Algin oligosaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to alginate oligosaccharide as well as a preparation method and application thereof. The preparation method comprises the following steps: by taking brown algae as a raw material, firstly performing impurity removal treatment on the brown algae, and then performing subcritical acid water extraction, neutralization, decoloration, heavy metal removal, membrane ultrafiltration / nanofiltration refining, spray drying and the like to obtain the high-purity saturated alginate oligosaccharide with specific polymerization degree distribution. The effect of relaxing bowel is achieved. According to the alginate oligosaccharide and the preparation method thereof provided by the invention, the brown algae is directly adopted as the raw material, so that the defect that the alginate oligosaccharide is prepared firstly and then hydrolyzed in the traditional method is overcome, the process steps are greatly shortened, and the production efficiency is improved. The alginate oligosaccharide provided by the invention is high in purity, and has wide application potential in preparation of intestinal health medicines, health care products or dietary supplements.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to alginate oligosaccharide, a preparation method and an application thereof. Background Art

[0002] Alginate oligosaccharide is a linear oligosaccharide containing 2 to 25 sugar units composed of β-D-mannuronate (M) and α-L-guluronate (G) through 1→4 glycosidic bonds. Studies have shown that alginate oligosaccharides have biological activities such as improving immunity, regulating intestinal flora, improving memory, promoting growth, anti-oxidation, and moisturizing. They have been widely used in the development of end products such as food, medicine, cosmetics, and agricultural products. However, alginate oligosaccharides are multi-component mixtures, which are a mixture of a group of oligosaccharide monomers with different degrees of polymerization; the degree of polymerization distribution of alginate oligosaccharides directly affects their biological activity. Therefore, for specific application scenarios, the discovery of alginate oligosaccharides with a special degree of polymerization distribution and superior efficacy and the study of preparation processes are important ways to accelerate the development of alginate oligosaccharide drugs or functional products.

[0003] In addition, the traditional method for preparing high-purity alginate oligosaccharides is to directly use commercial alginate as raw material and obtain it by acid degradation, oxidative degradation or enzyme degradation. Among them, the advantage of acid degradation is that the original structure of alginate can be retained (i.e., saturated alginate oligosaccharides are obtained), and oligosaccharides with different molecular weight fragments or wide polymerization distribution can be obtained by controlling the process conditions; the disadvantage is that the prepared alginate oligosaccharides have a high content of monosaccharides. Oxidative degradation can be used efficiently for the preparation of alginate oligosaccharides, especially hydrogen peroxide degradation combined with catalase post-treatment, which can relatively greenly manufacture alginate oligosaccharides; the disadvantage is that some reducing end residues in the prepared alginate oligosaccharides are oxidized and destroyed to form a diacid structure. Enzymatic degradation can achieve the preparation of alginate oligosaccharides under mild conditions, and the non-reducing end of the obtained oligosaccharides has a non-original structure of double bonds; the disadvantage is that in order to ensure the activity of alginate lyase during the degradation process, a buffer salt system is often used, resulting in a cumbersome desalting process in the later stage, and the enzymatically hydrolyzed alginate oligosaccharides generally have a low degree of polymerization, mostly about 2 to 4 sugars.

[0004] In view of this, the present invention provides a saturated alginate oligosaccharide with high purity and specific polymerization degree distribution directly prepared from brown algae. The alginate oligosaccharide is obtained by using brown algae as raw material, first removing impurities from the brown algae, and then extracting it through subcritical acid water, as well as neutralization, decolorization, heavy metal removal, membrane ultrafiltration / nanofiltration refining and spray drying. It has significant superiority in increasing the amount of butyrate produced by intestinal flora and exerting a laxative effect. The alginate oligosaccharide and its preparation method provided by the present invention directly use brown algae as raw materials, breaking through the disadvantages of first preparing alginate and then hydrolyzing alginate oligosaccharides in the traditional method, greatly shortening the process steps and improving production efficiency. The alginate oligosaccharide provided by the present invention has high purity and a specific polymerization degree distribution, excellent 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 the present invention is to provide an alginate oligosaccharide, a preparation method and an application thereof. A high-purity alginate oligosaccharide with a saturated structure is directly prepared from brown algae as a raw material, and the alginate oligosaccharide is used in the preparation of intestinal prebiotics and / or laxative drugs, health products or dietary supplements.

