Preparation method of aloe polysaccharide, aloe polysaccharide and application of aloe polysaccharide in antibacterial product

Abstract

The invention provides a preparation method of aloe polysaccharide, the aloe polysaccharide and application of the aloe polysaccharide in antibacterial products, and belongs to the field of biological medicine. According to the method disclosed by the invention, freeze thawing treatment is carried out in the aloe pretreatment stage, microwave treatment is adopted in the aloe polysaccharide extraction stage, and the aloe polysaccharide can be better dissolved out by controlling the type of a solvent and the power of microwaves and matching with the addition of a surfactant, so that the extraction efficiency of the aloe polysaccharide is remarkably improved. The molecular weight of the aloe polysaccharide obtained through the preparation method is 50-500 KDa, the content of O-acetyl is 30% or above, and it is accidentally found in the research process that the obtained aloe polysaccharide can selectively inhibit bacteria, has the inhibiting effect on harmful bacteria and does not have the inhibiting effect on beneficial bacteria.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a preparation method of aloe polysaccharide, aloe polysaccharide and its application in antibacterial products. Background Art

[0002] Aloe is a perennial succulent herbaceous plant of the Liliaceae family, and is a plant integrating ornamental, beauty, edible, medicinal and health care functions. Aloe has many pharmacological effects mainly because it contains a large amount of polysaccharide components. Compared with other plant polysaccharides, aloe polysaccharide has broad-spectrum pharmacological activities such as immunomodulation, anti-radiation, antibacterial, antiviral, anti-tumor, anti-ulcer, lipid-lowering, hypoglycemic, antioxidant, anti-aging, and prevention and treatment of AIDS. And its own toxic and side effects are small, and there has been no report of allergic reactions due to the consumption of aloe polysaccharide, so it is one of the key research directions of aloe in recent years.

[0003] At present, the methods for extracting aloe polysaccharide mainly include solvent extraction method, ultrasonic-assisted method, microwave-assisted method, enzyme method, supercritical CO2 extraction method, etc. Among them, the solvent extraction method has a relatively long extraction time, high cost, low extraction rate, and may have the problem of solvent residue. The microwave-assisted extraction process parameters are not easy to control, and ordinary laboratory equipment is not resistant to microwave and is easy to burst, which has certain risks; the supercritical CO2 extraction method is used less due to cost and operation convenience. The ultrasonic-assisted method uses the cavitation effect of ultrasonic waves to increase the movement frequency and speed of substance molecules and improve the dissolution speed of the extracted components, and has the advantages of short extraction time, high extraction efficiency, convenient operation and no pollution, and is often used to extract effective components in plants.

[0004] For example, Chinese Patent CN117050202A discloses a preparation method for ultrasonic-assisted stirring extraction of aloe crude polysaccharide, which includes the following steps: 1) Remove the yellow-spotted part from fresh aloe leaves, cut off the outer hard spines, wash and dry, cut into pieces and crush to obtain aloe pulp, and store it at 4°C for later use; 2) Add distilled water to the aloe pulp obtained in step 1), perform ultrasonic treatment at an ultrasonic temperature of 30-70°C and simultaneously start stirring, and after 1-3 hours of ultrasonic stirring treatment, obtain an extract; 3) Add ethanol to the extract obtained in step 2), let it stand in a 4°C refrigerator for 12-24 hours, and take the precipitate after centrifugation and dry it to obtain aloe crude polysaccharide.

[0005] The enzyme method utilizes the high efficiency and specificity of biological enzymes to accelerate the dissolution of intracellular substances by destroying plant cell walls and increasing the permeability of cell membranes, and has the advantages of mild reaction, short operation time, low cost, etc., and is gradually applied to the extraction research of polysaccharides.

[0006] As disclosed in Chinese Patent CN109320626A, a kind of aloe polysaccharide, its preparation method and application are provided. The aloe polysaccharide is prepared from fresh aloe leaves through processes such as pretreatment, pulping, passing through a colloid mill, double enzyme hydrolysis, solid-liquid separation, concentration, drying, etc. In the preparation method of this invention, cellulase and pectinase are added to the slurry for double enzyme hydrolysis. Cellulase destroys the aloe cell wall, facilitating the dissolution of aloe polysaccharide. Pectinase destroys the aloe gel, making it easier for subsequent processing. The aloe polysaccharide prepared by this method has significantly higher extraction yield, polysaccharide content and viscosity than the prior art.

[0007] However, the above method does not fractionate aloe polysaccharides with different molecular weights, so that the various effects of aloe polysaccharides are not better demonstrated. Although Chinese Patent CN100491404A discloses a method for fractionated extraction of aloe polysaccharides, that is, adding 8 - 10 times of 95% ethanol to the concentrated aloe product, stirring, standing for precipitation, filtering, recovering ethanol, collecting crude aloe polysaccharides, dissolving in water and passing through ultrafiltration membranes with a molecular weight cut-off of 200,000 - 4,000 step by step, and then concentrating through nanofiltration membranes to obtain aloe polysaccharides with different molecular weights. Although different molecular weight aloe polysaccharides can be obtained by this method, the content of aloe polysaccharides is not high.

