CN103588892A - Solvent extraction method for active polysaccharide of wheat bran - Google Patents
Solvent extraction method for active polysaccharide of wheat bran Download PDFInfo
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- 238000000638 solvent extraction Methods 0.000 title claims description 5
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- Polysaccharides And Polysaccharide Derivatives (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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Abstract
The invention discloses a method for extracting active polysaccharide-araboxylan from wheat bran through a compound solvent. The extraction method mainly comprises the following steps: the wheat bran is mildly hydrolyzed and preprocessed by a weak acid (citric acid); the target polysaccharide is extracted through a strong alkali (KOH); the extracting solution is centrifugated, neutralized, concentrated and subjected to ethanol precipitation; the ethanol precipitate is dried to obtain the target polysaccharide. The centrifuged supernate of the ethanol precipitate is concentrated and dried, and then can be used as an agricultural compound fertilizer for comprehensive utilization. According to the invention, a large quantity of the byproduct resource, namely the wheat bran is utilized, and the compound solvent is designed to extract the biological active polysaccharide, namely the araboxylan. Compared with the traditional alkali method, the extraction method improves the working efficiency, and reduces the environmental protection pressure. Animal experiments prove that the polysaccharide has prominent immunoregulatory activity, and the effect is comparable to that of lentinan. According to the target polysaccharide provided in the invention, the raw material is low in cost and rich, and the extraction method is environment-friendly, so that the industrialization development of the polysaccharide is promising in the future.
Description
Technical field
The present invention relates to a kind of from Testa Tritici green, environmentally friendly solvent extracting method extraction, that there is immunoregulatory activity araboxylan.
Background technology
Testa Tritici is large by product of wheat flour milling processing, and, as wheat main product and consumption big country, there is the wheat bran resource of 2,000 ten thousand tons of left and right in China every year.Testa Tritici mainly consists of the tissue sites such as kind skin, aleurone layer and remaining endosperm of wheat grain, in its composition, closely half belongs to fibre substrate, comprise main non-starch, non-cellulosic polysaccharide-araboxylan (or claiming piperylene) in Mierocrystalline cellulose, xylogen and cereal crop cell wall structure, wherein araboxylan accounts for the more than 60% of wheat bran fiber matrix.Early stage, large quantity research showed both at home and abroad, araboxylan in Testa Tritici is a kind of important function polysaccharide, except the machining functions of flour products is had material impact, it also has the common organism physiology regulatory function of bioactive polysaccharide, as relax bowel, reducing blood-fat, immunostimulant etc.Araboxylan mainly exists with insoluble form in Testa Tritici, and its functionally active need to be under solvable state competence exertion, therefore the araboxylan in Testa Tritici is extracted and separated from insoluble fibre substrate, change into soluble polysaccharide and just seem very necessary.At present in China, Testa Tritici more than 90%, still mainly as animal and fowl fodder, fermention medium, lacks the exploitation and deep processing conversion of high value added product, especially as the exploitation of this clear efficacy active ingredient of araboxylan.