[0006] In order to achieve the above-mentioned purpose of the present invention, the present invention adopts the following technical scheme:

[0007] An alginate oligosaccharide, wherein the component having the structural characteristics of formula (I) accounts for ≥90%,

[0008]

[0009] Among the components of the structural characteristics described in formula (I), m and g are natural numbers, 2≤m+g≤10, m / (m+g)≥65%, among which the component m+g=1 accounts for less than 1%, the component m+g=2 accounts for 13-16%, the component m+g=3 accounts for 22-25%, the component m+g=4 accounts for 23-26%, the component m+g=5 accounts for 18-21%, and the component m+g≥6 accounts for 16-19%.

[0010] The present invention also provides a preparation method and application of the above-mentioned alginate oligosaccharide, comprising the following steps:

[0011] S1: putting brown algae into an aqueous solution containing a de-impurity aid at a material-liquid ratio of 1:5-20 (m / v), de-impurizing at 60-90°C for 1-3h, filtering out algae residue, repeating the above de-impurity step 1-3 times, and then washing the algae residue with purified water;

[0012] S2: putting the algae residue after impurity removal in step S1 into an acid solution with a hydrogen ion concentration of 0.01 to 0.1 mol / L at a material-liquid ratio of 1:5 to 20 (m / v), extracting at 110 to 140° C. for 2 to 5 hours, and filtering to obtain the filtrate;

[0013] S3: neutralizing the filtrate extracted in step S2 to a pH of 5.5 to 6.5 using an alkaline neutralizer;

[0014] S4: adding activated carbon into the neutralized liquid in step S3 at a material-liquid ratio of 1:50-200 (m / v), decolorizing at 60-80° C. for 0.5-1.5 h, and circulating filtration to remove the activated carbon;

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

[0016] S6: spray-drying or freeze-drying the liquid purified in step S5 to obtain alginate oligosaccharides.

[0017] Preferably, in step S1, the aqueous solution of the de-doping aid is an acid solution with a hydrogen ion concentration of 0.01 to 0.5 mol / L or a calcium ion solution with a hydrogen ion concentration of 0.01 to 0.5 mol / L.

[0018] Further preferably, the acid solution is at least one of monobasic acids such as hydrochloric acid, nitric acid, and acetic acid;

[0019] Preferably, the alkaline neutralizing agent in step S3 is at least one of alkali metal-containing alkaline substances such as 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 use of the alginate oligosaccharide in the preparation of intestinal prebiotics and / or laxative medicines, health products or dietary supplements.

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

[0022] The alginate oligosaccharide is used in the preparation of a drug for increasing the fecal water content and the number of stool particles of constipated patients, increasing the small intestinal propulsion rate of constipated patients, promoting intestinal peristalsis, and improving the gastrointestinal transport function.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

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

[0025] (2) The method for preparing alginate oligosaccharides provided by the present invention not only removes non-target sugar components, water-soluble organic small molecules, inorganic salt components, etc. in the process of removing impurities from the raw materials, but also effectively removes the pigments in the brown algae. The algae body no longer needs to adopt the formaldehyde fixation process used in extracting alginate, so that the prepared alginate oligosaccharides have no formaldehyde residue problem.

