Preparation method and application of black garlic polysaccharide

Abstract

The invention belongs to the technical field of extraction and preparation of natural products, and particularly discloses a preparation method of black garlic polysaccharide, which comprises the steps of degreasing, water extraction, deproteinization, alcohol precipitation, drying, crushing and the like. The preparation method of the black garlic polysaccharide has the remarkable advantages of simple and convenient process, low cost, safety, high efficiency and the like. Pharmacological experiments prove that the black garlic polysaccharide prepared by the preparation method disclosed by the invention has a remarkable anti-constipation effect, and can be used for preparing anti-constipation healthy products, including medicines, health-care foods and foods.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to a preparation method of black garlic polysaccharide and its application in anti-constipation health products. Background Art

[0002] Constipation refers to a decrease in the frequency of defecation (<3 times / week), accompanied by difficult defecation and dry stools, which is mainly caused by bad living habits, psychosocial factors, intestinal or systemic diseases, and abuse of laxatives. Clinically, the intervention measures for constipation include drugs, massage, diet, etc., but all have various drawbacks. Drug intervention is suitable for severe constipation, but has low compliance, obvious side effects of some drugs, and is prone to recurrence after drug withdrawal. For example, commonly used traditional Chinese medicine tea drinks such as senna leaves damage the intestinal flora and spleen and stomach at the same time, and long-term use of drugs may even lead to intractable constipation. Massage and general diet intervention have a long cycle, unreliable curative effect, and are more difficult to last. Therefore, there is an urgent need to develop new constipation intervention products with good compliance, small side effects, and homology of medicine and food.

[0003] Black garlic is a fermented garlic, which is prepared by fermenting garlic under high temperature and high humidity conditions for a certain period of time (usually 90-120 days). China ranks first in the world in garlic production and the production is increasing year by year. In 2020, the output reached 24.028 million tons. Export and deep processing are the main ways of garlic consumption in China [Zheng Lan, Liang Jie, Zhao Yonglei, et al. Analysis of the components and antioxidant activities of single-clove and multi-clove black garlic before and after fermentation. Food and Fermentation Industries, 2022, 48(16): 95-102]. Fermenting garlic to prepare black garlic can greatly increase the added value of garlic and can be used as an industrial development direction for the huge garlic resources. Research shows that black garlic has a significant anti-constipation effect and has great potential in the treatment of constipation. Moreover, fermented black garlic not only removes the original unpleasant flavor and irritation of garlic, but also has a strong aroma that enhances appetite, and is gradually favored by consumers. However, the dosage of black garlic for anti-constipation is relatively large, with a daily dosage of more than 20 g, which is likely to lead to problems such as poor patient compliance and difficulty in long-term adherence [Lu Xiaoming, Tian Jixin, Li Ningyang, et al. Laxative function of black garlic on constipation model mice. Journal of Chinese Institute of Food Science and Technology, 2019, 19(12): 39-43]. Therefore, isolating and purifying the main active ingredients from black garlic and reducing the dosage while retaining the efficacy is an effective way to solve this problem.

[0004] The main chemical components of black garlic include melanoidins, proteins, polyphenols, sulfur-containing compounds, polysaccharides, etc. However, its active ingredients for efficacy are not clear. Therefore, the discovery, extraction, and preparation of its anti-constipation active ingredients are very difficult problems [Xiao Kefei, Zhang Qi, Wang Wei, et al. Comparison of the Contents of Nutritional Active Ingredients between Fresh Garlic and Black Garlic in Jinxiang. China Food Additives, 2018, (6): 141-144]. In recent years, the inventors have focused on solving this problem. Through repeated experimental research and process optimization, it has been clarified that black garlic polysaccharide is the main anti-constipation active ingredient in black garlic, and its extraction and preparation method has been constructed to confirm its efficacy. The content of the present invention lays a foundation for the research and development of new anti-constipation products and the high-value industrial development of black garlic. Summary of the Invention

[0005] Aiming at the above deficiencies of the prior art, according to the embodiments and test examples of the present invention, it is intended to provide a preparation method of black garlic polysaccharide that is simple in process, low in cost, safe and efficient, and to propose the application of black garlic polysaccharide in the preparation of anti-constipation health products.