In domestic publication, mostly to take Testa Tritici to prepare wheat-bran dietary fiber as raw material, as " extracting method of the Multifunctional Conversion-food fibre of wheat bran (200410006552.x) ", " the multienzyme method of fractional steps is prepared the method (200510019123.0) of Wheat bran food fibre powder ", the method (200610024903.9) of extraction of multifunctional diet fibre " a kind of from Wheat bran ", " a kind of method (200710135501.0) of utilizing ultrasound-assisted enzymolysis to prepare wheat-bran dietary fiber ", " a kind of preparation method of wheat-bran dietary fiber (201010185365.8) ", " a kind of method (201110152252.2) of utilizing Testa Tritici to prepare food fibre " etc., mostly product is insoluble food fibre.Also there are some patents to relate to and utilize enzymolysis process from Testa Tritici, to extract araboxylan (or claiming piperylene) or xylo-oligosaccharide, as " a kind of method for separating and preparing of Wheat Bran Pentosans and application (200410014376.4) ", " a kind of method of preparing feruoylated oligosaccharide by enzymolysis of wheat bran (200610037952.6) ", " a kind of method of preparing xylo-oligosaccharide by enzymolysis of wheat bran (200610037953.0) ", " a kind of extruding assisted enzymolysis wheat bran that utilizes is for the method (200710135300.0) of xylo-oligosaccharide ", " Testa Tritici enzyme engineering method transforms the method (200910064898.8) of piperylene and food fibre ", " a kind of method of preparing feruloyl oligosaccharides by wheat bran fermentation (201110060123.0) ", multinomial patents such as " a kind of methods (201210371793.9) of extracting xylan from Testa Tritici ", main employing can degrade zytase or the pentosanase of target polysaccharide-araboxylan, in addition, enzymolysis target also comprises the impurity such as the starch removed in Testa Tritici, protein, Mierocrystalline cellulose.In the research work in our early stage, find, the effect of amylase, proteolytic enzyme, cellulase is mainly to improve the purity of araboxylan, and for improving, the deliquescent influence of target polysaccharide is very not remarkable.In addition, although xylan or pentosanase can directly act on target polysaccharide, make it that degraded occur and reach solubilising object, but in practical application, be subject to the Mierocrystalline cellulose coexisting with target polysaccharide, the constraint of the components such as xylogen and steric restriction effect, actual enzymolysis solubilizing effect is not remarkable, and enzyme action time is conventionally longer, reach 20 hours above and even a couple of days (as " a kind of method of preparing feruoylated oligosaccharide by enzymolysis of wheat bran (200610037952.6) ", " a kind of method of preparing xylo-oligosaccharide by enzymolysis of wheat bran (200610037953.0) ", " a kind of method of biodegradation of wheat bran araboxylan (200810071343.1) ").Although acid system degradation of polysaccharide effect is better, controls improper being easy to polysaccharide hydrolysis is become to even monose (wood sugar, pectinose) of small molecular sugar (wood oligose), and make it lose the due biological activity of polysaccharide.
In research at home and abroad, the araboxylan extracting in Testa Tritici more commonly adopts basic solvent method, as sodium hydroxide (NaOH), calcium hydroxide (Ca (OH)) at present
2), ammoniacal liquor (NH
3) etc. strong or weak alkali lye, in room temperature (60-90 ℃) to comparatively high temps, extract, wherein the most frequently used is NaOH alkaline agent.This research team also finds out in early-stage Study, controlling higher NaOH concentration (0.1N), extracting under temperature (88 ℃) and the condition of abundant extraction time (2h), the extraction yield of wheat bran araboxylan can reach more than 70% (78.5%).But in following commercial application, high NaOH concentration, high temperature extraction can bring ecological, environmental protective problem.In NaOH and the sodium salt of rear generation, if directly discharge meeting causes severe contamination to environment, reclaim and lack again economic feasibility.Therefore the araboxylan of, choosing in practical, feasible method extraction Testa Tritici is the prerequisite that solves its outlet.
Through large quantity research in early stage, on the basis of extracting at traditional alkaline process, this patent has proposed a kind of NEW TYPE OF COMPOSITE solvent-extraction process, i.e. weak acid pre-treatment adds the method for extracting after alkaline agent.Weak acid pre-treatment be for substitute biological enzyme to the mild hydrolysis of polysaccharide chain to strengthen the effect of later stage alkaline extraction; Alkali used is the alkaline agent of relative environmental protection, and the product after neutralization reaction is recyclable as agrochemical use, can avoid thus the environmental protection pressure of Mirae Corp., therefore can be described as green, environmentally friendly solvent extracting method.
Summary of the invention
The object of this invention is to provide a kind of double solvents extracting method with immunoregulatory activity araboxylan that extracts from Testa Tritici.
The double solvents method of extraction polysaccharide provided by the present invention, mainly comprises the steps: first with acid solvent, to process Testa Tritici, obtains suitable acid-hydrolyzed Testa Tritici; With basic solvent, extract target polysaccharide wherein again.
In said process, describedly with acid solvent, process Testa Tritici and specifically comprise the steps: described Testa Tritici, water to mix with acid solvent, carry out acid hydrolytic reaction; Described acid solvent is the citric acid solution that contains 0.05M-0.15M, and acid-hydrolyzed temperature is 50.0 ℃-80.0 ℃, and be 1.0h-3.0h action time, and the proportioning of described Testa Tritici and water can be 1kg:(8-12) L.
