Preparation method of food-medicine source compound polysaccharide capable of improving glucose metabolism disorder

Abstract

The invention belongs to the technical field of polysaccharide extraction, and particularly relates to a preparation method of food-medicine source compound polysaccharide capable of improving glucose metabolism disorder, which comprises the following steps: (1) wet pulping; (2) preparing a deep eutectic solvent; (3) compound polysaccharide extraction; (4) compound polysaccharide separation; (5) directionally optimizing the compound polysaccharide; and (6) optimizing compound polysaccharide separation to obtain the food-medicine source compound polysaccharide capable of improving the glucose metabolism disorder. The compound polysaccharide with high yield and purity is obtained through the method, and the obtained polysaccharide has a good effect of improving glucose metabolism disorder.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide extraction, and particularly relates to a method for preparing a food and medicine-derived composite polysaccharide capable of improving sugar metabolism disorders. Background Art

[0002] Polygonatum kingianum (Polygonatum kingianum) is a plant of the genus Polygonatum in the Liliaceae family. It is a unique edible and medicinal resource in my country, combining medicinal, health-promoting, and edible uses. It possesses extremely high application value and broad market prospects. However, its processing and utilization are primarily developed into traditional or low-end foods such as decoction pieces and ultrafine powders, with a lack of innovative or highly refined products, which has severely hampered the healthy development of the Polygonatum industry.

[0003] Mesona chiensis Benth is an annual herbaceous plant of the genus Mesona in the family Lamiaceae. A unique food and medicinal resource in my country, Mesona chiensis is known for its cold, sweet, and mild properties. It has the benefits of clearing heat and relieving summer heat, as well as promoting diuresis. It is primarily used to treat ailments such as heatstroke, erysipelas, and diabetes. However, processing and utilization of Mesona chiensis have primarily resulted in traditional or low-end foods such as herbal tea and jelly, severely hindering the quality and efficiency of the Mesona chiensis industry.

[0004] Polysaccharides are the most important and abundant active ingredients in Polygonatum sibiricum and Ayurveda aurantifolia, possessing immune-boosting and anti-diabetic properties. Therefore, using Polygonatum sibiricum and Ayurveda aurantifolia as raw materials and aiming to improve glucose metabolism disorders, a targeted approach has been developed to prepare food- and drug-derived composite polysaccharides. These can be used as ingredients in functional food formulations that regulate glucose metabolism. This addresses the issues of low comprehensive utilization of these two food- and drug-derived resources and a short supply chain, providing consumers with more choices. Summary of the Invention

[0005] The present invention aims to solve the above technical problems and provide a method for preparing a food- and drug-derived composite polysaccharide capable of improving glucose metabolism disorders.

[0006] The technical solution of the present invention is:

[0007] A method for preparing a edible and medicinal composite polysaccharide capable of improving glucose metabolism disorders comprises the following steps:

[0008] (1) Wet pulping: Mix the herb Agar-agar and Polygonatum dahliae slices, add deionized water, soak, grind, and collect the composite pulp;

[0009] (2) Preparation of a deep eutectic solvent: betaine, proline, and tartaric acid are mixed to obtain a deep eutectic solvent, and distilled water is added to the deep eutectic solvent to form a deep eutectic solvent solution having a water content of 10-15 wt%;

[0010] (3) Extraction of complex polysaccharides: The complex slurry is mixed with a deep eutectic solvent solution, and then a complex enzyme is added at a volume of 2-3% of the total volume of the complex slurry and the deep eutectic solvent solution, mixed evenly, enzymatically hydrolyzed, filtered and separated, and the supernatant is collected and concentrated to obtain a concentrate;

[0011] (4) Separation of complex polysaccharides: add anhydrous ethanol to the concentrate until the ethanol volume fraction is 85-90%, stir evenly, precipitate with alcohol, centrifuge and collect the precipitate;

[0012] (5) Directed optimization of complex polysaccharides: deionized water was added to the precipitate of step (4) to dissolve it, and segmented interception was performed using a filter membrane with a molecular weight cutoff of 3-1000 kDa. The intercepted solutions of the corresponding filter membranes were collected and concentrated and freeze-dried to obtain complex polysaccharides of different molecular weights, and the complex polysaccharide with the highest polysaccharide yield was selected;

[0013] (6) Optimizing the separation of complex polysaccharides: adding anhydrous ethanol to the complex polysaccharide with the highest polysaccharide yield until the ethanol volume fraction is 85-90%, stirring thoroughly, precipitating with alcohol, centrifuging, collecting the precipitate, and freeze-drying to obtain a food and drug-derived complex polysaccharide with the ability to improve sugar metabolism disorders.

