CN112759664B - Preparation method of quinoa small-molecule heteropolysaccharide - Google Patents
Preparation method of quinoa small-molecule heteropolysaccharide Download PDFInfo
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Abstract
The invention discloses a preparation method of quinoa small-molecule heteropolysaccharide, which comprises the following steps: s1, selecting and crushing chenopodium quinoa willd raw materials, carrying out ethanol reflux extraction, and preparing a steamed material; s2, carrying out alpha-amylase treatment twice to obtain a heat preservation solution; s3, preparing a supernatant treatment solution; washing with absolute ethanol twice the volume of S4 to obtain refined quinoa non-starch polysaccharide; s5, performing online cyclic degradation by ultrasonic wave-hydrogen peroxide-Vc-ultrafiltration to obtain polysaccharide degradation liquid; s6 anion exchange resin adsorption, desorption, ultrafiltration with ultrafiltration membrane to obtain quinoa micromolecule heteropolysaccharide 150 g; the invention has the beneficial effects that: the quinoa micromolecule heteropolysaccharide with high purity and relatively uniform molecular mass is obtained by firstly crushing quinoa, then infiltrating with PBS buffer solution, carrying out alpha-amylase treatment twice, preorder treatment, absolute ethanol treatment twice, treatment with an online cyclic degradation method, and finally carrying out anion exchange resin treatment and ultrafiltration membrane treatment.
Description
Technical Field
The invention relates to the field of food and medicine, in particular to a preparation method of quinoa small-molecule heteropolysaccharide.
Background
Quinoa has physiological characteristics of drought resistance, saline-alkali resistance, barren resistance and the like, is called as perfect nutritional food, has rich nutritional value of grains, contains rich starch, protein, fat, soluble dietary fiber and polysaccharide, and also contains various vitamins, mineral substances, flavone, polyphenol and other functional components with biological activity. Quinoa carbohydrate accounts for about 60%, wherein starch accounts for about 40-50%, non-starch soluble polysaccharide and other dietary fibers account for about 20% of total carbohydrate, heteropolysaccharide composed of arabinose, xylose, galactose, rhamnose, glucose and the like, pectin polysaccharide and xylan with molecular weight of 20-500 KDa.
Quinoa seeds have high starch content and are not suitable for being eaten by obese patients, diabetic patients and old people with slow metabolism. Although the quinoa non-starch polysaccharide has important physiological functions, has activities of resisting oxidation, reducing blood fat and blood sugar, improving immunity and the like, the content of the quinoa non-starch polysaccharide is lower than that of starch, and the relative molecular mass of the quinoa non-starch polysaccharide is equivalent to that of starch, so that a non-starch polysaccharide product with uniform relative molecular mass distribution is difficult to obtain, and the fine and deep processing and deep application and development of the quinoa are limited. The small molecular heteropolysaccharide is low molecular weight polysaccharide polymerized by different monosaccharides; the polysaccharide has the characteristics of low viscosity, good solubility, easy digestion and absorption and the like, and in recent years, a large number of reports on the aspects of improving and repairing the intestinal microbial function, resisting oxidation, reducing blood fat and blood sugar, improving immunity and the like are provided, so that the polysaccharide has important research and development values.
Small molecule heteropolysaccharides can be prepared by degradation of polysaccharides. The existing preparation method mainly comprises an acid degradation method, an oxidative degradation method, a physical (ultrasonic wave and microwave) degradation method and an enzyme degradation method, wherein the simple acid degradation method and the oxidative degradation method have the disadvantages of violent reaction, difficulty in controlling degradation degree, high energy consumption of the physical degradation method, difficulty in controlling degradation degree and small sample amount in single degradation. The enzymatic degradation has high cost and harsh reaction conditions, and protein impurities are introduced. The small molecular heteropolysaccharide with uniform quality can be obtained by combining the oxidative degradation method and the physical degradation method and connecting the ultrafiltration devices in series, and the preparation method has low cost and can be used for large-scale preparation.
No report is found on the method for directly preparing the small-molecule heteropolysaccharide with high purity and uniform relative molecular mass distribution from chenopodium quinoa seeds.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation method of quinoa small-molecule heteropolysaccharide, so as to at least achieve the aims of high purity and uniform relative molecular mass.
The purpose of the invention is realized by the following technical scheme:
a preparation method of quinoa small-molecule heteropolysaccharide comprises the following steps:
s1, selecting and crushing chenopodium quinoa raw materials, screening, performing reflux extraction on the screened raw materials by using 75-85% by volume of ethanol, filtering after extraction, collecting filter residues, evaporating the ethanol, and collecting evaporated materials;
s2, soaking the steamed material by adopting a PBS buffer solution, wherein the volume ratio of the PBS buffer solution to the quinoa raw material powder is (5-10) ml: 1g, heating to 50-60 ℃, adding alpha-amylase, preserving heat for 1-3 hours, boiling, extracting for 1-3 hours, filtering an extracting solution, cooling the filtered liquid to the heat preservation temperature of 50-60 ℃, adding the alpha-amylase again, preserving heat for 1-3 hours at 50-60 ℃ to obtain a heat preservation liquid; wherein the addition amount of the alpha-amylase is that per 100g of quinoa raw material powder, enzyme preparation with the activity unit of 2500-15000U is added;
s3, centrifuging the obtained heat preservation solution at 8000 Xg for 10min at a high speed, treating the obtained heat preservation supernatant to obtain a treatment solution, and centrifuging the treatment solution at 8000 Xg for 10min at a high speed to obtain a supernatant treatment solution;
s4, adding absolute ethyl alcohol with the volume twice that of the supernatant into the obtained supernatant, precipitating overnight, then centrifuging at a high speed of 8000 Xg for 10min to obtain a precipitate, repeatedly washing the precipitate with the absolute ethyl alcohol, and volatilizing residual ethyl alcohol in the precipitate to obtain the refined quinoa non-starch polysaccharide;
s5, performing online cyclic degradation of the refined quinoa non-starch polysaccharide by using ultrasonic wave-hydrogen peroxide-Vc-ultrafiltration to obtain polysaccharide degradation liquid;
s6, adsorbing the obtained polysaccharide degradation liquid by using anion exchange resin, wherein the solid-to-liquid ratio of the ion exchange resin to the polysaccharide degradation liquid is 1 g: (5-10) ml, desorbing with 0.1-0.5 mol/L sodium chloride solution to obtain desorption solution, wherein the ratio of the volume of the sodium chloride solution to the mass of the anion exchange resin is (3-5) ml: 1g, carrying out ultrafiltration on the obtained desorption solution by using an ultrafiltration membrane tube 8 with the molecular weight cutoff of 10000Da, and carrying out vacuum freeze drying on the obtained ultrafiltrate to obtain the quinoa micromolecule heteropolysaccharide, wherein the ultrafiltration membrane is actually tubular, a jacket tube is arranged outside the ultrafiltration membrane, and the sample solution can be filled in the membrane tube only by reverse flow, so that no air bubbles exist in the whole ultrafiltration membrane tube, and the micromolecules can penetrate through the membrane and flow out from a flow outlet under a certain pressure condition to be collected, so that the ultrafiltration membrane can adopt an ultrafiltration membrane tube of an ultrafiltration system.