In said process, describedly with the target polysaccharide that basic solvent extracts wherein, specifically comprise the steps: the acid-hydrolyzed Testa Tritici mixing solutions of aforementioned process, add highly basic and adjust solution alkaline agent concentration; Described highly basic is potassium hydroxide (KOH), and alkaline agent concentration is controlled at 0.01M-0.08M, and the temperature that alkaline agent extracts is 40.0 ℃-60.0 ℃, and extraction time is 0.5h-1.5h.
In said process, before described acid solvent is processed, described method also can comprise the pre-treatment step that as follows described Testa Tritici is removed endosperm starch, obtains relatively pure fibre substrate Testa Tritici raw material.
The described method that removes endosperm starch can specifically comprise the steps: described commodity wheat wheat bran repeatedly to wash, sieve; Washing times is 3-5 time; The proportioning of described commodity wheat wheat bran and water can be 1kg:(15-25) L.
In said process, after described basic solvent extracts, described method also can comprise following purification step: described extract is centrifugal, collect supernatant liquor; Neutralization, concentrated; Enriched material adopts ethanol precipitation; Centrifugal, collection alcohol hypostasis.
In described centrifugation step, centrifugal force is 3000g-5000g, and centrifugation time is 15min-30min, collects described centrifuged supernatant;
In described neutralization procedure, in and supernatant liquor sour agent used be hydrochloric acid, concentration of hydrochloric acid is 2M-4M;
In described enrichment step, take cryogenic vacuum concentration technology, temperature is 45.0 ℃-55.0 ℃, and vacuum tightness is 0.003MPa-0.005MPa, and described concentrated solution volume is controlled at the 1/5-1/10 of described centrifuged supernatant volume;
In described ethanol settling step, described precipitation temperature is 4.0 ℃-25.0 ℃, and sedimentation time is 12h-24h, and it is 65% that final alcohol concn is controlled.
After the above process of experience, centrifugal, collect described alcohol hypostasis, adopt boulton process that its moisture is down to below 8%-10%, obtain described target polysaccharide.
At described alcohol precipitation, centrifugal, after collecting described alcohol hypostasis, centrifuged supernatant adopts vacuum concentration method to reclaim ethanol, and concentrated solution is finally dry, obtains described composite potassium fertilizer.
In above-mentioned concentrated solution dry, the final concentrated concentration of described concentrated solution should reach more than 35%;
In above-mentioned concentrated solution dry, drying means used is spray-drying process;
In above-mentioned concentrated solution dry, the final moisture content of described composite potassium fertilizer should be down to below 5%.
In said process, every proportioning that relates to Testa Tritici and something, all refers to the amount without the original Testa Tritici of any processing.
The polysaccharide that above-mentioned arbitrary described method obtains also belongs to protection scope of the present invention.
Described target polysaccharide main component is araboxylan; The weight-average molecular weight of described polysaccharide should be 5.0 * 10
4da-10.0 * 10
4da.
Main purpose of the present invention be take Testa Tritici as raw material obtains a kind of polyose product with enhancing body immunologic function.
The application of above-mentioned polysaccharide product in preparation enhancing body immunologic function medicine also belongs to protection scope of the present invention.
Another object of the present invention is to utilize weak acid-highly basic double solvents to extract target polysaccharide by product afterwards still can effectively be utilized and can not bring environmental protection pressure.
The present invention be take commodity wheat wheat bran as raw material, by processing steps such as destarch, the processing of weak acid (citric acid)-highly basic (KOH) double solvents, neutralization, alcohol precipitation, vacuum-dryings, obtain target polysaccharide, this polysaccharide yield is in more than 15.0% (mass percent of product and commodity wheat wheat bran), and wherein araboxylan content is more than 75.0% (in pentosan content in butt).
The inventive method is utilized this agricultural byproducts resource of Testa Tritici, can obtain in a large number that to take araboxylan (or claim piperylene) be main polysaccharide product by pre-treatment and solvent extraction.Another experimentation on animals shows that this polysaccharide has significant immunoloregulation function, and effect may compare favourably with commercially available medicinal fungi polysaccharide (lentinan).This target polysaccharide cost of material is cheap, source is abundant, compared with medicinal fungi polysaccharide, has more market competition advantage, in the environmental protection pressure Hou， Mirae Corp. development prospect that has solved extraction process, will be more wide.