[0006] The above object of the present invention is achieved by the following technical solutions.

[0007] According to an embodiment, a preparation method of black garlic polysaccharide provided by the present invention includes the following steps:

[0008] (A) Degreasing

[0009] Black garlic is impregnated with an ethanol aqueous solution overnight, filtered by suction, washed, and dried and pulverized to obtain defatted black garlic powder. The solvent used for degreasing in this step is an ethanol aqueous solution of 60%-95%, preferably an ethanol aqueous solution of 75%-90%, and most preferably an ethanol aqueous solution of 85%. The degreasing solvent in this step can also be selected from common organic solvents such as petroleum ether, ether, and acetone. Since the fat content of black garlic itself is relatively low, considering factors such as the simplicity of experimental operation, organic solvent recovery, production safety, and cost, it is most suitable to choose ethanol as the degreasing solvent. At the same time, by adjusting the concentration of the ethanol aqueous solution, most small-molecule sugars can be removed to achieve the purpose of partial impurity removal. Selecting different degreasing solvents can partially or fully realize the defatted black garlic powder described in this patent, but it does not constitute a substantial change to the present invention.

[0010] (B) Water extraction

[0011] The above-mentioned defatted black garlic powder is extracted with distilled water, filtered by suction, and the filtrate is concentrated to obtain a concentrated solution. In this step, extraction methods that comply with pharmaceutical specifications such as hot water extraction, heat reflux extraction, ultrasonic-assisted extraction, etc., as well as the corresponding extraction temperature, extraction time, solid-liquid ratio, etc. can be selected. For example, in hot water extraction, the solid-liquid ratio can be selected as 1:10 - 1:100, in heat reflux extraction, the extraction time can be selected as 1 - 3 h, and in ultrasonic-assisted extraction, the extraction time can be selected as 0.25 - 0.75 h. In addition, considering the simplicity of operation, high extraction rate, and the impact of too high temperature on the polysaccharide structure, hot water extraction method is the most commonly used. The preferred extraction temperature is 65 - 85 °C, the solid-liquid ratio is 1:30 - 1:70, and the extraction time is 0.5 - 1.5 h. Selecting different extraction and concentration methods does not constitute a substantial change to the present invention.

[0012] (C) Deproteinization

[0013] The above-mentioned concentrated solution is deproteinized by the trichloroacetic acid method, left standing at 4 °C, centrifuged to remove the precipitate, and the supernatant is obtained. In this step, conventional deproteinization methods that comply with pharmaceutical specifications such as the Sevage method, enzyme method, etc. can be selected. However, the Sevage method and enzyme method are more cumbersome in operation. The present invention uses the trichloroacetic acid method for deproteinization, which is simple in operation, safe and efficient. The standing time at 4 °C is 8 - 12 h, preferably 10 h. Selecting different deproteinization methods does not constitute a substantial change to the present invention.

[0014] (D) Ethanol precipitation

[0015] The above-mentioned supernatant is precipitated with an ethanol aqueous solution, left standing overnight at 4 °C, centrifuged to remove the supernatant, and the precipitate is obtained. The concentration of the ethanol aqueous solution in this step is 60% - 95% (V / V), preferably 75% - 85%.

[0016] (E) Drying

[0017] The above-mentioned precipitate is dried and pulverized to obtain black garlic polysaccharide. In this step, it can be washed successively by immersing in acetone, petroleum ether, and absolute ethanol, and the solvent is volatilized in an oven to achieve the purpose of rapid drying. Other drying methods that comply with pharmaceutical specifications such as freeze drying and spray drying can also be used. The pulverization method can use reasonable pulverization instruments such as a ball mill, a universal pulverizer, and an impact pulverizer. Among them, freeze drying and spray drying can achieve drying and pulverization simultaneously, which is simple and efficient.

