Hawthorn pectin oligosaccharide and its preparation method and application

Abstract

The invention discloses a hawthorn pectin oligosaccharide and a preparation method and application thereof. The preparation method comprises the following steps: adding hawthorn powder to water for homogenization, heating and leaching, and after the leaching is completed, centrifuging to obtain the supernatant and cooling; adding ethanol to the supernatant for alcohol precipitation to precipitate pectin; adjusting the pH value of the feed liquid to 3-4, physically treating the set time, and then adding endopolygalacturonase for hydrolysis; after the hydrolysis is completed, using a chromatography column for separation and purification, concentrating, and freeze-drying to obtain; the physical method is microwave, ultrasound or ultrahigh pressure. The hawthorn pectin oligosaccharide prepared by the present invention is different from the monosaccharide composition of ordinary hawthorn pectin oligosaccharides, mainly contains galacturonic acid, has anti-aging activity in vitro and in vivo, and has no toxic side effects, and is not only suitable for the elderly population, but also for the prevention of aging in normal populations.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to hawthorn pectin oligosaccharide and a preparation method and application thereof. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Aging is the gradual degenerative changes in the morphology and structure of cells, tissues, and organs that occur as an organism ages, impairing tissue function and regeneration capacity, leading to an increased risk of age-related neurodegenerative diseases. However, there is currently no effective treatment to delay aging. Diet is an important factor in aging, and a reasonable diet and nutritional management can reduce the incidence of age-related diseases and prolong life. Therefore, a healthy diet is a safe and effective option to alleviate aging.

[0004] At present, more and more food-derived functional ingredients have been discovered for delaying aging. For example, polysaccharides and oligosaccharides extracted from fruits, vegetables, seaweed, mushrooms and Chinese medicinal materials have received widespread attention. These sugars have good anti-aging effects and fewer side effects, and have potential clinical application value. The common anti-aging mechanisms of the above oligosaccharides and polysaccharides include direct or indirect effects, such as improving oxidative stress, inhibiting neuronal damage, reducing inflammation levels, regulating intestinal flora, etc. Exploring more food-derived active ingredients for delaying aging and in-depth research on the mechanism of action is of great significance to human health.

[0005] Hawthorn is a plant of the genus Crataegus in the Rosaceae family. It is both a medicine and food, rich in nutrients and contains functional factors such as hawthorn pectin, fruit acid, total flavonoids, vitamin C, etc. It is currently mainly used as food, such as preparing hawthorn candied fruits such as moutan bark and hawthorn cake. Hawthorn pectin oligosaccharides (HPOS) are oligosaccharides induced by hawthorn pectin. The known functions include antioxidant and anti-inflammatory effects; regulating cholesterol, blood sugar and lipid metabolism; and inhibiting the growth of Escherichia coli, Bacillus subtilis and Staphylococcus aureus.

[0006] At present, the preparation of HPOS mainly adopts the enzymatic hydrolysis method. At present, the preparation of HPOS by the enzymatic hydrolysis method has the problems of incomplete enzymatic hydrolysis, low preparation yield, complex monosaccharide composition and difficulty in separation and purification. Summary of the invention

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a hawthorn pectin oligosaccharide and a preparation method and application thereof. The present invention utilizes physical methods to assist pectinase, which can achieve efficient preparation and effective separation of HPOS, and has a single composition.

[0008] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0009] In a first aspect, the present invention provides a method for preparing hawthorn pectin oligosaccharide, comprising the following steps:

[0010] Add hawthorn powder into water for homogenization, heat and extract, after the extraction is completed, centrifuge to obtain the supernatant, and cool;

[0011] Add ethanol to the supernatant to perform alcohol precipitation to precipitate pectin;

[0012] The pH value of the feed solution is adjusted to 3-4, the physical method is used for a set time, and then endopolygalacturonase is added for hydrolysis;

[0013] After the hydrolysis is completed, the product is separated and purified by chromatography column, concentrated, and freeze-dried to obtain the product;

[0014] The physical method is microwave, ultrasonic or ultra-high pressure.

