CN114230684A - Purification method of alpha-1, 2 glucan, alpha-1, 2 glucan and application thereof - Google Patents
Purification method of alpha-1, 2 glucan, alpha-1, 2 glucan and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
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- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Abstract
A purification method of alpha-1, 2 glucan, alpha-1, 2 glucan and application thereof belong to the field of enzyme engineering and comprise the following steps: cracking blue algae, and collecting supernatant containing alpha-1, 2 glucan; adding ethanol, standing, collecting alpha-1, 2 glucan precipitate, and freeze-drying; dissolving the precipitate in water, dialyzing, adding ethanol, standing, collecting the precipitate of alpha-1, 2-glucan, and lyophilizing; DEAE-cellulose ion exchange chromatography; detecting the peak position of alpha-1, 2 glucan by thin layer chromatography; mixing the elution peaks containing alpha-1, 2 glucan, adding ethanol, standing, collecting alpha-1, 2 glucan precipitate, and freeze-drying; gel filtration chromatography; detecting the peak position of alpha-1, 2 glucan by thin layer chromatography; mixing the elution peaks containing alpha-1, 2 glucan, adding ethanol, standing, collecting alpha-1, 2 glucan precipitate, and freeze-drying; dissolving with water to obtain purified alpha-1, 2 glucan. The purification method is suitable for large-scale industrial purification of alpha-1, 2 glucan.
Description
Technical Field
The invention belongs to the technical field of enzyme engineering, and particularly relates to a purification method of alpha-1, 2 glucan, the alpha-1, 2 glucan and application thereof.
Background
Two articles (FEBS J.2006 Jan; 273(1):137-49. and Plant Science 172(2007) 29-35) have disclosed methods for purifying alpha-1, 2 glucan, but these methods use many expensive purification materials, including Bio-Gel P2(2cm 230cm), Bio-Gel P4(2.05cm 191cm), CarboPac PA1 column (4mm 250mm), Glass fiber filters (MN GF-1, Macherey-Nagel Co.), and the like. The chromatography columns or materials are very expensive, the amount of the separated alpha-1, 2 glucan is very small, only the alpha-1, 2 glucan with the gram level or below can be obtained, and the method is only suitable for small-scale production in a laboratory and is not suitable for industrial mass production. In addition, the purification method steps reported above and their complexity are not suitable for industrial production.
Up to now, no method suitable for the extraction and purification of α -1,2 glucan in large quantities has been disclosed, which is disadvantageous for the practical application of α -1,2 glucan.
Disclosure of Invention
The invention aims to provide a purification method of alpha-1, 2 glucan, the alpha-1, 2 glucan and application thereof.
The technical scheme adopted by the invention for solving the technical problem is as follows:
the purification method of the alpha-1, 2 glucan comprises the following steps:
mixing blue algae and water, and then carrying out cracking treatment, wherein the weight ratio of the blue algae to the water is 1: 10-10: 1;
step two, collecting the supernatant containing alpha-1, 2 glucan;
adding 90-100% ethanol into the supernatant, wherein the volume ratio of the supernatant to the ethanol is 1: 10-10: 1, standing for 1-48 h, and precipitating alpha-1, 2 glucan;
step four, collecting alpha-1, 2 glucan precipitate and freeze-drying;
dissolving the freeze-dried alpha-1, 2 glucan precipitate in water, and dialyzing in a dialysis membrane for 24-48 h;
step six, adding 90-100% ethanol into the solution, wherein the volume ratio of the solution to the ethanol is 1: 10-10: 1, standing for 1-48 hours, and precipitating alpha-1, 2 glucan;
collecting alpha-1, 2 glucan precipitate and freeze-drying;
step eight, removing impurities by DEAE-cellulose ion exchange chromatography, and separating alpha-1, 2 glucan;
step nine, detecting the peak position of the alpha-1, 2 glucan in DEAE-cellulose ion exchange chromatography by thin layer chromatography;
mixing elution peaks containing alpha-1, 2 glucan, adding 90-100% ethanol into the mixed solution, wherein the volume ratio of the mixed solution to the ethanol is 1: 10-10: 1, and standing for 1-48 hours to precipitate the alpha-1, 2 glucan;
step eleven, collecting alpha-1, 2 glucan precipitate and freeze-drying;
step twelve, further removing impurities by gel filtration chromatography, and separating and purifying the alpha-1, 2 glucan;
thirteen, detecting the peak position of the alpha-1, 2 glucan in the gel filtration chromatography by thin layer chromatography;
fourteen, mixing elution peaks containing alpha-1, 2 glucan, adding 90-100% ethanol into the mixed solution, wherein the volume ratio of the mixed solution to the ethanol is 1: 10-10: 1, standing for 1-48 hours, and precipitating the alpha-1, 2 glucan;
fifteen, collecting alpha-1, 2 glucan precipitate and freeze-drying; dissolving the freeze-dried alpha-1, 2 glucan precipitate in water to obtain purified alpha-1, 2 glucan.
In a preferred embodiment, in the first step, the method of the lysis treatment comprises: heating at 80-200 ℃ for 10 min-4 h, or carrying out ultrasonic crushing for 5-60 min by using an ultrasonic crusher with the power set to 10-80%, or carrying out high-pressure crushing by using an ultrahigh-pressure continuous flow cell crusher with the pressure set to 50-200 Mpa.
In a preferred embodiment, in the third step, 90% ethanol is added into the supernatant, the volume ratio of the supernatant to the 90% ethanol is 1:2, and the mixture is left for 1 hour to precipitate the alpha-1, 2 glucan.