[0018] The black garlic polysaccharide prepared by the preparation method of the present invention has been confirmed to have a significant anti-constipation effect through animal experiments, including the mouse small intestine ink propulsion experiment and the mouse defecation experiment. Moreover, the dosage is small, achieving good efficacy and reduction. The invention purpose has been achieved. Therefore, the black garlic polysaccharide prepared by the preparation method of the present invention can be used for the development of anti-constipation health products, including drugs, health foods, and foods, and has very good application prospects. Description of the Drawings

[0019] Figure 1 It is the experimental result diagram of the blank group in the ink propulsion experiment of the small intestine of mice in Test Example 1.

[0020] Figure 2 It is the experimental result diagram of the model group in the ink propulsion experiment of the small intestine of mice in Test Example 1.

[0021] Figure 3 It is the experimental result diagram of the positive group in the ink propulsion experiment of the small intestine of mice in Test Example 1.

[0022] Figure 4 It is the experimental result diagram of the low-dose group of black garlic polysaccharide in the ink propulsion experiment of the small intestine of mice in Test Example 1.

[0023] Figure 5 It is the experimental result diagram of the medium-dose group of black garlic polysaccharide in the ink propulsion experiment of the small intestine of mice in Test Example 1.

[0024] Figure 6 It is the experimental result diagram of the high-dose group of black garlic polysaccharide in the ink propulsion experiment of the small intestine of mice in Test Example 1. Specific Embodiments

[0025] The present invention will be further described below in conjunction with specific embodiments. These embodiments should be understood as being only for the purpose of illustrating the present invention and not for limiting the protection scope of the present invention. After reading the content described in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent changes and modifications also fall within the scope defined by the claims of the present invention.

[0026] Embodiment 1 Preparation Method of Black Garlic Polysaccharide

[0027] Take 2 kg of black garlic and process it according to the following steps: (1) Degreasing: Peel off the outer shell, grind it into a paste, impregnate it with 85% ethanol aqueous solution (V / V), let it stand at room temperature for 8 h, filter by suction, wash the filter residue with 85% ethanol aqueous solution twice, place it in an oven at 60 °C and dry it until there is no alcohol smell, and grind it thoroughly to obtain defatted black garlic powder. (2) Water extraction: The obtained defatted black garlic powder is extracted with hot water at 80 °C for 1 h, the solid-liquid ratio is 1:50, filtered by suction, and the filtrate is concentrated under reduced pressure to 1 / 2 of the original volume. (3) Deproteinization: Add an equal volume of 10% trichloroacetic acid aqueous solution to the concentrated solution, mix well, let it stand at 4 °C for 10 h, and centrifuge to take the supernatant. (4) Alcohol precipitation: Add anhydrous ethanol to the supernatant, adjust the concentration of the ethanol aqueous solution to 80%, mix well, let it stand at 4 °C for 12 h, and centrifuge to take the precipitate. (5) Drying and pulverization: The precipitate is successively immersed and washed with acetone, petroleum ether, and anhydrous ethanol, and the organic solvent is dried in an oven at 60 °C to obtain 163.21 g of dry black garlic polysaccharide powder. The polysaccharide content is determined by the phenol-sulfuric acid method (Standard of the General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China, Determination of Crude Polysaccharides in Exported Plant-derived Foods: Phenol-sulfuric Acid Method: SN / T 4260-2015.) to be 58.23%.

[0028] Preparation Method of Black Garlic Polysaccharide in Example 2

[0029] In the water extraction step, it was extracted with hot water at 60°C, and the remaining steps were the same as in Example 1. 129.32 g of dry powder of black garlic polysaccharide was obtained, and the polysaccharide content was determined to be 50.12% by the phenol-sulfuric acid method.

