rate (%) = Length of propulsion by activated charcoal / gum arabic solution ÷ Total length of small intestine × 100

[0085] Determination of gastric residue mass and gastrointestinal transit rate: Weigh and record the mass of the whole stomach of the mice. Wash the contents with physiological saline, blot dry on clean gauze, weigh and record the mass of the empty stomach. Calculate the gastric emptying rate and gastric residue mass using the following formulas:

[0086] Gastric residue mass (grams) = Whole stomach mass - Empty stomach mass

[0087] Gastrointestinal transit rate (%) = (mass of activated charcoal / gum arabic - mass of gastric residue) ÷ mass of activated charcoal / gum arabic × 100

[0088] Data processing: All experimental data are expressed as mean ± standard deviation (mean ± SEM), and analyzed by one-way ANOVA. P < 0.05 indicates that the difference between the two groups is statistically significant.

[0089] Results Analysis

[0090] Appendix Figures 8-13 This is a comparative analysis of the laxative effects of alginate oligosaccharides from Examples 1-(1) and comparative examples 2-(1-3) on mice. (See attached figure.) Figures 8-13 It can be seen that, compared with the comparative example, the alginate oligosaccharide prepared in Example 1, overall, can increase the fecal water content and number of fecal particles in constipated mice, increase the small intestinal propulsion rate, promote intestinal peristalsis, and improve gastrointestinal transit function. In summary, the alginate oligosaccharide provided by this patent has excellent laxative effects within its specified degree of polymerization distribution range.

Claims

1. A method for preparing alginate oligosaccharides, characterized in that, Includes the following steps: S1: Add brown algae to an aqueous solution containing a purification aid at a material-to-liquid ratio of 1:5 to 20, and remove impurities at 60 to 90°C for 1 to 3 hours. Filter and collect the algal residue. Repeat the above purification steps 1 to 3 times with the algal residue, and then wash the algal residue with purified water. S2: Add the algal residue after impurity removal in step S1 to an acidic solution with a hydrogen ion concentration of 0.01-0.1 mol / L at a material-to-liquid ratio of 1:5-20, extract at 110-140℃ for 2-5 hours, and filter to obtain the filtrate. S3: Neutralize the filtrate extracted in step S2 with an alkaline neutralizing agent to a pH of 5.5-6.5; S4: Add activated carbon to the liquid after neutralization in step S3 at a ratio of 1:50~200, decolorize at 60~80℃ for 0.5~1.5h, and remove activated carbon by circulating filtration. S5: The decolorized liquid in step S4 is first treated with an ultrafiltration membrane with a molecular weight cutoff of 2500~5000 Da, and the filtrate is collected. The filtrate is then treated with a nanofiltration membrane with a molecular weight cutoff of 300~800 Da, and the filtrate is collected. S6: Spray dry or freeze dry the retentate from step S5 to obtain alginate oligosaccharide; The above steps also include: in step S1, the aqueous solution of the impurity removal aid is a calcium ion solution of 0.01~0.5 mol / L; in step S3, the alkaline neutralizing agent is one or more of sodium hydroxide, lithium hydroxide, potassium hydroxide, sodium carbonate, lithium carbonate, potassium carbonate, sodium bicarbonate, lithium bicarbonate, and potassium bicarbonate.

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