[0008] The literature (Separation and Purification of Aloe Polysaccharides by ATP, Membrane Separation Technology and Polyamide Resin, Xing Jianmin, Master's Thesis) records that first, a ternary copolymer ultrafiltration membrane is prepared by solution copolymerization using three monomers of acrylonitrile, acrylamide and styrene, and this ultrafiltration membrane is used to separate and purify aloe polysaccharides. This ultrafiltration membrane allows aloe polysaccharides with a molecular weight less than 68,000 to pass through the membrane, resulting in a decreasing content of aloe polysaccharides with a molecular weight less than 68,000 in the membrane concentration, realizing the fractionated purification of aloe polysaccharides. However, there are few reports on the separation and purification of aloe polysaccharides with a molecular weight between 50KDa - 100KDa in the prior art, and the content and purity are not high.

[0009] Therefore, it is necessary to develop a preparation method and application of aloe polysaccharides with high aloe polysaccharide content and narrow molecular weight distribution. Summary of the Invention

[0010] Based on the deficiencies in the prior art, the present invention aims to provide a preparation method of aloe polysaccharides, aloe polysaccharides and their application in antibacterial products. In the aloe pretreatment stage of the present invention, freeze-thaw treatment is carried out, and in the aloe polysaccharide extraction stage, microwave treatment is adopted. By controlling the type of solvent and the power of microwave, and cooperating with the addition of surfactants, aloe polysaccharides can be better dissolved, thus significantly improving the extraction efficiency of aloe polysaccharides. And unexpectedly, it is found during the research process that the obtained aloe polysaccharides can selectively inhibit bacteria, have an inhibitory effect on harmful bacteria, and have no inhibitory effect on beneficial bacteria.

[0011] To achieve the above object, the present invention adopts the following technical solutions:

[0012] On the one hand, the present invention provides a preparation method of aloe polysaccharide, comprising the following steps:

[0013] (1) Aloe pretreatment: Take fresh aloe leaves, wash them clean, remove thorns, cut them into thin slices horizontally, dry them, then soak the dried aloe slices in water and freeze-dry them, and pulverize the dried aloe slices to obtain aloe powder;

[0014] (2) Soak the aloe powder in a solvent, add a composite enzyme for enzymatic hydrolysis, then perform microwave extraction, filter the extract and concentrate it, and then perform alcohol precipitation to obtain the precipitate after centrifugation; Take the precipitate and dissolve it in water to obtain a crude polysaccharide aqueous solution;

[0015] (3) Ultrafilter the crude polysaccharide aqueous solution in step (2) through an ultrafiltration membrane to obtain an aloe polysaccharide concentrate with a molecular weight of 50KDa - 500KDa, decolorize and sterilize the aloe polysaccharide concentrate, and then perform freeze-drying and pulverize it to obtain the aloe polysaccharide.

[0016] Among them,

[0017] The thickness of the thin slices in step (1) is 2 - 3 cm.

[0018] The specific steps of the freeze-drying in step (1) are as follows: Place the soaked aloe thin slices in a freeze-dryer and quickly freeze them at -60 - -80 °C for 3 - 5 hours, and then under a vacuum environment of 40 - 60 Pa and -30 °C - -50 °C, perform temperature-controlled sublimation drying, and the drying time is 20 - 28 hours.

[0019] Preferably, the specific steps of the freeze-drying are as follows: Place the soaked aloe thin slices in a freeze-dryer and quickly freeze them at -70 °C for 4 hours, and then under a vacuum environment of 50 Pa and -40 °C, perform temperature-controlled sublimation drying, and the drying time is 24 hours.

[0020] During the implementation of the present invention, it is found that in the pretreatment step, the aloe is first dried, then soaked in water and then subjected to freeze-drying treatment. By controlling the freezing temperature and time, the cells can be swollen and ruptured, so that the polysaccharide components in the aloe can be better dissolved, thereby improving the extraction efficiency of aloe polysaccharide.

[0021] The solvent in step (2) is a mixture of water and 1-butyl-3-methylimidazolium chloride, and the volume ratio of the two is 8 - 9:1 - 2; preferably 8:2.

[0022] The mass volume of the solvent in step (2) and the aloe powder is 1 g:15 - 20 mL; preferably 1 g:18 mL.

[0023] The complex enzyme described in step (2) is a mixture of cellulase and pectinase, and the mass ratio of the two is 1:1-3; preferably 1:2.

[0024] The enzyme addition amount in step (2) is 0.12-0.2% of the mass of aloe powder; preferably, the enzyme addition amount is 0.18% of the mass of aloe powder.

[0025] The enzymolysis time described in step (2) is 15-25 minutes; preferably 20 minutes.

[0026] In the aloe extraction step of the present invention, water + ionic solution is used as the solvent. It is unexpectedly found that the addition of the ionic solution can not only make the solvent better penetrate the cell wall into the cell interior, so that more polysaccharides in the cells are dissolved in water, thereby improving the dissolution of polysaccharides, but also can reduce the addition amount of the complex enzyme, especially the addition amount of cellulase, so that the enzyme addition amount is 0.12-0.2% of the mass of aloe powder, and the mass ratio of cellulase and pectinase is 1:1-3, which can achieve a higher polysaccharide extraction efficiency.

