Preparation method and application of black garlic polysaccharide

Abstract

The invention discloses a preparation method of black garlic polysaccharide, which comprises the steps of extraction, enzymolysis deproteinization, ultrafiltration, concentration, drying, crushing and the like, and'enzymolysis deproteinization + ultrafiltration 'is the core step of the method. Wherein the enzymolysis deproteinization can be selected from papain, neutral protease, acid protease, alkaline protease and the like or a combination of the papain, the neutral protease, the acid protease and the alkaline protease. In the ultrafiltration step, an organic ultrafiltration membrane or an inorganic ceramic membrane or a combination of the organic ultrafiltration membrane and the inorganic ceramic membrane can be selected. The method has the remarkable advantages of simple process flow, low cost, safety, high efficiency, good finished product quality and the like. Pharmacological experiments prove that the black garlic polysaccharide prepared by the 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 relates to a preparation method of black garlic polysaccharide and its application in the preparation of 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 of them have various drawbacks. Drug intervention is suitable for severe constipation, but the compliance is low, some drugs have obvious side effects, and it is easy to relapse after stopping the drug. For example, the commonly used anti-constipation traditional Chinese medicine, such as senna leaf, destroys the intestinal flora, damages the spleen and stomach, and long-term use of the drug 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 (generally 90 - 120 days). Studies have shown that black garlic has a significant anti-constipation effect, but its dosage is large, with a daily dosage of more than 20 g, which easily leads to problems such as poor compliance and difficulty in long-term adherence. Therefore, on the premise of retaining the anti-constipation effect of black garlic, it is necessary to extract the anti-constipation active ingredients from it to achieve the "reduction" of the dosage, which has a great practical need.

[0004] Previously, the inventor has extracted and prepared crude polysaccharide from black garlic through repeated experimental studies. On the basis of retaining the anti-constipation effect, the dosage can be reduced to 1 / 10 - 1 / 20 of the dosage of black garlic, which is an important progress in the in-depth development of black garlic, and a Chinese patent CN202410086068.X has been applied for. In this patent application, the inventor disclosed a preparation process method of black garlic polysaccharide, including steps such as degreasing, deproteinization, and ethanol precipitation. However, due to the large amount of ethanol used in this process, high risk, and the introduction of harmful impurities such as trichloroacetic acid, as well as many process steps and high production cost, there is still much room for improvement. Therefore, on the basis of the previous research, the inventor has innovated through repeated experimental studies and developed a new preparation process with "enzymatic deproteinization + ultrafiltration" as the core. Compared with the previously disclosed process method, it has significant advantages such as safety, simplicity, good quality, and low cost. 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 aims to provide a preparation method of black garlic polysaccharide with simple process, safety, high efficiency, good quality, and low cost.

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

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

[0008] (A) Extraction

[0009] Extract black garlic with a solvent to obtain an extract; in this step, water is preferably used as the extraction solvent, and an ethanol aqueous solution with a concentration not higher than 30% can also be selected. The extraction method can be selected from extraction methods that conform to pharmaceutical specifications such as leaching extraction, percolation extraction, heat reflux extraction, ultrasonic extraction, and / or grinding and pulping extraction (or an appropriate combination application of these methods), as well as the corresponding suitable extraction temperature, extraction time, solid-liquid ratio, etc. In addition, considering the simplicity of operation, high extraction rate, and the influence of temperature on the polysaccharide structure, hot water extraction and grinding and pulping extraction are preferably used, with a solid-liquid ratio of 1:5 - 1:100, preferably 1:10 - 1:50.

[0010] (B) Enzymatic deproteinization

[0011] Subject the above extract to enzymatic deproteinization to obtain an enzymatically deproteinized solution; this step is one of the core steps of the process route described in the present invention. In this step, the types of enzymes used are selected from papain, neutral protease, acidic protease, alkaline protease, etc., or their combined use. The specific application method can be to add an appropriate amount of protease to the extract obtained in the previous step, mix well, enzymatically hydrolyze for a certain time under the optimal temperature and pH conditions of the enzyme, and then inactivate the enzyme at 100°C for 1 - 30 min. Since papain has enzymatic activity in acidic, neutral, and alkaline environments, it can avoid the influence of pH adjustment on the physiological function of polysaccharides. Therefore, papain is preferably used for enzymatic deproteinization in this step, with a dosage of 500 - 10000 U per 1 g of black garlic raw material, preferably 2000 - 5000 U, and the enzymatic hydrolysis time is 0.5 - 5 h, preferably 1 - 2 h.

