CN103349775B - Method for preparing hypoglycemic agent or hypoglycemic food with natural biological active proteins of rice - Google Patents
Method for preparing hypoglycemic agent or hypoglycemic food with natural biological active proteins of rice Download PDFInfo
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Abstract
The invention discloses a method for preparing a hypoglycemic agent or hypoglycemic food. The method comprises the following steps: defatting unpolished rice or/and rice bran to obtain defatted rice flour; mixing the defatted rice flour and sodium chloride water uniformly, and then collecting a supernatant fluid through vibrating extraction and separation, wherein the supernatant fluid is a totally salt soluble protein solution; adding ethanol into the totally salt soluble protein solution, mixing uniformly, and removing the sediment part after standing and separation to obtain a biological active protein solution; obtaining the natural biological active proteins of rice through after-treatment of the biological active protein solution; taking the natural biological active proteins of rice as an active ingredient to prepare the hypoglycemic agent or the hypoglycemic food. According to the method provided by the invention, the natural biological active proteins of rice have the activity of inhibiting pancreatic Alpha-amylase in vitro and achieve a hypoglycemic effect; the prepared hypoglycemic agent or hypoglycemic food has a hypoglycemic efficacy.
Description
Technical field
The present invention relates to the preparation field of hypoglycemic drug or blood sugar reducing food, be specifically related to a kind of method utilizing rice natural bioactive albumen to prepare hypoglycemic drug or blood sugar reducing food.
Background technology
Diabetes are metabolic troubles of a kind of multi-pathogenesis, and the medicine for the treatment of diabetes is if Regular Insulin is mainly based on drug administration by injection at present.According to the WHO(World Health Organization) statistics, annual drug administration by injection 12,000,000,000 times in global range, because unsafe injection causes 1,500 ten thousand routine hepatitis B virus infections, 1,000,000 hepatitis c virus infections, 340,000 HIV (human immunodeficiency virus) infection, 3,000,000 bacteriological infection and 850,000 injection point abscess.Especially diabetic subject, accepting injection of insulin number of times in life may more than 60,000 time.Expert estimates, within 2012, China diabetic subject is more than 9,600 ten thousand, belongs to the whole world at most.Generation and the treatment of diabetes are all closely related with diet, and the sugar and starch particularly in food is directly related with glucostasis.Common starch very soon can saccharogenesis degraded by enzymes and absorbing rapidly in enteron aisle.Concerning diabetics, if with the equally edible general rice of normal people, due to rapid, high volume absorb the glucide of starch degradation, therefore, glycemic index can be made to raise rapidly, cause illness to be aggravated, even bring life danger.Here it is, and diabetics must strictly go on a diet, the least possible reason of eating sugar and starch based food.2008, Zhejiang The Incredible Hulk Bioisystech Co., Ltd make " should sugared rice " that sell derive from resistant starch content up to more than 10% activated water rice varieties, mainly improve the starch structure in rice, improve resistant starch content, after people is eaten suitable sugared rice, the speed of Amylase digestion starch slows down, thus is beneficial to the steady blood sugar of diabetic subject, and reaching control blood sugar increases too fast object.
Rice protein is a kind of food protein of high nutritive value, and mainly the Amino acid profile of rice protein is reasonable, its utilization ratio and biological value high, and having hypoallergenic, is uniquely can in order to avoid the com gluten protein of allergic experiment.According to Osborne based on deliquescent sorting technique, rice protein can rough segmentation be white protein, sphaeroprotein, prolamine and gluten.Wherein white protein, sphaeroprotein can dissolve with sodium-chlor the protein ingredient extracted in rice, also referred to as salting-in protein, be main physiologically active protein (Wang Liangdong etc. Advance of preparation of rice protein. grain processing, 2007,32 (1): 43-47) the rice protein enzymolysis polypeptide, reported and the physiological function of extract comprise hypotensive, hypoglycemic, removing cholesterol, anticancer change etc.