Preferably, for the purpose of further achieving high purity and relative molecular mass uniformity, the on-line cyclic degradation comprises the following specific steps:
a. re-dissolving the refined quinoa non-starch polysaccharide in the S2 with distilled water, and centrifuging the dissolved substance at high speed to remove precipitates to obtain supernatant;
b. adding distilled water into the obtained supernatant to dilute the supernatant to a concentration of 50-200 mg/ml, so as to obtain a diluent;
c. adding hydrogen peroxide and vitamin C reagents into the diluent respectively, wherein the adding volume of the hydrogen peroxide accounts for 1-5% (ml: mg) of the weight of the quinoa polysaccharide, the adding volume of the vitamin C accounts for 2-6% (mg: mg) of the weight of the quinoa polysaccharide, stirring uniformly, and performing ultrasonic-assisted cyclic degradation for 20-50 min in an ultrafiltration system;
the ultrafiltration system comprises a pump circulating pipe, a glass guide pipe, an ultrasonic cleaner, an ultrafiltration pump and an ultrafiltration membrane pipe, wherein two ends of the pump circulating pipe are fixed on the side surface of the ultrafiltration pump; the glass guide pipe is fixed on the outer side surface of the pump circulating pipe and is connected between the pump circulating pipe and the ultrafiltration pump in series; the pump circulating pipe is communicated with the glass guide pipe; an ultrasonic cleaner is arranged below the ultrafiltration pump; the glass conduit is completely submerged in a solvent in a cleaning tank of the ultrasonic cleaner to form an ultrasonic degradation tank; the top surface of the ultrafiltration pump is respectively and fixedly connected with the bottom ends of an ultrafiltration membrane tube and an ultrafiltration liquid storage cup in parallel; the top surface of the ultrafiltration liquid storage cup is communicated with the side surface of the ultrafiltration membrane tube through a connecting tube, and one end of the ultrafiltration membrane tube, which is far away from the connecting tube, is provided with a liquid outlet; the top end of the ultrafiltration membrane tube is provided with a pressure gauge;
the glass conduit is 2 mm thick, 5-8 cm long and 10-20 mm in inner diameter;
the ultrafiltration circulating system adopts a series-circulation mode, the series-circulation degradation time is 20-50 min, and an ultrasonic cleaner with the power of 280 plus 320w is adopted; the specific working principle is that a sample to be ultrafiltered is firstly led into an ultrafiltration pump, a liquid outlet is closed at the same time, liquid firstly enters a pump circulating pipe through the ultrafiltration pump and is subjected to ultrasound in a cleaning tank of an ultrasonic cleaner, at the moment, a glass guide pipe connected in series with the pump circulating pipe is in resonance with ultrasonic vibration, so that the sample in the pump circulating pipe is fully oscillated and dissolved, and then is led back into a filter pump through the pump circulating pipe, the filter pump leads the sample liquid into an ultrafiltration membrane pipe from low to high, the ultrafiltration membrane pipe filters the sample solution along with the change of the pump pressure and then leads the sample solution into an ultrafiltration liquid storage cup through a connecting pipe, at the moment, the liquid outlet is opened, a small molecular sample is collected, the ultrafiltration liquid storage cup fully collects the large molecular sample and then leads the large molecular sample into the ultrafiltration pump, the ultrafiltration pump returns into the glass guide pipe through the pump circulating pipe for ultrasonic vibration, and the pressure in the process is displayed on a pressure gauge at the top end of the ultrafiltration membrane pipe, the pressure in the filter pump can be adjusted according to the pressure gauge, so that the series-circulation process is realized, and after the process is finished, the liquid outlet is opened, so that the sample solution is discharged and collected;
by adopting the hydrogen peroxide and the vitamin C as degradation reagents of the quinoa non-starch polysaccharide, utilizing the hydrogen peroxide and the vitamin C under the assistance of ultrasonic waves and matching with an ultrafiltration system, carrying out serial circulating degradation, and utilizing the physical and chemical degradation method to be combined with membrane separation, the polysaccharide degradation efficiency and the target product separation efficiency are improved, and the purposes of high purity and uniform relative molecular quality are realized to a certain extent.
Preferably, for the purpose of further realizing high purity, the anion exchange resin is one of agarose gel resin, cellulose resin, polymethacrylic resin or polystyrene resin; the anion exchange resin is one of DEAE-Sepharose Fast Flow, DEAE-650M, Toyopearl HW-55F, Toyopearl HW-65F, D201 or D301 type ion exchange resin; the selected anion exchange resin is utilized, and the condition of the related ion exchange resin is limited, so that the polysaccharide degradation liquid can be fully adsorbed in the anion exchange resin, and further a cushion is laid for subsequent desorption, so that the polysaccharide degradation liquid can be fully adsorbed, and the aim of improving the high purity of the polysaccharide degradation liquid is fulfilled.
Preferably, in order to further achieve the purpose of high purity, the preorder treatment comprises the steps of adding calcium chloride solid powder into the obtained heat-preservation supernatant, boiling for 30min, cooling to the heat-preservation temperature, centrifuging at a high speed to obtain a mixed supernatant, and adding disodium ethylene diamine tetraacetate powder into the mixed supernatant to obtain a treatment solution; wherein the addition amount of the calcium chloride is 1-5% of the weight of the quinoa flour raw material, and the addition amount of the disodium edetate is 1-5% of the weight of the quinoa flour raw material; the heat preservation temperature is 50-60 ℃; the heat-preservation supernatant is subjected to impurity removal and decoloration treatment by adopting the calcium chloride solid and the disodium ethylene diamine tetraacetate, so that the interference of impurities can be fully eliminated after the heat-preservation supernatant is subjected to complexing treatment and decoloration treatment, and the aim of high purity is fulfilled.
The invention has the beneficial effects that:
1. the quinoa is firstly crushed and then soaked in PBS buffer solution, and then is treated by alpha-amylase, preorder and absolute ethyl alcohol twice, and then is treated by a linear circulation degradation method, and finally is treated by anion exchange resin and an ultrafiltration membrane, and the alpha-amylase is treated by the alpha-amylase twice, so that the alpha-amylase has high starch degradation activity, polysaccharide degrading enzymes such as cellulase, pectinase, glucoglycosidase and the like are prevented from degrading the quinoa non-starch polysaccharide, and simultaneously 2 times of volume of absolute ethyl alcohol is added to precipitate the non-starch polysaccharide, monosaccharide, disaccharide, oligosaccharide and starch degradation products (oligosaccharide) are easily separated from polysaccharide, so that the refined polysaccharide with high purity is obtained, and the purity and yield of the final product are improved.
2. By adopting hydrogen peroxide and vitamin C as degradation reagents of the quinoa non-starch polysaccharide, utilizing the hydrogen peroxide and the vitamin C under the assistance of ultrasonic waves and matching with an ultrafiltration system, utilizing the hydrogen peroxide and the vitamin C under the assistance of the ultrasonic waves and matching with the ultrafiltration system, serially connecting and circularly degrading, and utilizing the physical and chemical degradation method to be combined with membrane separation, the polysaccharide degradation efficiency and the target product separation efficiency are improved, and the purposes of high purity and uniform relative molecular mass are realized to a certain extent.
3. The selected anion exchange resin is utilized, and the condition of the related ion exchange resin is limited, so that the polysaccharide degradation liquid can be fully adsorbed in the anion exchange resin, and further a cushion is laid for subsequent desorption, so that the polysaccharide degradation liquid can be fully adsorbed, and the aim of improving the high purity of the polysaccharide degradation liquid is fulfilled.
4. The heat-preservation supernatant is subjected to impurity removal and decoloration treatment by adopting the calcium chloride solid and the disodium ethylene diamine tetraacetate, so that the interference of impurities can be fully eliminated after the heat-preservation supernatant is subjected to impurity removal and decoloration treatment, and the aim of high purity is fulfilled.
Drawings
FIG. 1 is a graph showing the results of purity and relative molecular mass detection of the product of the present invention;
FIG. 2 is a schematic diagram of monosaccharide composition analysis of the product of the present invention;
FIG. 3 is a schematic representation of infrared spectroscopic analysis of a product of the present invention;
FIG. 4 is a schematic view of an ultrafiltration system of the present invention,
the device comprises a pump circulation pipe 1, a glass guide pipe 2, an ultrasonic cleaner 3, an ultrafiltration pump 4, a pressure gauge 5, an ultrafiltration liquid storage cup 6, a connecting pipe 7, an ultrafiltration membrane pipe 8, a liquid outlet 81 and an ultrasonic degradation tank 9.
Detailed Description
The technical solutions of the present invention are further described in detail below with reference to the accompanying drawings, but the scope of the present invention is not limited to the following.