[0012] (C) Ultrafiltration

[0013] Filter the above enzymatically deproteinized solution through an ultrafiltration membrane to obtain a retentate; this step is another core step of the process route described in the present invention. In this step, the ultrafiltration membrane material and pore size have a great influence on the filtration efficiency and the quality of the final product. The ultrafiltration membrane material can be selected from organic ultrafiltration membranes or inorganic ceramic membranes, or a combined application of both. Inorganic ceramic membranes are preferably used, and their pore sizes are selected from 5 - 50 KD, preferably 10 - 25 KD pore sizes, and most preferably 15 KD pore sizes.

[0014] (D) Concentration, drying, and pulverization

[0015] Concentrate, dry, and pulverize the above-mentioned retentate to obtain black garlic polysaccharide. In this step, conventional vacuum concentration can be used. The drying method can be oven drying, or other drying methods that comply with pharmaceutical specifications such as freeze drying and spray drying. The pulverization method can be carried out using pulverization equipment such as a ball mill, a universal pulverizer, or an impact pulverizer. Among them, freeze drying and spray drying can achieve drying and pulverization simultaneously, which is relatively simple and efficient.

[0016] Compared with the preparation process disclosed in Chinese Patent CN202410086068.X, the advantages of the method of the present invention are that the process steps are simple, the ceramic membrane can be repeatedly used for a long time, the production throughput is large, the production efficiency is high, the cost is low, less or no ethanol solvent is used in the production process, no impurities such as trichloroacetic acid are introduced, the quality of the finished product is good, and the safety is also good. The black garlic polysaccharide prepared by the method of the present invention has been confirmed by animal experiments to have a significant anti-constipation effect and can be used in health-related products including drugs, health foods, and foods, and has very good application prospects. Description of the Drawings

[0017] Figure 1 It is a schematic diagram of the ceramic ultrafiltration membrane equipment used in the method of the present invention. Detailed Embodiments

[0018] The present invention will be further described below in conjunction with the drawings and specific embodiments. These embodiments should be understood as being only for illustrative purposes of the present invention and not for limiting the protection scope of the present invention. After reading the content recorded 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.

[0019] Example 1

[0020] Weigh 3 kg of black garlic and process it according to the following steps: (1) Extraction: Pulverize the black garlic and extract it by hydrothermal reflux with 10 times the amount of water, 1 hour each time, for a total of 2 extractions. Combine the extraction liquids to obtain the total extraction liquid; (2) Enzymatic deproteinization: Add papain to the water extract, with a dosage of 2500 U per 1 g of black garlic raw material, enzymatically hydrolyze at 55 °C for 1 h, heat to inactivate the enzyme for 5 min, and filter by suction to obtain the enzymatically deproteinized liquid; (3) Ultrafiltration: Filter the enzymatically hydrolyzed protein liquid through a 15 KD ceramic membrane (the equipment assembly is as Figure 1 shown), wash it with an equal amount of water to obtain the retentate. (4) Concentration, drying, and pulverization: Concentrate, dry, and pulverize the retentate to obtain 157.26 g of black garlic polysaccharide. The polysaccharide content is measured to be 73.84% by the phenol-sulfuric acid method (Standard of the General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China: SN / T 4260-2015 "Determination of Crude Polysaccharides in Export Plant-derived Foods: Phenol-Sulfuric Acid Method").

[0021] Example 2

[0022] The ultrafiltration step uses a 5KD inorganic ceramic membrane, and the rest is the same as in Example 1, obtaining 212.43 g of black garlic polysaccharide with a polysaccharide content of 53.72%.

[0023] Example 3

[0024] The ultrafiltration step uses a 10KD inorganic ceramic membrane, and the rest is the same as in Example 1, obtaining 186.19 g of black garlic polysaccharide with a polysaccharide content of 61.81%.