Accompanying drawing explanation
The schema that Fig. 1 Testa Tritici active polysaccharide extracts.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Testa Tritici can be bought and obtain from whole meal flour processing enterprise (as Guqiao Wheat Flour Group, Beijing).
The extract by solvents of embodiment 1, Testa Tritici active polysaccharide
One, polysaccharide extracts: the schema that polysaccharide extracts as shown in Figure 1.
1) Testa Tritici pre-treatment
Take Testa Tritici 100g, be scattered in 500mL water, under room temperature, stir 30min, cross 60 mesh sieves, collect screen overflow; Repeat this process 3-5 time, clear to water, obtain the Testa Tritici of de-endosperm starch.
2) acidolysis
The Testa Tritici that removes starch is dispersed in the citric acid solution that 800mL concentration is 0.15M, is placed in 50.0 ℃ of heat insulation tank stirring reaction 3.0h, obtain acidolysis Testa Tritici mixing solutions.
3) alkaline agent extracts
In acidolysis Testa Tritici mixing solutions, add KOH, adjust alkaline concentration to 0.08M; Be placed at 40.0 ℃ and stir and extract, extraction time is 1.0h.
4) centrifugal, concentrated
The alkaline agent that is cooled to room temperature is extracted to mixed solution centrifugal (3000rpm, 30min), collect supernatant liquor; With 2M hydrochloric acid soln, regulate pH value to neutral supernatant liquor; By neutralizer vacuum concentration at 45.0 ℃, vacuum tightness is 0.003MPa, liquor capacity is concentrated into 160mL left and right and stops (being concentrated into 1/5 left and right of original volume), and the product obtaining is denoted as concentrated solution.
5) ethanol precipitation
In concentrated solution, stir appropriate 95% edible ethanol of interpolation, regulation system alcohol concn reaches 65% and (under whipped state, slowly adds ethanol, avoid adding the too fast partial concn that causes too high and bring non-target compound precipitation as far as possible), form flocculation sediment, standing 12h at 4 ℃; Centrifugal (3000rpm, 30min), collects alcohol hypostasis.
6) dry
Gained ethanol throw out is dry under vacuum condition, be down to 10% to moisture content of material; Pulverize, cross 100 mesh sieves, obtain final polysaccharide product.
7) extracting solution reclaims
Collect centrifuged supernatant, concentrated under vacuum condition, reclaim ethanol; Continue concentratedly, reach 35% to concentrated solution solid content; Spraying is dried to moisture content of material and is down to below 5%, the composite potassium fertilizer that to obtain take Tripotassium Citrate, Repone K be main component.
More than experiment repeats 2 times.
Two, polysaccharide fraction identification
1) essentially consist detects
In product, the mensuration conventional method execution of GB/T21305-2007 cereal and grain products moisture is pressed in moisture content check, and pentosan content is pressed orcinol-salt acid system and carried out, and concrete steps are as follows:
1. the drafting of typical curve
Accurately weigh wood sugar (analytical pure, Sigma company, catalog number 95729) 0.100g, with distilled water, be settled to 100mL, therefrom pipette 10ml in 100ml volumetric flask, constant volume, be mixed with 100 μ g/ml wood sugar standardized solution.
Pipette respectively wood sugar standardized solution 0ml, 0.4ml, 0.8ml, 1.2ml, 1.6ml and 2.0ml, add successively in 6 test tubes, with distilled water, add volume to 3.0ml, adding respectively more successively 0.3ml massfraction is the FeCL that 1% orcinol (Aldrich company, catalog number 447420) ethanol solution and 3.0ml massfraction are 0.1%
3(analytical pure, traditional Chinese medicines group chemical reagent Beijing company limited) concentrated hydrochloric acid solution, mix, boiling water bath 30min, take out and be cooled to rapidly room temperature, with the blank tube that does not add xylose solution, compare, colorimetric under 670nm, 580nm wavelength, gained light absorption value is difference (the Δ A=A of two wavelength light absorption values
670-A
580), take Δ A as ordinate zou, the quality of corresponding xylose solution of take is X-coordinate, drawing standard curve.