Application publication number is that CN102225161A Chinese invention patent application discloses and utilizes rice bran to prepare method that is anti-oxidant, hypoglycemic, decreasing cholesterol three kinds of function ingredients, this invention take brown rice as raw material, husk rice sieves the rice bran that obtains through microwave deactivating enzyme, ethanol microwave-assisted prepares anti-oxidant function component, that is left with this process is precipitated as raw material, and employing amylase, proteinase hydrolization method prepare function of blood sugar reduction component.The major defect of this technology is: one is have employed microwave treatment and enzymolysis step, and cost is higher; Two are prepared active polypeptide is not natural being present in rice, and is through enzymolysis rice gluten and obtains, and complicated components is uncertain.Report is had no about rice natural bioactive albumen hypoglycemic activity.
Summary of the invention
The invention provides a kind of method utilizing rice natural bioactive albumen to prepare hypoglycemic drug or blood sugar reducing food, adopt sodium-chlor and ethanol two kinds of food grade materials, rice natural bioactive albumen can be extracted from brown rice or rice bran, low cost, pollution-free, again using rice natural bioactive albumen as effective constituent, prepare hypoglycemic drug or blood sugar reducing food, there is hypoglycemic effect.
Utilize rice natural bioactive albumen to prepare a method for hypoglycemic drug or blood sugar reducing food, comprise the following steps:
1) by brown rice or/and rice bran obtains degreasing ground rice through skimming treatment;
2) the degreasing ground rice in step 1) is mixed with sodium chloride aqueous solution, through mechanical shaking extraction, the first separated and collected supernatant liquor after mixing, be total salting-in protein solution;
3) to step 2) in total salting-in protein solution add ethanol, mix, through leave standstill, second be separated after remove sediment fraction, obtain solutions of biologically active proteins;
4) solutions of biologically active proteins in step 3) is removed ethanol, except obtaining rice natural bioactive albumen after salinity and drying through aftertreatment;
5) using the rice natural bioactive albumen in step 4) as activeconstituents, prepare hypoglycemic drug or blood sugar reducing food.
In the present invention, sodium chloride aqueous solution is first adopted to extract the total salting-in protein of rice, obtain total salting-in protein solution, ethanol is adopted to carry out removing impurity by means of precipitation to the foreign protein of total salting-in protein solution except rice natural bioactive albumen again, thus after aftertreatment, obtain the higher rice natural bioactive albumen of purity, finally using rice natural bioactive albumen as effective constituent, prepare hypoglycemic drug or blood sugar reducing food.
In step 1), degreasing ground rice can adopt commercially available prod also can prepare voluntarily, as preferably, described skimming treatment comprises: by brown rice or/and rice bran crosses 30 ~ 80 orders (further preferably 40 ~ 50 orders) sieve after pulverizing obtain powder material, in powder material, add acetone degreasing, after vibration, separation, drying, obtain degreasing ground rice.Through above-mentioned size screening and through the degreasing ground rice that acetone degreasing obtains, be conducive to extracting rice natural bioactive albumen from degreasing ground rice.Further preferably, the quality of described powder material and the volume ratio of acetone are 1:2 ~ 4g/mL, i.e. the solid-to-liquid ratio 1:2 ~ 4g/mL of powder material and acetone, more preferably 1:3g/mL, adopt the acetone of above-mentioned quality, be conducive to the extraction of follow-up rice natural bioactive albumen.
Step 2) in, sodium chloride aqueous solution is adopted to extract the total salting-in protein of degreasing ground rice, as preferably, the concentration of described sodium chloride aqueous solution is 0.5mol/L ~ 1.5mol/L, protein solubleness in water depends on the water molecules number on protein molecule around ionic group, namely depends on the hydration levels of protein.Adopt the neutral salt solution of different concns can control the hydration levels of rice protein, namely control the solubleness of protein in rice corn.When adding a small amount of salt in water, salt ion and water molecules are on the impact of the polar group of protein molecule, and protein solubleness in water is increased, and this i.e. the molten phenomenon of salt; But salt concn increase to a certain degree time, the activity of water reduces, and the electric charge of protein surface is neutralized in a large number, and hydration shell is destroyed, so protein is assembled and Precipitation mutually, Here it is saltouts.The neutral salt that the present invention selects is sodium-chlor, sodium chloride concentration suitable in the aqueous solution is the key affecting the total salting-in protein extraction yield of rice, the sodium chloride aqueous solution of 0.5mol/L ~ 1.5mol/L concentration is conducive to salting-in protein and extracts, and rice total salting-in protein yield is higher.