[0025] Example 4

[0026] The ultrafiltration step uses a 50KD inorganic ceramic membrane, and the rest is the same as in Example 1, obtaining 93.37 g of black garlic polysaccharide with a polysaccharide content of 78.48%.

[0027] Example 5

[0028] The extraction step uses 50-fold water extraction, and the rest is the same as in Example 1, obtaining 142.11 g of black garlic polysaccharide with a polysaccharide content of 70.33%.

[0029] Example 6

[0030] The extraction step uses 30-fold water grinding and pulping extraction, and the rest is the same as in Example 1, obtaining 162.53 g of black garlic polysaccharide with a polysaccharide content of 72.78%.

[0031] Example 7

[0032] For every 1 g of black garlic raw material, 1000 U of papain is used for enzymatic deproteinization, and the rest is the same as in Example 1, obtaining 192.64 g of black garlic polysaccharide with a polysaccharide content of 62.35%.

[0033] Example 8

[0034] For every 1 g of black garlic raw material, 5000 U of papain is used for enzymatic deproteinization, and the rest is the same as in Example 1, obtaining 136.73 g of black garlic polysaccharide with a polysaccharide content of 75.02%.

[0035] Example 9

[0036] For every 1 g of black garlic raw material, 10000 U of papain is used for enzymatic deproteinization, and the rest is the same as in Example 1, obtaining 132.14 g of black garlic polysaccharide with a polysaccharide content of 75.42%.

[0037] Example 10

[0038] For the enzymatic deproteinization step, neutral protease is selected, and the dosage per 1 g of black garlic raw material is 5000 U. The rest is the same as in Example 1, obtaining 173.61 g of black garlic polysaccharide with a polysaccharide content of 67.20%.

[0039] Test Example - Confirmation of the Anti-constipation Efficacy of Black Garlic Polysaccharide

[0040] To confirm the efficacy of the black garlic polysaccharide prepared by the method of the present invention, animal experiments were conducted for confirmation. The specific experimental content is as follows:

[0041] Test Example 1 - Small Intestine Ink Propulsion Experiment in Mice

[0042] 1 Experimental Purpose

[0043] Through this experiment, the effect of intragastric administration of black garlic polysaccharide for 1 week on the intestinal peristalsis of mice was observed.

[0044] 2 Test Drugs and Animals

[0045] Lactulose Oral Liquid (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).

[0046] SPF - level C57 male mice, weighing 18 - 22 g, were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd., license number: SCXK(Zhe)2019 - 00004.

[0047] 3 Test Articles

[0048] Black garlic polysaccharide: Prepared according to the method of Example 1.

[0049] 4 Experimental Method

[0050] The experiment was mainly carried out according to the method of the literature (Lu Xiaoming, Tian Jixin, Li Ningyang, etc. Laxative function of black garlic on constipation model mice. Journal of Chinese Institute of Food Science and Technology, 2019, 19(12): 39 - 43).

[0051] 4.1 Preparation of Test Drugs

[0052] Lactulose Oral Liquid: Take an appropriate amount of lactulose oral liquid and add distilled water to prepare a lactulose solution with a concentration of 200 mg / mL.

[0053] Compound Diphenoxylate Solution: Take an appropriate amount of compound diphenoxylate tablets, grind them finely, and add distilled water to prepare a compound diphenoxylate solution with a concentration of 0.5 mg / mL.

[0054] Low, medium, and high dose groups of black garlic polysaccharide: Weigh an appropriate amount of black garlic polysaccharide powder and add distilled water to prepare black garlic polysaccharide solutions with concentrations of 25, 50, and 100 mg / mL respectively.

[0055] Ink: Weigh 10 g of gum arabic, add 80 mL of distilled water, boil until the solution becomes transparent, add 5 g of activated carbon powder and boil three times, cool for 15 min in the middle, make up the volume to 100 mL, and store at 4 °C for later use.

[0056] Drug-containing ink: Take an appropriate amount of the test drug and prepare a drug-containing ink with the same dose as the above test drug by adding ink.

