CN101724666A - Process for extracting and purifying beta-glucan contained in highland barley - Google Patents
Process for extracting and purifying beta-glucan contained in highland barley Download PDFInfo
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- CN101724666A CN101724666A CN200810046375A CN200810046375A CN101724666A CN 101724666 A CN101724666 A CN 101724666A CN 200810046375 A CN200810046375 A CN 200810046375A CN 200810046375 A CN200810046375 A CN 200810046375A CN 101724666 A CN101724666 A CN 101724666A
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- 235000007340 Hordeum vulgare Nutrition 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title claims abstract description 49
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 title claims abstract description 28
- 229920002498 Beta-glucan Polymers 0.000 title claims abstract description 28
- 240000005979 Hordeum vulgare Species 0.000 title 1
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- 108010065511 Amylases Proteins 0.000 description 3
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- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 108010059820 Polygalacturonase Proteins 0.000 description 3
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 3
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Abstract
The invention discloses a process for extracting and purifying beta-glucan contained in highland barley, comprising the following steps of: (a) smashing highland barley, and sieving by a sieve of 60 meshes; (b) drying and inactivating enzymes at 85 DEG C for 2 hours, and adding water according to the material and water ratio of 1:22; (c) adding sodium carbonate, regulating the pH value to be 8.0, extracting at 80 DEG C for 1.5 hours, cooling and centrifugalizing so as to obtain supernate; (d) regulating the pH value of the supernate to be 6.0, adding high-temperature resistant a-amylases with 4 u/g, destarching for 30 minutes by carrying out enzymolysis at the water bath temperature of 90-95 DEG C till an iodine solution is checked without containing starch, and cooling to room temperature; (e) regulating the pH value to be 4.5, refrigerating filtering liquid and centrifugalizing so as to obtain the supernate; (f) regulating the pH value to be 8.0, adding 0.25 gram of pancreatic enzymes (after being dissolved), carrying out the enzymolysis at 47 DEG C for 2.5 hours, and centrifugalizing so as to obtain the supernate; (g) regulating the pH value of the supernate to be 7.0, adding ethanol till the content of the ethanol is 70 percent, and refrigerating the filtering liquid; (h) filtering to obtain sediments, adding 50 times of water to dissolve the sediments, adding the ethanol till the content of the ethanol is 75 percent, and refrigerating the filtering liquid; and (i) filtering to obtain sediments, freezing and drying.
Description
Technical field
The present invention relates to beta-glucan extraction and purifying process in the extracting method of plant constituent, particularly highland barley.
Background technology
Highland barley is a kind of cereal crop of Gramineae Hordeum, and because of coetonium shell in it separates, seed is exposed, so claim hull-less barley, highland barley, Mi Damai again.Highland barley has abundant nutritive value and special efficacy, and highland barley is the highest crop of β one dextran in the wheat crops in the world, is 6.57% according to detecting the highland barley Beta-dextran average content, and improved seeds highland barley 25 can 8.6%, is 50 times of wheat average content.Beta-glucan is used for preventing colorectal carcinoma by reducing intestinal mucosa with contacting with the carcinogenic thing of supporting one's family of indirect inhibition of carcinogenic substance; Synthetic preventing cardiovascular disease by reducing blood-fat and decreasing cholesterol: prevent and treat diabetes by controlling blood sugar.Has the effect that improves the body defence capability, regulates physiological rhythm.
The present invention extracts from highland barley has abundant nutritive value and special efficacy beta-glucan, has determined that first alkali carries the extraction and the purifying process of back enzymolysis starch.
Summary of the invention
The purpose of this invention is to provide in a kind of highland barley beta-glucan and extract and purifying process,, meet the big production requirement of industry so that beta-glucan extraction yield and purity are higher in the highland barley.
Purpose of the present invention is achieved through the following technical solutions: beta-glucan extracts and purifying process in a kind of highland barley, carries out in turn according to the following steps: a), get highland barley and pulverized 60 mesh sieves; B), go out enzyme 2 hours of 85 ℃ of dryings, add water at 1: 22 by material-water ratio, c), add yellow soda ash and transfer PH8.0,80 ℃ are extracted 1.5h, put cold, centrifugal, supernatant liquor.D), to transfer the supernatant liquor pH value be 6.0, adds high temperature resistant a-amylase by 4u/g, bath temperature is 90-95 ℃ and carries out enzymolysis destarching 30min, till the iodine fluid inspection is not starch-containing, is cooled to room temperature.E), to transfer PH be 4.5, refrigerated liquid, centrifugal, supernatant liquor.F), transfer PH8.0, add 0.25g pancreatin (the dissolving back adds), 47 ℃ of enzymolysis 2.5h, centrifugal supernatant liquor.G), to transfer PH be 7.0 to supernatant liquor, adding ethanol is 70% to containing the alcohol amount, refrigeration is spent the night.H), filter precipitation, precipitation adds 50 times of water dissolution, adding ethanol is 75% to containing the alcohol amount, refrigeration is spent the night.I), filter to such an extent that precipitate lyophilize.
The present invention is a foundation with the theory, and based on experiment, and through experiment checking repeatedly, the beta-glucan extraction yield is higher relatively in the highland barley of this technology, and satisfies the big production requirement of industry, can directly be applied in the industrial production.