Example 1
A preparation method of quinoa small-molecule heteropolysaccharide comprises the following steps:
s1, selecting and crushing 1kg of chenopodium quinoa willd raw material, screening, carrying out reflux extraction on the screened raw material for 3 hours at 80 ℃ by using 8L of 78% ethanol in volume percentage, filtering after extraction, collecting filter residues, steaming out the ethanol at 60 ℃, and collecting the steamed material;
s2, soaking the obtained steamed material by using 8L of PBS buffer solution, wherein the volume ratio of the PBS buffer solution to the quinoa wheat raw material powder is 8 ml: 1g, heating to 80 ℃, adding alpha-amylase, preserving heat for 1h, boiling and extracting for 2h, filtering an extracting solution, cooling the filtered liquid to the heat preservation temperature of 80 ℃, adding the alpha-amylase again, and preserving heat for 2h at the temperature of 80 ℃ to obtain a heat preservation liquid; wherein the addition amount of the alpha-amylase is that 2800U of enzyme preparation with activity unit is added into every 100g of quinoa raw material powder;
s3, centrifuging the obtained heat preservation solution at 8000 Xg for 10min at a high speed, treating the obtained heat preservation supernatant to obtain a treatment solution, and centrifuging the treatment solution at 8000 Xg for 10min at a high speed to obtain a supernatant treatment solution;
s4 adding anhydrous ethanol twice the volume of the supernatant into the supernatant, standing overnight for precipitation, centrifuging at 8000 Xg for 10min to obtain precipitate, washing the precipitate with anhydrous ethanol repeatedly, and volatilizing the residual precipitate to obtain refined quinoa non-starch polysaccharide;
s8, performing online cyclic degradation of the refined quinoa non-starch polysaccharide by using ultrasonic wave-hydrogen peroxide-Vc-ultrafiltration to obtain polysaccharide degradation liquid;
s6, adsorbing the obtained polysaccharide degradation liquid by using 200g of anion exchange resin, wherein the solid-to-liquid ratio of the ion exchange resin to the polysaccharide degradation liquid is 1 g: 8ml, desorbing by using a sodium chloride solution with the concentration of 0.1-0.8 mol/L to obtain a desorption solution, wherein the ratio of the volume of the sodium chloride solution to the mass of the anion exchange resin is 3 ml: 1g, namely 600ml, ultrafiltering the obtained desorption solution by using an ultrafiltration membrane tube 8 with the molecular weight cutoff of 10000Da, and carrying out vacuum freeze drying on the obtained ultrafiltrate to obtain 148g of quinoa small-molecular heteropolysaccharide.
In order to further realize the purposes of high purity and uniform relative molecular mass, the online cyclic degradation comprises the following specific steps:
a. re-dissolving the refined quinoa non-starch polysaccharide in the S2 with distilled water, and centrifuging the dissolved substance at high speed to remove precipitates to obtain supernatant;
b. diluting the obtained supernatant with distilled water to a concentration of 80mg/ml to obtain a diluted solution;
c. adding hydrogen peroxide and vitamin C reagent into the diluent respectively, wherein the volume of hydrogen peroxide is 100ml, the adding mass of vitamin C is 200mg, stirring for 0.8h for uniformity, and performing ultrasonic-assisted cyclic degradation for 80min in an ultrafiltration system;
the ultrafiltration system comprises a pump circulation pipe 1, a glass guide pipe 2, an ultrasonic cleaner 3, an ultrafiltration pump 4 and an ultrafiltration membrane pipe 8, wherein two ends of the pump circulation pipe 1 are fixed on the side surface of the ultrafiltration pump 4; the glass guide tube 2 is fixed on the outer side surface of the pump circulating tube 1 and is connected in series between the pump circulating tube 1 and the ultrafiltration pump 4; the pump circulation pipe 1 is communicated with the glass guide pipe 2; an ultrasonic cleaner 3 is arranged below the ultrafiltration pump 4; the glass conduit 2 is completely submerged in a solvent in a cleaning tank of the ultrasonic cleaner 3 to form an ultrasonic degradation tank 9; the top surface of the ultrafiltration pump 4 is respectively fixed and communicated with the bottom ends of an ultrafiltration membrane tube 8 and an ultrafiltration liquid storage cup 6; the top surface of the ultrafiltration liquid storage cup 6 is communicated with the side surface of the ultrafiltration membrane tube 8 through a connecting tube 7, and one end of the ultrafiltration membrane tube 8, which is far away from the connecting tube, is provided with a liquid outlet 81; the top end of the ultrafiltration membrane tube 8 is provided with a pressure gauge 5;
the glass conduit is 2 mm thick, 8-8 cm long and 10-20 mm in inner diameter;
the ultrafiltration circulating system adopts a series-circulation mode, the series-circulation degradation time is 20min, and an ultrasonic cleaner 3 with the power of 280w is adopted; by adopting hydrogen peroxide and vitamin C as degradation reagents of the quinoa non-starch polysaccharide, utilizing the hydrogen peroxide and the vitamin C under the assistance of ultrasonic waves and matching with an ultrafiltration system, carrying out serial cyclic degradation, and utilizing a physical and chemical degradation method to be combined with membrane separation, the aims of high purity and relative molecular mass uniformity are fulfilled to a certain extent.
For further high purity, the anion exchange resin is DEAE-680M; the selected anion exchange resin is utilized, and the condition of the related ion exchange resin is limited, so that the polysaccharide degradation liquid can be fully adsorbed in the anion exchange resin, and further a cushion is laid for subsequent desorption, so that the polysaccharide degradation liquid can be fully adsorbed, and the aim of improving the high purity of the polysaccharide degradation liquid is fulfilled.
In order to further realize the purpose of high purity, the preorder treatment comprises the steps of adding calcium chloride solid powder into the obtained heat preservation supernatant, boiling for 30min, cooling to the heat preservation temperature, centrifuging at a high speed to obtain mixed supernatant, and adding ethylene diamine tetraacetic acid powder into the mixed supernatant to obtain treatment liquid; wherein the addition amount of the calcium chloride is 1 percent of the weight of the quinoa powder raw material, namely 10g, and the addition amount of the disodium ethylene diamine tetraacetate is 1 percent of the weight of the quinoa powder raw material, namely 10 g; the heat preservation temperature is 80 ℃; by adopting the calcium chloride solid and the disodium ethylene diamine tetraacetate to carry out complexing treatment and decoloring treatment on the heavy metal of the heat-preservation supernatant respectively, impurities can be fully removed after the heat-preservation supernatant is subjected to complexing treatment and decoloring treatment, so that the aim of high purity is fulfilled.
Aiming at the obtained product, the HPSEC method is adopted for determination, and the method specifically comprises the following steps:
the chromatographic column is YMC-Pack Diol-200 chromatographic column (8 × 300mm), and the mobile phase is double distilled water; the flow rate is 0.8 mL/min; the sample volume is 10 mu L; the detector is RID; the temperature was room temperature.
The method comprises the following specific steps: (1) glucose, T10, T80 and T70 glycan standard substances are precisely weighed and prepared into a standard solution of 8 mg/mL.
(2) The relative molecular mass of the degradation product is measured according to the external standard method and is about 8000Da, and the purity reaches 98 percent.
The monosaccharide composition of the degradation product is determined by adopting a PMP pre-column derivatization high performance liquid chromatography, and specifically comprises the following steps:
column Diamonsil C18 column (4.6 x 280mm), mobile phase acetonitrile:
phosphate buffer (80:20) at a flow rate of 1 mL/min; the sample volume is 20 mu L; the detector is UV; the temperature was room temperature. The specific steps are as follows:
(1) weighing arabinose, galactose, glucose, rhamnose, xylose and galacturonic acid standard monosaccharide, preparing into a standard solution of 2mg/mL, performing PMP derivatization modification, weighing 10mg of degradation product freeze-dried powder, preparing into a solution of 1mg/mL, performing acid hydrolysis, and performing PMP derivatization.
(2) The small molecule heteropolysaccharide whose degradation product is composed of 6 monosaccharides is determined by external standard method, as shown in FIG. 2.
(3) The infrared spectroscopic analysis of the degradation product is shown in fig. 3, wherein 8mg of freeze-dried powder of the degradation product is weighed, mixed and ground with spectral grade potassium bromide, tabletted, and subjected to spectral scanning analysis, and the degradation product has a typical characteristic peak of heteropolysaccharide.
The analysis results showed that the degradation product has a purity of 98% and a relative molecular mass of about 8000Da, and is a small molecular weight heteropolysaccharide composed of 6 monosaccharides, as shown in FIG. 1.