The quality (g) of described degreasing ground rice and the volume (mL) of sodium chloride aqueous solution are than being 1:5 ~ 12g/mL, the i.e. solid-to-liquid ratio 1:5 ~ 12g/mL of degreasing ground rice and sodium chloride aqueous solution, solid-to-liquid ratio refers to the ratio of the quality (g) of degreasing ground rice and the volume (mL) of sodium chloride aqueous solution.When solid-liquid is smaller, a small amount of NaCl solution is unfavorable for the abundant dissolving of albumen; Too high solid-to-liquid ratio (higher than 1:12) is unfavorable for follow-up concentrated, dry, and energy consumption is increased.Under solid-to-liquid ratio 1:5 ~ 12g/mL, rice total salting-in protein yield is higher.
As preferably, the condition of described mechanical shaking extraction is: 20 ~ 40 DEG C of mechanical shaking extractions 2 ~ 4 hours, this vibration temperature can improve protein dissolution speed, and extracts 2 ~ 4 hours, makes rice total salting-in protein yield higher.
As preferably, described first condition be separated is: at the centrifugal 10 ~ 30min of 5000 ~ 8000rpm, first object be separated is separated with insoluble coarse rice powder residue by the total salting-in protein extracting solution being dissolved in sodium chloride aqueous solution, centrifugal force and centrifugation time can affect the effect that residue precipitates, centrifugal rotational speed is too low or the time is too short, residue precipitation not exclusively, extracting solution muddiness is caused to be unfavorable for that the later stage is separated, the too high overlong time of centrifugal rotational speed, energy consumption is increased and causes centrifugal heat production to cause protein denaturation even starch pasting, optimal separation effect can be ensured at the centrifugal 10 ~ 30min of 5000 ~ 8000rpm.
In step 3), adopt ethanol to carry out removing impurity by means of precipitation to the foreign protein of total salting-in protein solution except rice natural bioactive albumen, as preferably, described total salting-in protein solution and the volume ratio of ethanol are 1:1 ~ 3.Ethanol causes the reason of protein precipitation: be because ethanol is added to the water, solvent dielectric constant is reduced on the one hand, add the magnetism of opposite charges; Be on the other hand because ethanol is strong hydrophilicity reagent, the water of hydration on contention protein molecule surface, destroy the hydration layer of protein colloid molecular surface and molecular aggregates is precipitated, the precipitation selecting ethanol to carry out foreign protein can ensure the edible of protein isolate and application security.In general the concentration of ethanol is how many, relevant with the molecular weight of the foreign protein that can precipitate.When the volume ratio of total salting-in protein solution and ethanol is 1:1 ~ 3, in rice total salting-in protein extracting solution, the foreign protein precipitation of more than 20kDa is more and more complete, and there is bioactive 10 ~ 18kDa lower molecular weight salting-in-protein subfraction (the rice natural bioactive albumen that namely will extract) be retained in solution supernatant, purity is higher.Total salting-in protein solution can ensure the extraction effect of lower molecular weight biological activity protein subfraction in different brown rice or raw rice bran from the scope of volume ratio 1:1 ~ 3 of ethanol.
As preferably, the described condition left standstill is: 1 ~ 10 DEG C leaves standstill 0.5 ~ 1.5h, makes foreign protein form precipitation, is conducive to removing in follow-up process.Temperature reduces makes the reduction of the solvability of foreign protein more easily precipitate removal, is beneficial to the biological activity keeping target protein simultaneously; Time of repose is longer, and foreign protein sedimentation effect is better, leaves standstill 0.5 ~ 1.5h at 1 ~ 10 DEG C, can ensure that the foreign protein precipitation of more than 20kDa in rice is effectively removed.
As preferably, described second condition be separated is: at the centrifugal 10 ~ 30min of 7000 ~ 9000rpm, second object be separated is separated the foreign protein of more than the 20kDa of above-mentioned alcohol settling and the rice lower molecular weight biological activity protein be dissolved in supernatant (the rice natural bioactive albumen that namely will extract), centrifugal force and centrifugation time can affect the effect of precipitation, centrifugal rotational speed is too low or the time is too short, precipitation can not be separated completely with supernatant, cause part foreign protein to remain in supernatant, reduce target protein purity.The too high overlong time of centrifugal rotational speed, makes energy consumption increase, prolonged process, foreign protein can be made to precipitate better be separated with supernatant liquor, ensure the DNA purity of rice natural bioactive albumen at the centrifugal 10 ~ 30min of 5000 ~ 8000rpm.