Embodiment
The preparation technology of beta-glucan carries out according to the following steps in turn in the highland barley: a), get highland barley and pulverized 60 mesh sieves; B), go out enzyme 2 hours of 85 ℃ of dryings, add water at 1: 22 by material-water ratio, c), add yellow soda ash and transfer PH8.0,80 ℃ are extracted 1.5h, put cold, centrifugal, supernatant liquor.D), to transfer the supernatant liquor pH value be 6.0, adds high temperature resistant a-amylase by 4u/g, bath temperature is 90-95 ℃ and carries out enzymolysis destarching 30min, till the iodine fluid inspection is not starch-containing, is cooled to room temperature.E), to transfer PH be 4.5, refrigerated liquid, centrifugal, supernatant liquor.F), transfer PH8.0, add 0.25g pancreatin (the dissolving back adds), 47 ℃ of enzymolysis 2.5h, centrifugal supernatant liquor.G), to transfer PH be 7.0 to supernatant liquor, adding ethanol is 70% to containing the alcohol amount, refrigeration is spent the night.H), filter precipitation, precipitation adds 50 times of water dissolution, adding ethanol is 75% to containing the alcohol amount, refrigeration is spent the night.I), filter to such an extent that precipitate lyophilize.
Concrete experimental study is as follows:
1 materials and methods
1.1 main agents
95% ethanol (analytical pure); Yellow soda ash (analytical pure); Hydrochloric acid (analytical pure); Sodium hydroxide (analytical pure); High temperature resistant a-amylase; Pancreatin; Polygalacturonase.
1.2 main test apparatus
Water-bath; Whizzer; Ultraviolet spectrophotometer; The PH acidometer; Rotary Evaporators; Constant temperature jolting bed.
1.3 test method
1.3.1 investigate whether pulverize to extract and influence
Get each two parts of highland barley grain, highland barley flours, numbering is respectively 1,2; 3,4, every part of 80g adds water by material-water ratio at 1: 22, and transfers PH8.0, extracts 1.5h, puts cold, centrifugal.Get supernatant liquor.The high temperature resistant a-amylase liquid of new preparation 100u/ml, transferring the supernatant liquor pH value is 6.0, and bath temperature is 90-95 ℃, adds the high temperature resistant a-amylase liquid 5ml of new preparation, and destarching 30min till the iodine fluid inspection is not starch-containing, is cooled to room temperature.Transferring PH is 4.5, refrigerates liquid, centrifugal, gets supernatant liquor.Transfer PH8.0, add 0.25g pancreatin (the dissolving back adds), 47 ℃ of enzymolysis 2.5h.The centrifugal supernatant liquor that gets.It is 7.0 that supernatant liquor is transferred PH, and adding ethanol is 70% to containing the alcohol amount, and refrigeration is spent the night.Drying precipitated, measure content and write down measurement result.
1.3.2 baking, dry enzyme inactivating method are relatively
Get wherein that 90g is divided into even 3 parts, toast go out enzyme 5min, 15min, 30min, observing effect respectively with the high fire screen of microwave oven.100g pulverized and sieved rearmounted baking oven (80 ℃) the drying enzyme 2h that goes out for No. 3.At any time observing effect.
1.3.3 investigate the influence of whether stirring to extracting
Get 4 parts of Rikaze highland barley flours, every part of 40g is numbered 1,2,3,4, adds water at 1: 22 by material-water ratio, and transfers PH8.0, and (not stirring) 1.5h is extracted in 1,2 water-baths, and 3,4 stir extraction (agitator) extracts 1.5h, puts cold, centrifugal.Get supernatant liquor.The high temperature resistant a-amylase liquid of new preparation 100u/ml, transferring the supernatant liquor pH value is 6.0, and bath temperature is 90-95 ℃, adds the high temperature resistant a-amylase liquid 5ml of new preparation, and destarching 30min till the iodine fluid inspection is not starch-containing, is cooled to room temperature.Transferring PH is 4.5, refrigerates liquid, centrifugal, gets supernatant liquor.Transfer PH8.0, add 0.25g pancreatin (the dissolving back adds), 47 ℃ of enzymolysis 2.5h.The centrifugal supernatant liquor that gets.It is 7.0 that supernatant liquor is transferred PH, and adding ethanol is 70% to containing the alcohol amount, and refrigeration is spent the night.Drying precipitated, measure content and write down measurement result.
1.3.4 investigate the reflux enzyme inactivating method
Before extracting, this The effects highland barley the dextran extraction effect is influenced through the processing of 95% alcohol heating reflux.
It is an amount of to take by weighing highland barley flour, adds 95% alcohol heating reflux 2h by 1: 6 times of amount, and gained highland barley slag is waved the ethanol after drying, is divided into three parts, every part of 30g.Add water at 1: 22 by solid-liquid ratio, it is 8.0 that 20% sodium carbonate solution is transferred PH, extracts 1.5h in 70 ℃ of water-baths, the centrifugal supernatant liquor that gets.Transferring PH is 6.0, adds high temperature resistant a-amylase by 4u/g, in 90～95 ℃ of water enzyme digestion 30min.The gained extracting solution adds water to 600ml, and transferring PH is 7.0.