Example 2
A preparation method of quinoa small-molecule heteropolysaccharide comprises the following steps:
s1, selecting and crushing 1kg of chenopodium quinoa willd raw material, screening, performing reflux extraction on the screened raw material by using 10L of 78% ethanol in volume percentage, filtering and collecting filter residue after extraction, and collecting a steamed material after ethanol is steamed out;
s2, soaking the steamed material by adopting a PBS buffer solution, wherein the ratio of the volume of the PBS buffer solution to the mass of the quinoa raw material powder is 10 ml: 1g of the extract is 10L, heating to 80 ℃, adding alpha-amylase, preserving heat for 3h, boiling and extracting for 3h, filtering the extracting solution, cooling the filtered liquid to 80 ℃, adding the alpha-amylase again, preserving heat for 3h at 80 ℃ to obtain a heat preservation liquid; wherein the addition amount of the alpha-amylase is that an enzyme preparation with the activity unit of 8000U is added into every 100g of quinoa raw material powder;
s3, centrifuging the obtained heat preservation solution at 8000 Xg for 10min at a high speed, treating the obtained heat preservation supernatant to obtain a treatment solution, and centrifuging the treatment solution at 8000 Xg for 10min at a high speed to obtain a supernatant treatment solution;
s4, adding absolute ethyl alcohol with the volume twice that of the supernatant into the obtained supernatant, precipitating overnight, then centrifuging at a high speed of 8000 Xg for 10min to obtain a precipitate, repeatedly washing the precipitate with the absolute ethyl alcohol, and volatilizing residual ethyl alcohol in the precipitate to obtain the refined quinoa non-starch polysaccharide;
s8, performing online cyclic degradation of the refined quinoa non-starch polysaccharide by using ultrasonic wave-hydrogen peroxide-Vc-ultrafiltration to obtain polysaccharide degradation liquid;
s6, adsorbing the obtained polysaccharide degradation liquid by using 300g of anion exchange resin, wherein the solid-to-liquid ratio of the ion exchange resin to the polysaccharide degradation liquid is 1 g: 8ml, desorbing with 0.2mol/L sodium chloride solution to obtain desorption solution, wherein the volume of the sodium chloride solution is 1L, ultrafiltering the obtained desorption solution with an ultrafiltration membrane tube 8 with the molecular weight cutoff of 10000Da, and vacuum freeze-drying the obtained ultrafiltrate to obtain 186g of quinoa micromolecular heteropolysaccharide.
In order to further realize the purposes of high purity and uniform relative molecular mass, the on-line cyclic degradation comprises the following specific steps:
a. re-dissolving the refined quinoa non-starch polysaccharide in the S2 with distilled water, and centrifuging the dissolved substance at high speed to remove precipitates to obtain supernatant;
b. diluting the obtained supernatant with distilled water to a concentration of 200mg/ml to obtain a diluted solution;
c. adding hydrogen peroxide and vitamin C reagents into the diluent respectively, wherein the hydrogen peroxide is added in an amount of 120ml, the vitamin C is added in an amount of 220mg, stirring for 0.8h uniformly, and performing ultrasonic-assisted cyclic degradation for 230min in an ultrafiltration system;
the ultrafiltration system comprises a pump circulation pipe 1, a glass guide pipe 2, an ultrafiltration pump 4 and an ultrafiltration membrane pipe 8, wherein both ends of the pump circulation pipe 1 are fixed on the side surface of the ultrafiltration pump 4; the glass guide tube 2 is fixed on the outer side surface of the pump circulating tube 1 and is connected between the pump circulating tube 1 and the ultrafiltration pump 4 in series; the pump circulating pipe 1 is communicated with the glass guide pipe 2; an ultrasonic cleaner 3 is arranged below the ultrafiltration pump 4; the glass conduit 2 is completely submerged in a solvent in a cleaning tank of the ultrasonic cleaner 3 to form an ultrasonic degradation tank 9; the top surface of the ultrafiltration pump 4 is respectively and fixedly connected with the bottom ends of an ultrafiltration membrane tube 8 and an ultrafiltration liquid storage cup 6 in parallel; the top surface of the ultrafiltration liquid storage cup 6 is communicated with the side surface of the ultrafiltration membrane tube 8 through a connecting tube 7, and one end of the ultrafiltration membrane tube 8, which is far away from the connecting tube, is provided with a liquid outlet 81; the top end of the ultrafiltration membrane tube 8 is provided with a pressure gauge 5;
the glass conduit is 2 mm thick, 8-8 cm long and 10-20 mm in inner diameter;
the ultrafiltration circulating system adopts a series-circulation mode, the series-circulation degradation time is 30min, and an ultrasonic cleaner 3 with the power of 300w is adopted; by adopting hydrogen peroxide and vitamin C as degradation reagents of the quinoa non-starch polysaccharide, utilizing the hydrogen peroxide and the vitamin C under the assistance of ultrasonic waves and matching with an ultrafiltration system, carrying out serial circulating degradation, and utilizing a physical and chemical degradation method to be combined with membrane separation, the aims of high purity and uniform relative molecular mass are fulfilled to a certain extent.
For further high purity, the anion exchange resin is DEAE-Sepharose Fast Flow; the selected anion exchange resin is utilized, and the condition of the related ion exchange resin is limited, so that the polysaccharide degradation liquid can be fully adsorbed in the anion exchange resin, and further a cushion is laid for subsequent desorption, so that the polysaccharide degradation liquid can be fully adsorbed, and the aim of improving the high purity of the polysaccharide degradation liquid is fulfilled.
In order to further realize the purpose of high purity, the preorder treatment comprises the steps of adding calcium chloride solid powder into the obtained heat-preservation supernatant, boiling for 30min, cooling to the heat-preservation temperature of 80 ℃, centrifuging at a high speed to obtain mixed supernatant, and adding ethylene diamine tetraacetic acid powder into the mixed supernatant to obtain treatment liquid; wherein the addition amount of the calcium chloride is 8 percent of the weight of the quinoa powder raw material, namely 18g, and the addition amount of the disodium ethylene diamine tetraacetate is 8 percent of the weight of the quinoa powder raw material, namely 18 g; the heat preservation temperature is 80 ℃; the heat-preservation supernatant is subjected to complexing treatment and decoloring treatment of heavy metal by adopting the calcium chloride solid and the disodium ethylene diamine tetraacetate, so that impurities can be fully removed after the heat-preservation supernatant is subjected to complexing treatment and decoloring treatment, and the aim of high purity is fulfilled.
The product of example 1 has a purity of 94%, a relative molecular mass of about 8000Da, and a low molecular weight heteropolysaccharide composed of 6 monosaccharides
Example 3
A preparation method of quinoa small-molecule heteropolysaccharide comprises the following steps:
s1, selecting and crushing 1kg of chenopodium quinoa willd raw material, screening, performing reflux extraction on the screened raw material by using 10L of 88% ethanol in volume percentage, filtering and collecting filter residue after extraction, evaporating the ethanol at 60 ℃, and collecting the evaporated material;
s2, soaking the steamed material by adopting a PBS buffer solution, wherein the ratio of the volume of the PBS buffer solution to the mass of the quinoa raw material powder is 10 ml: 1g of the extract is 10L, heating to 80 ℃, adding alpha-amylase, preserving heat for 3h, boiling and extracting for 2h, filtering the extracting solution, cooling the filtered liquid to 80 ℃, adding the alpha-amylase again, preserving heat for 2h at 80 ℃ to obtain a heat preservation liquid; wherein, the addition amount of the alpha-amylase is that an enzyme preparation with the activity unit of 8000U is added into every 100g of the chenopodium quinoa raw material powder;
s3, centrifuging the obtained heat preservation solution at 8000 Xg for 10min at a high speed, treating the obtained heat preservation supernatant to obtain a treatment solution, and centrifuging the treatment solution at 8000 Xg for 10min at a high speed to obtain a supernatant treatment solution;
s4, adding absolute ethyl alcohol with the volume twice that of the supernatant into the obtained supernatant, precipitating overnight, then centrifuging at a high speed of 8000 Xg for 10min to obtain a precipitate, repeatedly washing the precipitate with the absolute ethyl alcohol, and volatilizing residual ethyl alcohol in the precipitate to obtain the refined quinoa non-starch polysaccharide;
s8, performing online cyclic degradation of the refined quinoa non-starch polysaccharide by using ultrasonic wave-hydrogen peroxide-Vc-ultrafiltration to obtain polysaccharide degradation liquid;
s6, adsorbing the obtained polysaccharide degradation liquid by using 300g of anion exchange resin, wherein the solid-to-liquid ratio of the ion exchange resin to the polysaccharide degradation liquid is 1 g: 8ml, desorbing with 0.3mol/L sodium chloride solution to obtain desorption solution, wherein the volume of the sodium chloride solution is 1.8L, ultrafiltering the obtained desorption solution with an ultrafiltration membrane tube 8 with the molecular weight cutoff of 10000Da, and carrying out vacuum freeze drying on the obtained ultrafiltrate to obtain 148g of quinoa micromolecular heteropolysaccharide.