In a preferred embodiment, in the sixth step, the solution is an aqueous solution outside the dialysis bag, and the aqueous solution contains alpha-1, 2 glucan; adding 90% ethanol into the solution, wherein the volume ratio of the solution to the 90% ethanol is 1:2, and standing for 1h to precipitate alpha-1, 2 glucan.
In step eight, the DEAE-cellulose ion exchange chromatography is performed by the following steps:
DEAE-cellulose was subjected to pretreatment before use according to the specification of DEAE-cellulose; dissolving the alpha-1, 2 glucan precipitate, and centrifuging to remove insoluble precipitate; loading the supernatant into a DEAE-cellulose ion exchange column; cleaning a DEAE-cellulose ion exchange column by double distilled water at the flow rate of 1 ml/min; the elution method is sodium chloride gradient elution, the concentration of sodium chloride is 0-0.5M, the flow rate is 1ml/min, the sodium chloride is collected every 5ml, and the collected samples are detected by thin layer chromatography.
In the step nine and the step thirteen, the specific operation steps of the thin layer chromatography are as follows:
cutting the silica gel plate with four parallel sides; marking a line of the pencil, wherein the distance from the lower edge is 10 mm; loading sample by a capillary tube, loading 1-10 mu l of sample, vertically loading the sample, and naturally drying; developing with a developing agent, wherein the developing agent consists of n-butanol, acetone and water in a volume ratio of 4:3:1, the developing agent is not higher than the lower edge marking line, and after the development is finished, taking out and naturally drying; and (2) putting the silica gel plate into a color developing agent which is composed of 2% aniline acetone solution, 2% diphenylamine acetone solution and 85% phosphoric acid in a volume ratio of 5:5:1, removing, naturally drying, and putting the silica gel plate into an oven at 70-85 ℃ for color development for 10-15 min.
In a preferred embodiment, in step twelve, the specific operation steps of the gel filtration chromatography are as follows:
pretreating gel filtration column Sepharose CL-6B before use according to the specification of gel filtration column Sepharose CL-6B; dissolving the alpha-1, 2 glucan precipitate, and centrifuging to remove insoluble precipitate; loading the supernatant onto gel filtration column Sepharose CL-6B; eluting with 0.15M sodium chloride at flow rate of 2ml/min, collecting every 5ml, and detecting the collected sample by thin layer chromatography.
In a preferred embodiment, in the fourteenth step, the elution peak containing α -1, 2-glucan is mixed, 90% ethanol is added to the mixture, the volume ratio of the mixture to the 90% ethanol is 1:2, and the mixture is left to stand for 1 hour to precipitate α -1, 2-glucan.
The invention also provides alpha-1, 2 glucan obtained by the purification method.
The invention also provides application of the alpha-1, 2 glucan obtained in the step of preparing a food thickener, a food additive, a blood sugar reducing product, a blood fat reducing product, a blood pressure reducing product, a fatty liver eliminating product, an intestinal flora regulating product, a moisturizing cosmetic, a wound healing promoting cosmetic, an ultraviolet-resistant cosmetic, an anti-aging cosmetic, an antioxidant cosmetic, an immunity improving product, a sleep quality improving product, an anti-tumor product, an anti-cancer medicine, a sticking agent, mucus, glue and paste.
The invention has the beneficial effects that:
according to the purification method of alpha-1, 2 glucan, the purification materials mainly used comprise DEAE-cellulose, Sepharose CL-6B, ethanol and the like, the price of the materials is very low, and the cost for producing and purifying the alpha-1, 2 glucan is greatly reduced. The cost of purifying alpha-1, 2 glucan according to the invention is calculated to be only about 10% of the cost of the two articles in the background art. In addition, the experimental method is simple and is suitable for industrial production.
The purification method of alpha-1, 2 glucan has the advantages of simple method, simple and convenient operation, low prices of the adopted chromatographic column and experimental materials, and capability of purifying a large amount of alpha-1, 2 glucan.
The invention extracts and purifies the alpha-1, 2 glucan from the blue algae, and the obtained alpha-1, 2 glucan is applied to the food industry, the health product industry, the cosmetic industry, the pharmaceutical industry and the industry, and obtains better application effect.
The purified alpha-1, 2 glucan prepared by the invention can form a transparent, elastic and breathable film on the skin, can effectively isolate the damage of harmful substances in the environment to the skin, can fully lock water by a macromolecular structure, prevents water loss, and has high-efficiency moisture retention, so that the skin is moist and smooth.
The purified alpha-1, 2 glucan prepared by the invention is completely composed of glucose molecules, has certain sweetness and can be directly eaten as food; meanwhile, the mixture of the purified alpha-1, 2 glucan and water prepared by the invention has certain viscosity and can be used as a food thickener and a food additive.
Because the animal body is lack of enzymes for digesting alpha-1, 2 glycosidic bonds in the alpha-1, 2 glucan, glucose molecules are slowly released after the purified alpha-1, 2 glucan prepared by the invention is eaten, so that the blood sugar can be reduced, the blood fat can be reduced, the fatty liver can be eliminated, and the blood pressure can be reduced; in addition, the purified alpha-1, 2 glucan prepared by the invention has the function of regulating the species and the composition of intestinal bacteria, can increase the number of beneficial bacteria, reduce the number of harmful bacteria, relieve flatulence and treat dyspepsia.
The purified alpha-1, 2 glucan prepared by the invention has the effects of moisturizing, ultraviolet resistance, aging resistance and oxidation resistance, and can be used as a component of cosmetics.
The purified alpha-1, 2 glucan prepared by the invention has the effect of promoting wound healing, and can be prepared into ointments, wound dressings and the like.