[0057] 4.2 Animal grouping

[0058] Sixty mice were adaptively fed for 1 week (temperature 25 ± 2 °C, light 12 h / d, humidity 40%-45%), fed with standard feed, and allowed free access to water. Weigh them successively one day before dosing, and randomly divide them into 6 groups according to body weight, with 10 mice in each group, namely the blank group, the model group, the positive group, and the low, medium, and high dose groups of black garlic polysaccharide.

[0059] 4.3 Drug administration and index determination

[0060] Each group was given the corresponding test drug by gavage at 0.1 mL / 10 g, specifically: the mice in the blank group and the model group were given distilled water, the mice in the positive group were given lactulose oral liquid, and the mice in the low, medium, and high dose groups of black garlic polysaccharide were given black garlic polysaccharide solution. Administer the drug once a day for 1 week continuously, observe the general condition, and record the change in the body weight of the mice. After the last dose, fast the mice for 16 h without water restriction. Except for the mice in the blank group being given distilled water by gavage, the mice in other groups were given compound diphenoxylate solution by gavage to establish a model. 30 min after modeling, the mice in the blank group and the model group were given ink by gavage, and the mice in other groups were given the corresponding drug-containing ink by gavage. Sacrifice the mice by cervical dislocation 25 min later, dissect the small intestine from the pylorus to the cecum of the mice, gently stretch it flat into a straight line, measure the total length of the small intestine and the advancing length of the ink, and calculate the ink propulsion rate of the small intestine.

[0061] Calculation formula: Ink propulsion rate of small intestine (%) = Total length of ink (cm) / Total length of small intestine (cm) × 100%

[0062] 5 Experimental results

[0063] Table 1. Effect of black garlic polysaccharide on the body weight of mice (n = 10,

[0064]

[0065] Note: Compared with the blank control group, * P < 0.05, ** P < 0.01, *** P < 0.001

[0066] Table 2. Effect of black garlic polysaccharide on the ink propulsion of the small intestine of mice (n = 10,

[0067]

[0068] 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

[0069] As can be seen from Table 1, compared with the blank group, there was no significant difference in body weight among the groups, indicating that black garlic polysaccharide had 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.

[0070] As can be seen from Table 2, 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, showing a significant difference, indicating that black garlic polysaccharide had a promoting effect on the small intestine motility of constipation model mice and was dose-dependent.

[0071] Test Example 2 - Mouse defecation experiment

[0072] 1 Experimental purpose

[0073] Through this experiment, observe the effects of intragastric administration of black garlic polysaccharide for 1 week on the defecation and fecal water content of mice.

[0074] 2 Test drugs and animals

[0075] Same as Test Example 1.

[0076] 3 Test articles

[0077] Black garlic polysaccharide: Prepared according to the method of Example 1.

[0078] 4 Experimental method

[0079] The experiment was mainly carried out according to the method in the literature (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.).

[0080] 4.1 Preparation of test drugs

[0081] Same as Test Example 1.

[0082] 4.2 Animal grouping

[0083] Same as Test Example 1.

[0084] 4.3 Administration and index determination

[0085] Each group was intragastrically administered the corresponding test drug at a dose of 0.1 mL / 10 g, specifically as follows: mice in the blank group and the model group were given distilled water, mice in the positive group were given lactulose oral liquid, and mice in the low, medium, and high-dose groups of black garlic polysaccharide were given black garlic polysaccharide solution. The drug was administered once a day for 1 week. The general condition was observed, and the body weight changes of the mice were recorded. After the last administration, the mice were fasted but allowed to drink water for 16 h. Except for the mice in the blank group that were intragastrically administered distilled water, the mice in other groups were intragastrically administered compound diphenoxylate solution for modeling. 30 minutes after modeling, the mice in the blank group and the model group were intragastrically administered ink, and the mice in other groups were intragastrically administered the corresponding ink containing the drug. The time for each mouse to first defecate black feces, the number, weight, and characteristics (elliptical, hemispherical granular feces, whether in series, whether loose feces) of black feces excreted within 8 h were recorded.

[0086] Determination of fecal water content: The EP tube was dried in advance and weighed. The fresh black feces of the mice were placed in the dried EP tube, weighed, and then placed in an oven and dried until the weight no longer changed, and the weights of the black feces before and after drying were recorded.