[0015] In some embodiments, the hawthorn powder is obtained by crushing hawthorn and passing it through a 20-mesh sieve.

[0016] In some embodiments, during homogenization, the mass ratio of hawthorn powder to water is 1:10-20. Specifically, it can be 1g:10mL, 1g:10-15mL, 1g:10-20mL or 1g:12-15mL.

[0017] Preferably, the temperature of the heating leaching is 85-95° C., and the time of the heating leaching is 60-120 min.

[0018] In some embodiments, during alcohol precipitation, the volume ratio of the supernatant to ethanol is 1:2-6.

[0019] Preferably, the alcohol precipitation temperature is 2 to 5° C., and the alcohol precipitation time is 10 to 24 hours.

[0020] In some embodiments, the power of ultrasonic treatment is 100-800 W, and the time of ultrasonic treatment is 10-30 min.

[0021] In some embodiments, the power of the microwave treatment is 100-800 W, and the time of the microwave treatment is 10-30 min.

[0022] In some embodiments, the pressure of the ultra-high pressure treatment is 100-600 MPa, and the time of the ultra-high pressure treatment is 5-20 min.

[0023] In some embodiments, after the endopolygalacturonase is added, the concentration of the endopolygalacturonase is 0.1 to 2.0 U / mL, preferably 0.1 to 1.0 U / mL, and more preferably 0.2 U / mL.

[0024] Preferably, the endopolygalacturonase is selected from any one of Aspergillus niger, Rhizopus or thermophilic fungi; preferably, it is an endopolygalacturonase derived from Aspergillus niger.

[0025] Preferably, the hydrolysis temperature is 45-55° C. and the hydrolysis time is 1-3 h.

[0026] More preferably, the hydrolysis temperature is 50° C. and the hydrolysis time is 2 h.

[0027] In some embodiments, the chromatography column is a DEAE-cellulose chromatography column or a dextran gel chromatography column, and during elution, distilled water is used for elution first, and then 0.3 mol / L NH4HCO3 is used for elution. Anionic sugars have an opposite charge to that of the filler, so they are bound to the filler due to electrostatic action, while neutral polysaccharides do not bind to the filler. Therefore, distilled water is used for washing, and neutral polysaccharides are eluted to obtain neutral polysaccharides, and then 0.3 mol / L NH4HCO3 is used to elute acidic sugars and other sugars, in order to improve the purity of sugars and separate neutral sugars from other types of sugars.

[0028] In a second aspect, the present invention provides a hawthorn pectin oligosaccharide prepared by the preparation method.

[0029] In a third aspect, the present invention provides the use of the hawthorn pectin oligosaccharide in at least one of the following aspects:

[0030] 1) Application in the preparation of drugs for preventing and / or treating aging;

[0031] 2) Application in the preparation of drugs for assisting in improving memory;

[0032] 3) Application in the preparation of drugs for inhibiting apoptosis of senescent cells;

[0033] 4) Application in the preparation of drugs for increasing bacterial diversity;

[0034] 5) Application in the preparation of drugs for promoting the production of short-chain fatty acids.

[0035] In some embodiments, the aging is shortened lifespan, neuronal damage, apoptosis, or cognitive impairment.

[0036] In some embodiments, the senescent cells are induced fibroblasts, neuronal cells, smooth muscle cells, or endothelial cells;

[0037] Preferably, the senescent cells are H2O2-induced fibroblasts, such as human embryonic lung fibroblasts.

[0038] In some embodiments, the increasing of the diversity of the microbiota is to increase the diversity of the intestinal microbiota of the elderly or naturally aged animals, or to promote the growth and proliferation of probiotics in vitro. The animal may be a mouse.

[0039] Preferably, the intestinal tract is specifically the cecum;

[0040] The probiotics are lactobacillus or bifidobacterium.

[0041] In some embodiments, the promotion of short-chain fatty acid production is promoting the production of short-chain fatty acids in the intestines of the elderly or naturally aged animals, or promoting the production of short-chain fatty acids in in vitro probiotic fermentation broth. The animal may be a mouse.