2. the mensuration of sample
Get appropriate (0.010-0.050g) and extract polysaccharide sample, add 20ml2M HCl solution, in sealing, in vitro in 100 ℃ of water-baths, be hydrolyzed 3h, cooling rear with filter paper filtering; Hydrolyzed solution is suitably accurately pipetted to 1ml in tool plug test tube after dilution, add successively the orcinol ethanol solution of 2ml distilled water, 0.3ml massfraction 1%, the FeCL3 concentrated hydrochloric acid solution of 3.0ml massfraction 0.1%, mix boiling water bath 30min; Be cooled to rapidly room temperature, under 670nm, 580nm wavelength, measure absorbancy, other is same 1..Calculation formula:
Pentosan content (%)=C*n*0.88*100/m/10
6
In formula, C represents to obtain wood sugar quality (μ g) by typical curve; N is extension rate; M is sample dry weight (g).
Product yield calculation formula: the Testa Tritici butt quality (g) * 100% of yield (%)=products obtained therefrom butt quality (g)/for extracting; Wherein, something butt quality: refer to by after the moisture removal in something the weight of this material.
The composition evaluation of the polysaccharide product that each extraction is obtained all repeats 3 times, and result is taken the mean.
Experimental result shows, the inventive method gained target polysaccharide yield is 16.5%, and wherein pentosan content is 78.7% (in wood sugar).
2) monose compositional analysis: high performance liquid chromatography (HPLC) method
Get 10.0mg gained polysaccharide of the present invention solid phase prod, at 20ml, 1M H
2sO
4100 ℃ of hydrolysis 3h in solution; Cooling, add BaCO
3be neutralized to pH7.0; Filter, collect filtrate; Micro-filtration (φ 0.45 μ m), filtrate for later use.
HPLC condition: Sugar pak1,6.8 * 300mm chromatographic column (Waters company); Moving phase is ultrapure water; Flow velocity 0.5mL/min; 85 ℃ of column temperatures; 30 ℃ of pond temperature; Sample size 10 μ L; Detector is differential refraction detector.
Dextrose standard sample is purchased from Sigma company, and catalog number is G5400; Wood sugar standard substance are purchased Sigma company, and catalog number is 95729; Pectinose standard substance are purchased from Sigma company, and catalog number is 10860.
Under as above chromatographic condition, the retention time of dextrose standard sample is 13.023min; The retention time of wood sugar standard substance is 15.008min; The retention time of pectinose standard substance is 16.285min.
Two parallel repetitions are established in experiment.In gained polysaccharide product of the present invention, syrup hydrolysis products is respectively after measured: glucose, wood sugar, pectinose; The quality percentage composition of glucose in three kinds of sugar is 3.06%, and the quality percentage composition of wood sugar in three kinds of sugar is 62.18%, and the quality percentage composition of pectinose in three kinds of sugar is 34.76%; Result shows, in the sugar unit composition of gained polysaccharide product of the present invention, more than 95% is wood sugar and these two kinds of five-carbon sugars of pectinose, and glucose content is very low, judges that product of the present invention is as a kind of araboxylan (or piperylene) product.
3) molecular weight of product measure of spread: multi-angle laser light scattering-high performance gel filtration chromatography (MALLS-HPSEC)
It is the solution of 5mg/mL that product configuration of the present invention is become to polysaccharide concentration, and solvent is 0.01M, pH7.2 phosphoric acid buffer; Micro-filtration (φ 0.45 μ m), filtrate for later use.
Condition determination: DAWN-EOS multiple angle laser light scattering instrument (U.S. Wyatt company), HPLC system (Waters company), TSK-gel G4000HHR post (Japanese TOSOH company), moving phase 0.01M, pH7.2 phosphoric acid buffer, flow velocity 0.5mL/min, chromatographic column temperature is room temperature, optical maser wavelength 690.0nm, working time 30min.Data processing adopts Astra software.
Two parallel repetitions are done in experiment.
Detected result shows: target polysaccharide main ingredient of the present invention is that a kind of weight-average molecular weight is 7.123 * 10
4da, the araboxylan (or claiming piperylene) being formed by pectinose and wood sugar.
The extract by solvents of embodiment 2, Testa Tritici active polysaccharide
One, polysaccharide extracts: the schema that polysaccharide extracts as shown in Figure 1.