[0014] In order to obtain a complex polysaccharide with high yield and strong α-glucosidase inhibition ability, preferably, in step (1) of the present invention, the weight ratio of the herb Agar-agar and the Polygonatum sibiricum tablets is 2-3:1. Due to the differences in polysaccharide content and extraction difficulty between the two, if the weight ratio of the two is unbalanced, it will affect the dissolution or release of the polysaccharides of the herb Agar-agar and the Polygonatum sibiricum tablets, and reduce the yield of the polysaccharide. In addition, if the ratio is not suitable, it will also lead to the weakening of the efficacy of the extracted polysaccharides.

[0015] In order to better release the polysaccharide components of the herb Agar-agar and Polygonatum yunnanensis slices, preferably, in the step (1) of the present invention, deionized water is added at a material-liquid ratio of 1:40-50 g / mL, in the step (1), the mixture is immersed at a constant temperature of 55-60°C for 10-13 hours, and in the step (1), the mixture is ground at a pressure of 50-60 MPa for 15-30 minutes.

[0016] In order to obtain a complex polysaccharide with high yield and strong α-glucosidase inhibition ability, preferably, in step (2) of the present invention, the molar ratio of betaine, proline and tartaric acid is 2: (1-2): 1. The low eutectic solvent of the present invention has a stable hydrogen bond network, which can improve the solubility of the complex polysaccharide of the present invention and can selectively extract polysaccharide components with strong ability to improve sugar metabolism and stable activity, thereby improving the efficacy of the polysaccharide in improving sugar metabolism disorders.

[0017] Preferably, in step (3) of the present invention, the complex enzyme is a mixture of pectinase, cellulase and papain in a weight ratio of 2:1:1; in step (3), the enzymatic hydrolysis is carried out at 50-55° C. for 2-2.5 h;

[0018] Preferably, in step (5) of the present invention, a filter membrane with a molecular weight cut-off of 50-70 kDa is used for segmented interception. The polysaccharide yield in this range is the highest and the polysaccharide has a better ability to improve sugar metabolism.

[0019] Preferably, in steps (4) and (6) of the present invention, the alcohol precipitation is carried out by standing in an environment of 4°C for 12-15 hours.

[0020] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0021] 1. The present invention utilizes a characteristic low eutectic solvent in conjunction with a complex enzyme system to carry out targeted extraction of the Yunnan Polygonatum-Agar-agar composite pulp, thereby increasing the yield and content of the complex polysaccharide that has the effect of improving sugar metabolism disorders, and making the activity more stable while reducing energy consumption. The preparation process is simple and the operation is convenient.

[0022] 2. The present invention adopts wet grinding to process dry herb Agar-agar and dry Polygonatum sibiricum slices to obtain composite slurry, which can improve the subsequent yield of composite polysaccharides.

[0023] 3. The method of the present invention uses a characteristic low eutectic solvent to simultaneously extract the polysaccharide components of Polygonatum yunnanensis and Herba Arydioscenicae, without the need for segmented extraction, thereby simplifying the process and shortening the preparation cycle.

[0024] 4. The present invention utilizes a characteristic deep eutectic solvent in conjunction with a complex enzyme system to ensure the stability and activity of the extract.