In order to further realize the purposes of high purity and uniform relative molecular mass, the online cyclic degradation comprises the following specific steps:
a. redissolving the refined quinoa non-starch polysaccharide in the S2 by adding distilled water, and centrifuging the dissolved substance at high speed to remove precipitates to obtain supernatant;
b. diluting the obtained supernatant with distilled water to a concentration of 200mg/ml to obtain a diluted solution;
c. adding hydrogen peroxide and vitamin C reagents into the diluent respectively, wherein 180ml of hydrogen peroxide and 280mg of vitamin C are added, stirring for 0.8h uniformly, and performing ultrasonic-assisted cyclic degradation for 30min in an ultrafiltration system;
the ultrafiltration system comprises a pump circulation pipe 1, a glass guide pipe 2, an ultrafiltration pump 4 and an ultrafiltration membrane pipe 8, wherein both ends of the pump circulation pipe 1 are fixed on the side surface of the ultrafiltration pump 4; the glass guide pipe 2 is fixed on the outer side surface of the pump circulating pipe 1 and is connected between the pump circulating pipe 1 and the ultrafiltration pump 4 in series; the pump circulating pipe 1 is communicated with the glass guide pipe 2; an ultrasonic cleaner 3 is arranged below the ultrafiltration pump 4; the glass conduit 2 is completely submerged in a solvent in a cleaning tank of the ultrasonic cleaner 3 to form an ultrasonic degradation tank 9; the top surface of the ultrafiltration pump 4 is respectively and fixedly connected with the bottom ends of an ultrafiltration membrane tube 8 and an ultrafiltration liquid storage cup 6 in parallel; the top surface of the ultrafiltration liquid storage cup 6 is communicated with the side surface of the ultrafiltration membrane tube 8 through a connecting tube 7, and one end of the ultrafiltration membrane tube 8, which is far away from the connecting tube, is provided with a liquid outlet 81; the top end of the ultrafiltration membrane tube 8 is provided with a pressure gauge 5;
the glass conduit is 2 mm thick, 8-8 cm long and 10-20 mm in inner diameter;
the ultrafiltration circulating system adopts a series-circulation mode, the series-circulation degradation time is 30min, and an ultrasonic cleaner 3 with the power of 300w is adopted; by adopting hydrogen peroxide and vitamin C as degradation reagents of the quinoa non-starch polysaccharide, utilizing the hydrogen peroxide and the vitamin C under the assistance of ultrasonic waves and matching with an ultrafiltration system, carrying out serial circulating degradation, and utilizing a physical and chemical degradation method to be combined with membrane separation, the aims of high purity and uniform relative molecular mass are fulfilled to a certain extent.
In order to further realize the purpose of high purity, the anion exchange resin is one of agarose gel resin, cellulose resin, polymethacrylic resin or polystyrene resin; the anion exchange resin is one of D201 type ion exchange resins; the selected anion exchange resin is utilized, and the condition of the related ion exchange resin is limited, so that the polysaccharide degradation liquid can be fully adsorbed in the anion exchange resin, and then a cushion is laid for subsequent desorption, so that the polysaccharide degradation liquid can be fully adsorbed, and the aim of improving the high purity of the polysaccharide degradation liquid is fulfilled.
In order to further realize the purpose of high purity, the preorder treatment comprises the steps of adding calcium chloride solid powder into the obtained heat-preservation supernatant, boiling for 30min, cooling to the heat-preservation temperature, centrifuging at a high speed to obtain mixed supernatant, and adding ethylene diamine tetraacetic acid powder into the mixed supernatant to obtain treatment liquid; wherein the addition amount of the calcium chloride is 20g, and the addition amount of the disodium ethylenediamine tetraacetate is 20 g; the heat preservation temperature is 80 ℃; by adopting the calcium chloride solid and the disodium ethylene diamine tetraacetate to carry out complexing treatment and decoloring treatment on the heavy metal of the heat-preservation supernatant respectively, impurities can be fully removed after the heat-preservation supernatant is subjected to complexing treatment and decoloring treatment, so that the aim of high purity is fulfilled.
The purity of the degradation product reaches 92 percent by the same method as that of the product in the example 1, the relative molecular mass is about 8000Da, and the degradation product is a small molecular weight heteropolysaccharide consisting of 6 monosaccharides.
Example 4
A preparation method of quinoa small-molecule heteropolysaccharide comprises the following steps:
s1, selecting and crushing 1kg of chenopodium quinoa willd raw material, screening, performing reflux extraction on the screened raw material for 3 hours at 80 ℃ by using 8L of 78% ethanol in volume percentage, filtering and collecting filter residue after extraction, and collecting a steamed material after ethanol is steamed at 60 ℃;
s2, soaking the steamed material by adopting a PBS buffer solution, wherein the ratio of the volume of the PBS buffer solution to the mass of the quinoa raw material powder is 8 ml: heating to 60 ℃, adding alpha-amylase, keeping the temperature for 3 hours, boiling and extracting for 1 hour, filtering the extracting solution, cooling the filtered liquid to the temperature of 60 ℃, adding the alpha-amylase again, keeping the temperature for 3 hours at 60 ℃ to obtain heat preservation liquid, wherein the volume of the liquid is 1L; wherein, the addition amount of the alpha-amylase is that 6000U of enzyme preparation with the activity unit is added into every 100g of the chenopodium quinoa willd raw material powder;
s3, centrifuging the obtained heat preservation solution at 8000 Xg for 10min at a high speed, treating the obtained heat preservation supernatant to obtain a treatment solution, and centrifuging the treatment solution at 8000 Xg for 10min at a high speed to obtain a supernatant treatment solution;
s4, adding absolute ethyl alcohol with the volume twice that of the supernatant into the obtained supernatant, precipitating overnight, then centrifuging at a high speed of 8000 Xg for 10min to obtain a precipitate, repeatedly washing the precipitate with the absolute ethyl alcohol, and volatilizing residual ethyl alcohol in the precipitate to obtain the refined quinoa non-starch polysaccharide;
s8, performing online cyclic degradation of the refined quinoa non-starch polysaccharide by using ultrasonic wave-hydrogen peroxide-Vc-ultrafiltration to obtain polysaccharide degradation liquid;
s6, adsorbing the obtained polysaccharide degradation liquid by using 280g of anion exchange resin, wherein the solid-to-liquid ratio of the ion exchange resin to the polysaccharide degradation liquid is 1 g: 8ml, desorbing with 0.4mol/L sodium chloride solution to obtain desorption solution, wherein the volume of the sodium chloride solution is 1L, ultrafiltering the obtained desorption solution with an ultrafiltration membrane tube 8 with the molecular weight cutoff of 10000Da, and vacuum freeze-drying the obtained ultrafiltrate to obtain 180g of quinoa micromolecular heteropolysaccharide.