[0027] However, it is found in the research process that the addition amount of the ionic solution will affect the purity of aloe polysaccharide, because the addition of the ionic solution will increase the solubility of flavonoid components. By controlling the volume ratio of water and 1-butyl-3-methylimidazolium chloride, controlling the addition ratio of the ionic solution can not only improve the dissolution degree of aloe polysaccharide, but also reduce the dissolution of flavonoid components, and improve the purity of the product on the basis of ensuring the dissolution degree of aloe polysaccharide.

[0028] The enzymolysis temperature described in step (2) is 40-50 °C, preferably 45 °C.

[0029] The power of the microwave extraction described in step (2) is 400-700 W, and the time is 1-3 min.

[0030] The microwave extraction in step (2) is intermittent extraction, and the specific operation is as follows:

[0031] Soak the aloe powder in the solvent and add the complex enzyme, turn on the microwave and microwave-treat at a power of 400-500 W for 1-3 minutes, stop the microwave treatment, wait for 5-10 minutes, then turn on the microwave and treat at a power of 600-700 W for 1-3 minutes, stop the microwave treatment, and repeat 1-2 times.

[0032] The present invention uses microwaves with different powers to treat aloe, so that different energy collisions and squeezes occur between molecules, thereby effectively dissolving polysaccharides with different molecular weights in the cells and improving the extraction of active ingredients.

[0033] In the alcohol precipitation step described in step (2), the volume fraction of ethanol is 90-95%, and the addition amount of ethanol is 5-8 times the volume of the concentrated solution.

[0034] In step (2), the rotation speed of the centrifugation is 4000 - 5000 r / min, and the time is 10 - 15 min.

[0035] The specific steps of ultrafiltration in step (3) are as follows: The filtrate is ultrafiltered through an ultrafiltration membrane with a molecular weight cut-off of 500 KDa, and the obtained dialysis solution is ultrafiltered again through an ultrafiltration membrane with a molecular weight cut-off of 50 KDa. The ultrafiltration is concentrated 10 - 30 times, and then concentrated 5 - 10 times with a nanofiltration membrane, thus obtaining an aloe polysaccharide concentrate with a molecular weight between 50 KDa and 500 KDa.

[0036] As some preferred embodiments, the solvent in the above step (2) further contains surfactants, and the surfactants are dodecyldimethylbetaine and hydroxypropyl-β-cyclodextrin, and the mass ratio of the two is 1:3 - 5.

[0037] The addition amount of the surfactant is 5% of the aloe powder.

[0038] In the implementation process of the present invention, it is found that adding surfactants in the extraction step can accelerate the extraction efficiency of polysaccharides. The use of surfactants can increase the speed of the solvent entering the cell wall, accelerate the dissolution of the cell wall, so that the active ingredients can be better dissolved, and the extraction efficiency is improved.

[0039] On the other hand, the present invention also provides aloe polysaccharide prepared by the above method.

[0040] On yet another aspect, the present invention also provides the application of the aloe polysaccharide prepared by the above method in the preparation of products with antibacterial efficacy.

[0041] An antibacterial composition, and the composition includes the aloe polysaccharide prepared by the above method.

[0042] The dosage form of the composition is a gel ointment, a cream, a powder or a tablet.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0044] (1) In the pretreatment step of the present invention, the aloe is first dried, then soaked in water and then freeze-dried. By controlling the freezing temperature and time, the cells can be swollen and ruptured, so that the polysaccharide components in the aloe can be better dissolved, thereby improving the extraction efficiency of aloe polysaccharide.

[0045] (2) In the aloe extraction step of the present invention, water + ionic solution is used as the solvent. It is unexpectedly found that the addition of the ionic solution can not only make the solvent better penetrate the cell wall into the interior of the cell, so that more polysaccharides in the cell dissolve in water, thereby improving the dissolution of polysaccharides, but also reduce the addition amount of the complex enzyme, especially the addition amount of cellulase, so that the enzyme addition amount is 0.12 - 0.2%, and the mass ratio of cellulase to pectinase is 1:1 - 3, which can achieve a relatively high polysaccharide extraction efficiency.

[0046] (3) During the research process, it is found that the addition amount of the ionic solution will affect the purity of aloe polysaccharide, because the addition of the ionic solution will increase the solubility of flavonoid components. By controlling the volume ratio of water to 1-butyl-3-methylimidazolium chloride, controlling the addition ratio of the ionic solution can not only improve the dissolution degree of aloe polysaccharide, but also reduce the dissolution of flavonoid components, and improve the purity of the product on the basis of ensuring the dissolution degree of aloe polysaccharide.

[0047] (4) During the implementation process of the present invention, it is unexpectedly found that in the extraction step, using dodecyl dimethyl betaine and hydroxypropyl-β-cyclodextrin with a mass ratio of 1:3 - 5 as surfactants and controlling the addition amount to 5% of the aloe powder can accelerate the polysaccharide extraction efficiency. The use of surfactants can increase the speed of the solvent entering the cell wall, accelerate the dissolution of the cell wall, so that the active ingredients dissolve better, and improve the extraction efficiency.