1) Testa Tritici pre-treatment
Take Testa Tritici 1000g, be scattered in 6L water, under room temperature, stir 30min, cross 60 mesh sieves, collect screen overflow; Repeat this process 4 times, clear to water, obtain the Testa Tritici of de-endosperm starch.
2) acidolysis
The Testa Tritici that removes starch is dispersed in the citric acid solution that 10L concentration is 0.05M, is placed in 80.0 ℃ of heat insulation tank stirring reaction 1.0h, obtain acidolysis Testa Tritici mixing solutions.
3) alkaline agent extracts
In acidolysis Testa Tritici mixing solutions, add KOH, adjust alkaline concentration to 0.05M; Be placed at 50.0 ℃ and stir and extract, extraction time is 0.5h.
4) centrifugal, concentrated
The alkaline agent that is cooled to room temperature is extracted to mixed solution centrifugal (3000rpm, 15min), collect supernatant liquor; With 4M hydrochloric acid soln, regulate pH value to neutral supernatant liquor; By neutralizer vacuum concentration at 55.0 ℃, vacuum tightness is 0.005MPa, liquor capacity is concentrated into 1L left and right and stops (being concentrated into 1/10 left and right of original volume), and the product obtaining is denoted as concentrated solution.
5) ethanol precipitation
In concentrated solution, stir appropriate 95% edible ethanol of interpolation, regulation system alcohol concn reaches 65% and (under whipped state, slowly adds ethanol, avoid adding the too fast partial concn that causes too high and bring non-target compound precipitation as far as possible), form flocculation sediment, standing 24h at 4 ℃; Centrifugal (3000rpm, 20min), collects alcohol hypostasis.
6) dry
Gained ethanol throw out is dry under vacuum condition, be down to 8% to moisture content of material; Pulverize, cross 100 mesh sieves, obtain final polysaccharide product.
7) extracting solution reclaims
Collect centrifuged supernatant, concentrated under vacuum condition, reclaim ethanol; Continue concentratedly, reach 36% to concentrated solution solid content; Spraying is dried to moisture content of material and is down to below 5%, the composite potassium fertilizer that to obtain take Tripotassium Citrate, Repone K be main component.
More than experiment repeats 2 times.
Two, polysaccharide fraction identification
1) essentially consist detects
Method is with consistent described in embodiment 1.
Experimental result shows, the inventive method gained target polysaccharide yield is 17.1%, and wherein pentosan content is 75.3% (in wood sugar).
2) monose compositional analysis: high performance liquid chromatography (HPLC) method
Method is with consistent described in embodiment 1.
In gained polysaccharide product of the present invention, syrup hydrolysis products is respectively after measured: glucose, wood sugar, pectinose, the quality percentage composition of three kinds of monose is followed successively by 3.06%, 60.15%, 36.79%.The sugar unit of gained polysaccharide product of the present invention more than 95% is wood sugar and these two kinds of five-carbon sugars of pectinose in forming, and glucose content is very low, judges that thus product of the present invention is as a kind of araboxylan (or piperylene) product.
3) molecular weight of product measure of spread: multi-angle laser light scattering-high performance gel filtration chromatography (MALLS-HPSEC)
Method is with consistent described in embodiment 1.
Detected result shows: target polysaccharide main ingredient of the present invention is that a kind of weight-average molecular weight is 5.218 * 10
4da, the araboxylan (or claiming piperylene) being formed by pectinose and wood sugar.
The extract by solvents of embodiment 3, Testa Tritici active polysaccharide
One, polysaccharide extracts: the schema that polysaccharide extracts as shown in Figure 1.
1) Testa Tritici pre-treatment
Take Testa Tritici 10kg, be scattered in 4.5L water, under room temperature, stir 30min, cross 60 mesh sieves, collect screen overflow; Repeat this process 5 times, clear to water, obtain the Testa Tritici of de-endosperm starch.
2) acidolysis
The Testa Tritici that removes starch is dispersed in the citric acid solution that 120L concentration is 0.10M, is placed in 60.0 ℃ of heat insulation tank stirring reaction 2.4h, obtain acidolysis Testa Tritici mixing solutions.