[0042] Preferably, the short-chain fatty acid comprises acetic acid, propionic acid or butyric acid.

[0043] In some embodiments, the product is a medicament or a formulation.

[0044] In clinical application, as a conventional preparation process, it is used alone or combined with other drugs to prepare different dosage forms of drugs that can be used clinically. For example, excipients or flavoring agents and preservatives can be added to make anti-aging capsules or anti-aging oral liquids.

[0045] In some embodiments, the dosage form of the drug includes capsules, oral solutions, injection solutions, tablets, powders, and granules.

[0046] The beneficial effects achieved by one or more embodiments of the present invention are as follows:

[0047] The hawthorn pectin oligosaccharide prepared by the invention is prepared by a physical method assisted by an enzyme method. The physical method is used to break pectin molecules, which is helpful for further enzymolysis, thereby obtaining hawthorn pectin oligosaccharides with high preparation yield, high enzymolysis efficiency and single composition. The operation is simple and the oligosaccharide is suitable for industrial production.

[0048] The hawthorn pectin oligosaccharide prepared by the present invention is different from the monosaccharide composition of common hawthorn pectin oligosaccharide (containing galacturonic acid, rhamnose, arabinose, glucose or galactose, etc.), mainly contains galacturonic acid, has anti-aging activity in vitro and in vivo, and has no toxic side effects. It is not only suitable for the elderly population, but also suitable for aging prevention for normal people.

[0049] In addition, the hawthorn pectin oligosaccharide of the present invention increases the cell viability of human embryonic lung fibroblasts induced by H2O2, inhibits their apoptosis and reduces their β-galactosidase activity. The effect of hawthorn pectin oligosaccharide on the proliferation of probiotics was evaluated by in vitro fermentation, and it was found that the hawthorn pectin oligosaccharide significantly promoted the growth and proliferation of lactobacillus and bifidobacterium, and significantly promoted the production of short-chain fatty acids (such as acetic acid), and had the effect of improving intestinal health.

[0050] The water maze experiment was used to evaluate the effect of hawthorn pectin oligosaccharide on the active learning and memory ability of mice. The results showed that the relative swimming time and relative distance of the target quadrant and the number of crossing the platform of the mice in the hawthorn pectin oligosaccharide intervention group were significantly increased, with the increases of 53.4%, 124.6% and 500.0% respectively (p<0.01). The present invention provides a new option for treating or preventing aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0052] Figure 1 It is composed of hawthorn pectin oligosaccharide monosaccharide.

[0053] Figure 2 The effect of hawthorn pectin oligosaccharide on MRC-5 cell viability; cell experiment design ideas (A), toxicity experiment (B), H2O2 modeling concentration (C) and cell viability (D). Compared with the control group, *p<0.05 is significant, **p<0.01 is extremely significant; compared with the model group, # p<0.05 was significant. ## p<0.01 was extremely significant.

[0054] Figure 3 The effect of hawthorn pectin oligosaccharide on the mortality rate and β-galactosidase activity of MRC-5 cells; mortality rate (A) and β-galactosidase activity (B). Compared with the control group, *p<0.05 is significant, **p<0.01 is extremely significant; compared with the model group, # p<0.05 was significant. ## p<0.01 was extremely significant.

[0055] Figure 4 The effect of hawthorn pectin oligosaccharide on probiotic growth and acetic acid production in vitro; probiotic growth (A) and acetic acid content (B). Compared with the control group, *p<0.05 is significant, **p<0.01 is extremely significant; compared with the model group, # p<0.05 was significant. ## p<0.01 was extremely significant.

[0056] Figure 5 This is a trajectory diagram of the effect of hawthorn pectin oligosaccharides on the active learning and memory ability of mice, where A is the normal group; B is the NMN group; C is the hawthorn pectin oligosaccharide group.