Precision is measured 1ml to 25ml volumetric flask respectively, adds suitable quantity of water, in 70 ℃ of water-bath dissolvings fully, adds water to scale after the placement room temperature, to refrigerator cold-storage.Precision is measured 1ml to 10ml measuring bottle, adds water and mends to 2ml, adds the Congo red solution reaction 10min of 4ml, surveys maximum absorption band, optical density under the 545nm.Calculate beta-glucan content.
1.3.5 investigate the supersound extraction of beta-glucan in the highland barley and decoct the water extracting method
Get highland barley and choose removal of impurities in right amount, suitably broken, take by weighing two parts, each 100g respectively adds the 600ml95% alcohol dipping and spends the night.The steeping fluid that inclines is got highland barley and is added 95% alcohol heating reflux three times, each 1 hour.Leach highland barley, wave the ethanol after drying.Two parts are respectively A 94g; B 93.1g.Get A part, add the 1000mlPH8.5 sodium carbonate solution, add 1/150 times amylase 0.627g, in ultrasonic three times of ultrasonic machine (60 ℃～70 ℃), each 1h.Filtered through gauze, refrigeration.Add dilute hydrochloric acid and transfer PH to 4.5, add the 0.3g saccharifying enzyme, 60 ℃ of water enzyme digestions 40 minutes.Centrifugal.Get B part, add the 1000mlPH8.5 sodium carbonate solution, add 1/150 times amylase 0.627g, water is carried and is decocted each 1 hour three times.Filtered through gauze, refrigeration.Add dilute hydrochloric acid and transfer PH to 4.5, add the 0.3g saccharifying enzyme, 60 ℃ of water enzyme digestions 40 minutes.Centrifugal.Alcohol precipitation A, B are to containing alcohol amount 60%, and standing over night is filtered, and gets the resolution of precipitate moon 5 times of water gagings, adds 20% ammonium sulfate, reacts 6 hours, and is centrifugal.The gained precipitation is measured washed with isopropyl alcohol 2 times, 60 ℃ of vacuum-dryings with 2 times.
1.3.6 investigation water-bath alkali is carried and is rotated alkali and carry
Get highland barley and choose removal of impurities in right amount, suitably broken, cross sieve No. 2, get the 215g highland barley flour.Add 800ml80% ethanol, stir, add 100ml80% ethanol again, reflux 2 hours.Wave ethanol, get highland barley flour, drying.Respectively get totally two parts of 50g highland barley flours, add 15 times of water respectively, transfer PH to 8,80 ℃ are extracted twice, each 1.5h, and portion is a heating in water bath, a rotation heating.Filtered through gauze gets filtrate A, B.Transferring PH respectively is 6, adds 65 ℃ of water-bath 0.5h of 1.69g amylase, is concentrated into 250ml, and alcohol is sink to and contains alcohol amount 75% again, spends the night.Reclaim ethanol, precipitation vacuum-drying.
1.3.7 investigate high temperature resistant α-Dian Fenmei add-on
Take by weighing the about 1000g of highland barley, the selection removal of impurities.Pulverized and sieved rearmounted baking oven (85 ℃) the drying enzyme 1h that goes out for No. 3, pulverized sieve No. 1.Got 120 order highland barley flour 600g, and be divided into even two parts, every part of 300g uses 1000ml95% alcohol reflux 2h respectively.Controlled temperature is about 80 ℃.Leave standstill and put cold back and reclaim ethanol, the highland barley slag volatilizes ethanol, puts 60 ℃ in baking oven to complete drying.Collection and weighing are standby.
(1) gets highland barley slag, weighing and record.Be divided into first 1/3, second 1/3, the third 1/3 respectively, all add 10 times of water gagings.Wherein first is 7.0 with 20% sodium carbonate solution accent pH value, and the second pH value is adjusted to 8.0, the third pH values and is adjusted to 9.0, and solution colour changed after pH value was transferred in observation.Extracting method: extract twice in 80 ℃ of water-baths, each 1h.United extraction liquid, centrifugal, collect supernatant liquor.It is 6.0 that first, second, third liquid are all transferred pH value with dilute hydrochloric acid, behind 90 ℃～95 ℃ water-bath preheating 3min, add high temperature resistant a-amylase liquid (considering to press respectively 4u/g, 8u/g, 12u/g enzyme add-on) according to previous step starch content result, sampling is surveyed starch, colorimetric with the iodine test fluid behind the enzymolysis 30min.Again whether consideration according to circumstances enzymolysis 30min.Enzymolysis finishes the back in 100 ℃ of boiling water enzyme 10min that goes out.Survey starch, colorimetric with the iodine test fluid.
(2) get highland barley slag, weighing and record.Be divided into I 1/3, II 1/3, III 1/3 respectively, all add 5 times of water gagings, in 65 ℃ of gelatinization 1h, (13: 15) transfer pH value again is 6.0, add high temperature resistant a-amylase liquid (pressing 15u/g, 100u/g, 200u/g respectively) in 90 ℃～95 ℃ water enzyme digestion 1h, enzymolysis finishes the back in 100 ℃ of boiling water enzyme 10min that goes out.Survey starch number in the liquid with the iodine test fluid, investigate hydrolysis result.