In step 4), described aftertreatment comprises except ethanol, except salinity and drying: centrifugal vacuum method of enrichment removes ethanol, dialysis except freshen and lyophilize.Through the rice natural bioactive albumen that aftertreatment obtains, be lower molecular weight biological activity protein dry powder, convenient storage, transport and use.Rice natural bioactive albumen, for lower molecular weight biological activity protein dry powder, in white or yellow-white, tasteless, through SDS(sodium lauryl sulphate) to detect protein molecular weight range be 10kDa ~ 18kDa(10000 dalton ~ 18000 dalton in-polyacrylate hydrogel electrophoretic analysis).
In step 5), using rice natural bioactive albumen as activeconstituents, prepare hypoglycemic drug or blood sugar reducing food.Described activeconstituents is the composition useful to human body, namely in the present invention, for playing the composition of effect of lowering blood sugar to human body.Rice natural bioactive albumen has the activity suppressing pancreaticα-amylase in vitro, level of postprandial blood sugar peak value can be made to slow down and reduce, have hypoglycemic activity.Namely when preparing hypoglycemic drug, using the effective constituent of rice natural bioactive albumen as hypoglycemic drug, auxiliary material can prepare hypoglycemic drug in addition, hypoglycemic drug can be pulvis, granule, capsule, tablet, pill or oral liquid, wherein, auxiliary material can adopt usual component of the prior art, and auxiliary material carries out different selections according to the difference of formulation, during as selected tablet, auxiliary material can adopt as weighting agent, softening agent etc.When preparing blood sugar reducing food, using the effective constituent of rice natural bioactive albumen as blood sugar reducing food, blood sugar reducing food comprises hypoglycemia healthcare food and hypoglycemic common delicatessen food, hypoglycemia healthcare food can be pulvis, granule, capsule, tablet, pill or oral liquid, and hypoglycemic common delicatessen food is starchbased product, milk-product, bean product, bakery or beverage.
Compared with prior art, tool of the present invention has the following advantages:
The present invention utilizes rice natural bioactive albumen to prepare the method for hypoglycemic drug or blood sugar reducing food, adopt sodium-chlor and ethanol two kinds of food grade materials, rice natural bioactive albumen can be extracted from brown rice or rice bran, low cost, pollution-free, rice natural bioactive albumen has the activity suppressing pancreaticα-amylase in vitro, level of postprandial blood sugar peak value can be made to slow down and reduce, there is hypoglycemic activity, finally using rice natural bioactive albumen as effective constituent, prepare hypoglycemic drug or blood sugar reducing food, there is hypoglycemic effect.
The present invention utilizes rice natural bioactive albumen to prepare the method for hypoglycemic drug or blood sugar reducing food, and preparation is simple, and controllability is good, easy handling and enforcement, is easy to industrialization scale operation, has broad application prospects and good economic benefit.
Embodiment
By following specific embodiment the present invention done and further illustrate, but should be appreciated that the present invention not limited by these contents.
Embodiment 1:
1) the organic brown rice of Great Northern Wilderness green field adopting commercially available Great Northern Wilderness green food dispensing company limited is raw material, 40 mesh sieves are crossed after pulverizing, obtain powder material, acetone is selected to carry out degreasing to powder material, powder material (g) and acetone (mL) solid-to-liquid ratio 1:3g/mL, the centrifugal 20min collecting precipitation of 6000rpm after vibration 2h, is placed in stink cupboard natural air drying and can obtains degreasing ground rice.