The purified alpha-1, 2 glucan prepared by the invention has the function of regulating immunity, and can improve immunity, treat tumors, treat cancers, resist oxidation and improve sleep quality after being prepared into injection or tablets and injected subcutaneously or orally.
The mixture of the purified alpha-1, 2 glucan and water prepared by the invention has viscosity and can be used for preparing adhesive, mucilage, glue, paste and the like.
Drawings
FIG. 1 is a photograph of cyanobacteria collected in example 1.
Figure 2 is a photograph of the lyophilized alpha-1, 2 glucan precipitate collected in example 1.
FIG. 3 shows the DEAE-cellulose ion exchange chromatography results of α -1, 2-glucan in example 1. In the figure, 3 to 13 represent the 3-sugar to 13-sugar, respectively.
FIG. 4 is a Thin Layer Chromatography (TLC) profile of α -1,2 glucan in example 1.
FIG. 5 is a gel filtration chromatography (Sepharose CL-6B) pattern of α -1,2 glucan in example 1. In the figure, 3 to 13 represent the 3-sugar to 13-sugar, respectively.
FIG. 6 is a photograph of the purified α -1,2 glucan of example 1.
FIG. 7 shows the results of the hypoglycemic functionality test of alpha-1, 2 glucan in example 3.
FIG. 8 shows the results of the blood lipid-lowering functional test of alpha-1, 2 glucan in example 3.
FIG. 9 shows the results of fatty liver function elimination test of α -1,2 glucan in example 3.
FIG. 10 shows the results of the blood pressure lowering functionality test of alpha-1, 2 glucan in example 3.
FIG. 11 shows the results of functional tests of alpha-1, 2 glucan for regulating the composition of intestinal bacterial species in example 3.
FIG. 12 shows the results of the moisturizing functionality test of α -1,2 glucan in example 4.
FIG. 13 shows the results of the wound healing promoting functional test of alpha-1, 2 glucan in example 4.
FIG. 14 shows the results of the sleep time improving functional test of the α -1,2 glucan group in example 5.
FIG. 15 shows the results of functional tests of α -1,2 glucan for cancer treatment and tumor treatment in example 5.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 extraction and purification of alpha-1, 2 Glucan
(1) Obtaining blue algae from nature, culture pond or laboratory. The water in rivers, lakes and seas can be used for culturing blue algae; the culture medium for culturing blue algae in culture pond or laboratory is BG11 culture medium (see Ricppka, R.1988.isolation and purification of cyanobacteria. methods Enzymol.167: 3-27.) and is cultured at room temperature under normal sunlight.
(2) The blue-green algae is collected by carrying out stress treatment (high salt stress (0.1M-4M sodium chloride, 12-48 hours), high temperature stress (40-60 ℃, 12-48 hours), high pressure stress (2-10 atmospheric pressure, 12-48 hours) or low temperature stress (0.5-10 ℃, 12-48 hours) on the blue-green algae or not carrying out any treatment, and adopting a centrifugation (centrifugal force 3000 plus 6000g) or filtration (0.2-1 mu M filter membrane) method, wherein the collected blue-green algae is shown in figure 1.
(3) Adding water into the collected blue algae, mixing the blue algae and the water according to the weight ratio of 1:1, and heating the mixture for 10 minutes at 100 ℃.
(4) Separating the supernatant from the precipitate by centrifugation or filtration, and collecting the supernatant containing alpha-1, 2-glucan.
(5) Adding 90% ethanol into the supernatant containing the alpha-1, 2 glucan, wherein the volume ratio of the supernatant to the 90% ethanol is 1:2, and standing for 1 hour to precipitate the alpha-1, 2 glucan.
(6) Separating the supernatant from the alpha-1, 2 glucan precipitate by centrifugation or filtration, collecting the alpha-1, 2 glucan precipitate, and lyophilizing the alpha-1, 2 glucan precipitate, wherein the collected lyophilized alpha-1, 2 glucan precipitate is shown in FIG. 2: the collected lyophilized alpha-1, 2 glucan precipitate was yellow-white and appeared to be somewhat sticky.
(7) Adding a proper amount of water, dissolving the freeze-dried alpha-1, 2 glucan precipitate, and dialyzing in a dialysis membrane (cut off is more than or equal to 3kDa) for 24 hours.
(8) Water is dialyzed as a dialysate. Alpha-1, 2-glucan having a molecular weight of less than 3kDa is dispersed from the dialysis bag and dissolved in the dialysate.
(9) Adding 90% ethanol into the solution, wherein the volume ratio of the solution to the 90% ethanol is 1:2, and standing for 1 hour to precipitate alpha-1, 2 glucan.
(10) After the alpha-1, 2 glucan is freeze-dried and precipitated, a proper amount of water is added to dissolve the alpha-1, 2 glucan precipitate.
(11) The results of DEAE-cellulose ion exchange chromatography for separating α -1, 2-glucan and α -1, 2-glucan by removing impurities using DEAE-cellulose ion exchange chromatography are shown in FIG. 3, in which 3 to 13 represent 3-13 sugars, respectively. As can be seen from fig. 3: DEAE-cellulose ion exchange chromatography can be used to purify alpha-1, 2 glucan and can separate different sizes of alpha-1, 2 glucan.
The DEAE-cellulose ion exchange chromatography comprises the following specific operation steps:
DEAE-cellulose was subjected to pretreatment before use according to the specification of DEAE-cellulose; dissolving the alpha-1, 2 glucan precipitate, and centrifuging to remove insoluble precipitate; loading the supernatant into a DEAE-cellulose ion exchange column; cleaning a DEAE-cellulose ion exchange column by double distilled water at the flow rate of 1 ml/min; the elution method comprises gradient elution with sodium chloride at concentration of 0-0.5M and flow rate of 1ml/min, collecting every 5ml, and detecting the collected sample by Thin Layer Chromatography (TLC).