1.3.8 investigate first alkali carry again enzymolysis starch extracting method and first gelatinization enzymolysis starch again alkali carry extracting method
Take by weighing the about 150g of highland barley, the selection removal of impurities.Put baking oven (85 ℃) the drying enzyme 1h that goes out.Pulverize.Cross sieve No. 4.The alcohol reflux degrease is dezymotized.Get highland barley flour, with 600ml 95% alcohol reflux 2h.Controlled temperature is about 80 ℃.Leave standstill and put cold back and reclaim ethanol, suitably wave ethanol, put 80 ℃ in baking oven again to complete drying.Weighing is standby.
Take by weighing 120g, be divided into 2 parts of A, B, every part of 60g.Again A is divided into 3 parts of a, b, c, every part of 20g; B is divided into d, e, every part of 20g of f.
(1) first alkali is carried enzymolysis starch extracting method again
1, gets highland barley slag (20g), add 10 times of water gagings (200ml).Transferring pH value with 20% sodium carbonate solution is 8.0
Extracting method: extract 2h in 80 ℃ of water-baths.Centrifugal 10min (4500r/min) collects supernatant liquor.
2, transferring the supernatant liquor pH value with dilute hydrochloric acid is 6.0, behind 90 ℃～95 ℃ water-bath preheating 3min, adds high temperature resistant a-amylase liquid (press 4u/g enzyme add-on), and enzymolysis 1h, enzymolysis finish afterwards in 100 ℃ of boiling water enzyme 10min that goes out.
3, a, b, c liquid filter respectively, and putting cold back survey pH value and transferring pH value with 20% sodium carbonate solution is 8.0.
4, stir and to add ethanol and reach 75%, alcohol precipitation spend the night (4 ℃ in refrigerator) to containing the alcohol amount.
5, filter precipitation solution, collecting precipitation.
(2) first gelatinization enzymolysis starch again alkali carry extracting method
1, get highland barley slag (20g), add 5 times of water gagings (100ml), in 65 ℃ of gelatinization 1h, transferring pH value with dilute hydrochloric acid again is 6.0, adds high temperature resistant a-amylase liquid (press 100u/g) in 90 ℃～95 ℃ water enzyme digestion 1h, and enzymolysis finishes afterwards in 100 ℃ of boiling water enzyme 10min that goes out.
2, centrifugal must the precipitation adds 10 times of water gagings, and transferring pH value with 20% sodium carbonate solution is 8.0, and 2h is extracted in 80 ℃ of rotations of water-bath.Centrifugal again collection supernatant liquor filters.
3, add ethanol to the alcohol amount of containing and reach 55% alcohol precipitation spend the night (4 ℃ in refrigerator).
4, filter precipitation solution, collecting precipitation.
1.3.9 screening alkali lye extraction process condition, preferred extracting parameter.
(1) orthogonal test
Get the highland barley crushing screening, in 80 ℃ of enzyme 1h that go out.Take by weighing nine parts, every part of 30g, numbering 1-9 puts the 1000ml beaker, carries out alkali lye by orthogonal test level of factor table and extracts (transferring PH with 20% sodium carbonate solution).The gained extracting solution gets supernatant liquor through the centrifugal 10min of 5000r/min, transfers volume to 600ml, transfers pH value to 6 with dilute hydrochloric acid, in 90～95 ℃ of water-baths, adds high temperature resistant enzymolyzing alpha-amylase starch 30min by 4u/g.The gained extracting solution is transferred PH to 7.
Precision is measured each extracting solution 1ml to 25ml volumetric flask, adds suitable quantity of water, in 70 ℃ of water-bath dissolvings fully, adds water to scale after the placement room temperature, to refrigerator cold-storage.Precision is measured 1ml to 10ml measuring bottle, adds water and mends to 2ml, adds the Congo red solution reaction 10min of 4ml, surveys maximum absorption band place wavelength, and optical density under the 545nm, calculates beta-glucan content.Formula: C=144.3091A-3.2990 R=0.9894.
Table 2-1 orthogonal test level of factor table
(2) orthogonal test checking
1 to No. 9 extracting solution of above-mentioned gained is concentrated into 200ml, adds 95% ethanol alcohol and be sink to that to contain alcohol amount be 75%, refrigeration is spent the night, and vacuum filtration must precipitate, drying.Weigh, precision takes by weighing 0.01g respectively, to the 25ml volumetric flask, adds suitable quantity of water and dissolves in 70 ℃ of water-baths, be placed to room temperature, precision is measured 1ml to 10ml measuring bottle, and moisturizing adds and newly joins Congo red solution 4ml to 2ml, reaction 10min surveys maximum absorption band place wavelength, and optical density under the 545nm, calculates content.
1.3.10 ethanol alcohol precipitation concentration
This experiment relatively alcohol precipitation contains best alcohol precipitation concentration in alcohol amount 50%～80% scope.
Get highland barley and see and must pulverize sieve No. 4 by after the removal of impurities, 80 ℃ of dry 1h of baking oven are 1: 22 by solid-liquid ratio, and pH value is adjusted to 8.0, extract 1.5 hours in 80 ℃ of water-baths.Add high temperature resistant enzymolyzing alpha-amylase starch by 4u/g, be concentrated in right amount, be divided into 4 parts, alcohol is sink to 50%, 60%, 70%, 80% respectively, and refrigeration is spent the night.Centrifugal must the precipitation, dry back congo red method is surveyed B-Glucan purity.