[0025] 5. The present invention makes full use of resources, effectively improves the processing efficiency of Chinese characteristic edible and medicinal resources such as Polygonatum sibiricum and Achyranthes bidentata, and increases the economic value of their planting industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the infrared spectrum of the complex polysaccharide obtained in step (6) of Example 1 of the present invention;

[0027] Figure 2 This is an electron microscope scanning image of the complex polysaccharide obtained in step (6) of Example 1 of the present invention (left: 1.00KX; right: 10KX). DETAILED DESCRIPTION

[0028] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0029] Example 1

[0030] (1) Wet pulping: Add Herba Aurantii and Polygonatum yunnanensis slices (2:1, w / w) to deionized water at a solid-liquid ratio of 1:45 (g / mL), soak overnight at 60°C (12 h), grind by wet method (pressure 55 MPa), and collect the composite pulp;

[0031] (2) Preparation of a characteristic deep eutectic solvent: betaine, proline, and tartaric acid were mixed in a molar ratio of 2:1:1 to obtain a novel deep eutectic solvent; distilled water was added to the obtained deep eutectic solvent to form a deep eutectic solvent solution having a water content of 13%, which was set aside;

[0032] (3) Extraction of complex polysaccharides: The complex slurry was mixed with the deep eutectic solvent solution at a solid-liquid ratio of 110:1 (g / L), and 2.5% complex enzyme (pectinase: cellulase: papain = 2:1:1, w / w / w) was added and mixed thoroughly. The extraction temperature was 50°C and the extraction time was 2.5 h. The mixture was filtered and separated, and the supernatant was collected and concentrated to 1 / 8 of the original volume to obtain a concentrate.

[0033] (4) Separation of complex polysaccharides: add anhydrous ethanol to the concentrate to a volume fraction of 90%, stir thoroughly, and allow to stand at 4°C for 13 h for alcohol precipitation. Centrifuge and collect the precipitate.

[0034] (5) Targeted optimization of complex polysaccharides: Deionized water was added to the precipitate of step (4) to dissolve it, and ultrafiltration membranes with molecular weight cutoffs of 3, 5, 10, 30, 50, 70, 100, 300, 500, and 1000 kDa were used for segmented interception, and the solutions were collected and concentrated and freeze-dried to obtain complex polysaccharides of different molecular weights. Among them, polysaccharides in the range of 50-70 kDa had the highest yield and better ability to improve sugar metabolism;

[0035] (6) Optimizing the separation of complex polysaccharides: adding anhydrous ethanol to the complex polysaccharides in the range of 50-70 kDa until the ethanol volume fraction is 90%, stirring thoroughly, standing at 4°C for 13 h for alcohol precipitation, centrifuging, collecting the precipitate, and freeze-drying to obtain the complex polysaccharide.

[0036] Example 2

[0037] (1) Wet pulping: Add Herba Aurantii and Polygonatum yunnanensis slices (3:1, w / w) to deionized water at a solid-liquid ratio of 1:50 (g / mL), soak overnight at 60°C (12 h), grind by wet method (pressure 60 MPa), and collect the composite pulp;

[0038] (2) Preparation of a characteristic deep eutectic solvent: betaine, proline, and tartaric acid were mixed in a molar ratio of 2:1:1 to obtain a novel deep eutectic solvent; distilled water was added to the obtained deep eutectic solvent to form a deep eutectic solvent solution having a water content of 10 wt %, which was set aside;

[0039] (3) Extraction of complex polysaccharides: The complex slurry was mixed with the deep eutectic solvent solution at a solid-liquid ratio of 100:1 (g / L), and then 3% complex enzyme (pectinase: cellulase: papain = 2:1:1, w / w / w) was added and mixed thoroughly. The extraction temperature was 50°C and the extraction time was 2 h. The mixture was filtered and separated, and the supernatant was collected and concentrated to 1 / 5 of the original volume to obtain a concentrate;

[0040] (4) Separation of complex polysaccharides: add anhydrous ethanol to the concentrate until the ethanol volume fraction is 85%, stir thoroughly, let it stand at 4°C for 15 h for alcohol precipitation, centrifuge and collect the precipitate;

[0041] (5) Targeted optimization of complex polysaccharides: Deionized water was added to the precipitate of step (4) to dissolve it, and ultrafiltration membranes with molecular weight cutoffs of 3, 5, 10, 30, 50, 70, 100, 300, 500, and 1000 kDa were used for segmented interception, and the solutions were collected and concentrated and freeze-dried to obtain complex polysaccharides of different molecular weights. Among them, polysaccharides in the range of 50-70 kDa had the highest yield and better ability to improve sugar metabolism;

[0042] (6) Optimizing the separation of complex polysaccharides: adding anhydrous ethanol to the complex polysaccharides in the range of 50-70 kDa until the ethanol volume fraction is 85%, stirring thoroughly, standing at 4 ° C for 15 h for alcohol precipitation, centrifuging, collecting the precipitate, and freeze-drying to obtain the complex polysaccharide.