(12) Thin Layer Chromatography (TLC) was used to detect the peak position of α -1, 2-glucan on DEAE-cellulose ion exchange chromatography. The collected sample was subjected to Thin Layer Chromatography (TLC) after DEAE-cellulose ion exchange chromatography, and its TLC pattern is shown in FIG. 4: sugars 3, 4 and 5 could be developed on TLC and could be well separated; from the depth of the band, it can be seen that the amount of 3 saccharides is more than that of other oligosaccharides. Other high molecular weight alpha-1, 2 glucans than the 3-, 4-and 5-saccharides do not perform well due to limitations of TLC technology.
The Thin Layer Chromatography (TLC) comprises the following specific operation steps:
cutting a silica gel plate with a proper size, wherein four sides are parallel; pencil lines (take care not to break the silicone, light lines) approximately 10mm from the lower edge; loading sample by a capillary tube, loading 1-10 mu l of sample, vertically loading the sample, and naturally drying; developing with a developing agent (the volume ratio of n-butanol to acetone to water is 4:3:1), wherein the developing agent is not higher than the lower edge line, and taking out and naturally drying after the development is finished; and (3) completely immersing the silica gel plate in a color developing agent (2% aniline acetone solution, 2% diphenylamine acetone solution and 85% phosphoric acid in a volume ratio of 5:5:1), removing, naturally drying, and placing in an oven at 70-85 ℃ for color development for 10-15 min.
(13) Mixing elution peaks containing alpha-1, 2 glucan, adding 90% ethanol into the mixed solution, wherein the volume ratio of the mixed solution to the 90% ethanol is 1:2, and standing for 1 hour to precipitate the alpha-1, 2 glucan.
(14) After the alpha-1, 2 glucan is freeze-dried and precipitated, a proper amount of water is added to dissolve the alpha-1, 2 glucan precipitate.
(15) Further removing impurities by gel filtration chromatography (Sepharose CL-6B), and separating and purifying alpha-1, 2-glucan, wherein the eluent is sodium chloride with the concentration of 0.15M. The gel filtration chromatography (Sepharose CL-6B) pattern is shown in FIG. 5: gel filtration chromatography can be used to purify alpha-1, 2 glucan and can separate alpha-1, 2 glucans of different molecular weights.
The gel filtration chromatography (Sepharose CL-6B) comprises the following specific operation steps:
pretreating gel filtration column Sepharose CL-6B before use according to the specification of gel filtration column Sepharose CL-6B; dissolving the alpha-1, 2 glucan precipitate, and centrifuging to remove insoluble precipitate; loading the supernatant onto gel filtration column Sepharose CL-6B; elution was performed with 0.15M sodium chloride at a flow rate of 2ml/min, collected every 5ml and the collected samples were checked by Thin Layer Chromatography (TLC).
(16) Thin Layer Chromatography (TLC) was used to detect the peak positions of α -1, 2-glucan on gel filtration chromatography.
The Thin Layer Chromatography (TLC) comprises the following specific operation steps:
cutting a silica gel plate with a proper size, wherein four sides are parallel; pencil lines (take care not to break the silicone, light lines) approximately 10mm from the lower edge; loading sample by a capillary tube, loading 1-10 mu l of sample, vertically loading the sample, and naturally drying; developing with a developing agent (the volume ratio of n-butanol to acetone to water is 4:3:1), wherein the developing agent is not higher than the lower edge line, and taking out and naturally drying after the development is finished; and (3) completely immersing the silica gel plate in a color developing agent (2% aniline acetone solution, 2% diphenylamine acetone solution and 85% phosphoric acid in a volume ratio of 5:5:1), removing, naturally drying, and placing in an oven at 70-85 ℃ for color development for 10-15 min.
(17) Mixing elution peaks containing alpha-1, 2 glucan, adding 90% ethanol into the mixed solution, wherein the volume ratio of the mixed solution to the 90% ethanol is 1:2, and standing for 1 hour to precipitate the alpha-1, 2 glucan.
(18) After the alpha-1, 2 glucan precipitate is freeze-dried, a proper amount of water is added to dissolve the alpha-1, 2 glucan precipitate to prepare purified alpha-1, 2 glucan, the obtained purified alpha-1, 2 glucan is shown in figure 6, and finally the obtained alpha-1, 2 glucan precipitate is white and can absorb a certain amount of moisture from the air to present a certain viscosity.
Example 2 application of purified alpha-1, 2 glucan prepared according to the invention in the food industry
The purified alpha-1, 2 glucan prepared by the invention is glucan formed by alpha-1, 2 glycosidic bonds, can be directly eaten and has certain sweetness. The specific sweetness of the purified alpha-1, 2 glucan prepared according to the invention was 0.2-0.6 (table 1).
TABLE 1
| Item | Specific sweetness |
| Sucrose | 1.00 |
| Alpha-1, 2 glucan | 0.2-0.6 |
The purified alpha-1, 2 glucan prepared by the invention has certain viscosity and can be used as a food thickener and a food additive. The specific viscosities of sucrose (50g/l) and alpha-1, 2 glucan (50g/l) were measured at 25 ℃ using a rotational (Brookfield) viscometer to determine rheology. The specific viscosity of the purified alpha-1, 2 glucan prepared by the invention at 25 ℃ is 50-200 (Table 2).