[0048] (5) The molecular weight of the aloe polysaccharide prepared by the present invention is between 50KDa - 500KDa, the O-acetyl group content in the aloe polysaccharide is above 30%, and it is unexpectedly found during the research process that the obtained aloe polysaccharide can selectively inhibit bacteria, has an inhibitory effect on harmful bacteria, and has no inhibitory effect on beneficial bacteria. Description of the Drawings

[0049] Figure 1 High performance gel permeation chromatogram of aloe polysaccharide prepared in Example 5;

[0050] Figure 2 Comparison diagram of antibacterial test of aloe polysaccharide prepared in Example 5 against Escherichia coli;

[0051] Among them, A, B, and C are respectively positive control tests; D is a negative control test. Detailed Embodiments

[0052] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the tables in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0053] Next, examples and comparative examples of the present invention are shown to describe the composition of the present invention in more detail, but the present invention is not limited thereto.

[0054] The present invention does not limit the source of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all common commercially available products in the technical field.

[0055] The aloe polysaccharide prepared in the present invention is named ACE, and specific details can be found in the examples and comparative examples.

[0056] Example 1 Preparation method of an aloe polysaccharide

[0057] It includes the following steps:

[0058] (1) Aloe pretreatment: Take fresh aloe leaves, wash them clean, remove thorns, cut them horizontally into slices 2-3 cm thick, dry them, then soak the dried aloe slices in water and freeze-dry them. Crush the dried aloe slices to obtain aloe powder;

[0059] The specific steps of freeze-drying are as follows: Place the soaked aloe slices in a freeze-dryer and quickly freeze them at -60°C for 5 hours, then carry out temperature-controlled sublimation drying in a vacuum environment of 40 Pa and -50°C, and the drying time is 28 hours.

[0060] (2) Soak 200 g of aloe powder in 1.5 L of solvent, add 0.24 g of composite enzyme, enzymatically hydrolyze at 40°C for 15 minutes, then turn on the microwave and microwave-treat at a power of 400 W for 3 minutes. Stop the microwave treatment. After 10 minutes, turn on the microwave and treat at a power of 600 W for 3 minutes. Stop the microwave treatment and repeat the microwave treatment 2 times to obtain an enzymolysis solution; Filter and concentrate the enzymolysis solution, then carry out alcohol precipitation with ethanol with a volume fraction of 90%, and the addition amount of ethanol is 8 times the volume of the concentrated solution. Centrifuge the precipitate at a speed of 4000 r / min for 15 minutes; Take the precipitate, dissolve it in water to obtain a crude polysaccharide aqueous solution;

[0061] The solvent is a mixture of water and 1-butyl-3-methylimidazolium chloride, and the volume ratio of the two is 9:1; The composite enzyme is a mixture of cellulase and pectinase, and the mass ratio of the two is 1:1;

[0062] (3) Ultrafilter the crude polysaccharide aqueous solution in step (2) through an ultrafiltration membrane with a molecular weight cut-off of 500 KDa. The obtained dialysis solution is ultrafiltered again with an ultrafiltration membrane with a molecular weight cut-off of 50 KDa, ultrafiltered and concentrated 20 times, and then concentrated 8 times with a nanofiltration membrane to obtain an aloe polysaccharide concentrate with a molecular weight between 50 KDa and 500 KDa. Decolorize and sterilize the aloe polysaccharide concentrate, then carry out freeze-drying and pulverize to obtain the aloe polysaccharide (ACE-1).

[0063] Example 2 A preparation method of aloe polysaccharide

[0064] It includes the following steps:

[0065] (1) Aloe pretreatment: Take fresh aloe leaves, wash them clean, remove the thorns, cut them horizontally into thin slices of 2-3 cm, dry them, then soak the dried aloe slices in water and freeze-dry them. Crush the dried aloe slices to obtain aloe powder;

[0066] The specific steps of the freeze-drying are as follows: Place the soaked aloe thin slices in a freeze-dryer and quickly freeze them at -80°C for 3 hours, then carry out temperature-controlled sublimation drying in a vacuum environment of 60 Pa and -50°C, and the drying time is 20 hours.

[0067] (2) Soak 200 g of aloe powder in 4 L of solvent, add 0.4 g of composite enzyme, enzymolyze at 50°C for 25 minutes, then turn on the microwave and microwave-treat at a power of 500 W for 1 minute, stop the microwave treatment. After 5 minutes, turn on the microwave and treat at a power of 700 W for 1 minute, stop the microwave treatment, and repeat the microwave treatment 2 times to obtain an enzymolysis solution; Filter and concentrate the enzymolysis solution, then carry out alcohol precipitation with ethanol with a volume fraction of 95%, the addition amount of ethanol is 5 times the volume of the concentrated solution, and centrifuge the precipitate at a speed of 5000 r / min for 10 minutes; Take the precipitate, dissolve it in water to obtain a crude polysaccharide aqueous solution;

[0068] The solvent is a mixture of water and 1-butyl-3-methylimidazolium chloride, and the volume ratio of the two is 8.5:1.5; The composite enzyme is a mixture of cellulase and pectinase, and the mass ratio of the two is 1:3;

[0069] (3) Ultrafilter the crude polysaccharide aqueous solution in step (2) through an ultrafiltration membrane with a molecular weight cut-off of 500 KDa, and then ultrafilter the obtained dialysis solution again through an ultrafiltration membrane with a molecular weight cut-off of 50 KDa. Ultrafilter and concentrate by 20 times, and then concentrate by 8 times with a nanofiltration membrane to obtain an aloe polysaccharide concentrate with a molecular weight between 50 KDa and 500 KDa. Decolorize and sterilize the aloe polysaccharide concentrate, and then carry out freeze-drying and crushing to obtain the aloe polysaccharide (ACE-2).