[0043] Example 3

[0044] (1) Wet pulping: Add Herba Aurantii and Polygonatum yunnanensis slices (2:1, w / w) to deionized water at a solid-liquid ratio of 1:40 (g / mL), soak overnight at 55°C (12 h), grind by wet method (pressure 50 MPa), and collect the composite pulp;

[0045] (2) Preparation of a characteristic deep eutectic solvent: betaine, proline, and tartaric acid were mixed in a molar ratio of 2:2:1 to obtain a novel deep eutectic solvent; distilled water was added to the obtained deep eutectic solvent to form a deep eutectic solvent solution having a water content of 15 wt %, which was set aside;

[0046] (3) Extraction of complex polysaccharides: The complex slurry was mixed with the deep eutectic solvent solution at a solid-liquid ratio of 120:1 (g / L), and then 2% complex enzyme (pectinase: cellulase: papain = 2:1:1, w / w / w) was added and mixed thoroughly. The extraction temperature was 55°C and the extraction time was 2.5 h. The mixture was filtered and separated, and the supernatant was collected and concentrated to 1 / 10 of the original volume to obtain a concentrate;

[0047] (4) Separation of complex polysaccharides: add anhydrous ethanol to the concentrate to a volume fraction of 90%, stir thoroughly, let stand at 4°C for 12 h for alcohol precipitation, centrifuge and collect the precipitate;

[0048] (5) Targeted optimization of complex polysaccharides: Deionized water was added to the precipitate of step (4) to dissolve it, and ultrafiltration membranes with molecular weight cutoffs of 3, 5, 10, 30, 50, 70, 100, 300, 500, and 1000 kDa were used for segmented interception, and the solutions were collected and concentrated and freeze-dried to obtain complex polysaccharides of different molecular weights. Among them, polysaccharides in the range of 50-70 kDa had the highest yield and better ability to improve sugar metabolism;

[0049] (6) Optimizing the separation of complex polysaccharides: adding anhydrous ethanol to the complex polysaccharides in the range of 50-70 kDa until the ethanol volume fraction is 90%, stirring thoroughly, standing at 4°C for 12 h for alcohol precipitation, centrifuging, collecting the precipitate, and freeze-drying to obtain the complex polysaccharide.

[0050] Characteristic functional groups and microstructure analysis of the complex polysaccharide with the effect of improving glucose metabolism disorders prepared in Example 1 of the present invention:

[0051] 1. Characteristic functional group analysis

[0052] 1 mg of the composite polysaccharide sample obtained in step (6) of Example 1 was mixed with 200 mg of potassium bromide and pressed into a sheet with a thickness of 1 mm. The sheet was then tested using a Nicolet iZ-10 Fourier transform infrared spectrometer with an instrument resolution of 4.00 cm -1 , scanning range is 4000-450cm -1 Scan number: 32. Sampling gain: 8.0; mirror speed: 0.4747; aperture: 80.00; DTGS KBr detector; KBr beam splitter; infrared light source.

[0053] The infrared spectrum of the complex polysaccharide obtained in step (6) of Example 1 is shown in FIG. Figure 1 As shown, the absorption band is at 3600-3200 cm -1 It is the stretching vibration absorption peak of -OH. The absorption peak in this area is the characteristic peak of sugars. -1 It is the stretching vibration absorption peak of OH, which is the characteristic peak of sugars. -1 The absorption peak at 1021.44 cm is attributed to CH stretching vibration. -1 There is an absorption peak at , which is attributed to the stretching vibration of CO.