In order to further realize the purposes of high purity and uniform relative molecular mass, the online cyclic degradation comprises the following specific steps:
a. re-dissolving the refined quinoa non-starch polysaccharide in the S2 with distilled water, and centrifuging the dissolved substance at high speed to remove precipitates to obtain supernatant;
b. diluting the obtained supernatant with distilled water to a concentration of 200mg/ml to obtain a diluted solution;
c. adding hydrogen peroxide and vitamin C reagent into the diluent respectively, wherein 180ml of hydrogen peroxide and 280mg of vitamin C are added, stirring for 0.8h uniformly, and performing ultrasonic-assisted cyclic degradation for 80min in an ultrafiltration system;
the ultrafiltration system comprises a pump circulation pipe 1, a glass guide pipe 2, an ultrafiltration pump 4 and an ultrafiltration membrane pipe 8, wherein both ends of the pump circulation pipe 1 are fixed on the side surface of the ultrafiltration pump 4; the glass guide tube 2 is fixed on the outer side surface of the pump circulating tube 1 and is connected between the pump circulating tube 1 and the ultrafiltration pump 4 in series; the pump circulating pipe 1 is communicated with the glass guide pipe 2; an ultrasonic cleaner 3 is arranged below the ultrafiltration pump 4; the glass conduit 2 is completely submerged in a solvent in a cleaning tank of the ultrasonic cleaner 3 to form an ultrasonic degradation tank 9; the top surface of the ultrafiltration pump 4 is respectively and fixedly connected with the bottom ends of an ultrafiltration membrane tube 8 and an ultrafiltration liquid storage cup 6 in parallel; the top surface of the ultrafiltration liquid storage cup 6 is communicated with the side surface of the ultrafiltration membrane tube 8 through a connecting tube 7, and one end of the ultrafiltration membrane tube 8, which is far away from the connecting tube, is provided with a liquid outlet 81; the top end of the ultrafiltration membrane tube 8 is provided with a pressure gauge 5;
the glass conduit is 2 mm thick, 8-8 cm long and 10-20 mm in inner diameter;
the ultrafiltration circulating system adopts a series-circulation mode, the series-circulation degradation time is 30min, and an ultrasonic cleaner 3 with the power of 320w is adopted; by adopting hydrogen peroxide and vitamin C as degradation reagents of the quinoa non-starch polysaccharide, utilizing the hydrogen peroxide and the vitamin C under the assistance of ultrasonic waves and matching with an ultrafiltration system, carrying out serial circulating degradation, and utilizing a physical and chemical degradation method to be combined with membrane separation, the aims of high purity and uniform relative molecular mass are fulfilled to a certain extent.
In order to further realize the purpose of high purity, the anion exchange resin is one of agarose gel resin, cellulose resin, polymethacrylic resin or polystyrene resin; the anion exchange resin is Toyopearl HW-88F anion exchange resin; the selected anion exchange resin is utilized, and the condition of the related ion exchange resin is limited, so that the polysaccharide degradation liquid can be fully adsorbed in the anion exchange resin, and further a cushion is laid for subsequent desorption, so that the polysaccharide degradation liquid can be fully adsorbed, and the aim of improving the high purity of the polysaccharide degradation liquid is fulfilled.
In order to further realize the purpose of high purity, the preorder treatment comprises the steps of adding calcium chloride solid powder into the obtained heat-preservation supernatant, boiling for 30min, cooling to the heat-preservation temperature, centrifuging at a high speed to obtain mixed supernatant, and adding ethylene diamine tetraacetic acid powder into the mixed supernatant to obtain treatment liquid; wherein the addition amount of the calcium chloride is 28g, and the addition amount of the disodium ethylenediamine tetraacetate is 28 g; the heat preservation temperature is 80 ℃; by adopting the calcium chloride solid and the disodium ethylene diamine tetraacetate to carry out complexing treatment and decoloring treatment on the heavy metal of the heat-preservation supernatant respectively, impurities can be fully removed after the heat-preservation supernatant is subjected to complexing treatment and decoloring treatment, so that the aim of high purity is fulfilled.
The purity of the degradation product reaches 93 percent by the same method for measuring the product in the example 1, the relative molecular mass is about 8000Da, and the degradation product is a small molecular weight heteropolysaccharide consisting of 6 monosaccharides.
Example 8
A preparation method of quinoa small-molecule heteropolysaccharide comprises the following steps:
s1, selecting and crushing 1kg of chenopodium quinoa willd raw material, screening, performing reflux extraction on the screened raw material for 3 hours at 80 ℃ by using 10L of 78% ethanol in volume percentage, filtering and collecting filter residue after extraction, and collecting a steamed material after ethanol is steamed at 60 ℃;
s2, soaking the steamed material by adopting a PBS buffer solution, wherein the volume of the PBS buffer solution and the mass ratio of the chenopodium quinoa raw material powder are 10 ml: 1g of the extract is 10L, heating to 60 ℃, adding alpha-amylase, preserving heat for 3h, boiling and extracting for 1h, filtering the extracting solution, cooling the filtered liquid to the heat preservation temperature of 80 ℃, adding the alpha-amylase again, preserving heat for 3h at the temperature of 60 ℃ to obtain heat preservation liquid; wherein the addition amount of the alpha-amylase is that an enzyme preparation with the activity unit of 8000U is added into every 100g of quinoa raw material powder;
s3, centrifuging the obtained heat preservation solution at 8000 Xg for 10min at a high speed, treating the obtained heat preservation supernatant to obtain a treatment solution, and centrifuging the treatment solution at 8000 Xg for 10min at a high speed to obtain a supernatant treatment solution;
s4, adding absolute ethyl alcohol with the volume twice that of the supernatant into the obtained supernatant, precipitating overnight, then centrifuging at a high speed of 8000 Xg for 10min to obtain a precipitate, repeatedly washing the precipitate with the absolute ethyl alcohol, and volatilizing residual ethyl alcohol in the precipitate to obtain the refined quinoa non-starch polysaccharide;
s8, performing online cyclic degradation of the refined quinoa non-starch polysaccharide by using ultrasonic wave-hydrogen peroxide-Vc-ultrafiltration to obtain polysaccharide degradation liquid;
s6, adsorbing the obtained polysaccharide degradation liquid by using 300g of anion exchange resin, wherein the solid-to-liquid ratio of the ion exchange resin to the polysaccharide degradation liquid is 1 g: 8ml, desorbing with 0.1mol/L sodium chloride solution to obtain desorption solution, wherein the volume of the sodium chloride solution is 1.8L, ultrafiltering the obtained desorption solution with an ultrafiltration membrane tube 8 with the molecular weight cutoff of 10000Da, and carrying out vacuum freeze drying on the obtained ultrafiltrate to obtain 148g of quinoa micromolecular heteropolysaccharide.
In order to further realize the purposes of high purity and uniform relative molecular mass, the online cyclic degradation comprises the following specific steps:
a. re-dissolving the refined quinoa non-starch polysaccharide in the S2 with distilled water, and centrifuging the dissolved substance at high speed to remove precipitates to obtain supernatant;
b. diluting the obtained supernatant with distilled water to a concentration of 200mg/ml to obtain a diluted solution;
c. adding hydrogen peroxide and vitamin C reagent into the diluent respectively, wherein 180ml of hydrogen peroxide and 280mg of vitamin C are added, stirring for 0.8h uniformly, and performing ultrasonic-assisted cyclic degradation for 80min in an ultrafiltration system;
the ultrafiltration system comprises a pump circulation pipe 1, a glass guide pipe 2, an ultrafiltration pump 4 and an ultrafiltration membrane pipe 8, wherein both ends of the pump circulation pipe 1 are fixed on the side surface of the ultrafiltration pump 4; the glass guide tube 2 is fixed on the outer side surface of the pump circulating tube 1 and is connected between the pump circulating tube 1 and the ultrafiltration pump 4 in series; the pump circulating pipe 1 is communicated with the glass guide pipe 2; an ultrasonic cleaner 3 is arranged below the ultrafiltration pump 4; the glass conduit 2 is completely submerged in a solvent in a cleaning tank of the ultrasonic cleaner 3 to form an ultrasonic degradation tank 9; the top surface of the ultrafiltration pump 4 is respectively and fixedly connected with the bottom ends of an ultrafiltration membrane tube 8 and an ultrafiltration liquid storage cup 6 in parallel; the top surface of the ultrafiltration liquid storage cup 6 is communicated with the side surface of the ultrafiltration membrane tube 8 through a connecting tube 7, and one end of the ultrafiltration membrane tube 8, which is far away from the connecting tube, is provided with a liquid outlet 81; the top end of the ultrafiltration membrane tube 8 is provided with a pressure gauge 5;
the glass conduit is 2 mm thick, 8-8 cm long and 10-20 mm in inner diameter; the ultrafiltration circulating system adopts a series-circulation mode, the series-circulation degradation time is 30min, and an ultrasonic cleaner 3 with the power of 320w is adopted; by adopting hydrogen peroxide and vitamin C as degradation reagents of the quinoa non-starch polysaccharide, utilizing the hydrogen peroxide and the vitamin C under the assistance of ultrasonic waves and matching with an ultrafiltration system, carrying out serial cyclic degradation and utilizing a physical and chemical degradation method to be separated and combined with a membrane, the aims of high purity and uniform relative molecular mass are fulfilled to a certain extent.