[0070] Example 3 A preparation method of aloe polysaccharide

[0071] It includes the following steps:

[0072] (1) Aloe pretreatment: Take fresh aloe leaves, wash them clean, remove the thorns, cut them horizontally into thin slices of 2-3 cm, dry them, then soak the dried aloe slices in water and freeze-dry them. Crush the dried aloe slices to obtain aloe powder;

[0073] The specific steps of freeze-drying are as follows: Place the soaked aloe vera slices in a freeze-dryer and quickly freeze them at -70°C for 4 hours. Then, carry out temperature-controlled sublimation drying in a vacuum environment of 50 Pa and -40°C for 24 hours.

[0074] (2) Soak 200 g of aloe vera powder in 3.6 L of solvent, add 0.36 g of composite enzyme, and carry out enzymatic hydrolysis at 45°C for 20 minutes. Then, turn on the microwave and carry out microwave treatment at a power of 400 W for 2 minutes. Stop the microwave treatment. After waiting for 5 minutes, turn on the microwave and treat it at a power of 700 W for 2 minutes. Stop the microwave treatment and repeat the microwave treatment 2 times to obtain an enzymatic hydrolysate. Filter and concentrate the enzymatic hydrolysate, and then carry out alcohol precipitation with ethanol having a volume fraction of 90%. The addition amount of ethanol is 6 times the volume of the concentrated solution, and the precipitate after centrifuging at a speed of 5000 r / min for 10 minutes is taken. Dissolve the precipitate in water to obtain a crude polysaccharide aqueous solution;

[0075] The solvent described is a mixture of water and 1-butyl-3-methylimidazolium chloride, and the volume ratio of the two is 8:2; the composite enzyme is a mixture of cellulase and pectinase, and the mass ratio of the two is 1:2;

[0076] (3) Ultrafilter the crude polysaccharide aqueous solution in step (2) through an ultrafiltration membrane with a molecular weight cut-off of 500 KDa. The obtained dialysis solution is ultrafiltered again with an ultrafiltration membrane with a molecular weight cut-off of 50 KDa, ultrafiltered and concentrated 20 times, and then concentrated 8 times with a nanofiltration membrane to obtain an aloe vera polysaccharide concentrated solution with a molecular weight between 50 KDa and 500 KDa. After decolorizing and sterilizing the aloe vera polysaccharide concentrated solution, carry out freeze-drying and pulverize it to obtain the aloe vera polysaccharide (ACE-3).

[0077] Example 4 A method for preparing aloe vera polysaccharide

[0078] The difference from Example 3 is only that a surfactant is added to the solvent in step (2). The surfactant is dodecyldimethylbetaine and hydroxypropyl-β-cyclodextrin, and the mass ratio of the two is 1:3; the addition amount of the surfactant is 10 g. Others are the same as in Example 3, and finally the aloe vera polysaccharide (ACE-4) is obtained.

[0079] Example 5 A method for preparing aloe vera polysaccharide

[0080] The difference from Example 3 is only that a surfactant is added to the solvent in step (2). The surfactant is dodecyldimethylbetaine and hydroxypropyl-β-cyclodextrin, and the mass ratio of the two is 1:4; the addition amount of the surfactant is 10 g. Others are the same as in Example 3, and finally the aloe vera polysaccharide (ACE-5) is obtained.

[0081] The aloe polysaccharide obtained in this example was detected by high performance gel permeation chromatography (HPGPC), as shown in Figure 1 , where two main peaks are shown in the figure, labeled as Mw (weight-average molecular weight) and Mn (number-average molecular weight) respectively. Both can characterize the molecular weight of the tested aloe polysaccharide. Generally, the narrower the peak, the more uniform the molecular weight distribution.

[0082] Example 6 A method for preparing aloe polysaccharide

[0083] The difference from Example 3 is only that a surfactant is added to the solvent in step (2). The surfactants are dodecyldimethylbetaine and hydroxypropyl-β-cyclodextrin, and the mass ratio of the two is 1:5; the addition amount of the surfactant is 10 g. Others are the same as in Example 3, and finally the aloe polysaccharide (ACE-6) is obtained.

[0084] Comparative Example 1

[0085] The difference from Example 3 is that in step (2), the solvent is only water and 1-butyl-3-methylimidazolium chloride is not added. Others are the same as in Example 3, and finally the aloe polysaccharide (ACE-7) is obtained.