[0054] 2. Microstructure analysis

[0055] The results were determined using a Zeiss Merlin Compact high-resolution field emission scanning electron microscope. Figure 2 It can be seen that under the conditions of magnification of 1 and 10K, the surface of the polysaccharide sample was observed to be rough and filamentous.

[0056] Experimental Example 1: Effects of different mass ratios of dried jelly grass and dried polygonatum slices on the yield of complex polysaccharides and the ability to improve glucose metabolism

[0057] 1. Calculate the yield of complex polysaccharides at different mass ratios of dried jelly grass and dried polygonatum slices. The polysaccharide yield is calculated according to formula (1):

[0058]

[0059] 2. Measure the α-glucosidase inhibition ability of dried jelly grass and dried Polygonatum dahliae tablets at different mass ratios

[0060] The α-glucosidase inhibition activity test was used to compare the glucose metabolism improving abilities of different complex polysaccharides, as shown in Table 1.

[0061] Calculate the α-glucosidase inhibitory capacity IC 50 The results are shown in Table 2. The specific experimental steps are as follows:

[0062] Take a 2 mL centrifuge tube and add the reagents in the order listed in the table below. Measure the absorbance at a wavelength of 405 nm and calculate the inhibition rate according to formula (2).

[0063]

[0064] Where, W is the α-glucosidase inhibition rate, A c is the blank absorbance value, A S is the sample or positive absorbance value, A b is the blank absorbance of the sample.

[0065] Table 2 Table of reagent addition for determining the ability to inhibit α-glucosidase activity

[0066]

[0067]

[0068] Table 2 Yield of complex polysaccharides extracted from different mass ratios of Herba Aurantii and Polygonatum sibiricum and their inhibition of α-glucosidase activity

[0069]

[0070] Note: Except for the different weight ratios of dried Herba Amaranthus and dried Polygonatum daturae slices, the above groups are the same as Example 1.

[0071] It can be seen from Table 2 that when the weight ratio of dry agar-agar grass and dry polygonatum sibiricum slices is 2-3:1, the yield of complex polysaccharides is the highest and the α-glucosidase inhibition ability is the strongest. When the weight ratio of agar-agar grass and polygonatum sibiricum slices exceeds the range of the present invention, the yield of complex polysaccharides is lower and the α-glucosidase inhibition ability is also poor.

[0072] Experimental Example 2: Effects of different deep eutectic solvent compositions on the yield of complex polysaccharides and the ability to improve sugar metabolism

[0073] As in Experimental Example 1, the yield of complex polysaccharides extracted with different deep eutectic solvent compositions and their α-glucosidase inhibitory abilities were determined. The polysaccharide yield was calculated according to Formula (1) of Experimental Example 1, and the α-glucosidase inhibitory ability IC 50 (μg / mL) was calculated according to formula (2) of Test Example 1. The results are shown in Table 3.

[0074] Table 3 Yield of composite polysaccharides and inhibition of α-glucosidase activity by different deep eutectic solvents

[0075]

[0076]

[0077] Note: Except for the different compositions of the deep eutectic solvents, the other components of the above groups are the same as those of Example 2.

[0078] As can be seen from Table 3, the deep eutectic solvent of the present invention requires specific raw materials and composition to obtain a complex polysaccharide with high yield and strong α-glucosidase inhibition ability.

[0079] Experimental Example 3: Effects of different molecular weight cut-offs on optimizing the yield of complex polysaccharides and improving sugar metabolism

[0080] As in Experimental Example 1, the yield of complex polysaccharides extracted with different deep eutectic solvent compositions and their α-glucosidase inhibitory abilities were determined. The polysaccharide yield was calculated according to Formula (1) of Experimental Example 1, and the α-glucosidase inhibitory ability IC 50 (μg / mL) was calculated according to formula (2) of Test Example 1. The results are shown in Table 4.

[0081] Table 4 Yield of composite polysaccharides with different molecular weights and inhibition of α-glucosidase activity

[0082]

[0083]

[0084] Note: Except for the different molecular weight cut-offs, the other conditions in the above groups are the same as those in Example 3.

[0085] As can be seen from Table 4, when the filter membrane with a molecular weight cut-off of 50-70 kDa is used for segmented interception, a complex polysaccharide with high yield and strong α-glucosidase inhibition ability is obtained.