2) the total salting-in protein of rice extracts: take degreasing ground rice 15g and add 1.0mol/L NaCl aqueous solution 120mL, fully after mixing, and 40 DEG C of mechanical shaking extraction 2h, the centrifugal 20min of 6000rpm collects supernatant liquor, repeating step 2) once, merge supernatant liquor, obtain total salting-in protein solution;
3) alcohol settling removal of impurities: to step 2) in total salting-in protein solution add dehydrated alcohol, the volume ratio of total salting-in protein solution and dehydrated alcohol is 1:2, mix by magnetic stirrer simultaneously, 1h is precipitated under placing 4 DEG C of conditions, the centrifugal 20min of 8000rpm, remove sediment fraction, collect supernatant and namely obtain solutions of biologically active proteins;
4) aftertreatment is except ethanol, except salinity and drying: above-mentioned steps 3) in the solutions of biologically active proteins that obtains adopt ethanol in centrifugal vacuum method of enrichment removing solution, again the solution removing ethanol is loaded dialysis tubing and freshen was removed in 3 hours to distill water dialysis, obtaining rice natural bioactive albumen finally by lyophilize, is the dry powder of yellow-white.The yield of rice natural bioactive albumen is 1.5mg/g degreasing ground rice.
Embodiment 2:
1) the organic brown rice of Great Northern Wilderness green field adopting commercially available Great Northern Wilderness green food dispensing company limited is raw material, 50 mesh sieves are crossed after pulverizing, obtain powder material, acetone is selected to carry out degreasing to powder material, powder material (g) and acetone (mL) solid-to-liquid ratio 1:3g/mL, the centrifugal 20min collecting precipitation of 6000rpm after vibration 2h, is placed in stink cupboard natural air drying and can obtains degreasing ground rice;
2) the total salting-in protein of rice extracts: claim 15g degreasing ground rice to add 1.0mol/L NaCl aqueous solution 150mL, fully after mixing, and 30 DEG C of mechanical shaking extraction 2h, the centrifugal 20min of 6000rpm collects supernatant liquor, repeating step 2) once, merge supernatant liquor, obtain total salting-in protein solution;
3) alcohol settling removal of impurities: to step 2) in total salting-in protein solution add dehydrated alcohol, the volume ratio of total salting-in protein solution and dehydrated alcohol is 1:2, mix by magnetic stirrer simultaneously, 1h is precipitated under placing 4 DEG C of conditions, the centrifugal 20min of 8000rpm, remove sediment fraction, collect supernatant and namely obtain solutions of biologically active proteins;
4) aftertreatment is except ethanol, except salinity and drying: above-mentioned steps 3) in the solutions of biologically active proteins that obtains adopt ethanol in centrifugal vacuum method of enrichment removing solution, again the solution removing ethanol is loaded dialysis tubing and freshen was removed in 2 hours to distill water dialysis, obtaining rice natural bioactive albumen finally by lyophilize, is the dry powder of yellow-white.The yield of rice natural bioactive albumen is 1.8mg/g degreasing ground rice.
Embodiment 3:
1) the meticulous defatted rice bran powder adopting commercially available Tian Yu grease company limited of Jiangxi Province to produce is raw material, as degreasing ground rice;
2) the total salting-in protein of rice extracts: accurately take 15g degreasing ground rice and add 1.0mol/L NaCl aqueous solution 150mL, fully after mixing, and 30 DEG C of mechanical shaking extraction 2h, the centrifugal 20min of 6000rpm collects supernatant liquor, repeating step 2) once, merge supernatant liquor, obtain total salting-in protein solution;
3) alcohol settling removal of impurities: to step 2) in total salting-in protein solution add dehydrated alcohol, the volume ratio of total salting-in protein solution and dehydrated alcohol is 1:2.5, mix by magnetic stirrer simultaneously, 1h is precipitated under placing 4 DEG C of conditions, the centrifugal 20min of 8000rpm, remove sediment fraction, collect supernatant and namely obtain solutions of biologically active proteins;
4) aftertreatment is except ethanol, except salinity and drying: above-mentioned steps 3) in the solutions of biologically active proteins that obtains adopt ethanol in centrifugal vacuum method of enrichment removing solution, again the solution removing ethanol is loaded dialysis tubing and freshen was removed in 3 hours to distill water dialysis, obtaining rice natural bioactive albumen finally by lyophilize, is the dry powder of yellow-white.The yield of rice natural bioactive albumen is 6.2mg/g degreasing ground rice (i.e. meticulous defatted rice bran powder).