1.3.11 compare Different Extraction Method
Get 3 parts of Rikaze highland barley flours, every part of 40g is numbered 1,2,3, adds water at 1: 22 by material-water ratio, and transfers PH8.0, and 1 one times alkali is carried 1.5h, and 2 secondary alkali are carried (1.5h * 2), and 3 microwave extraction (12min), put cold, centrifugal by 810w.Get supernatant liquor.The high temperature resistant a-amylase liquid of new preparation 100u/ml, transferring the supernatant liquor pH value is 6.0, and bath temperature is 90-95 ℃, adds the high temperature resistant a-amylase liquid 5ml of new preparation, and destarching 30min till the iodine fluid inspection is not starch-containing, is cooled to room temperature.Transferring PH is 4.5, refrigerates liquid, centrifugal, gets supernatant liquor.It is 7.0 that supernatant liquor is transferred PH, and adding ethanol is 70% to containing the alcohol amount, and refrigeration is spent the night.Drying precipitated, measure content and write down measurement result.
1.4 highland barley Beta-dextran purifying research
1.4.1 the removal protein research of highland barley Beta-dextran
This The effects pancreatin enzymolysis protein matter is in conjunction with isoelectric point method isolating protein effect.
(1) Xylene Brilliant Cyanine G method typical curve is painted precision and is taken by weighing bovine serum albumin 0.00502g, adds the water constant volume in the 50ml measuring bottle.The 100ug/ml bovine serum albumin solution.Precision takes by weighing Coomassie brilliant blue G250 0.05g, adds 90% ethanol 25ml, and 85% phosphoric acid 50ml dissolving, moisturizing be to 500ml, Xylene Brilliant Cyanine G solution.According to the form below is surveyed ultraviolet absorptivity.
Table 2-2 Xylene Brilliant Cyanine G method standard curve determination is table as a result
Typical curve: C=203.5046+3.9062 R=0.9818
(2) get highland barley, press optimum extraction process and extract, press 0.25g/40g pancreatin amount again, ph8, enzymolysis 2.5h under 47 ℃ of water-baths surveys protein, glucan content, is transferring ph4.5, and refrigeration is spent the night, and surveys protein, glucan content.
1.4.2 highland barley Beta-dextran hydrogen peroxide, activated carbon decolorizing are investigated
Press optimised process and extract highland barley two parts of A, B, every part of 50g, gained extracting solution, refrigerate in conjunction with the isoelectric point method isolating protein with pancreatin.Centrifugal, get supernatant A liquid and B liquid.
A liquid is 900ml altogether, measures 450ml, is divided into 9 parts, every part of 50ml.According to the form below carries out decolorization experiment.Dilute 10 times, congo red method is surveyed glucan content.
B liquid is 500ml altogether, is divided into 5 parts, every part of 100ml, and according to the form below decolours, and the dextran extraction yield is surveyed in the decolouring back.
1.4.3 macroporous resin purification method
Get extraction crude product 5g and be dissolved in 500ml, transfer PH4.5,4 ℃ are spent the night.And then transfer PH7.0 to get the direct alcohol precipitation of 250ml.250ml crosses macroporous resin column in addition.
(1) macroporous resin pre-treatment
At first use saturated aqueous common salt (36-100) to soak 18-20h, drain salt solution then, the water that makes discharge only with the clear water rinsing is displaing yellow not, uses 2%-4%NaoH solution soaking 2-4h again, and it is stand-by near neutrality to water to wash resin after draining again.
(2) cross post
Cross the D101 macroporous adsorbent resin, be washed till in the elutriant with distilled water again and add 95% ethanol till do not have a precipitation, four sections collections of the dense branch of elutriant are respectively 100ml, 100ml, 70ml, 100ml.Therefrom respectively getting 1ml respectively adds water 1ml and carries out assay.
1.4.4 the removal of pectin in the highland barley Beta-dextran crude product
The accurate title, decided Semen avenae nudae extract crude product 1g (purity is 34%), adds 50ml water, presses 120u/g, adds polygalacturonase, and transferring pH value is 4.5, jolting 5h in the constant temperature shaking table, and temperature is 50-55 ℃, 120r/min observes the dissolving situation, cooling, transferring pH value is 7.0, alcohol precipitation spends the night.Suction filtration and drying precipitated recovery ethanol.Measure content.
1.4.5 ammonium sulfate precipitation purifying
Get extract crude product 5g and be dissolved in 500ml water after, precipitation once more in the ammoniumsulphate soln of 20-30%, 4 ℃ of refrigerations are spent the night, filtering precipitation, precipitation with the dialysis tubing dialysis after, it is 75% to containing the alcohol amount that solution adds ethanol, alcohol precipitation spends the night, and must precipitate, and measures its content.