[0030] Preparation Method of Black Garlic Polysaccharide in Example 3

[0031] In the water extraction step, it was extracted with hot water at 100°C, and the remaining steps were the same as in Example 1. 152.92 g of dry powder of black garlic polysaccharide was obtained, and the polysaccharide content was determined to be 53.19% by the phenol-sulfuric acid method.

[0032] Preparation Method of Black Garlic Polysaccharide in Example 4

[0033] In the water extraction step, the solid-liquid ratio was 1:10, and the remaining steps were the same as in Example 1. 134.48 g of dry powder of black garlic polysaccharide was obtained, and the polysaccharide content was determined to be 53.43% by the phenol-sulfuric acid method.

[0034] Preparation Method of Black Garlic Polysaccharide in Example 5

[0035] In the water extraction step, the solid-liquid ratio was 1:70, and the remaining steps were the same as in Example 1. 148.34 g of dry powder of black garlic polysaccharide was obtained, and the polysaccharide content was determined to be 53.21% by the phenol-sulfuric acid method.

[0036] Preparation Method of Black Garlic Polysaccharide in Example 6

[0037] In the water extraction step, the hot water extraction time was 0.5 h, and the remaining steps were the same as in Example 1. 148.55 g of dry powder of black garlic polysaccharide was obtained, and the polysaccharide content was determined to be 52.15% by the phenol-sulfuric acid method.

[0038] Preparation Method of Black Garlic Polysaccharide in Example 7

[0039] In the water extraction step, the hot water extraction time was 1.5 h, and the remaining steps were the same as in Example 1. 156.21 g of dry powder of black garlic polysaccharide was obtained, and the polysaccharide content was determined to be 53.19% by the phenol-sulfuric acid method.

[0040] Preparation Method of Black Garlic Polysaccharide in Example 8

[0041] In the water extraction step, ultrasonic-assisted extraction was used, and it was extracted for 0.5 h at 60°C and 400 W. The remaining steps were the same as in Example 1. 149.56 g of dry powder of black garlic polysaccharide was obtained, and the polysaccharide content was determined to be 50.12% by the phenol-sulfuric acid method.

[0042] Preparation Method of Black Garlic Polysaccharide in Example 9

[0043] The water extraction step was carried out by reflux extraction with boiling water, and the remaining steps were the same as those in Example 1. 150.51 g of dried black garlic polysaccharide powder was obtained, and the polysaccharide content was determined to be 50.68% by the phenol-sulfuric acid method.

[0044] Example 10 Preparation method of black garlic polysaccharide

[0045] For the protein removal step, the static precipitation time was 8 h, and the remaining steps were the same as those in Example 1. 162.41 g of dried black garlic polysaccharide powder was obtained, and the polysaccharide content was determined to be 50.26% by the phenol-sulfuric acid method.

[0046] Example 11 Preparation method of black garlic polysaccharide

[0047] For the protein removal step, the static precipitation time was 12 h, and the remaining steps were the same as those in Example 1. 150.21 g of dried black garlic polysaccharide powder was obtained, and the polysaccharide content was determined to be 52.89% by the phenol-sulfuric acid method.

[0048] Example 12 Preparation method of black garlic polysaccharide

[0049] For the protein removal step, the Sevage method was used. A chloroform-n-butanol (V / V = 4:1) mixed solution was added to the concentrated solution, shaken well, and the precipitate was removed by centrifugation. The above operation was repeated until no precipitate was produced. The remaining steps were the same as those in Example 1. 149.48 g of dried black garlic polysaccharide powder was obtained, and the polysaccharide content was determined to be 51.41% by the phenol-sulfuric acid method.

[0050] Example 13 Preparation method of black garlic polysaccharide

[0051] For the alcohol precipitation step, a 60% ethanol aqueous solution was used, and the remaining steps were the same as those in Example 1. 104.41 g of dried black garlic polysaccharide powder was obtained, and the polysaccharide content was determined to be 52.13% by the phenol-sulfuric acid method.