[0086] The above description is a detailed description of the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A method for preparing a food-based composite polysaccharide capable of improving glucose metabolism disorders, characterized in that: The following steps are involved: (1) Wet pulping: Mix the herb Agar-agar and Polygonatum dahliae slices, add deionized water, soak, grind, and collect the composite pulp; (2) Preparation of a deep eutectic solvent: betaine, proline, and tartaric acid are mixed to obtain a deep eutectic solvent, and distilled water is added to the deep eutectic solvent to form a deep eutectic solvent solution having a water content of 10-15 wt%; (3) Extraction of complex polysaccharides: The complex slurry is mixed with a deep eutectic solvent solution, and then a complex enzyme is added at a volume of 2-3% of the total volume of the complex slurry and the deep eutectic solvent solution, mixed evenly, enzymatically hydrolyzed, filtered and separated, and the supernatant is collected and concentrated to obtain a concentrate; (4) Separation of complex polysaccharides: add anhydrous ethanol to the concentrate until the ethanol volume fraction is 85-90%, stir evenly, precipitate with alcohol, centrifuge and collect the precipitate; (5) Directed optimization of complex polysaccharides: deionized water was added to the precipitate of step (4) to dissolve it, and segmented interception was performed using a filter membrane with a molecular weight cutoff of 3-1000 kDa. The intercepted solutions of the corresponding filter membranes were collected and concentrated and freeze-dried to obtain complex polysaccharides of different molecular weights, and the complex polysaccharide with the highest polysaccharide yield was selected; (6) Optimizing the separation of complex polysaccharides: adding anhydrous ethanol to the complex polysaccharide with the highest polysaccharide yield until the ethanol volume fraction is 85-90%, stirring, alcohol precipitation, centrifugation, collecting the precipitate, and freeze-drying to obtain a food and drug-derived complex polysaccharide with the ability to improve sugar metabolism disorders.

2. The method for preparing the edible and medicinal composite polysaccharide capable of improving glucose metabolism disorders according to claim 1, wherein: In the step (1), the weight ratio of the herb Agar-agar and the Polygonatum yunnanensis slices is 2-3:

1.

3. The method for preparing the edible and medicinal composite polysaccharide capable of improving glucose metabolism disorder according to claim 1, characterized in that: In the step (1), deionized water is added at a material-liquid ratio of 1:40-50 g / mL.

4. The method for preparing the edible and medicinal composite polysaccharide capable of improving glucose metabolism disorder according to claim 1, wherein: In the step (1), the mixture is immersed at a constant temperature of 55-60° C. for 10-13 hours.

5. The method for preparing the edible and medicinal composite polysaccharide capable of improving glucose metabolism disorder as claimed in claim 1, characterized in that: In the step (1), grinding is carried out at a pressure of 50-60 MPa for 15-30 minutes.

6. The method for preparing the edible and medicinal composite polysaccharide capable of improving glucose metabolism disorders according to claim 1, wherein: In the step (2), the molar ratio of betaine, proline and tartaric acid is 2:(1-2):

1.

7. The method for preparing the edible and medicinal composite polysaccharide capable of improving glucose metabolism disorder as claimed in claim 1, characterized in that: In the step (3), the complex enzyme is a mixture of pectinase, cellulase and papain in a weight ratio of 2:1:

1.

8. The method for preparing the edible and medicinal composite polysaccharide capable of improving glucose metabolism disorder as claimed in claim 1, characterized in that: In the step (3), the enzymatic hydrolysis is carried out at 50-55° C. for 2-2.5 hours.

9. The method for preparing the edible and medicinal composite polysaccharide capable of improving glucose metabolism disorder as claimed in claim 1, characterized in that: In the step (5), a filter membrane with a molecular weight cut-off of 50-70 kDa is used for segmented interception.

10. The method for preparing the edible and medicinal composite polysaccharide capable of improving glucose metabolism disorder as claimed in claim 1, characterized in that: In the steps (4) and (6), the mixture is allowed to stand at 4° C. for 12-15 hours for alcohol precipitation.

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