TABLE 2
| Item | Specific viscosity |
| Sucrose | 1.00 |
| Alpha-1, 2 glucan | 50-200 |
Example 3 application of purified alpha-1, 2 glucan prepared according to the present invention in the healthcare industry
1. The purified alpha-1, 2 glucan prepared by the invention has the function of reducing blood sugar
Firstly, fasting mice are not forbidden for 12 hours, tail breaking and blood taking are carried out to measure fasting blood glucose value, secondly, a plurality of mice with blood glucose concentration of 3.5-5.5mmol/L are selected and randomly divided into two groups, the two groups of mice are respectively perfused with starch (2g/kg) and alpha-1, 2 glucan (2g/kg), the blood glucose changes of the two groups of mice are recorded at different time periods, and the result is shown in figure 7: the blood sugar of the mice perfused with the purified alpha-1, 2 glucan prepared by the invention is increased by a lower amount than that of the mice perfused with starch in the same time period, thereby proving that the purified alpha-1, 2 glucan prepared by the invention has the function of reducing blood sugar.
2. The purified alpha-1, 2 glucan prepared by the invention has the function of reducing blood fat
Selecting a plurality of normal healthy mice randomly, continuously feeding high-fat feed (1% of cholesterol, 10% of yolk powder, 10% of lard and 79% of common feed) for 4 weeks, cutting off tails, taking blood, and detecting Total Cholesterol (TC), Triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in serum. Next, these mice were fed with purified α -1,2 glucan prepared according to the present invention for a certain period of time, and blood was taken again to examine the above index, and the results are shown in fig. 8: the TG value of a mouse fed with high fat by using the prepared purified alpha-1, 2 glucan can be increased, and TC and LDL-C, HDL-C values are reduced, so that the purified alpha-1, 2 glucan has the function of reducing blood fat.
3. The purified alpha-1, 2 glucan prepared by the invention has the function of eliminating fatty liver
Modeling a plurality of mice by using high-fat feed containing sodium cholate and methyl thiouracil, detecting data of liver homogenate Malondialdehyde (MDA), liver homogenate superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) of fatty liver mice, feeding the mice with the purified alpha-1, 2 glucan prepared by the invention for a period of time, and detecting the indexes again, wherein the result is shown in figure 9: after the purified alpha-1, 2 glucan prepared by the invention is used for feeding fatty liver mice, the MDA value is reduced, and the SOD and GSH-PX values are increased, thereby proving that the purified alpha-1, 2 glucan prepared by the invention has the effect of eliminating fatty liver.
4. The purified alpha-1, 2 glucan prepared by the invention has the function of reducing blood pressure
Ligating one kidney of a normal healthy mouse by adopting a double-kidney single-clamp method to cause unilateral renal artery stenosis, measuring the blood pressure of the mouse by using a non-invasive blood pressure measuring system after feeding for three weeks, and determining whether the modeling is successful or not; after successful modeling, the purified α -1,2 glucan prepared according to the present invention was fed to the mice for a period of time, and then the blood pressure of the mice was measured again, and the results are shown in fig. 10: the blood pressure of hypertensive mice fed with the purified α -1,2 glucan prepared according to the present invention was reduced over the same period of time, thus demonstrating that the purified α -1,2 glucan prepared according to the present invention has a blood pressure lowering effect.
5. The purified alpha-1, 2 glucan prepared by the invention has the function of regulating intestinal flora
Firstly, taking feces of a normal healthy mouse, sequencing 16srRNA by adopting a high-throughput sequencing technology, and detecting the composition and the quantity of partial bacteria in the intestinal tract of the mouse; then, feeding the mice with alpha-1, 2 glucan for a period of time under conditions that maintain basic vital signs; finally, the composition and the number of partial bacteria in the intestinal tract of the mice are detected, and the results are shown in fig. 11: compared with normal feeding mice, the purified alpha-1, 2 glucan prepared by the invention is used for feeding the mice with increased quantity of beneficial intestinal bacteria such as bifidobacterium, bacteroides, eubacterium, lactobacillus and the like and decreased quantity of harmful intestinal bacteria such as clostridium perfringens, pseudomonas aeruginosa and the like. The purified alpha-1, 2 glucan prepared by the invention can increase the number of beneficial bacteria, reduce the number of harmful bacteria, relieve flatulence and treat dyspepsia, and can be used for regulating intestinal flora.
Example 4 use of purified alpha-1, 2 glucans prepared according to the invention in cosmetics
1. The purified alpha-1, 2 glucan prepared by the invention can form a transparent, elastic and breathable film on the skin, can effectively isolate the damage of harmful substances in the environment to the skin, can fully lock water by a macromolecular structure, prevents water loss, and has high-efficiency moisture retention, so that the skin is moist and smooth.
The samples were weighed according to the data shown in table 3 to make moisturizing creams with different contents of α -1,2 glucan, and the water loss rate was calculated to verify the moisturizing effect.
TABLE 3
| Item | Glycerol | Water (W) | EDTA disodium salt | Alpha-1, 2 |
| Formulation | ||||
| 1 | 3g | 100g | 0.05 |
0 |
| |
3g | 100g | 0.05 | 1g |
| Formulation | ||||
| 3 | 3g | 100g | 0.05g | 5g |
An in-vitro weighing test method is adopted, 5.2g of each formula in the table is accurately weighed respectively, the materials are placed in a 100g beaker, the temperature is 25-26 ℃, the relative humidity is 58-65%, the materials are weighed and recorded every 4h, and the water loss rate is obtained by using the following formula.