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

[0027] (4) The alginate oligosaccharides obtained by the method provided by the present invention have excellent efficacy in intestinal prebiotics and / or intestinal moistening biological activities. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a monosaccharide component analysis diagram of alginate oligosaccharides in Example 1-(1);

[0029] Figure 2 This is the infrared spectrum of alginate oligosaccharide of Example 1-(1);

[0030] Figure 3 This is the H NMR spectrum of alginate oligosaccharide of Example 1-(1);

[0031] Figure 4 This is a diagram showing the degree of polymerization of alginate oligosaccharides in Example 1-(1);

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

[0033] Figure 6 This is a comparative analysis chart of the effects of alginate oligosaccharides of Example 1-(1) and a comparative example on the production of propionic acid by human intestinal flora;

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

[0035] Figure 8 This is a comparative analysis chart of the number of feces particles of mice in Example 1-(1) alginate oligosaccharide and the comparative example;

[0036] Fig. 9 This is a comparative analysis of the time to first black stool excretion in mice of alginate oligosaccharide of Example 1-(1) and the comparative example;

[0037] Fig.10 This is a comparative analysis chart of the water content of mouse feces of alginate oligosaccharide in Example 1-(1) and the comparative example;

[0038] Fig.11 This is a comparative analysis chart of the propulsion rate of alginate oligosaccharide in Example 1-(1) and the comparative example on the small intestine of mice;

[0039] Fig.12 This is a comparative analysis chart of the gastrointestinal transit rate of mice by alginate oligosaccharide of Example 1-(1) and the comparative example;

[0040] Fig.13 This is a comparative analysis chart of the gastric residue quality of mice in Example 1-(1) alginate oligosaccharide and the comparative example. Specific implementation plan

[0041] Embodiments of the present invention are described in detail below in conjunction with specific examples. However, it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. In the examples, if specific conditions are not specified, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be purchased commercially.

[0042] Example 1 Preparation Example of Alginate Oligosaccharide

[0043] (1) Preparation Example 1

[0044] Take Antarctic sea shrub (Durvillaea antarctica (Chamisso) Hariot) and crush it, and pass it through a 20-mesh sieve. Take 1kg of algae powder and put it into 10L of 0.15mol / L calcium chloride solution, and extract it at 70℃ for 2.5h; after the extraction, filter the liquid, and repeat the above steps twice for the algae residue; after the impurity removal, rinse the algae residue with an appropriate amount of purified water to remove the residual calcium chloride. The algae residue is again put into 15L of 0.03mol / L hydrochloric acid solution and extracted at 120℃ for 3h; after the extraction, filter the liquid. The filtrate is adjusted to pH 6 with sodium hydroxide solution, and then 100g of activated carbon is added, decolorized at 70℃ for 1h, and the liquid is filtered. The above-mentioned liquid is ultrafiltered with a 5000Da ultrafiltration membrane, and the collected liquid is filtered; purified water is added repeatedly for ultrafiltration until the filtrate is tested to have no sugar content, and the filtrate is combined. The obtained filtrate was nanofiltered with a 500Da nanofiltration membrane, and purified water was repeatedly added for nanofiltration until the filtrate was free of sugar content, and the retentate was collected. The obtained retentate was directly spray-dried with a spray dryer, with the inlet temperature set at 180°C and the outlet temperature set at 80°C. Alginate oligosaccharides were obtained after spray drying.

[0045] (2) Preparation Example 2

[0046] Take Antarctic sea shrub (Durvillaea antarctica (Chamisso) Hariot) and crush it, and pass it through a 20-mesh sieve. Take 1kg of algae powder and put it into 10L of 0.1mol / L hydrochloric acid solution, and extract it at 50℃ for 2.5h; after the extraction, filter the liquid, and repeat the above steps 3 times for the algae residue; after the impurity removal, rinse the algae residue with an appropriate amount of purified water to pH 3-4 to remove most of the residual hydrochloric acid. The algae residue is again put into 10L of 0.04mol / L hydrochloric acid solution and extracted at 130℃ for 2h; after the extraction, filter the liquid. The filtrate is adjusted to pH 5.5 with sodium hydroxide solution, and then 80g of activated carbon is added, decolorized at 65℃ for 0.5h, and the liquid is filtered. The above-mentioned liquid is ultrafiltered with a 5000Da ultrafiltration membrane, and the collected liquid is filtered; purified water is added repeatedly for ultrafiltration until the filtrate is tested to have no sugar content, and the filtrate is combined. The obtained filtrate was nanofiltered with a 500Da nanofiltration membrane, and purified water was repeatedly added for nanofiltration until the filtrate was free of sugar content, and the retentate was collected. The obtained retentate was directly spray-dried with a spray dryer, with the inlet temperature set at 180°C and the outlet temperature set at 80°C. Alginate oligosaccharides were obtained after spray drying.