[0057] Figure 6The effect of hawthorn pectin oligosaccharide on the active learning and memory ability of mice; relative distance (A) and relative time (B) of swimming in the target quadrant, and the number of times of crossing the platform (C), the data are expressed as mean ± standard deviation. Compared with the control group, *p<0.05 is significant, **p<0.01 is extremely significant; compared with the model group, # p<0.05 was significant. ## p<0.01 was extremely significant. DETAILED DESCRIPTION

[0058] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0059] The present invention utilizes a physical method to assist endopolygalacturonase derived from Aspergillus niger to hydrolyze hawthorn pectin to prepare pectin oligosaccharides from hawthorn (HPOS), and explores its anti-aging activity in vivo and in vitro.

[0060] The present invention will be further described below in conjunction with the embodiments.

[0061] Example 1: Preparation of Hawthorn Pectin Oligosaccharides by Ultrasonic-Assisted Pectinase

[0062] The hawthorn pectin oligosaccharide is prepared by ultrasonic-assisted pectinase. The preparation method is briefly described as follows: homogenization, hot water extraction and ethanol precipitation, physical method-assisted enzymatic hydrolysis, separation and purification, elution, rotary evaporation or thin film concentration, and drying. The specific preparation method is as follows:

[0063] Take 10kg of crushed hawthorn powder that has been passed through a 20-mesh sieve and add it to a water reaction container, add 100L of distilled water to homogenize, set the temperature of the reaction container to 90°C, keep it warm for 120 minutes after reaching the temperature, release the material after the insulation time is reached, centrifuge at 5000r / min for 10 minutes after the extraction is completed, retain the supernatant, and cool to room temperature.

[0064] Add twice the volume of ethanol and stir slowly at 500 r / min for 15 min. Allow to precipitate overnight at 4°C and centrifuge at 500 r / min for 10 min to obtain the precipitate, which is pectin.

[0065] The pH of the feed solution was adjusted to 3.5 with 0.02 mol / L acetate buffer, and the solution was treated with ultrasound (800 W, 20 kHz) for 20 min. Then, 0.2 U / mL endopolygalacturonase from Aspergillus niger was added and hydrolyzed at 50° C. for 2 h.

[0066] The samples were separated by DEAE-Sephadex A-25 dextran gel chromatography column, which was first eluted with distilled water and then with 0.3 mol / L NH4HCO3.

[0067] After appropriate concentration, the mixture is freeze-dried to obtain sugar powder (yield: 3.6%), and the hawthorn pectin oligosaccharide (such as Figure 1 shown).

[0068]

[0069] Effects of Hawthorn Pectin Oligosaccharide on Vitality of Senescent Cells

[0070] Human embryonic lung fibroblasts (MRC-5) were selected, and a senescent cell model was established by inducing MRC-5 cells with H2O2. The effect of hawthorn pectin oligosaccharides on the viability of senescent cells was investigated to evaluate its in vitro anti-aging activity.

[0071] The specific implementation scheme is as follows: the growth medium of MRC-5 cells is MEM medium, 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin are added to the medium, and the culture conditions are set to 5% CO2 and 37°C.

[0072] When the confluence of MRC-5 cells was about 70%, the cells were cultured at a rate of 2×10 4 The cells were inoculated in 96-well plates and divided into the following groups: normal control group (MEM medium); aging model group (MEM medium); positive control group (30 μM resveratrol concentration in MEM medium); intervention group (0.2 mg / mL, 0.8 mg / mL and 2.0 mg / mL hawthorn pectin oligosaccharide concentrations in MEM medium). After 24 hours of culture, fresh serum-free medium was replaced and cultured for another 4 hours. Then H2O2 (200 μM) was added to both the aging model group and the sample addition group, and cultured in a CO2 incubator for another 24 hours. Cell morphology was observed and recorded, and cell viability was measured.