[0086] Comparative Example 2

[0087] The difference from Example 3 is that the volume ratio of water to 1-butyl-3-methylimidazolium chloride is 7:3. Others are the same as in Example 3, and finally the aloe polysaccharide (ACE-8) is obtained.

[0088] Comparative Example 3

[0089] The difference from Example 3 is that the microwave treatment conditions in step (2) are different, that is, the microwave is turned on and microwave-treated at a power of 500 W for 2 minutes, the microwave treatment is stopped, after 5 minutes, the microwave is turned on and treated at a power of 500 W for 2 minutes, the microwave treatment is stopped, and the microwave treatment is repeated 2 times. Others are the same as in Example 3, and finally the aloe polysaccharide (ACE-9) is obtained.

[0090] Comparative Example 4

[0091] The difference from Example 3 is that the microwave treatment conditions in step (2) are different, that is, the microwave is turned on and microwave-treated at a power of 200 W for 2 minutes, the microwave treatment is stopped, after 5 minutes, the microwave is turned on and treated at a power of 800 W for 2 minutes, the microwave treatment is stopped, and the microwave treatment is repeated 2 times. Others are the same as in Example 3, and finally the aloe polysaccharide (ACE-10) is obtained.

[0092] Comparative Example 5

[0093] The difference from Example 3 is that microwave treatment is omitted. That is, 200 g of aloe powder is soaked in 3.6 L of solvent, 0.36 g of composite enzyme is added, and enzymatic hydrolysis is carried out at 45 °C for 20 minutes to obtain an enzymatic hydrolysate; after filtering and concentrating the enzymatic hydrolysate, ethanol precipitation is carried out with ethanol having a volume fraction of 90%, and the addition amount of ethanol is 6 times the volume of the concentrated solution, and the precipitate after centrifuging at a speed of 5000 r / min for 10 minutes is taken; the precipitate is dissolved in water to obtain a crude polysaccharide aqueous solution; the others are the same as in Example 3, and the aloe polysaccharide (ACE-11) is finally obtained.

[0094] Comparative Example 6

[0095] The difference from Example 5 is that the surfactant is only dodecyl dimethyl betaine, and the others are the same as in Example 5, and the aloe polysaccharide (ACE-12) is finally obtained.

[0096] Effect experiment:

[0097] 1. Yield and purity detection of aloe polysaccharide

[0098] The calculation method of the yield is: yield% = (mass of the finally obtained aloe polysaccharide powder / mass of aloe powder) × 100%; purity: detected by high performance gel permeation chromatography (HPGPC). The specific detection method is: using dextran as a standard sample, two columns of Agilent PL aquagel-OH 60 (8 μm, 300 mm × 7.5 mm) and Agilent PL aquagel-OH40 (8 μm, 300 mm × 7.5 mm) are connected in series, purified water is used as the mobile phase, and the flow rate is 0.6 mL·min -1 , the column temperature is 30 °C; detected by a differential refractive index detector (RID), the detection temperature is 40 °C, and the injection volume is 50 μL. The detection results are shown in Table 1 below.

[0099] Table 1

[0100] Yield % Purity % Example 1 (ACE-1) 6.53 87.1 Example 2 (ACE-2) 6.62 86.9 Example 3 (ACE-3) 6.81 88.2 Example 4 (ACE-4) 6.89 88.4 Example 5 (ACE-5) 6.94 89.2 Example 6 (ACE-6) 6.85 88.6 Comparative Example 1 (ACE-7) 5.65 87.5 Comparative Example 2 (ACE-8) 6.88 73.4 Comparative Example 3 (ACE-9) 5.84 82.5 Comparative Example 4 (ACE-10) 5.62 79.2 Comparative Example 5 (ACE-11) 4.85 87.5 Comparative Example 6 (ACE-12) 6.80 86.8

[0101] According to the detection results in Table 1 above, it can be known that:

[0102] In Examples 1-3, the same method was used for the extraction of aloe polysaccharide, and the yields of the obtained aloe polysaccharides were all above 6.5%, and the purities were all above 86%. In particular, in Example 3, the processing parameters of each step were controlled and the ratio between components was reasonably controlled, which significantly improved the extraction effect of aloe polysaccharide, making the yield of aloe polysaccharide reach 6.81% and the purity reach 87.5%.

[0103] Example 4 - 5: Based on Example 3, surfactants were added to the solvent in step (2). Dodecyl dimethyl betaine and hydroxypropyl - β - cyclodextrin with mass ratios of 1:3, 1:4, and 1:5 were used as surfactants respectively, and the addition amount was controlled at 5% of the aloe powder, which significantly improved the extraction efficiency of aloe polysaccharide. Especially in Example 5, when dodecyl dimethyl betaine and hydroxypropyl - β - cyclodextrin with a mass ratio of 1:5 were used as surfactants, the yield of aloe polysaccharide reached 6.94% and the purity reached 89.2%. The reason is that the use of surfactants can increase the speed of the solvent entering the cell wall, accelerate the dissolution of the cell wall, so that the active ingredients can be better dissolved, and the extraction efficiency is improved.