[0047] (3) Preparation Example 3

[0048] Take kelp (Laminaria japonica) and crush it, and pass it through a 2-mesh sieve. Take 1kg of algae powder and put it into 15L of 0.4mol / L calcium chloride solution, and extract it at 80℃ for 1.5h; after the extraction, filter the liquid, and repeat the above steps 3 times for the algae residue; after the impurity removal, rinse the algae residue with an appropriate amount of purified water to remove the residual calcium chloride. The algae residue is again put into 17L of 0.05mol / L hydrochloric acid solution and extracted at 125℃ for 4h; after the extraction, filter the liquid. The filtrate is adjusted to pH 5.5 with sodium hydroxide solution, and then 120g of activated carbon is added, decolorized at 70℃ for 0.5h, and the liquid is filtered. The above-mentioned liquid is ultrafiltered with a 2500Da ultrafiltration membrane, and the collected liquid is filtered; purified water is added repeatedly for ultrafiltration until the filtrate is tested to have no sugar content, and the filtrate is combined. The obtained filtrate was nanofiltered with a 300Da nanofiltration membrane, and purified water was repeatedly added for nanofiltration until the filtrate was free of sugar content, and the retentate was collected. The obtained retentate was directly spray-dried with a spray dryer, with the inlet temperature set at 180°C and the outlet temperature set at 80°C. Alginate oligosaccharides were obtained after spray drying.

[0049] Example 2 Comparative Sample Preparation Example

[0050] (1) Comparative Example 1

[0051] Take Antarctic sea shrub (Durvillaea antarctica (Chamisso) Hariot) and crush it, and pass it through a 20-mesh sieve. Take 1kg of algae powder and put it into 10L of 0.15mol / L calcium chloride solution, and extract it at 70℃ for 2.5h; after the extraction, filter the feed liquid, and repeat the above steps twice for the obtained algae residue; after the impurity removal, rinse the algae residue with an appropriate amount of purified water to remove the residual calcium chloride. The obtained algae residue is again put into 15L of 0.3mol / L sodium carbonate solution and extracted at 90℃ for 3h; after the extraction, filter the feed liquid. The obtained filtrate is adjusted to pH 6 with hydrochloric acid solution, and then 100g of activated carbon is added, decolorized at 70℃ for 1h, and the feed liquid is filtered. The above-mentioned feed liquid is ultrafiltered with a 5000Da ultrafiltration membrane, and the retentate is collected. The obtained retentate is directly spray-dried with a spray dryer, the inlet temperature is set to 180℃, the outlet temperature is set to 80℃, and the sample of comparative example 1 is obtained after spray drying.

[0052] (2) Comparative Example 2

[0053] Take Antarctic sea shrub (Durvillaea antarctica (Chamisso) Hariot) and crush it, and pass it through a 20-mesh sieve. Take 1kg of algae powder and put it into 10L of 0.15mol / L calcium chloride solution, and extract it at 70℃ for 2.5h; after the extraction, filter the liquid, and repeat the above steps twice for the algae residue; after the impurity removal, rinse the algae residue with an appropriate amount of purified water to remove the residual calcium chloride. The algae residue is again put into 15L of 0.1mol / L hydrochloric acid solution and extracted at 100℃ for 3h; after the extraction, filter the liquid. The filtrate is adjusted to pH 6 with sodium hydroxide solution, and then 100g of activated carbon is added, decolorized at 70℃ for 1h, and the liquid is filtered. The above-mentioned liquid is ultrafiltered with a 1000Da ultrafiltration membrane, and the retentate is collected. The retentate is directly spray-dried with a spray dryer, the inlet temperature is set to 180℃, the outlet temperature is set to 80℃, and the sample of comparative example 2 is obtained after spray drying.