In order to further realize the purpose of high purity, the anion exchange resin is one of agarose gel resin, cellulose resin, polymethacrylic resin or polystyrene resin; the anion exchange resin is D301 anion exchange resin; the selected anion exchange resin is utilized, and the condition of the related ion exchange resin is limited, so that the polysaccharide degradation liquid can be fully adsorbed in the anion exchange resin, and further a cushion is laid for subsequent desorption, so that the polysaccharide degradation liquid can be fully adsorbed, and the aim of improving the high purity of the polysaccharide degradation liquid is fulfilled.
In order to further realize the purpose of high purity, the preorder treatment comprises the steps of adding calcium chloride solid powder into the obtained heat-preservation supernatant, boiling for 30min, cooling to the heat-preservation temperature, centrifuging at a high speed to obtain mixed supernatant, and adding ethylene diamine tetraacetic acid powder into the mixed supernatant to obtain treatment liquid; wherein the addition amount of the calcium chloride is 80g, and the addition amount of the disodium ethylenediamine tetraacetate is 80 g; the heat preservation temperature is 80 ℃; by adopting the calcium chloride solid and the disodium ethylene diamine tetraacetate to carry out complexing treatment and decoloring treatment on the heavy metal of the heat-preservation supernatant respectively, impurities can be fully removed after the heat-preservation supernatant is subjected to complexing treatment and decoloring treatment, so that the aim of high purity is fulfilled.
The purity of the degradation product reaches 98 percent by the same method for measuring the product in the example 1, the relative molecular mass is about 8000Da, and the degradation product is a small molecular weight heteropolysaccharide consisting of 6 monosaccharides.
Example 6
A preparation method of quinoa small-molecule heteropolysaccharide comprises the following steps:
s1, selecting and crushing 1kg of chenopodium quinoa willd raw material, screening, performing reflux extraction on the screened raw material for 3 hours at 80 ℃ by using 10L of 78% ethanol in volume percentage, filtering and collecting filter residue after extraction, and collecting a steamed material after ethanol is steamed at 60 ℃;
s2, soaking the steamed material by adopting a PBS buffer solution, wherein the ratio of the volume of the PBS buffer solution to the mass of the quinoa raw material powder is 10 ml: 1g of the extract is 10L, heating to 60 ℃, adding alpha-amylase, preserving heat for 3h, boiling and extracting for 1h, filtering the extracting solution, cooling the filtered liquid to 80 ℃, adding the alpha-amylase again, preserving heat for 3h at 60 ℃ to obtain a heat preservation liquid; wherein the addition amount of the alpha-amylase is that an enzyme preparation with the activity unit of 8000U is added into every 100g of quinoa raw material powder;
s3, centrifuging the obtained heat preservation solution at 8000 Xg for 10min at a high speed, treating the obtained heat preservation supernatant to obtain a treatment solution, and centrifuging the treatment solution at 8000 Xg for 10min at a high speed to obtain a supernatant treatment solution;
s4, adding absolute ethyl alcohol with the volume twice that of the supernatant into the obtained supernatant, precipitating overnight, then centrifuging at a high speed of 8000 Xg for 10min to obtain a precipitate, repeatedly washing the precipitate with the absolute ethyl alcohol, and volatilizing residual ethyl alcohol in the precipitate to obtain the refined quinoa non-starch polysaccharide;
s8, performing online cyclic degradation of the refined quinoa non-starch polysaccharide by using ultrasonic wave-hydrogen peroxide-Vc-ultrafiltration to obtain polysaccharide degradation liquid;
s6, adsorbing the obtained polysaccharide degradation liquid by using 300g of anion exchange resin, wherein the solid-to-liquid ratio of the ion exchange resin to the polysaccharide degradation liquid is 1 g: 8ml, desorbing with 0.8mol/L sodium chloride solution to obtain desorption solution, wherein the volume of the sodium chloride solution is 1L, ultrafiltering the obtained desorption solution with an ultrafiltration membrane tube 8 with the molecular weight cutoff of 10000Da, and vacuum freeze-drying the obtained ultrafiltrate to obtain 180g of quinoa micromolecular heteropolysaccharide.
In order to further realize the purposes of high purity and uniform relative molecular mass, the online cyclic degradation comprises the following specific steps:
a. re-dissolving the refined quinoa non-starch polysaccharide in the S2 with distilled water, and centrifuging the dissolved substance at high speed to remove precipitates to obtain supernatant;
b. diluting the obtained supernatant with distilled water to a concentration of 200mg/ml to obtain a diluted solution;
c. adding hydrogen peroxide and vitamin C reagent into the diluent respectively, wherein 180ml of hydrogen peroxide and 280mg of vitamin C are added, stirring for 0.8h uniformly, and performing ultrasonic-assisted cyclic degradation for 80min in an ultrafiltration system;
the ultrafiltration system comprises a pump circulation pipe 1, a glass guide pipe 2, an ultrafiltration pump 4 and an ultrafiltration membrane pipe 8, wherein both ends of the pump circulation pipe 1 are fixed on the side surface of the ultrafiltration pump 4; the glass guide tube 2 is fixed on the outer side surface of the pump circulating tube 1 and is connected between the pump circulating tube 1 and the ultrafiltration pump 4 in series; the pump circulating pipe 1 is communicated with the glass guide pipe 2; an ultrasonic cleaner 3 is arranged below the ultrafiltration pump 4; the glass conduit 2 is completely submerged in a solvent in a cleaning tank of the ultrasonic cleaner 3 to form an ultrasonic degradation tank 9; the top surface of the ultrafiltration pump 4 is respectively and fixedly connected with the bottom ends of an ultrafiltration membrane tube 8 and an ultrafiltration liquid storage cup 6 in parallel; the top surface of the ultrafiltration liquid storage cup 6 is communicated with the side surface of the ultrafiltration membrane tube 8 through a connecting tube 7, and one end of the ultrafiltration membrane tube 8, which is far away from the connecting tube, is provided with a liquid outlet 81; the top end of the ultrafiltration membrane tube 8 is provided with a pressure gauge 5;
the glass conduit is 2 mm thick, 8-8 cm long and 10-20 mm in inner diameter;
the ultrafiltration circulating system adopts a series-circulation mode, the series-circulation degradation time is 30min, and an ultrasonic cleaner 3 with the power of 320w is adopted; by adopting hydrogen peroxide and vitamin C as degradation reagents of the quinoa non-starch polysaccharide, utilizing the hydrogen peroxide and the vitamin C under the assistance of ultrasonic waves and matching with an ultrafiltration system, carrying out serial circulating degradation, and utilizing a physical and chemical degradation method to be combined with membrane separation, the aims of high purity and uniform relative molecular mass are fulfilled to a certain extent.
For the purpose of further realizing high purity, the anion exchange resin is Toyopearl HW-68F anion exchange resin; the selected anion exchange resin is utilized, and the condition of the related ion exchange resin is limited, so that the polysaccharide degradation liquid can be fully adsorbed in the anion exchange resin, and further a cushion is laid for subsequent desorption, so that the polysaccharide degradation liquid can be fully adsorbed, and the aim of improving the high purity of the polysaccharide degradation liquid is fulfilled.
In order to further realize the purpose of high purity, the preorder treatment comprises the steps of adding calcium chloride solid powder into the obtained heat-preservation supernatant, boiling for 30min, cooling to the heat-preservation temperature, centrifuging at a high speed to obtain mixed supernatant, and adding ethylene diamine tetraacetic acid powder into the mixed supernatant to obtain treatment liquid; wherein the addition amount of the calcium chloride is 80g, and the addition amount of the disodium ethylenediamine tetraacetate is 80 g; the heat preservation temperature is 80 ℃; by adopting the calcium chloride solid and the disodium ethylene diamine tetraacetate to carry out complexing treatment and decoloring treatment on the heavy metal of the heat-preservation supernatant respectively, impurities can be fully removed after the heat-preservation supernatant is subjected to complexing treatment and decoloring treatment, so that the aim of high purity is fulfilled.
The purity of the degradation product reaches 93 percent by the same method for measuring the product in the example 1, the relative molecular mass is about 8000Da, and the degradation product is a small molecular weight heteropolysaccharide consisting of 6 monosaccharides.
The purity of the degradation product reaches 98 percent by the same method for measuring the product in the example 1, the relative molecular mass is about 8000Da, and the degradation product is a small molecular weight heteropolysaccharide consisting of 6 monosaccharides.