[0104] In Comparative Example 1, only water was used as the solvent, and the effect of the solvent penetrating the cell wall was weakened, which would affect the dissolution of polysaccharides in the cells, reduce the extraction effect of aloe polysaccharide, and thus lead to a decrease in the yield of aloe polysaccharide, but had little effect on the purity.

[0105] In Comparative Example 2, the volume ratio of water to 1 - butyl - 3 - methylimidazolium chloride was changed to 7:3, which had little effect on the yield of aloe polysaccharide, but significantly affected the purity of aloe polysaccharide, resulting in a significant decrease in purity. Because the addition of ionic solution would increase the solubility of flavonoid components, thus affecting the purity of aloe polysaccharide.

[0106] In Comparative Examples 3 - 4, changing the treatment power of microwave would affect the dissolution effect of polysaccharides and the dissolution of other components, resulting in different degrees of decrease in the yield and purity of aloe polysaccharide.

[0107] In Comparative Example 5, not using microwave treatment would seriously affect the dissolution of aloe polysaccharide and significantly reduce the yield.

[0108] In Comparative Example 6, only one surfactant was used for treatment, which would affect the dissolution of aloe polysaccharide to a certain extent, and thus affect the yield and purity.

[0109] 2. Detection of O - acetyl group content in aloe polysaccharide

[0110] Detection method: Spectrophotometry was used for detection. For the specific detection method, refer to "Determination Method of O - acetyl Group Content in Aloe Products, Yunnan Yuanjiang Wanlv Biology (Group) Co., Ltd., Luo Bingjun et al., "Aloe Industry".

[0111] The detection principle is as follows:

[0112] By using the acetyl groups of acetylated mannan in aloe products, in an alkaline hydroxylamine solution, a complex containing acetyl groups can be formed, which reacts with a ferric chloride-hydrochloric acid solution under acidic conditions to condense into a colored compound (the color of aloe products is generally light yellow-brown, and the rest of the color development is the normal color development of non-aloe O-acetyl groups). There is an absorption peak at 540 nm, and the O-acetyl group content (mass-volume percentage) can be determined by the absorbance. The test results are shown in Table 2 below.

[0113] Table 2

[0114] O-acetyl content % Example 1 (ACE-1) 31.3 Example 2 (ACE-2) 31.5 Example 3 (ACE-3) 32.3 Example 4 (ACE-4) 32.9 Example 5 (ACE-5) 33.4 Example 6 (ACE-6) 32.6 Comparative Example 1 (ACE-7) 30.5 Comparative Example 2 (ACE-8) 28.5 Comparative Example 3 (ACE-9) 26.1 Comparative Example 4 (ACE-10) 26.3 Comparative Example 5 (ACE-11) 30.3 Comparative Example 6 (ACE-12) 30.1

[0115] It can be seen from the test results in Table 2 above that the O-acetyl group content in the aloe polysaccharides obtained by the methods provided in Examples 1-6 of the present invention is all above 30%. In Comparative Example 1, water was used as the solvent, and although the O-acetyl group content in the obtained aloe polysaccharide was above 30%, the yield of the aloe polysaccharide was relatively low; in Comparative Example 2, the volume ratio of water to 1-butyl-3-methylimidazolium chloride was changed to 7:3, resulting in a decrease in the purity of the aloe polysaccharide, and further causing the O-acetyl group content in the aloe polysaccharide to decrease to below 30%; in Comparative Examples 3-4, the treatment power of microwave was changed, also resulting in a decrease in the purity of the aloe polysaccharide, and further causing the O-acetyl group content in the aloe polysaccharide to decrease to below 30%; in Comparative Examples 5-6, although the O-acetyl group content in the obtained aloe polysaccharide was above 30%, the yield of the aloe polysaccharide was also relatively low. Therefore, only the aloe polysaccharide obtained by the method provided in the present invention has relatively high yields, purities, and O-acetyl group contents.

[0116] 3. Detection of the antibacterial effect of aloe polysaccharide

[0117] Detection method:

[0118] For Staphylococcus aureus, Escherichia coli, Lactobacillus, Saccharomyces cerevisiae, and Streptococcus, the carrier immersion quantitative antibacterial test in WS / T 650-2019 "Evaluation of Antibacterial and Bacteriostatic Effects" 5.1.2 was used: The test product was prepared into a mass fraction of 3%, the bacterial liquid concentration was 1.6 - 1.8×10 7 CFU / mL, the action time was 2 min. If the inhibition rate was between 50 - 90%, it was determined to have an antibacterial effect, indicated by the symbol "+"; if the inhibition rate was greater than 90%, it was determined to have a strong antibacterial effect, indicated by the symbol "++"; if the inhibition rate was less than 10%, it was determined to have no obvious antibacterial effect, indicated by the symbol "-".

[0119] Antibacterial rate (%) = (number of colonies in the control group - number of colonies in the experimental control group) / number of colonies in the control group * 100; Each group of experiments was repeated at least 3 times, and the average value was taken.

[0120] The test results are shown in Table 3 below.