[0087] Calculation formula: Fecal water content (%) = (M1 - M2) / M1 × 100%

[0088] (Note: M1: Mass of black feces before drying, M2: Mass of black feces after drying)

[0089] 5 Experimental results

[0090] Table 3. Effects of black garlic polysaccharide on the body weight of mice (n = 10,

[0091]

[0092] Note: Compared with the blank control group, * P < 0.05, ** P < 0.01, *** P < 0.001

[0093] Table 4. Effects of black garlic polysaccharide on defecation of mice (n = 10,

[0094]

[0095] 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

[0096] Table 5. Results of fecal water content of mice (n = 10,

[0097]

[0098] 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

[0099] As can be seen from Table 3, 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, the mice in each group had normal diet and water intake, were in good condition, and had no adverse reactions such as diarrhea.

[0100] As can be seen from Table 4, compared with the blank group, the first black stool time of the model group was extremely significantly prolonged, and the number and weight of stools excreted at 10 h were extremely significantly reduced, indicating that the constipation model of mice in this experiment was successfully established; compared with the model group, the first black stool time of mice in each dose group of black garlic polysaccharide was significantly prolonged, and the number and weight of stools excreted at 10 h were significantly increased, indicating that black garlic polysaccharide had a laxative effect on constipation model mice.

[0101] As can be seen from Table 5, 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 each dose group of black garlic polysaccharide was significantly increased, and the feces were oblong, obviously moist and smooth, with a soft texture, and there was no diarrhea phenomenon, indicating that black garlic polysaccharide could increase the water content of feces in constipation model mice to play a laxative effect.

[0102] The experimental results of Test Examples 1 and 2 show that the black garlic polysaccharide prepared by the method of the present invention can significantly promote the intestinal peristalsis of constipation model mice, shorten the defecation time, and increase the water content of feces, so as to play an anti-constipation effect. While retaining the efficacy, the purpose of reducing the dosage is achieved, and it can be used to prepare anti-constipation health products with good prospects.

Claims

1. A preparation method of black garlic polysaccharide, characterized in that, It includes the following steps: (A) Extraction Extract black garlic with a solvent to obtain an extract; (B) Enzymatic deproteinization Subject the above-mentioned extract to enzymatic digestion for deproteinization to obtain an enzymatically deproteinized solution; (C) Ultrafiltration Filter the above-mentioned enzymatically deproteinized solution through an ultrafiltration membrane to obtain a retentate; (D) Concentration, drying, and pulverization Concentrate, dry, and pulverize the above-mentioned retentate to obtain the product.

2. The preparation method according to claim 1, characterized in that, In the step (A), the extraction solvent used is water or an ethanol aqueous solution with a concentration not higher than 30%.

3. The preparation method according to claim 1, characterized in that, In the step (A), the extraction method is maceration extraction, percolation extraction, heat reflux extraction, ultrasonic extraction, and / or grinding and pulping extraction, and the solid-liquid ratio is 1:5 - 1:

100.

4. The preparation method according to claim 1, characterized in that, In the step (B), papain is selected for enzymatic deproteinization, with a dosage of 500 - 10000 U per 1 g of black garlic raw material, and the enzymatic hydrolysis time is 0.5 - 5 h.

5. The preparation method according to claim 1, wherein In the step (C), the ultrafiltration membrane used is an organic ultrafiltration membrane or an inorganic ceramic membrane, or a combination of both.

6. The preparation method according to claim 1, characterized in that, In the step (C), the ultrafiltration membrane used is selected from inorganic ceramic membranes with a pore size of 5 - 50 KD.

7. The preparation method according to claim 1, wherein, In the step (C), the ultrafiltration membrane used is a 15 KD inorganic ceramic membrane.

8. The preparation method according to claim 1, characterized in that In the step (D), the drying method used is vacuum concentration and spray drying.

9. The black garlic polysaccharide prepared by the preparation method according to any one of claims 1 - 8 is applied to the preparation of a health product for treating constipation.

10. The application according to claim 9, wherein The health product for treating constipation is a medicine, a health food, or a general food.

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