3) alkaline agent extracts
In acidolysis Testa Tritici mixing solutions, add KOH, adjust alkaline concentration to 0.01M; Be placed at 60.0 ℃ and stir and extract, extraction time is 1.0h.
4) centrifugal, concentrated
The alkaline agent that is cooled to room temperature is extracted to mixed solution centrifugal (3000rpm, 20min), collect supernatant liquor; With 2.5M hydrochloric acid soln, regulate pH value to neutral supernatant liquor; By neutralizer vacuum concentration at 50.0 ℃, vacuum tightness is 0.004MPa, liquor capacity is concentrated into 15L left and right and stops (being concentrated into 1/8 left and right of original volume), and the product obtaining is denoted as concentrated solution.
5) ethanol precipitation
In concentrated solution, stir appropriate 95% edible ethanol of interpolation, regulation system alcohol concn reaches 65% and (under whipped state, slowly adds ethanol, avoid adding the too fast partial concn that causes too high and bring non-target compound precipitation as far as possible), form flocculation sediment, standing 15h at 4 ℃; Centrifugal (3000rpm, 15min), collects alcohol hypostasis.
6) dry
Gained ethanol throw out is dry under vacuum condition, be down to 7% to moisture content of material; Pulverize, cross 100 mesh sieves, obtain final polysaccharide product.
7) extracting solution reclaims
Collect centrifuged supernatant, concentrated under vacuum condition, reclaim ethanol; Continue concentratedly, reach 35.5% to concentrated solution solid content; Spraying is dried to moisture content of material and is down to below 5%, the composite potassium fertilizer that to obtain take Tripotassium Citrate, Repone K be main component.
More than experiment repeats 2 times.
Two, polysaccharide fraction identification
1) essentially consist detects
Method is with consistent described in embodiment 1.
Experimental result shows, the inventive method gained target polysaccharide yield is 15.8%, and wherein pentosan content is 78.6% (in wood sugar).
2) monose compositional analysis: high performance liquid chromatography (HPLC) method
Method is with consistent described in embodiment 1.
In gained polysaccharide product of the present invention, syrup hydrolysis products is respectively after measured: glucose, wood sugar, pectinose, the quality percentage composition of three kinds of monose is followed successively by 2.54%, 61.02%, 35.14%.The sugar unit of gained polysaccharide product of the present invention more than 95% is wood sugar and these two kinds of five-carbon sugars of pectinose in forming, and glucose content is very low, judges that thus product of the present invention is as a kind of araboxylan (or piperylene) product.
3) molecular weight of product measure of spread: multi-angle laser light scattering-high performance gel filtration chromatography (MALLS-HPSEC)
Method is with consistent described in embodiment 1.
Detected result shows: target polysaccharide main ingredient of the present invention is that a kind of weight-average molecular weight is 6.744 * 10
4da, the araboxylan (or claiming piperylene) being formed by pectinose and wood sugar.
Embodiment 4, the experiment of Testa Tritici active polysaccharide immunoloregulation function
Experimental arrangement carries out immunoloregulation function mensuration with reference to the check of < < protective foods and the method for evaluation technique standard > > (Ministry of Health 2003) to Kunming mouse.Detect index comprise 1. immune organ dirty/body weight ratio; 2. sheep red blood cell (SRBC) (SRBC) inducing mouse DTH (the sufficient sole of the foot thickens method) delayed allergy (DTH); 3. Hemagglutination Hemolysin product (Hemagglutination Method); 4. Turnover of Mouse Peritoneal Macrophages is engulfed chicken red blood cell experiment (half intracorporal method) mensuration phagocytic rate and phagocytic index.The visible above-mentioned standard of concrete grammar and calculation formula.
Adopt the prepared polysaccharide of embodiment 1 to carry out functional experiment.By polysaccharide according to 50,100, the basic, normal, high dosage of 150mg/kg joins in basal diet.Experiment mice is divided into 5 groups, every group 10, positive controls (lentinan as a control group,, 100mg/kg, Puluokangyu Natural Drug Co., Ltd., Zhejiang), low dose group (50mg/kg), middle dosage group (100mg/kg), high dose group (150mg/kg), feed respectively basal diet and the test daily ration of homopolysaccharide dosage not.
All experimental datas are used after SPSS8.0 statistical software processes
represent, between group, relatively adopt t check.Result is as shown in table 1-table 4.