[0073] The anti-aging effect was evaluated by measuring the viability of MRC-5 senescent cells using the MTT method. Figure 2 As shown. Figure 2 B shows that the viability of normal MRC-5 cells did not change significantly at a concentration of 0.01-2.0 mg / mL, indicating that it would not have a toxic effect on cells. The MRC-5 senescent cell model was established using 200 μM H2O2 ( Figure 2 C), after intervention with higher concentrations of hawthorn pectin oligosaccharides (0.8 mg / mL and 2.0 mg / mL), the viability of MRC-5 senescent cells was significantly improved. This shows that hawthorn pectin oligosaccharides will not have toxic effects on cells and can improve the viability of senescent cells.

[0074] Effects of hawthorn pectin oligosaccharide on cell death rate and β-galactosidase activity in senescent cells

[0075] Cell culture, grouping and intervention were the same as above. Dead cells can be stained blue by trypan blue, so the apoptosis of MRC-5 senescent cells was measured by trypan blue staining to evaluate the anti-aging effect. Figure 3 As shown in A. As can be seen from the figure, compared with the blank control group, the number of cell deaths increased significantly after H2O2 treatment. However, the death rate of MRC-5 cells was significantly reduced after intervention with different concentrations of hawthorn pectin oligosaccharides, especially at a concentration of 0.8 mg / mL.

[0076] Cells in the senescent state will produce β-galactosidase with high enzymatic activity, which will be stained dark blue by the kit. Figure 3 As shown in B, compared with the blank control group, the β-galactosidase activity of cells treated with H2O2 increased significantly. The hawthorn pectin oligosaccharide concentration was significantly reduced after treatment at 0.8 mg / mL.

[0077] Effects of Hawthorn Pectin Oligosaccharide on Proliferation and Short-Chain Fatty Acid Production of Probiotics in Vitro

[0078] Two strains of lactobacillus and two strains of bifidobacterium were activated and passaged three times in MRS liquid culture medium. The third-generation bacterial solution was centrifuged (10,000 r / min, 10 min), the bacteria were collected, and the bacteria were washed three times with PBS buffer. The bacteria were re-dissolved in MRS basic liquid culture medium to obtain seed liquid. The probiotic culture was carried out in an anaerobic bottle containing 50 mL of liquid culture medium. The negative control culture medium was MRS basic liquid culture medium without glucose. The experimental group added 10 g / L of hawthorn pectin oligosaccharide to the basic culture medium, and the positive control added 10 g / L of oligofructose. The growth amount was determined by turbidimetry at 595 nm. The seed liquid was inoculated into the culture medium. The initial turbidity after inoculation (OD 595 ) was 0.1-0.2, and the culture was incubated at 37°C for 48 h. Samples were taken at 0, 2, 4, 6, 8, 10, 12, 24, 36, and 48 h to measure the OD of the culture solution. 595 , draw a growth curve.

[0079] At 48h of cultivation, the fermentation broth was sampled and the acetic acid content was determined. The acetic acid content was determined by high performance liquid chromatography. The mobile phase was 5mM H2SO4 aqueous solution, the flow rate was 0.6mL / min, the column temperature was 55°C, and the injection volume was 20μL. A standard curve was prepared using 1-100mM acetic acid standards, and the acetic acid concentration in the sample was calculated using the standard curve.

[0080] like Figure 4As shown, hawthorn pectin oligosaccharides significantly promoted the growth and proliferation of Lactobacillus reuteri CICC-6132, Lactobacillus casei NRRLB-1922, Bifidobacterium longum NRRL B-41409 and Bifidobacterium adolescentis ATCC 15703, and significantly promoted the production of acetic acid.

[0081] Effects of hawthorn pectin oligosaccharide on active learning and memory ability of mice

[0082] Four-week-old male Kunming mice were adaptively fed for one week in an SPF animal room (temperature: 22±2℃; humidity: 50±10%; lighting: 12h light-dark alternation, 8:00-20:00 as daytime). The normal group was gavaged with distilled water (10mL / kg·bw / d) daily, the NMN group was gavaged with NMN (300mg / kg·bw / d) daily, and the hawthorn pectin oligosaccharide group was gavaged with hawthorn pectin oligosaccharide (1000mg / kg·bw / d) daily. The bedding and water bottles were changed once a week, and the experimental animals were free to drink water and eat. The water maze test was performed after 30 consecutive days of gavage.