W ═ (W1-W2) (/ W1-W0) × 100%, where W is the water loss rate of the sample; w0-beaker mass; w1 — mass of sample and beaker before experiment; w2 — mass of sample and beaker after specified time interval.
The results are shown in FIG. 12: the water loss rates of three kinds of moisturizing creams with different alpha-1, 2 glucan contents measured by an in-vitro weighing test method are different in different time periods, the water loss rate of the moisturizing cream without the alpha-1, 2 glucan is obviously higher than that of the moisturizing cream containing the alpha-1, 2 glucan, and the water loss rate of the moisturizing cream containing 1% of the alpha-1, 2 glucan is higher than that of the moisturizing cream containing 5% of the alpha-1, 2 glucan, so that the purified alpha-1, 2 glucan prepared by the invention has a good moisturizing effect.
2. The purified alpha-1, 2 glucan prepared by the invention has the function of promoting wound healing
Dividing mice of 8-12 weeks into normal group and subcutaneous alpha-1, 2 dextran injection group by random digital table method, each group contains 10 mice; after all mice are fed for 1 week adaptively, the mice are anesthetized by isoflurane inhalation, 2 full skin-lacking wound surfaces with the diameter of 8mm are respectively made along two sides of a spine after the back area of the mice is shaved, and the wound surfaces are covered by a semipermeable membrane to prevent the wound surfaces from drying; mice were all given local subcutaneous injections, with the normal group injected with an equal amount of isotonic saline. On days 1, 3, 5, and 7, the wound area was counted on the recorded photographs, with mice having a consistent local wound healing recorded under the same conditions. And calculating the wound healing rate by taking the original wound area after the operation as a reference. The results are shown in FIG. 13: on the 1 st day after operation, the wound surface of the alpha-1, 2 glucan group mouse is bright red, round and has no scars, and the wound periphery is clear and regular. On the 3 rd day after the operation, the wound edges of the mice in the normal group begin to be fuzzy, the color of the wound on the back of the mice in the alpha-1, 2 glucan group begins to deepen, the wound slowly shrinks, and scabs can be seen at the wound edges. On the 4 th day after operation, the wound surface of the normal group is reduced, the color is deepened to be dark brown, the wound area of the alpha-1, 2 glucan group is obviously reduced, the wound surface is scabbed and covered, and the healing area is increased. And the wound healing rate of the alpha-1, 2 glucan treatment group is obviously lower than that of the control group after four days of operation. Thus, it was demonstrated that the purified alpha-1, 2 glucan prepared by the present invention can enhance wound healing ability.
3. The purified alpha-1, 2 glucan prepared by the invention has the functions of resisting ultraviolet rays, aging and oxidation
(1) Ultraviolet resistance functional test of the purified alpha-1, 2 glucan prepared by the invention
Firstly, carrying out unhairing treatment on two sides of the belly of 20 male mice, wherein the volume of the unhaired epidermis is kept consistent; then, the sunscreen cream of the hollow white control group in the table 4 is uniformly smeared on the right belly of the mouse, and the sunscreen cream of the test group is uniformly smeared on the left belly of the mouse; then using a solar simulator to uniformly irradiate the white mouse with ultraviolet rays to induce erythema; the skin color was measured at 8h, 16h, 24h and 48h of irradiation to verify the anti-UV effect of alpha-1, 2 glucan.
TABLE 4
As shown in Table 5, the erythema index of the mice irradiated from the right side is 5 times of that of the sunscreen cream applied from the left side and containing 10% of the alpha-1, 2 glucan, thereby confirming that the purified alpha-1, 2 glucan prepared by the present invention has a significant anti-ultraviolet effect.
TABLE 5
| Item | 8h | 16h | 24h | 48h |
| Erythema index (control group) | 51 | 69 | 81 | 104 |
| Erythema index (Experimental group) | 12 | 15 | 19 | 21 |
(2) Anti-aging and anti-oxidation functional experiment of purified alpha-1, 2 glucan prepared by the invention
Firstly, depilating the skin folds of 15 male mice, performing a photoaging test for 8 weeks, and treating the skin folds of the mice twice a day with a cream containing 10% of alpha-1, 2 glucan in table 6 and a blank control; digital image analysis is carried out on wrinkles, and the anti-aging and anti-oxidation effects of the alpha-1, 2 glucan are detected by measuring the skin elasticity.
TABLE 6
| Item | Water (W) | Glycerol | EDTA disodium salt | Alpha-1, 2 glucan |
| Blank control group | 100g | 10g | 0.05g | 0g |
| Test group | 100g | 10g | 0.05g | 10g |
As shown in Table 7, the skin elastic modulus of mice using the cream containing the purified α -1,2 glucan prepared in the present invention was significantly higher than that of the cream containing no α -1,2 glucan. Therefore, the purified alpha-1, 2 glucan prepared by the invention can increase epidermal growth factors on wrinkled skin, promote the increase of collagen and elastin in the skin, improve the appearance of the skin and remove wrinkles, and has better anti-aging and anti-oxidation effects.
TABLE 7
| Item | Blank control group | Test group |
| Mean coefficient of skin elasticity | 0.658 | 0.898 |
Example 5 application of purified alpha-1, 2 glucan prepared according to the invention in the pharmaceutical industry
1. The purified alpha-1, 2 glucan prepared by the invention has the function of improving the immunity
The purified alpha-1, 2 glucan prepared by the invention can increase the phagocytic activity of macrophages by activating the macrophages, and plays a role in immunoregulation in resisting infection.