2 results and analysis
2.1 extract experimental result and analysis
2.1.1 whether pulverize the result is investigated in the influence of extracting
Table 2-3 pulverizes table is as a result investigated in the influence of extracting
| Numbering | Dry thing weight | Maximum absorption band | Purity (%) | Extraction yield (%) |
| ??1 | ??0.5392 | ??545.5nm | ??51.15 | ??1.01 |
| ??2 | ??0.6910 | ??545nm | ??52.66 | ??1.15 |
| ??3 | ??1.0766 | ??545nm | ??72.95 | ??2.03 |
| ??4 | ??1.4328 | ??545nm | ??69.92 | ??2.14 |
Experimental result shows: pulverizing can improve dna purity and extraction yield.
Enzyme is put the method comparative result 2.1.2 baking, drying are gone out
Experimental result shows: get wherein and toast go out enzyme 5min, 15min, 30min, observing effect respectively with the high fire screen of microwave oven.100g pulverized and sieved rearmounted baking oven (80 ℃) the drying enzyme 2h that goes out for No. 3.Result according to the observation selects baking oven (80 ℃) the drying enzyme 2h roasting mode that goes out.
2.1.3 whether stir the result is investigated in the influence of extracting
Table 2-4 does not stir table is as a result investigated in the influence of extracting
| Numbering | Dry thing weight | Maximum absorption band | Purity (%) | Extraction yield (%) |
| ??1 | ??0.5492 | ??545.5nm | ??81.15 | ??1.11 |
| ??2 | ??0.6810 | ??545nm | ??72.66 | ??1.24 |
| Numbering | Dry thing weight | Maximum absorption band | Purity (%) | Extraction yield (%) |
| ??3 | ??1.1666 | ??545nm | ??72.95 | ??2.21 |
| ??4 | ??1.3327 | ??545nm | ??69.92 | ??2.44 |
Experimental result shows that stirring can improve extraction yield and dna purity.
2.1.4 the reflux enzyme inactivating method is investigated the result
Table 2-5 reflux enzyme inactivating method is investigated table as a result
| Sequence number | ??A | ??B | ??C | Mean value |
| Maximum absorption band place wavelength (nm) | ??545 | ??545.5 | ??546 | |
| 545nm place optical density (ABS) | ??0.1717 | ??0.1964 | ??0.1843 | |
| Content (ug/ml) | ??21.48 | ??25.04 | ??23.30 | |
| β-Glucan extraction yield (%) | ??2.1 | ??2.5 | ??2.3 | ??2.3 |
Data and the table 5 not direct alkali of reflux are carried the alkali lye extraction highland barley that the data comparative descriptions is crossed with 95% alcohol heating reflux, and the beta-glucan extraction yield is lower, loses bigger.And it is big to consume amount of reagent, will not adopt.
2.1.5 the supersound extraction of beta-glucan and decoction water extracting method are investigated the result in the highland barley
Experimental result: the drying precipitated A 4.3g of supersonic extracting method, water is carried and is decocted the drying precipitated 4.5g of extracting method.
2.1.6 water-bath alkali is carried and is rotated alkali and put forward the investigation result
Experimental result: get dry thing water-bath alkali and carry 0.9g; Rotation alkali is carried 0.7g.Select water-bath alkali to carry and be extracting method.
2.1.7 high temperature resistant α-Dian Fenmei add-on is investigated the result
Experimental result:
Corresponding first, the second of method (1), third enzymolysis solution add the equal nondiscoloration of iodine liquid, and the third minimum amount 4u/g reaches the complete effect of enzymolysis.
The enzymolysis solution of the corresponding I of method (2), II, III adds iodine liquid I variable color, corresponding enzyme dosage 18,107,204u/g, and promptly the 100u/g enzymolysis is complete.
2.1.8 first alkali carry again enzymolysis starch extracting method and first gelatinization enzymolysis starch again alkali carry extracting method and investigate the result
Experimental result: precipitation after the drying a 0.7g b 1.0g c 0.9g d 0.2g (e, f thing precipitation) respectively get 0.01g, constant volume is in 100ml, the congo red method colorimetric, the 545nm place, optical density is respectively: 0.403,0.406,0.424,0.315.Then method (1) content is than method (2) height.It is better that elder generation's alkali is carried back enzymolysis starch.
2.1.9 orthogonal experiments
(1) Orthogonal experiment results
Table 2-6 orthogonal experiment data results table
Table 2-7 orthogonal experiment analytical table
Experimental data result shows: in the alkali lye leaching process, and factor B＞D＞A＞C, i.e. solid-liquid ratio＞extraction time＞extraction temperature＞alkali lye pH value, best of breed is A3B3C1D2, be that solid-liquid ratio is 1: 22, the extracting solution pH value is adjusted to 8.0, extracts 1.5 hours in 80 ℃ of water-baths.