Embodiment 4:
1) the meticulous defatted rice bran powder adopting commercially available Tian Yu grease company limited of Jiangxi Province to produce is raw material, as degreasing ground rice;
2) the total salting-in protein of rice extracts: accurately take 15g degreasing ground rice and add 0.5mol/L NaCl aqueous solution 75mL, fully after mixing, and 20 DEG C of mechanical shaking extraction 4h, the centrifugal 30min of 5000rpm collects supernatant liquor, repeating step 2) once, merge supernatant liquor, obtain total salting-in protein solution;
3) alcohol settling removal of impurities: to step 2) in total salting-in protein solution add dehydrated alcohol, the volume ratio of total salting-in protein solution and dehydrated alcohol is 1:1, mix by magnetic stirrer simultaneously, 0.5h is precipitated under placing 1 DEG C of condition, the centrifugal 30min of 7000rpm, remove sediment fraction, collect supernatant and namely obtain solutions of biologically active proteins;
4) aftertreatment is except ethanol, except salinity and drying: above-mentioned steps 3) in the solutions of biologically active proteins that obtains adopt ethanol in centrifugal vacuum method of enrichment removing solution, again the solution removing ethanol is loaded dialysis tubing and freshen was removed in 3 hours to distill water dialysis, obtaining rice natural bioactive albumen finally by lyophilize, is the dry powder of yellow-white.The yield of rice natural bioactive albumen is 4.1mg/g degreasing ground rice (i.e. meticulous defatted rice bran powder).
Embodiment 5:
1) the meticulous defatted rice bran powder adopting commercially available Tian Yu grease company limited of Jiangxi Province to produce is raw material, as degreasing ground rice;
2) the total salting-in protein of rice extracts: accurately take 15g degreasing ground rice and add 0.5mol/L NaCl aqueous solution 180mL, fully after mixing, and 40 DEG C of mechanical shaking extraction 2h, the centrifugal 10min of 8000rpm collects supernatant liquor, repeating step 2) once, merge supernatant liquor, obtain total salting-in protein solution;
3) alcohol settling removal of impurities: to step 2) in total salting-in protein solution add dehydrated alcohol, the volume ratio of total salting-in protein solution and dehydrated alcohol is 1:3, mix by magnetic stirrer simultaneously, 1.5h is precipitated under placing 10 DEG C of conditions, the centrifugal 10min of 9000rpm, remove sediment fraction, collect supernatant and namely obtain solutions of biologically active proteins;
4) aftertreatment is except ethanol, except salinity and drying: above-mentioned steps 3) in the solutions of biologically active proteins that obtains adopt ethanol in centrifugal vacuum method of enrichment removing solution, again the solution removing ethanol is loaded dialysis tubing and freshen was removed in 3 hours to distill water dialysis, obtaining rice natural bioactive albumen finally by lyophilize, is the dry powder of yellow-white.The yield of rice natural bioactive albumen is 4.7mg/g degreasing ground rice (i.e. meticulous defatted rice bran powder).
One, molecular weight ranges is determined:
Adopt ordinary method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method of protein molecule flow measurement, operation is with reference to the document (Jiang Shaomei of Jiang Shaomei etc., Zhu Susong, Liu family, Deng. the screening of Rice Glutelin mutant and genetic analysis. Acta Genetica Sinica, 2003,30 (7): 641-645) method listed by, carries out molecular weight analyse to the rice natural bioactive albumen prepared by embodiment.Wherein, not specified, percentage ratio is mass percent.
SDS-PAGE gel strength: separation gel 15%, concentrated glue 5%;
Sample preparation: get and dissolve in distilled water according to the rice natural bioactive albumen prepared by the embodiment of the present invention and make the solution that protein content is 0.5 μ g/ μ L, getting 100 μ L protein solutions (protein content is 50 μ g) with 25 μ L albumen loading mixed solutions [is the beta-mercaptoethanol aqueous solution of 5% by 5 μ L mass concentrations, 5 μ L mass concentrations are the tetrabromophenol sulfonphthalein aqueous solution of 0.5%, 5 μ L mass concentrations are the SDS aqueous solution of 10%, 5 μ L mass concentrations be 50% aqueous glycerin solution and 5 μ L concentration be that 250mmol/L Tris-HCl damping fluid (pH6.8) is mixed to get] mixing, boil the centrifugal 15min of 15000rpm after 5min, get 20 μ L supernatant application of samples and carry out electrophoresis on gel, simultaneously by protein standard marker solution, step 2 in embodiment) in total salting-in protein solution and step 3) solutions of biologically active proteins respectively application of sample electrophoresis, the protein content added in each sample well is 50 μ g.