Firstly, randomly selecting a male mouse to separate macrophages in the abdominal cavity of the male mouse in vitro; transferring the separated macrophages into a culture dish for adherent culture, and using a DMEM complete culture medium to adjust the cell density; adding alpha-1, 2 glucan into the cells, and setting a blank control group; culturing the cells in an incubator for 24, 48 and 72 hours; the test is repeated three times, and macrophage activity is detected to calculate the average value and standard deviation.
TABLE 8
The results are shown in table 8, after 24h and 48h of drug action, the alpha-1, 2 glucan group shows weaker enhancement effect on mouse abdominal cavity macrophage activity, and the drug action lasts for 72h, thereby proving that the purified alpha-1, 2 glucan prepared by the invention obviously enhances the mouse abdominal cavity macrophage activity.
2. The purified alpha-1, 2 glucan prepared by the invention has the function of resisting oxidation
According to the cell density, HACAT cells (human immortalized epidermal cells) (American ATCC cell bank) in logarithmic phase are inoculated into a 60mm cell culture dish and cultured in a constant temperature incubator for 24h, and after the test group is added with alpha-1, 2 glucan and cultured for 48h, the total antioxidant capacity of the cells is detected by using a kit.
TABLE 9
| Item | Blank control group | Test |
| Antioxidant capacity | ||
| 100％ | 120-200％ |
The results are shown in Table 9, and the total antioxidant capacity of the cells detected by the kit is 120-200% after the test group is cultured for 48h by adding alpha-1, 2-glucan. Therefore, the purified alpha-1, 2 glucan prepared by the invention has a certain antioxidation effect.
3. The purified alpha-1, 2 glucan prepared by the invention has the function of improving the sleep quality
Male mice were subjected to a pentobarbital-induced sleep test. In the mouse experiment, subcutaneous injection of alpha-1, 2-glucan, gavage alpha-1, 2-glucan and physiological saline were given for 30 consecutive days, respectively. The sleep time of the mice was measured when pentobarbital-induced alpha-1, 2 glucan was sleeping. The results are shown in FIG. 14: compared with a control group (normal saline), the purified alpha-1, 2 glucan prepared by the invention obviously increases the sleep time of mice, and the effect of the group injected with the alpha-1, 2 glucan subcutaneously is stronger than that of the group injected with the alpha-1, 2 glucan by intragastric administration. Therefore, the purified alpha-1, 2 glucan prepared by the invention can improve the sleep quality of mice.
4. The purified alpha-1, 2 glucan prepared by the invention has the function of treating cancers and tumors
A plurality of male mice are randomly divided into 3 groups of tumor groups, tumor + alpha-1, 2 glucan injection groups and tumor + alpha-1, 2 glucan intragastric groups, and each group comprises 8 mice. Placing mouse Lewis lung cancer cell in complete culture medium containing 10% fetal calf serum and high-sugar DMEM, placing at 37 deg.C and 5% CO2Culturing in incubator, changing culture solution 1 time every 1-2 days, and digesting with 0.25% pancreatin once every 3 days. When the cell confluence rate reaches 90%, collecting cells, centrifuging to remove supernatant, adding PBS to blow and beat, suspending the cells in the PBS, measuring the activity of trypan blue stained cells to be more than 95%, counting the cells, and adjusting the cell suspension with the cell concentration of 4X 107ml for later use.
The lung cancer cell suspension is inoculated to the right scapular subcutaneous part of the mouse. Rice-sized tumors can be observed in 7-10 days, and the tumor volume is measured at day 7 and is 100-150mm3Beginning daily gavage of α -1,2 glucan (gavage 5mg/kg) and intraperitoneal injection of α -1,2 glucan (injection 5mg/kg), respectively, tumor volumes were measured in mice every 3 days until day 21, and the results are shown in fig. 15: the tumors of the tumor + alpha-1, 2 glucan injection group and the tumor + alpha-1, 2 glucan intragastric group are obviously smaller than those of the control group, and the effect is more obvious along with the increase of the medication time, so that the purified alpha-1, 2 glucan prepared by the invention can obviously inhibit tumor cells and can be used for treating cancers and tumors.
Example 6 application of purified alpha-1, 2 glucan prepared by the present invention to industry
The purified alpha-1, 2 glucan prepared by the invention has certain viscosity and can be used for preparing adhesive, mucilage, glue, paste and the like. The specific viscosities of the pastes and alpha-1, 2 glucan (100g/l) were determined at 25 ℃ using a rotary (Brookfield) viscometer for rheological measurements. The purified α -1,2 glucan prepared by the present invention had a specific viscosity of 0.8 to 1.5 at 25 ℃ (table 10).
The invention discloses a purification method of alpha-1, 2 glucan, alpha-1, 2 glucan and application thereof, and a person skilled in the art can use the contents to appropriately improve process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the technology can be practiced and applied by modifying or appropriately combining the products described herein without departing from the spirit and scope of the invention.