The impact of table 1 wheat bran polysaccharide on mouse thymus and spleen/body weight
Note: the significance of difference of comparing with control group, * p<0.05, * * p<0.01.
Table 2 wheat bran polysaccharide is to mouse delayed allergy (DTH)
Note: the significance of difference of comparing with control group, * p<0.05, * * p<0.01.
The impact of table 3 wheat bran polysaccharide on mice serum hemolytic antibody product
Note: each dosage group has the trend of increasing, but difference does not reach conspicuous level.
The impact of table 4 wheat bran polysaccharide on mouse monokaryon macrophage phagocytic function
Note: the significance of difference of comparing with control group, * p<0.05, * * p<0.01.
By the above results, shown, compare with control group, the middle and high dosage group of wheat bran polysaccharide all can significantly improve Thymus and Spleen index, enhancing delayed allergy (DTH) and the mononuclear-macrophage phagocytic function (p<0.05, p<0.01) of mouse; And high dose group is suitable with commercially available lentinan (positive controls) functionally active, can judge by the prepared wheat bran polysaccharide of the inventive method and have immunoloregulation function thus.
Because embodiment 2 and 3 prepared target polysaccharide and embodiment 1 product are without the significant difference on raw material, method and product content and composition, can think that function and embodiment 1 product, without significant difference, have immunoloregulation function equally.
Claims (9)
1. a double solvents method of extracting active polysaccharide from Testa Tritici, comprises the steps: first with acid solvent, to process Testa Tritici, obtains suitable acid-hydrolyzed Testa Tritici, then obtains target polysaccharide wherein by basic solvent extraction, ethanol precipitation.
2. method according to claim 1, it is characterized in that: describedly first by the method that acid solvent is processed Testa Tritici, comprise the steps: that acid solvent is the citric acid solution of 0.05M-0.15M, acid-treated temperature is 50.0 ℃-80.0 ℃, time 1.0h-3.0h, the proportioning of described Testa Tritici and water is 1kg:(8-12) L.
3. according to the method described in claim 1-2, it is characterized in that: before described acid treatment, comprise as follows described Testa Tritici is carried out to pretreated step: described commodity wheat wheat bran is washed, sieved; Washing times is 3-5 time; The proportioning of described commodity wheat wheat bran and water can be 1kg:(15-25) L, obtain removing starch, relatively pure fibre substrate Testa Tritici raw material.
4. according to arbitrary described method in claim 1-3, it is characterized in that: in described method, describedly with basic solvent, extract and should comprise the steps: to add potassium hydroxide (KOH) highly basic to acid hydrolysis Testa Tritici mixing solutions, alkaline agent concentration is controlled at 0.01M-0.08M, extract 40.0 ℃-60.0 ℃ of temperature, time 0.5h-1.5h.
5. according to arbitrary described method in claim 1-4, it is characterized in that: in described method, after described basic solvent extracts, before described ethanol precipitation, comprise as follows described extract is carried out to centrifugal, neutralization, concentrated step: described alkaline extract is carried out centrifugal, collect supernatant liquor; Described supernatant liquor is added to acid for adjusting pH to neutral; Vacuum concentration, to the 1/5-1/10 of described centrifuged supernatant original volume, obtains concentrated extracting solution.
6. according to arbitrary described method in claim 1-5, it is characterized in that: described centrifugal, neutralization, concentrated after, regulate 65% alcohol concn to precipitate target polysaccharide, described precipitation temperature is 4.0 ℃-25.0 ℃, time 12h-24h.
7. according to arbitrary described method in claim 1-6, it is characterized in that: the supernatant liquor after described ethanol precipitation, after reclaiming ethanol, can be rich in the composite potassium fertilizer of Tripotassium Citrate, Repone K, without discarded discharge through vacuum concentration, the dry preparation of spraying.
8. the polysaccharide that arbitrary described method obtains in claim 1-7.
9. polysaccharide according to claim 8, is characterized in that: described target polysaccharide is mainly araboxylan; The molecular weight of described polysaccharide should described polysaccharide weight-average molecular weight should be 5.0 * 10
4da-10.0 * 10
4within the scope of Da; Described polysaccharide has significant immune-enhancing activity.
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