(2) quadrature confirmatory experiment result
Data results table after the table 2-8 drying
| Sequence number | The amount of taking by weighing (g) | Maximum absorption band wavelength (nm) | Optical density under the 545nm (ABS) | Beta-glucan content (ug/ml) in the 25ml measuring bottle trial-product | β-glucan content in the crude product (%) | Beta-glucan extraction yield (%) in the highland barley |
| ??1 | ??0.0100 | ??549.0 | ??0.3617 | ??97.80 | ??38.04 | ??0.90 |
| ??2 | ??0.0100 | ??547.0 | ??0.5547 | ??153.50 | ??24.45 | ??1.41 |
| ??3 | ??0.0104 | ??545.0 | ??0.5134 | ??141.58 | ??38.37 | ??1.13 |
| ??4 | ??0.0105 | ??545.0 | ??0.5059 | ??139.41 | ??34.03 | ??0.33 |
| ??5 | ??0.0103 | ??544.0 | ??0.4740 | ??130.21 | ??33.19 | ??0.53 |
| ??6 | ??0.0100 | ??545.5 | ??0.5198 | ??143.43 | ??31.60 | ??1.67 |
| ??7 | ??0.0103 | ??544.0 | ??0.4728 | ??129.86 | ??35.86 | ??1.16 |
| ??8 | ??0.0104 | ??544.0 | ??0.4594 | ??125.99 | ??31.52 | ??1.21 |
| ??9 | ??0.0101 | ??545.5 | ??0.4481 | ??122.73 | ??30.29 | ??1.62 |
Orthogonal experiment design result analytical table after the table 2-9 drying
Analysis of experimental data result shows: best of breed still is A3B3C1D2.Be that solid-liquid ratio is 1: 22, the extracting solution pH value is 8.0, extracts 1.5 hours in 80 ℃ of water-baths.
2.1.10 the ethanol alcohol precipitation concentration is investigated the result
Table 2-10 ethanol alcohol precipitation concentration is investigated table as a result
| Alcohol precipitation concentration | ??50％ | ??60％ | ??70％ | ??80％ |
| Maximum absorption band place wavelength (nm) | ??545.0 | ??545.0 | ??544.5 | ??544.0 |
| 545nm place optical density (ABS) | ??0.4979 | ??0.4774 | ??0.5223 | ??0.4993 |
| Content (ug/ml) | ??68.55 | ??65.59 | ??72.07 | ??68.75 |
| B-Glucan extraction yield (%) | ??2.29 | ??2.19 | ??2.40 | ??2.29 |
Experimental result shows that alcohol precipitation concentration 70% is best.
2.1.11 Different Extraction Method comparative result
Table 2-11 Different Extraction Method comparative result table
| Extracting method | Absorbance | Maximum absorption band (nm) | Purity (%) |
| Microwave extraction | ??0.1922 | ??545 | ??67.04 |
| One time alkali is carried | ??0.2064 | ??545.5 | ??65.32 |
| Secondary alkali is carried | ??0.1786 | ??545 | ??58.71 |
Experimental result shows: microwave extraction result is best, but to extract difficulty bigger, secondary alkali put forward not only trouble and purity undesirable, so alkali of employing is carried.
2.2 highland barley purifying experimental studies results
2.2.1 the investigation result of the beta-glucan isolating protein of highland barley
Table 2-12 deproteinize experimental result table
Experimental result shows: the isolating protein effect is obvious, and little to dextran shadow extraction yield is rung.
2.2.2 hydrogen peroxide, activated carbon decolorizing are investigated the result
9 parts of experimental result tables of table 2-13A liquid
5 parts of experimental result tables of table 2-14B liquid
Test-results shows: 10% hydrogen peroxide add-on 1.8ml/g, extraction yield is maximum.Remove 2 extras, mixes 1-9 number, be concentrated into 200ml, alcohol precipitation 75%, centrifugal must the precipitation, the dry purity of surveying, after the precipitation drying after the hydrogen peroxide decolouring, look yellowish-white, and survey purity 4.83% is lost excessively, and this method is inadvisable.The 1-4 of mixed active carbon decoloring number, be concentrated into 170ml, this moment, extracting solution became muddy, used activated carbon decolorizing again, alcohol precipitation, precipitation is dry, 0.8227g, recording average purity is 21.1%, and certain loss is arranged, and the dry thing of gained color depth still, decolouring to no effect.
2.2.3 macroporous resin purification experimental result
The measuring of table 2-15 macroporous resin purification is table as a result
| Sequence number | Volume (ml) | Absorbancy | Maximum absorption band |
| ??1 | ??100 | ??0.7883 | ??541 |
| ??2 | ??100 | ??0.7840 | ??540.5 |
| ??3 | ??70 | ??0.7902 | ??541 |
| ??4 | ??100 | ??0.4808 | ??545 |
The dried cream rate of table 2-16 assay result (getting 50ml surveys)
| Sequence number | Furnace pot weight | Furnace pot weight | Weight | Purity |
| ??1 | ??58.1588 | ??58.4778 | ??0.638 | ??3.462736 |
| ??2 | ??65.9153 | ??66.2990 | ??0.7674 | ??2.862673 |
| ??3 | ??56.4208 | ??56.8041 | ??0.53662 | ??2.889003 |
| ??4 | ??63.2491 | ??63.2956 | ??0.093 | ??14.28646 |
Experimental result shows that the macroporous resin purification method is infeasible, and content descends on the contrary.Will not adopt.
2.2.4 the result is investigated in the removal of pectin in the highland barley Beta-dextran crude product
Table is as a result investigated in the removal of pectin in the table 2-17 highland barley Beta-dextran crude product
| Sequence number | ??1 | ??2 | ??3 |
| Maximum absorption band (nm) | ??0.1375 | ??0.1115 | ??0.1531 |
| Absorbancy (ABS) | ??542 | ??544 | ??543 |
| Purity (%) | ??9.96 | ??8.65 | ??10.24 |
It is undesirable that experimental result shows that polygalacturonase is removed pectin, and cause beta-glucan heavy losses in the highland barley.Will not adopt.