Claims (10)
1. A method for purifying α -1,2 glucan, comprising the steps of:
mixing blue algae and water, and then carrying out cracking treatment, wherein the weight ratio of the blue algae to the water is 1: 10-10: 1;
step two, collecting the supernatant containing alpha-1, 2 glucan;
adding 90-100% ethanol into the supernatant, wherein the volume ratio of the supernatant to the ethanol is 1: 10-10: 1, standing for 1-48 h, and precipitating alpha-1, 2 glucan;
step four, collecting alpha-1, 2 glucan precipitate and freeze-drying;
dissolving the freeze-dried alpha-1, 2 glucan precipitate in water, and dialyzing in a dialysis membrane for 24-48 h;
step six, adding 90-100% ethanol into the solution, wherein the volume ratio of the solution to the ethanol is 1: 10-10: 1, standing for 1-48 hours, and precipitating alpha-1, 2 glucan;
collecting alpha-1, 2 glucan precipitate and freeze-drying;
step eight, removing impurities by DEAE-cellulose ion exchange chromatography, and separating alpha-1, 2 glucan;
step nine, detecting the peak position of the alpha-1, 2 glucan in DEAE-cellulose ion exchange chromatography by thin layer chromatography;
mixing elution peaks containing alpha-1, 2 glucan, adding 90-100% ethanol into the mixed solution, wherein the volume ratio of the mixed solution to the ethanol is 1: 10-10: 1, and standing for 1-48 hours to precipitate the alpha-1, 2 glucan;
step eleven, collecting alpha-1, 2 glucan precipitate and freeze-drying;
step twelve, further removing impurities by gel filtration chromatography, and separating and purifying the alpha-1, 2 glucan;
thirteen, detecting the peak position of the alpha-1, 2 glucan in the gel filtration chromatography by thin layer chromatography;
fourteen, mixing elution peaks containing alpha-1, 2 glucan, adding 90-100% ethanol into the mixed solution, wherein the volume ratio of the mixed solution to the ethanol is 1: 10-10: 1, standing for 1-48 hours, and precipitating the alpha-1, 2 glucan;
fifteen, collecting alpha-1, 2 glucan precipitate and freeze-drying; dissolving the freeze-dried alpha-1, 2 glucan precipitate in water to obtain purified alpha-1, 2 glucan.
2. The method for purifying alpha-1, 2 glucan according to claim 1, wherein in the first step, the method for the lysis treatment is as follows: heating at 80-200 ℃ for 10 min-4 h, or carrying out ultrasonic crushing for 5-60 min by using an ultrasonic crusher with the power set to 10-80%, or carrying out high-pressure crushing by using an ultrahigh-pressure continuous flow cell crusher with the pressure set to 50-200 Mpa.
3. The method for purifying alpha-1, 2 glucan according to claim 1, wherein in the third step, 90% ethanol is added to the supernatant, the volume ratio of the supernatant to the 90% ethanol is 1:2, and the mixture is left standing for 1 hour to precipitate the alpha-1, 2 glucan.
4. The method for purifying alpha-1, 2 glucan according to claim 1, wherein in the sixth step, the solution is an aqueous solution outside the dialysis bag, and the aqueous solution contains the alpha-1, 2 glucan; adding 90% ethanol into the solution, wherein the volume ratio of the solution to the 90% ethanol is 1:2, and standing for 1h to precipitate alpha-1, 2 glucan.
5. The method for purifying α -1, 2-glucan according to claim 1, wherein in step eight, the DEAE-cellulose ion exchange chromatography is performed by the following steps:
DEAE-cellulose was subjected to pretreatment before use according to the specification of DEAE-cellulose; dissolving the alpha-1, 2 glucan precipitate, and centrifuging to remove insoluble precipitate; loading the supernatant into a DEAE-cellulose ion exchange column; cleaning a DEAE-cellulose ion exchange column by double distilled water at the flow rate of 1 ml/min; the elution method is sodium chloride gradient elution, the concentration of sodium chloride is 0-0.5M, the flow rate is 1ml/min, the sodium chloride is collected every 5ml, and the collected samples are detected by thin layer chromatography.
6. The method for purifying alpha-1, 2 glucan according to claim 1, wherein in step nine and step thirteen, the thin layer chromatography is performed by the following specific steps:
cutting the silica gel plate with four parallel sides; marking a line of the pencil, wherein the distance from the lower edge is 10 mm; loading sample by a capillary tube, loading 1-10 mu l of sample, vertically loading the sample, and naturally drying; developing with a developing agent, wherein the developing agent consists of n-butanol, acetone and water in a volume ratio of 4:3:1, the developing agent is not higher than the lower edge marking line, and after the development is finished, taking out and naturally drying; and (2) putting the silica gel plate into a color developing agent which is composed of 2% aniline acetone solution, 2% diphenylamine acetone solution and 85% phosphoric acid in a volume ratio of 5:5:1, removing, naturally drying, and putting the silica gel plate into an oven at 70-85 ℃ for color development for 10-15 min.
7. The method for purifying alpha-1, 2 glucan according to claim 1, wherein in the twelfth step, the specific operation steps of the gel filtration chromatography are as follows:
pretreating gel filtration column Sepharose CL-6B before use according to the specification of gel filtration column Sepharose CL-6B; dissolving the alpha-1, 2 glucan precipitate, and centrifuging to remove insoluble precipitate; loading the supernatant onto gel filtration column Sepharose CL-6B; eluting with 0.15M sodium chloride at flow rate of 2ml/min, collecting every 5ml, and detecting the collected sample by thin layer chromatography.
8. The method for purifying α -1,2 glucan according to claim 1, wherein in the fourteenth step, the elution peak containing α -1,2 glucan is mixed, 90% ethanol is added to the mixture, the mixture and 90% ethanol are allowed to stand for 1 hour at a volume ratio of 1:2, and the α -1,2 glucan is precipitated.
9. Alpha-1, 2 glucan obtainable by the purification method according to any one of claims 1 to 8.
10. Use of the α -1,2 glucan according to claim 9 in the preparation of food thickeners, food additives, blood glucose lowering products, blood lipid lowering products, blood pressure lowering products, fatty liver elimination products, intestinal flora regulating products, moisturizing cosmetics, wound healing promoting cosmetics, ultraviolet light resistant cosmetics, anti-aging cosmetics, antioxidant cosmetics, immunity enhancing products, sleep quality enhancing products, anti-tumor products, anti-cancer drugs, stickers, mucilages, glues and pastes.
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