[0052] Example 14 Preparation method of black garlic polysaccharide

[0053] For the alcohol precipitation step, an 85% ethanol aqueous solution was used, and the remaining steps were the same as those in Example 1. 159.95 g of dried black garlic polysaccharide powder was obtained, and the polysaccharide content was determined to be 51.99% by the phenol-sulfuric acid method.

[0054] Experimental example Confirmation of the anti-constipation effect of black garlic polysaccharide

[0055] To confirm the efficacy of the black garlic polysaccharide of the present invention, animal experiments were carried out for confirmation. The results showed that it could significantly promote the intestinal peristalsis of constipation model mice, shorten the defecation time, and increase the water content of feces, thereby exerting an anti-constipation effect. Moreover, the dosage was small, achieving the purpose of reducing the dosage while retaining the efficacy. The specific experimental content is as follows:

[0056] Experimental example 1 - Small intestine ink propulsion experiment in mice

[0057] 1. Experimental Purpose

[0058] Through this experiment, observe the effect of intragastric administration of black garlic polysaccharide for 1 week on the intestinal peristalsis of mice.

[0059] 2. Reagents, Animals

[0060] Lactulose oral solution (specification 667 mg / mL, Beijing Hanmi Pharmaceutical Co., Ltd., batch number: 22110047), activated carbon powder (Shanghai Macklin Biochemical Co., Ltd., batch number: C14853603), gum arabic powder (Shanghai Macklin Biochemical Co., Ltd., batch number: C15109301), compound diphenoxylate (2.5 mg / tablet, Renhetang Pharmaceutical Chain Co., Ltd., batch number: 210804), normal saline (Beijing Regene Biotechnology Co., Ltd., batch number: 230327042), 4% paraformaldehyde (Shanghai Bogen Biotechnology Co., Ltd., batch number: HP184401).

[0061] 60 SPF-grade C57 male mice, weighing 18 - 22 g, were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd., license number: SCXK(Zhe)2019 - 00004.

[0062] 3. Test Articles

[0063] Black garlic polysaccharide: Prepared according to the method of Example 1.

[0064] 4. Solution Preparation

[0065] Lactulose oral solution: The normal adult dosage is 15 mL / d. Take 6 mL of lactulose oral solution and add 14 mL of distilled water to prepare a 200 mg / mL lactulose oral solution.

[0066] Compound diphenoxylate solution: Take 4 tablets of compound diphenoxylate tablets, grind them into fine powder, add 20 mL of distilled water to prepare a 0.5 mg / mL compound diphenoxylate solution.

[0067] Low, medium, and high dose groups of black garlic polysaccharide: Weigh 0.5 g, 1 g, and 2 g of black garlic polysaccharide powder respectively, add 20 mL of distilled water to each to prepare 25 mg / mL, 50 mg / mL, and 100 mg / mL black garlic polysaccharide solutions.

[0068] Ink: Weigh 10 g of gum arabic, add 80 mL of distilled water and boil until the solution becomes transparent. Add 5 g of accurately weighed activated carbon powder and boil 3 times, cool for 15 min in the middle, make up the volume to 100 mL, and store at 4℃ for later use.

[0069] Drug-containing ink: Take an appropriate amount of the test drug, add ink, and prepare drug-containing ink with the same dose as the above test drug.

[0070] 5. Animal grouping and drug administration method

[0071] The mice were adaptively fed for 1 week (temperature 25±2°C, light 12 h / d, humidity 40%-45%), fed with standard feed, and given free access to water. One day before drug administration, they were weighed in turn and randomly divided into 6 groups of 10 each, namely the blank group, the model group, the positive group, and the low, medium, and high-dose groups of black garlic polysaccharide. The drug administration doses for each group are shown in Table 1.