[0054] (3) Comparative Example 3

[0055] Take Antarctic sea shrub (Durvillaea antarctica (Chamisso) Hariot) and crush it, and pass it through a 20-mesh sieve. Take 1kg of algae powder and put it into 10L of 0.15mol / L calcium chloride solution, and extract it at 70℃ for 2.5h; after the extraction, filter the liquid, and repeat the above steps twice for the algae residue; after the impurity removal, rinse the algae residue with an appropriate amount of purified water to remove the residual calcium chloride. The algae residue is again put into 15L of 0.04mol / L hydrochloric acid solution and extracted at 120℃ for 3h; after the extraction, filter the liquid. The filtrate is adjusted to pH 6 with sodium hydroxide solution, and then 100g of activated carbon is added, decolorized at 70℃ for 1h, and the liquid is filtered. The above-mentioned liquid is ultrafiltered with a 1000Da ultrafiltration membrane, and the collected liquid is filtered; purified water is added repeatedly for ultrafiltration until the filtrate is detected to have no sugar content, and the filtrate is combined. The obtained filtrate was nanofiltered with a 150Da nanofiltration membrane, and purified water was repeatedly added for nanofiltration until the filtrate was free of sugar content, and the retentate was collected. The obtained retentate was directly spray-dried with a spray dryer, with the inlet temperature set to 180°C and the outlet temperature set to 80°C. After spray drying, the comparative example 3 sample was obtained.

[0056] Example 3 Analysis Method Example and Results

[0057] (1) Monosaccharide composition analysis

[0058] After a proper amount of the test sample was completely hydrolyzed into monosaccharides using trifluoroacetic acid, it was derivatized using 1-phenyl-3-methyl-5-pyrazolone (PMP); and then liquid phase analysis was performed according to the following chromatographic conditions: C18 chromatographic column (4.6×250mm); mobile phase was acetonitrile: 0.1mol / L PBS solution (pH=7.0)=17:83(v / v); flow rate was 0.8mL / min; detector was UV detector (254nm); column temperature was 30℃. Taking the alginate oligosaccharide prepared in Example 1-(1) as an example, the monosaccharide composition analysis was performed, and the results are shown in the attached figure. 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 is calculated according to the relative peak area, P2 / (P1+P2) is 79.07%; the content of alginate oligosaccharides calculated according to the monosaccharide peak area is (P1+P1) / (P1+P2+P3)=94.03%. Using the same method, the content of alginate oligosaccharides prepared in Example 1-(2) is 95.11%, and the content of alginate oligosaccharides prepared in Example 1-(3) is 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 structures of the alginate oligosaccharides prepared in Example 1-(1) were characterized by infrared spectroscopy and nuclear magnetic resonance spectroscopy. The results were consistent with the spectral characteristics of high-purity alginate oligosaccharides. Figure 2 and attached Figure 3 The above results further prove that the method provided by the present invention can directly extract high-purity alginate oligosaccharides from brown algae.

[0061] (3) Analysis of degree of polymerization composition

[0062] The polymerization degree was analyzed by high performance gel chromatography under the following chromatographic conditions: superdex30 column, 0.1 mol / L ammonium bicarbonate as mobile phase, flow rate of 0.3 mL / min, column temperature of 30°C, and detection time of 110 min. Figure 4 This is an example of the degree of polymerization analysis of the alginate oligosaccharide prepared in Example 1-(1). The degree of polymerization of the examples and comparative examples was analyzed by the above method, and the results are shown in the following table. The above results show that the method provided by the present invention can control the degree of polymerization and the corresponding content of the obtained alginate oligosaccharide within the expected range.

[0063]

[0064]

[0065]

[0066] Example 3 Activity Evaluation Example

[0067] (1) Prebiotic effects of the test products on intestinal flora

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

[0069] The final concentration of the trace elements is 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, respectively. The pH of the medium was adjusted to 6.5 before autoclaving.