Comparative example 1
S2 in example 1 was replaced with only one-time α -amylase, only distilled water, first α -amylase and then distilled water, or first distilled water and then α -amylase, respectively, and the polysaccharide extraction rate, starch content, and non-starch polysaccharide content of the refined quinoa polysaccharide and the refined quinoa non-starch polysaccharide of example 1 were measured, respectively, to obtain table 1.
TABLE 1 polysaccharide extraction rate, starch content and non-starch polysaccharide content in different extraction methods
| Extraction method | Polysaccharide extraction ratio% | Starch content% | Non-starch polysaccharide% |
| Enzyme | 28 | 0 | 100 |
| Water (W) | 26 | 46 | 8 |
| Enzyme + water | 38 | 36 | 8 |
| Water + enzyme | 32 | 0 | 100 |
| Enzyme + water + |
81 | 0 | 100 |
Wherein, the extraction rate of polysaccharide is polysaccharide mass ÷ quinoa powder mass × 100%,
the content of starch is divided by the mass of starch in polysaccharide and the mass of total polysaccharide is multiplied by 100 percent,
the percent of non-starch polysaccharide is the mass of non-starch polysaccharide in polysaccharide ÷ the mass of total polysaccharide multiplied by 100%.
Comparative example 2
Aiming at the method of online cyclic degradation by adopting ultrasonic wave-hydrogen peroxide-Vc-ultrafiltration, the uniformity, the polysaccharide degradation efficiency and the time for obtaining the micromolecular polysaccharide are analyzed for the degradation liquid obtained respectively and the degradation liquid obtained in comparative example 1 by adopting only ultrasonic degradation, only hydrogen peroxide, only vitamin C, first hydrogen peroxide and then vitamin C or first ultrasonic degradation and then hydrogen peroxide, and the table 2 is obtained.
TABLE 2 Table of homogeneity, polysaccharide degradation efficiency and time profiles for obtaining small polysaccharides in different degradation solutions
Wherein the polysaccharide degradation efficiency is defined as: 1g of polysaccharide is degraded in 1 hour to a ratio of the amount of polysaccharide fragments having a relative molecular mass of more than 5000Da and less than 10000Da to the initial polysaccharide mass.
The time for obtaining the micromolecular polysaccharide refers to: the time required for separating the degradation products from undegraded polysaccharides and the monosaccharides and oligosaccharides produced by degradation.
As can be shown by the data in tables 1 and 2 and various examples, the purity of the quinoa small-molecular heteropolysaccharide obtained by the invention is above 90%, the polysaccharide extraction rate is 81% and the polysaccharide degradation rate is 70%, and the relative molecular mass of the obtained product is about 8000Da and the obtained product is a small-molecular heteropolysaccharide composed of 6 monosaccharides.
The foregoing is illustrative of the preferred embodiments of this invention, and it is to be understood that the invention is not limited to the precise form disclosed herein and that various other combinations, modifications, and environments may be resorted to, falling within the scope of the concept as disclosed herein, either as described above or as apparent to those skilled in the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (2)
1. A preparation method of quinoa micromolecular heteropolysaccharide is characterized by comprising the following steps: the method comprises the following steps:
s1, selecting and crushing chenopodium quinoa willd raw materials, screening, performing reflux extraction on the screened raw materials by using ethanol, filtering after extraction, collecting filter residues, evaporating the ethanol, and collecting evaporated materials;
s2, soaking the steamed material by adopting a PBS buffer solution, wherein the volume ratio of the PBS buffer solution to the quinoa raw material powder is 5-10 ml: 1g, heating to 50-60 ℃, adding alpha-amylase, preserving heat for 1-3 hours, boiling, extracting for 1-3 hours, filtering an extracting solution, cooling the filtered liquid to the heat preservation temperature of 50-60 ℃, adding the alpha-amylase again, preserving heat for 1-3 hours at 50-60 ℃ to obtain a heat preservation liquid; wherein the addition amount of the alpha-amylase is that per 100g of quinoa raw material powder, enzyme preparation with the activity unit of 2500-15000U is added;
s3, centrifuging the obtained heat preservation solution at 8000 Xg for 10min at a high speed, processing the obtained heat preservation supernatant to obtain a processing solution, and centrifuging the processing solution at 8000 Xg for 10min at a high speed to obtain a supernatant processing solution; the preorder treatment comprises the steps of adding calcium chloride solid powder into the obtained heat-preservation supernatant, boiling for 30min, cooling to the heat-preservation temperature, centrifuging at a high speed to obtain mixed supernatant, and adding ethylene diamine tetraacetic acid powder into the mixed supernatant to obtain treatment liquid;
s4, adding absolute ethyl alcohol with the volume twice that of the supernatant into the obtained supernatant, precipitating overnight, then centrifuging at a high speed of 8000 Xg for 10min to obtain a precipitate, repeatedly washing the precipitate with the absolute ethyl alcohol, and volatilizing residual ethyl alcohol in the precipitate to obtain refined quinoa non-starch polysaccharide;
s5, performing online cyclic degradation of the refined quinoa non-starch polysaccharide by using ultrasonic wave-hydrogen peroxide-Vc-ultrafiltration to obtain polysaccharide degradation liquid;
s6, adsorbing the obtained polysaccharide degradation liquid by using anion exchange resin, wherein the solid-to-liquid ratio of the ion exchange resin to the polysaccharide degradation liquid is 1 g: 5-10 ml, desorbing with 0.1-0.5 mol/L sodium chloride solution to obtain desorption solution, wherein the ratio of the volume of the sodium chloride solution to the mass of the anion exchange resin is 3-5 ml: 1g, ultrafiltering the obtained desorption solution by using an ultrafiltration membrane tube with the molecular weight cutoff of 10000Da, and carrying out vacuum freeze drying on the obtained ultrafiltrate to obtain quinoa micromolecule heteropolysaccharide, wherein the ultrafiltration membrane is actually tubular, a jacketed pipe is arranged outside the ultrafiltration membrane tube, and a sample solution can be filled in the membrane tube only by reverse flow, so that no air bubbles exist in the whole ultrafiltration membrane tube, and the micromolecules can penetrate through the membrane and flow out from an outflow port under a certain pressure condition to be collected, so that the ultrafiltration membrane adopts an ultrafiltration membrane tube of an ultrafiltration system;
the online cyclic degradation comprises the following specific steps:
a. re-dissolving the refined quinoa non-starch polysaccharide in the S4 with distilled water, and centrifuging the dissolved substance at high speed to remove precipitates to obtain supernatant;
b. adding distilled water into the obtained supernatant to dilute the supernatant to a concentration of 50-200 mg/ml, so as to obtain a diluent;
c. respectively adding hydrogen peroxide and a vitamin C reagent into the diluent, uniformly stirring, and performing ultrasonic assisted degradation for 20-50 min in an ultrafiltration system;
the ultrafiltration system comprises a pump circulating pipe and a glass guide pipe, wherein the glass guide pipe is fixed on the outer side surface of the pump circulating pipe; one end of the glass guide pipe, which is far away from the pump circulating pipe, is abutted with the top end of an ultrasonic generator, and forms an ultrasonic degradation pool with the top end of the ultrasonic generator; the pump circulating pipe is fixed on the side surface of the ultrafiltration pump, and an ultrafiltration membrane pipe and an ultrafiltration liquid storage cup are respectively fixed on the top surface of the ultrafiltration pump; the ultrafiltration pump is fixed on the top surface of the ultrafiltration liquid storage cup of the ultrasonic explaining pool and is connected with the side surface of the ultrafiltration membrane tube through a connecting tube, and a liquid outlet is formed in one end, far away from the connecting tube, of the ultrafiltration membrane tube; the top end of the ultrafiltration membrane tube is provided with a pressure gauge;
the ultrafiltration system adopts a series-circulation mode, and the series-circulation degradation time is 20-50 min;
the anion exchange resin is one of DEAE-Sepharose Fast Flow, DEAE-650M, Toyopearl HW-55F, Toyopearl HW-65F, D201 or D301 type ion exchange resin.
2. The method for preparing quinoa small-molecule heteropolysaccharide according to claim 1, wherein the method comprises the following steps: the heat preservation temperature is 50-60 ℃.
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