[0121] Table 3

[0122]

[0123] According to the detection results in the above Table 3, it can be seen that the antibacterial rates of the aloe polysaccharide samples prepared in Examples 1-6 of the present invention against Staphylococcus aureus and Escherichia coli both reach more than 90%, and both have strong antibacterial effects, while there is no obvious antibacterial effect on Lactobacillus, Saccharomyces cerevisiae and Streptococcus.

[0124] In addition, according to Figure 2 it can also be seen that the number of colonies in the three groups of positive control groups (4 samples in each group) is significantly less than that in the negative control group, indicating that the extracted aloe polysaccharide has an obvious inhibitory effect on Escherichia coli.

[0125] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing aloe polysaccharide, characterized in that: The steps include: (1) Aloe pretreatment: fresh aloe leaves are washed and thorn-free, sliced ​​and dried, and then the dried aloe slices are soaked in water and freeze-dried, and the dried aloe slices are crushed to obtain aloe powder; (2) soaking aloe vera powder in a solvent, adding a composite enzyme for enzymolysis, then performing microwave extraction, filtering and concentrating the extract, and then performing alcohol precipitation, and centrifuging the precipitate; taking the precipitate and dissolving it in water to obtain a crude polysaccharide aqueous solution; (3) ultrafiltration of the crude polysaccharide aqueous solution in step (2) through an ultrafiltration membrane to obtain an aloe polysaccharide concentrate with a molecular weight of 50 KDa-500 KDa, decolorizing and sterilizing the aloe polysaccharide concentrate, freeze-drying it, and crushing it to obtain the aloe polysaccharide; The solvent is a mixture of water and 1-butyl-3-methylimidazolium chloride; the power of microwave extraction is 400-700W, and the time is 1-3min; the microwave extraction is intermittent extraction.

2. The preparation method according to claim 1, characterized in that: The specific steps of freeze drying described in step (1) are: placing the soaked aloe slices in a freeze dryer for quick freezing at -60--80°C for 3-5 hours, and then performing temperature-controlled sublimation drying at 40-60Pa and -30°C--50°C vacuum environment for 20-28 hours.

3. The preparation method according to claim 1, characterized in that: The volume ratio of water and 1-butyl-3-methylimidazolium chloride in step (2) is 8-9:1-2; the mass volume of the solvent and the aloe powder is 1g:15-20mL.

4. The preparation method according to claim 3, characterized in that: The volume ratio of water and 1-butyl-3-methylimidazolium chloride in step (2) is 8:2; the mass volume of the solvent and the aloe powder is 1g:18mL.

5. The preparation method according to claim 1, characterized in that: The complex enzyme in step (2) is a mixture of cellulase and pectinase, and the mass ratio of the two is 1:1-3; the amount of enzyme added is 0.12-0.2% of the mass of the aloe powder.

6. The preparation method according to claim 5, characterized in that: The complex enzyme described in step (2) is a mixture of cellulase and pectinase, and the mass ratio of the two is 1:2; the amount of enzyme added is 0.18% of the mass of the aloe powder.

7. The preparation method according to claim 1, characterized in that: The intermittent extraction is specifically performed as follows: Soak aloe vera powder in a solvent, add complex enzyme, turn on the microwave and process it at a power of 400-500 W for 1-3 minutes, stop the microwave treatment, wait for 5-10 minutes, turn on the microwave and process it at a power of 600-700 W for 1-3 minutes, stop the microwave treatment, and repeat 1-2 times.

8. The preparation method according to claim 7, characterized in that: The intermittent extraction is specifically performed as follows: Soak aloe vera powder in a solvent, add complex enzyme, turn on the microwave and process it at 400 W for 2 minutes, stop the microwave treatment, wait for 5 minutes, turn on the microwave and process it at 700 W for 2 minutes, stop the microwave treatment, and repeat the microwave treatment twice.

9. The preparation method according to claim 1, characterized in that: The specific steps of ultrafiltration described in step (3) are as follows: ultrafiltration is performed on the filtrate through an ultrafiltration membrane with a molecular weight cutoff of 500 KDa, and the dialysate is ultrafiltered again with an ultrafiltration membrane with a molecular weight cutoff of 50 KDa, and the ultrafiltration is concentrated by 10-30 times, and then concentrated by 5-10 times with a nanofiltration membrane to obtain an aloe polysaccharide concentrate with a molecular weight within 50 KDa-500 KDa.

10. The preparation method according to claim 1, characterized in that: The solvent described in step (2) also contains a surfactant.

11. The preparation method according to claim 10, characterized in that: The surfactant is dodecyl dimethyl betaine and hydroxypropyl-β-cyclodextrin, and the mass ratio of the two is 1:3-5.

12. The preparation method according to claim 10, characterized in that: The added amount of the surfactant is 5% of the mass of the aloe powder.

13. Aloe polysaccharide prepared by the method according to any one of claims 1 to 12.

14. Use of the aloe polysaccharide prepared by the preparation method according to any one of claims 1 to 12 in preparing products with antibacterial efficacy.

15. A composition with antibacterial effect, characterized in that: The composition comprises aloe polysaccharide prepared by the preparation method according to any one of claims 1 to 12.

16. The composition according to claim 15, characterized in that: The dosage form of the composition is a gel, cream, powder or tablet.

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