[0083] like Figure 5 and Figure 6 As shown, compared with the normal group, the relative swimming time and distance in the target quadrant and the number of times crossing the platform in the hawthorn pectin oligosaccharide intervention group were significantly increased, with increases of 53.4%, 124.6% and 500.0% respectively (p<0.01).

[0084] Example 2: Preparation of Hawthorn Pectin Oligosaccharides by Microwave-Assisted Pectinase

[0085] The hawthorn pectin oligosaccharide is prepared by microwave-assisted pectinase. The preparation method is briefly described as follows: homogenization, hot water extraction and ethanol precipitation, physical method-assisted enzymatic hydrolysis, separation and purification, elution, rotary evaporation or thin film concentration, and drying. The specific preparation method is as follows:

[0086] Take 10kg of crushed hawthorn powder that has been passed through a 20-mesh sieve and add it to a water reaction container, add 120L of distilled water to homogenize, set the temperature of the reaction container to 95°C, keep it warm for 100 minutes after reaching the temperature, release the material after the insulation time is reached, centrifuge at 5000r / min for 10 minutes after the extraction is completed, retain the supernatant, and cool to room temperature.

[0087] Add twice the volume of ethanol and stir slowly at 500 r / min for 15 min. Allow to precipitate overnight at 4°C and centrifuge at 500 r / min for 10 min to obtain the precipitate, which is pectin.

[0088] The pH of the feed solution was adjusted to 3.5 with 0.02 mol / L acetate buffer, treated with microwaves (800 W, 915 MHz) for 30 min, and then 0.2 U / mL endopolygalacturonase from Aspergillus niger was added and hydrolyzed at 55° C. for 1.5 h.

[0089] The samples were separated by DEAE-Sephadex A-25 dextran gel chromatography column, which was first eluted with distilled water and then with 0.3 mol / L NH4HCO3.

[0090] After appropriate concentration, the mixture is freeze-dried to obtain sugar powder (yield: 3.2%), i.e., hawthorn pectin oligosaccharide with a polymerization degree of 2-5 and a galacturonic acid content of 99.4%.

[0091] Example 3: Preparation of Hawthorn Pectin Oligosaccharides by Ultrahigh Pressure-Assisted Pectinase

[0092] The preparation method of hawthorn pectin oligosaccharide using ultrahigh pressure assisted pectinase is briefly described as follows: homogenization, hot water extraction and ethanol precipitation, physical method assisted enzymatic hydrolysis, separation and purification, elution, rotary evaporation or film concentration, and drying. The specific preparation method is as follows:

[0093] Take 10 kg of crushed hawthorn powder that has been passed through a 20-mesh sieve and add it to a water reaction container, add 150 L of distilled water to homogenize, set the temperature of the reaction container to 85°C, keep it warm for 100 minutes after reaching the temperature, release the material after the insulation time is reached, centrifuge at 5000 r / min for 10 minutes after the extraction is completed, retain the supernatant, and cool to room temperature.

[0094] Add twice the volume of ethanol and stir slowly at 500 r / min for 15 min. Allow to precipitate overnight at 4°C and centrifuge at 500 r / min for 10 min to obtain the precipitate, which is pectin.

[0095] The pH of the feed solution was adjusted to 3.5 with 0.02 mol / L acetate buffer, treated with ultrahigh pressure (500 MPa) for 20 min, and then 0.2 U / mL endopolygalacturonase from Aspergillus niger was added and hydrolyzed at 50° C. for 3 h.

[0096] The samples were separated by DEAE-Sephadex A-25 dextran gel chromatography column, which was first eluted with distilled water and then with 0.3 mol / L NH4HCO3.

[0097] After appropriate concentration, the mixture is freeze-dried to obtain sugar powder (yield: 3.8%), i.e., hawthorn pectin oligosaccharide with a polymerization degree of 2-5 and a galacturonic acid content of 99.9%.