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

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

[0073] Analysis of short-chain fatty acids: The fermentation medium liquid was collected and centrifuged at 12000rpm for 10min 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, with a sample volume of 20μL, an Aminex HPX-87H Exclusion Column, a mobile phase of 5mM H2SO4, a flow rate of 0.6mL / min, isocratic elution, and a UV detector at 210nm.

[0074] Results Analysis

[0075] Promoting the production of short-chain fatty acids such as propionic acid and butyric acid by intestinal flora, especially butyric acid, is an important evaluation indicator of the effect of prebiotics. Figures 5 to 7 The following is a comparative analysis of the effects of the alginate oligosaccharide of Example 1-(1) and the comparative examples of Example 2-(1-3) on the production of acetic acid, propionic acid and butyric acid by human intestinal flora. The alginate oligosaccharide prepared in Example 1 and the comparative examples prepared in Example 2-(1-3) have different polymerization degrees. Figures 5 to 7 It can be seen that compared with the comparative example, the alginate oligosaccharide prepared in Example 1 can significantly increase the butyric acid content produced by the intestinal flora, increase the propionic acid content to a certain extent, and significantly reduce the acetic acid content. Literature reports that butyric acid in the intestine is synthesized using acetic acid as a raw material. The above results show that the alginate oligosaccharide prepared in Example 1 can promote the conversion of acetic acid in the intestine into butyric acid. In summary, the alginate oligosaccharide provided by this patent has the superiority of intestinal prebiotic effect within the specified degree of polymerization distribution range.

[0076] (2) Therapeutic effect of the test article on loperamide hydrochloride-induced constipation in mice

[0077] Sixty SPF-grade BALB / c male mice, weighing 21±2 g, were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. and randomly divided into a normal control group, a model control group, a comparative example group 1, a comparative example group 2, an alginate oligosaccharide group, and a comparative example group 3, for a total of 6 groups. The body weight and food intake of the mice were recorded every day.

[0078] Solvent preparation: the modeling drug loperamide hydrochloride was 10 mg / kg / d, and the oral administration doses of comparative example 1, comparative example 2, comparative example 3 and alginate oligosaccharide were all 166.65 mg / kg / d.

[0079] Model establishment: During the entire experiment, except for the normal group, 0.2 mL of loperamide hydrochloride was gavaged every day. After 5 days of gavage, the feces morphology of the mice was observed, and it was found that the mice had obvious constipation. After gavage with loperamide hydrochloride, control group 1, control group 2, alginate oligosaccharide and control group 3 were gavaged at intervals of 5 hours for 16 days.

[0080] Time of first black stool and number of stool particles: On the 15th day, all mice were fasted for 12 h but not water, and then gavaged with activated carbon / gum arabic solution. The time of first black stool for each mouse within 6 h after gavage was observed and recorded. The feces of mice were collected and placed in 5 mL centrifuge tubes, and the number of stool particles was recorded.

[0081] Determination of fecal water content: The collected mouse feces were weighed and the wet weight of the feces was recorded. The feces were placed in an oven for 72 hours, and the dry weight of the feces was recorded. The fecal water content was calculated by the following formula:

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

[0083] Determination of small intestinal propulsion rate: On the 16th day, all mice were fasted for 12 hours but not water, and then gavaged with activated carbon / gum arabic solution. The mice were killed after 30 minutes of accurate timing. The total length of the small intestine from the upper end to the pylorus and the lower end to the ileocecal part, as well as the propulsion length of the activated carbon solution were measured. The small intestinal propulsion rate was calculated using the following formula:

[0084] Small intestine propulsion rate (%) = activated carbon / gum arabic solution propulsion length ÷ total small intestine length × 100

[0085] Gastric residual mass and gastrointestinal transit rate determination: weigh and record the whole stomach mass of the mouse, clean the contents with physiological saline, dry the water on a clean gauze, weigh and record the empty stomach mass, and calculate the gastric emptying rate and gastric residual mass according to the following formula:

[0086] Gastric residual mass (g) = whole stomach mass - empty stomach mass

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

[0088] Experimental data processing: The experimental data of each group were expressed as mean ± SEM, and one-way analysis of variance was performed. P < 0.05 indicated that the difference between the two groups was statistically significant.