2.2.5 ammonium sulfate precipitation purification result
Comparison sheet before and after the table 2-18 ammonium sulfate precipitation
| Maximum absorption band (nm) | Purity (%) | |
| Before the purifying | ??545 | ??42.5 |
| Behind the purifying | ??545 | ??43.6 |
Experimental result shows: behind ammonium sulfate precipitation, beta-glucan crude product purity improves little, and the dialysis procedure complexity, will not adopt.
3 discuss
By experiment of single factor, the heating alcohol reflux enzyme beta-glucan extraction yield that goes out is lower, loses bigger.And it is big to consume amount of reagent, will not adopt.Pulverizing is bigger to the influence of extraction effect, after the pulverizing not only extraction yield increase dna purity and also increase, so thing to be extracted needs to pulverize.Pulverize the back and adopt 80 ℃ of oven dry 2h, extracting method selects alkali to carry, and it is best to put forward effect with alkali.It is better that first alkali was carried back enzymolysis starch during alkali was carried, and high temperature resistant α-Dian Fenmei add-on 4u/g can remove starch.The extracting solution alcohol precipitation, alcohol concn is 70%, effect is best.
Determined the processing condition of extraction beta-glucan in the highland barley by orthogonal experiment, for solid-liquid ratio is 1: 22, the extracting solution pH value is 8.0, extracts 1.5 hours in 80 ℃ of water-baths.
Shows that by purifying experiment enzyme process and iso-electric point remove protein and can both reach good effect, adopt the two to remove protein, but decolorization experiment shows that decolouring to no effect and influences the beta-glucan extraction yield, can not adopt.Macroporous resin purification experiment and ammonium sulfate precipitation purification result are all undesirable, do not adopt.
Claims (2)
1. beta-glucan extracts and purifying process in the highland barley, it is characterized in that: carry out in turn according to the following steps:
A), get highland barley and pulverized 60 mesh sieves;
B), go out enzyme 2 hours of 85 ℃ of dryings, add water at 1: 22 by material-water ratio;
C), add yellow soda ash and transfer PH8.0,80 ℃ are extracted 1.5h, put cold, centrifugal, supernatant liquor;
D), to transfer the supernatant liquor pH value be 6.0, adds high temperature resistant a-amylase by 4u/g, bath temperature is 90-95 ℃ and carries out enzymolysis destarching 30min, till the iodine fluid inspection is not starch-containing, is cooled to room temperature;
E), to transfer PH be 4.5, refrigerated liquid, centrifugal, supernatant liquor.F), transfer PH8.0, add 0.25g pancreatin (the dissolving back adds), 47 ℃ of enzymolysis 2.5h, centrifugal supernatant liquor;
G), to transfer PH be 7.0 to supernatant liquor, adding ethanol is 70% to containing the alcohol amount, refrigeration is spent the night;
H), filter precipitation, precipitation adds 50 times of water dissolution, adding ethanol is 75% to containing the alcohol amount, refrigeration is spent the night.
I), filter to such an extent that precipitate lyophilize.
Described b), go out enzyme 2 hours of 85 ℃ of dryings 2. extract and purifying process according to beta-glucan in the described highland barley of claim 1, it is characterized in that:, add water at 1: 22 by material-water ratio: c), add yellow soda ash and transfer PH8.0,80 ℃ are extracted 1.5h, put cold, centrifugal, must supernatant liquor; D), to transfer the supernatant liquor pH value be 6.0, adds high temperature resistant a-amylase by 4u/g, bath temperature is 90-95 ℃ and carries out enzymolysis destarching 30min, till the iodine fluid inspection is not starch-containing, is cooled to room temperature.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102206292A (en) * | 2011-04-14 | 2011-10-05 | 青海省花宝蜂业股份合作公司 | Barley beta-glucan preparation method |
| CN104012877A (en) * | 2014-06-25 | 2014-09-03 | 叶炳年 | Preparation method of sweet pickled highland barley |
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| CN102206292A (en) * | 2011-04-14 | 2011-10-05 | 青海省花宝蜂业股份合作公司 | Barley beta-glucan preparation method |
| CN104012877A (en) * | 2014-06-25 | 2014-09-03 | 叶炳年 | Preparation method of sweet pickled highland barley |
| CN104286687A (en) * | 2014-10-11 | 2015-01-21 | 天津北洋百川生物技术有限公司 | Method for preparing beta-glucan malt extract |
| CN106318775A (en) * | 2016-08-24 | 2017-01-11 | 西藏月王生物技术有限公司 | Preparing technology of red yeast rice highland barley wine |
| CN106749750A (en) * | 2016-12-12 | 2017-05-31 | 上海理工大学 | A kind of preparation method of highland barley grain beta glucan |
| CN107216405A (en) * | 2017-05-26 | 2017-09-29 | 苏州大学 | The preparation technology and its structure sequence of a kind of highland barley beta glucan |
| CN108866122A (en) * | 2018-07-02 | 2018-11-23 | 青海华实科技投资管理有限公司 | A kind of method of water-soluble dietary fiber in extraction highland barley bran |
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