[0098] Comparative Example 1: Preparation of Hawthorn Pectin Oligosaccharides by Pectinase

[0099] The hawthorn pectin oligosaccharide is prepared by using pectinase. The preparation method is briefly described as follows: homogenization, hot water extraction and ethanol precipitation, pectinase hydrolysis, separation and purification, elution, rotary evaporation or film concentration, and drying. The specific preparation method is as follows:

[0100] Take 10kg of crushed hawthorn powder that has been passed through a 20-mesh sieve and add it to a water reaction container, add 100L of distilled water to homogenize, set the temperature of the reaction container to 90°C, keep it warm for 120 minutes after reaching the temperature, release the material after the insulation time is reached, centrifuge at 5000r / min for 10 minutes after the extraction is completed, retain the supernatant, and cool to room temperature.

[0101] Add twice the volume of ethanol and stir slowly at 500 r / min for 15 min. Allow to precipitate overnight at 4°C and centrifuge at 500 r / min for 10 min to obtain the precipitate, which is pectin.

[0102] The pH of the feed solution was adjusted to 3.5 with 0.02 mol / L acetate buffer, and then 0.2 U / mL endopolygalacturonase from Aspergillus niger was added to hydrolyze at 50°C for 2 h.

[0103] The samples were separated by DEAE-cellulose chromatography column, which was first eluted with distilled water and then with 0.3 mol / L NH4HCO3.

[0104] After appropriate concentration, the mixture was freeze-dried to obtain sugar powder (yield: 1.4%), i.e., hawthorn pectin oligosaccharide, the monosaccharide composition of which is shown in Table 1.

[0105] Table 1 Hawthorn pectin oligosaccharide monosaccharide composition

[0106]

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing hawthorn pectin oligosaccharide, characterized in that: The steps include: Add hawthorn powder into water for homogenization, heat and extract, after the extraction is completed, centrifuge to obtain the supernatant, and cool; The hawthorn powder is obtained by crushing hawthorn and passing it through a 20-mesh sieve; When homogenizing, the mass ratio of hawthorn powder to water is 1:15; The temperature of the heating extraction is 85°C, and the time of the heating extraction is 100 min; Add ethanol to the supernatant to perform alcohol precipitation to precipitate pectin; The pH value of the feed solution was adjusted to 3.5, and the ultrahigh pressure treatment was performed for a set time, and then endopolygalacturonase was added for hydrolysis; The pressure of the ultrahigh pressure treatment is 500 MPa, and the time of the ultrahigh pressure treatment is 20 min; The endopolygalacturonase is derived from Aspergillus niger; The concentration of endopolygalacturonase is 0.1-2.0 U / mL; The hydrolysis temperature is 45-55°C and the hydrolysis time is 1-3h; After the hydrolysis is completed, the product is separated and purified by chromatography column, concentrated, and freeze-dried to obtain the product; The chromatography column is a DEAE-Sephadex A-25 dextran gel chromatography column; When eluting the DEAE-Sephadex A-25 dextran gel chromatography column, first elute with distilled water and then elute with 0.3 mol / L NH4HCO3; The hawthorn pectin oligosaccharide has a polymerization degree of 2-5.

2. The method for preparing hawthorn pectin oligosaccharide according to claim 1, characterized in that: During alcohol precipitation, the volume ratio of supernatant to ethanol is 1:2-6.

3. The method for preparing hawthorn pectin oligosaccharide according to claim 1, characterized in that: The temperature of alcohol precipitation is 2-5°C, and the alcohol precipitation time is 10-24h.

4. The method for preparing hawthorn pectin oligosaccharide according to claim 1, characterized in that: After the endopolygalacturonase is added, the concentration of the endopolygalacturonase is 0.1 to 1.0 U / mL.

5. The method for preparing hawthorn pectin oligosaccharide according to claim 1, characterized in that: After the endopolygalacturonase was added, the concentration of the endopolygalacturonase was 0.2 U / mL.

6. The method for preparing hawthorn pectin oligosaccharide according to claim 1, characterized in that: The hydrolysis temperature was 50°C and the hydrolysis time was 2 h.

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