[0072] Table 1 Grouping and drug administration method of experimental animals

[0073]

[0074] 6. Experimental method

[0075] For preventive drug administration for 7 days, after drug administration on the 7th day, the mice were fasted and water-deprived for 16 h, and then intragastrically administered with compound diphenoxylate solution to establish a model. 30 min later, the corresponding drug-containing ink was intragastrically administered, and the dosage was 0.1 mL / 10 g for all. After 25 min of therapeutic drug administration, the mice were sacrificed by cervical dislocation. The whole small intestine from the pylorus to the cecum was dissected, cut off, taken out, laid flat, and gently pulled to a straight state to measure the total length of the small intestine and the length from the pylorus to the ink front, and the intestinal ink propulsion rate was calculated.

[0076] Calculation formula: Intestinal ink propulsion rate (%) = total length of ink (cm) / total length of small intestine (cm) × 100%

[0077] 7. Experimental results

[0078] Table 2 Effect of black garlic polysaccharide on the body weight of mice

[0079]

[0080] Note: Compared with the blank control group, * P<0.05, ** P<0.01, *** P<0.001

[0081] Table 3 Effect of black garlic polysaccharide on the intestinal ink propulsion of mice

[0082]

[0083] Note: Compared with the blank control group, * P<0.05, ** P<0.01, *** P<0.001; compared with the model group # P<0.05, ## P<0.01, ### P<0.001

[0084] The experimental results show that: From Figure 1-6As can be seen from Table 2, compared with the blank group, there was no significant difference in the body weight of each group, indicating that black garlic polysaccharide had no effect on the body weight of normal mice. During the experiment, each group of mice had normal diet and water intake, were in good condition, and had no adverse reactions such as diarrhea, providing a prerequisite guarantee for the subsequent experiment.

[0085] From Figure 1-6 As can be seen from Table 3, compared with the blank group, the ink propulsion rate of the model group decreased extremely significantly, indicating that the constipation model of mice in this experiment was successfully established. Compared with the model group, the ink propulsion rates of mice in each dose group of black garlic polysaccharide increased significantly, with significant differences, indicating that black garlic polysaccharide had a promoting effect on the small intestine motility of constipation model mice and showed a dose-dependent relationship.

[0086] Test Example 2 - Mouse defecation experiment

[0087] 1. Experimental purpose

[0088] Through this experiment, the effects of intragastric administration of black garlic polysaccharide for 1 week on the defecation and fecal water content of mice were observed.

[0089] 2. Reagents, animals

[0090] Same as Test Example 1.

[0091] 3. Test article

[0092] Black garlic polysaccharide: Prepared according to the method of Example 1.

[0093] 4. Solution preparation

[0094] Same as Test Example 1.

[0095] 5. Animal grouping and administration method

[0096] Same as Test Example 1.

[0097] 6. Experimental method

[0098] Preventive administration for 7 days. After administration on the 7th day, the mice were fasted and watered for 16 h, and then intragastrically administered with compound diphenoxylate solution to establish a model. 30 minutes later, the corresponding drug-containing ink was intragastrically administered, and the dosage was 0.1 mL / 10 g for all. Record the time of the first black stool excretion of each mouse, the number, weight and characteristics (elliptical, hemispherical granular stool, whether in series, whether loose stool) of black stools excreted within 8 h.

[0099] Determination of fecal water content: The EP tube was dried in advance and weighed. The fresh black stools of mice were placed in the dried EP tube, weighed, and then placed in an oven to dry until the weight no longer changed, and the weights of the black stools before and after drying were recorded.