[0089] Results Analysis

[0090] Attached Figures 8 to 13 The following is a comparative analysis of the effects of alginate oligosaccharide in Example 1-(1) and comparative examples 2-(1-3) on the intestinal moistening and laxative effect on mice. Figures 8 to 13 It can be seen that compared with the comparative example, the alginate oligosaccharide prepared in Example 1 can increase the fecal water content and the number of stool particles of constipated mice, increase the small intestinal propulsion rate of constipated mice, promote intestinal peristalsis, and improve gastrointestinal transit function. In summary, the alginate oligosaccharide provided by this patent has the superior effect of moisturizing the intestine and relieving constipation within the specified degree of polymerization distribution range.

Claims

1. An alginate oligosaccharide, wherein the component having the structural characteristics of formula (I) accounts for ≥ 90%, Features: Among the components of the structural feature described in formula (I), m and g are natural numbers, 2≤m+g≤10, m / (m+g)≥65%, among which the component with m+g=1 accounts for less than 1%, the component with m+g=2 accounts for 13-16%, the component with m+g=3 accounts for 22-25%, the component with m+g=4 accounts for 23-26%, the component with m+g=5 accounts for 18-21%, and the component with m+g≥6 accounts for 16-19%.

2. A method for preparing alginate oligosaccharides, characterized in that: The following steps are involved: S1: putting brown algae into an aqueous solution containing a de-impurity aid at a material-liquid ratio of 1:5-20 (m / v), de-impurizing at 60-90°C for 1-3h, filtering out algae residue, repeating the above de-impurity step 1-3 times, and then washing the algae residue with purified water; S2: putting the algae residue after impurity removal in step S1 into an acid solution with a hydrogen ion concentration of 0.01 to 0.1 mol / L at a material-liquid ratio of 1:5 to 20 (m / v), extracting at 110 to 140° C. for 2 to 5 hours, and filtering to obtain the filtrate; S3: neutralizing the filtrate extracted in step S2 to a pH of 5.5 to 6.5 using an alkaline neutralizer; S4: adding activated carbon into the neutralized liquid in step S3 at a material-liquid ratio of 1:50-200 (m / v), decolorizing at 60-80° C. for 0.5-1.5 h, and circulating filtration to remove the activated carbon; S5: The decolorized liquid in step S4 is first subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff of 2500 to 5000 Da, and the filtrate is taken. The obtained filtrate is then subjected to nanofiltration treatment using a nanofiltration membrane with a molecular weight cutoff of 300 to 800 Da, and the retentate is taken; S6: spray-drying or freeze-drying the liquid purified in step S5 to obtain alginate oligosaccharides.

3. The method for preparing alginate oligosaccharides according to claim 2, characterized in that: In the step S1, the aqueous solution of the de-doping aid is an acid solution with a hydrogen ion concentration of 0.01 to 0.5 mol / L or a calcium ion solution with a concentration of 0.01 to 0.5 mol / L.

4. The method for preparing alginate oligosaccharides according to claim 3, characterized in that: The acid solution is one or more of hydrochloric acid, nitric acid and acetic acid.

5. The method for preparing alginate oligosaccharide according to claim 2, characterized in that: 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.

6. Use of the alginate oligosaccharide according to claim 1 or the alginate oligosaccharide prepared by the preparation method according to any one of claims 2 to 5 in the preparation of intestinal prebiotics and / or laxative drugs, health products or dietary supplements.

7. Use of the alginate oligosaccharide according to claim 1 or the alginate oligosaccharide prepared by the preparation method according to any one of claims 2 to 5 in specifically increasing the butyrate production of intestinal flora.

8. Use of the alginate oligosaccharide according to claim 1 or the alginate oligosaccharide prepared by the preparation method according to any one of claims 2 to 5 in the preparation of a drug for increasing the fecal water content and the number of stool particles in patients with constipation, increasing the small intestinal propulsion rate of patients with constipation, promoting intestinal peristalsis, and improving gastrointestinal transport function.

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