[0100] Calculation formula: Fecal water content (%) = (M1 - M2) / M1 × 100%

[0101] (Note: M1: Mass of black feces before drying, M2: Mass of black feces after drying)

[0102] 7. Experimental results

[0103] Table 4 Effects of black garlic polysaccharide on body weight of mice

[0104]

[0105] Note: Compared with the blank control group, * P < 0.05, ** P < 0.01, *** P < 0.001

[0106] Table 5 Effects of black garlic polysaccharide on defecation of mice

[0107]

[0108] Note: Compared with the blank control group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with the model group # P < 0.05, ## P < 0.01, ### P < 0.001

[0109] Table 6 Results of water content in mouse feces

[0110]

[0111] Note: Compared with the blank control group, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with the model group # P < 0.05, ## P < 0.01, ### P < 0.001

[0112] The experimental results show that: As can be seen from Table 4, compared with the blank group, there is no significant difference in body weight among the groups, indicating that black garlic polysaccharide has no effect on the body weight of normal mice. During the experiment, the mice in each group had normal diet and water intake, were in good condition, and had no adverse reactions such as diarrhea, providing a prerequisite guarantee for the subsequent experiment.

[0113] As can be seen from Table 5, compared with the blank group, the first black feces time of the model group was significantly prolonged, and the number and weight of feces excreted at 10 h were significantly reduced, indicating that the constipation model of mice in this experiment was successfully established; compared with the model group, the first black feces time of the low, medium, and high dose groups of black garlic polysaccharide was significantly prolonged, and the number and weight of feces excreted at 10 h in the medium and high dose groups were significantly increased, indicating that black garlic polysaccharide has a promoting effect on defecation in constipation model mice.

[0114] As can be seen from Table 6, compared with the blank group, the water content of feces in the model group was extremely significantly reduced, and the feces were spherical or hemispherical granular, with a dry and hard surface in series, indicating that the constipation model of mice in this experiment was successfully established; compared with the model group, the water content of feces in the medium- and high-dose groups of black garlic polysaccharide was significantly increased, the feces were oblong, significantly moist and smooth, with a soft texture, and there was no diarrhea phenomenon, indicating that black garlic polysaccharide can increase the water content of feces in constipation model mice and play a role in promoting defecation.

[0115] In summary, the present invention prepared a kind of black garlic polysaccharide, and confirmed that black garlic polysaccharide can significantly promote small intestine peristalsis in constipation model mice, shorten the defecation time, increase the water content of feces, so as to play an anti-constipation role, and has the prospect of further developing anti-constipation products of black garlic polysaccharide.

[0116] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of black garlic polysaccharide, characterized in that, The preparation method is as follows: (A) Degreasing Black garlic is impregnated with an ethanol aqueous solution overnight, filtered by suction, washed with the ethanol aqueous solution, dried and pulverized to obtain degreased black garlic powder; (B) Water extraction The above-mentioned degreased black garlic powder is extracted with distilled water, filtered by suction, and the filtrate is concentrated to obtain a concentrated solution; (C) Protein removal The above-mentioned concentrated solution is deproteinized with an aqueous trichloroacetic acid solution, the precipitate is removed, and the supernatant is obtained; (D) Ethanol precipitation The above-mentioned supernatant is precipitated with an ethanol aqueous solution, the supernatant is removed, and a precipitate is obtained; (E) Drying and pulverizing The above-mentioned precipitate is dried and pulverized to obtain black garlic polysaccharide.

2. The preparation method according to claim 1, characterized in that, In the step (A), the concentration of the ethanol aqueous solution is 60%-95% (V / V).

3. The preparation method according to claim 1, characterized in that, In the step (B), the water extraction temperature is 60-100 °C.

4. The preparation method according to claim 1, characterized in that, In the step (B), the solid-liquid ratio of water extraction is 1:10-1:

100.

5. The preparation method according to claim 1, characterized in that, In the step (B), the water extraction time is 0.25-3 h.

6. The preparation method according to claim 1, characterized in that, In the step (C), the method of protein precipitation is to stand at 4 °C for 8-12 h.

7. The preparation method according to claim 1, characterized in that, In the step (D), the concentration of the ethanol aqueous solution is 60%-95% (V / V).

8. Black garlic polysaccharide prepared by the preparation method according to any one of claims 1-7.

9. Use of the black garlic polysaccharide according to claim 8 in the preparation of a health product for treating constipation.

10. The application according to claim 9, characterized in that, The health product for treating constipation is a drug, a health food or a common food.

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