Deposition condition: constant voltage 150V, electrophoresis 1.5 ~ 2 hours;
Gel-colored and decolouring: gel coomassie brilliant blue R_250 staining fluid after electrophoresis (takes in the destainer that 0.6g coomassie brilliant blue R_250 is dissolved in containing 25% dehydrated alcohol, 8% acetic acid and is made into staining fluid, use after filter paper filtering) dye 4 hours, use destainer (25% dehydrated alcohol, 8% acetic acid) to decolour again to spend the night, and observe gel electrophoresis figure.
According to its gel electrophoresis figure, by contrasting with protein standard marker, rice natural bioactive albumen that the embodiment of the present invention 1 ~ 5 obtains can be arrived at 13kDa ~ 16kDa, wherein, in step 2) in total salting-in protein solution in, it is except rice natural bioactive albumen is in addition higher than the foreign protein of 16kDa molecular weight, and step 3) solutions of biologically active proteins is exactly mainly the rice natural bioactive albumen of molecular weight at 13kDa ~ 16kDa.
Two, rice natural bioactive albumen suppresses the determination of pancreaticα-amylase activity
The rice natural bioactive albumen (dry powder) adopting above-described embodiment 1 to prepare is mixed with the protein solution of 2mg/mL with sterile distilled water, get 100 μ l as mensuration liquid, to publish thesis (Xu Qinmin etc. according to Xu Qinmin etc., the screening of pancreaticα-amylase inhibitor in plant, Chinese Journal of Pharmaceuticals, 1991,22(7): the method described 302-303) measures protein solution to the inhibit activities of ox pancreaticα-amylase.
The preparation of pancreaticα-amylase enzyme liquid: take Niu Xinxian pancreas 50g and rub in mincer, add in 50mL0.01mol/L phosphate buffered saline buffer, stirs homogenate, add acetone 500mL, continue stirring 30 minutes, then leave standstill 10 minutes, removing supernatant, add 250mL acetone again, stir 20 minutes, double gauze filters, must precipitate, precipitation is placed on suction filtration on filter paper dry, is placed into dry sterile glass vials airtight, preserves in refrigerator.Get ox pancreas acetone dry powder 100mg, mix with the 0.02mol/L phosphoric acid buffer (containing 6mmol/L NaCl) of 10mL pH6.9, centrifugal 10 minutes of 8000rpm under 4 DEG C of conditions, obtain supernatant and be pancreaticα-amylase stoste, diluting 10 times with the 0.02mol/L phosphoric acid buffer (containing 6mmol/LNaCl) of pH6.9 is ox pancreaticα-amylase working fluid afterwards.
Mensuration system: get ox pancreaticα-amylase working fluid 0.1mL, testing protein solution 0.1mL, in 37 DEG C of insulation 10min, cooled on ice to 0 DEG C, (getting iodine 1.27g and KI3.0g is dissolved in appropriate distilled water to add iodine liquid, add concentrated hydrochloric acid 1mL, with distilled water diluting to 100mL) 1mL and distilled water 5mL, under 660nm wavelength, measure absorbance immediately.In addition respectively using ox pancreaticα-amylase working fluid 0.1mL+ distilled water 0.1mL as without testing sample control group A, using the 0.02mol/L phosphoric acid buffer of pH6.9 (containing 6mmol/L NaCl)+distilled water 0.1mL as the blank group B without enzyme.
The calculating of testing protein sample pancreaticα-amylase inhibit activities:
Inhibiting rate=[(experimental group absorbancy S-blank B absorbancy)/(control group A absorbancy-blank B absorbancy)] × 100%;
Result shows, when protein concentration is 2.0mg/mL, inhibiting rate is 76%.
Rice natural bioactive albumen prepared by embodiment 2 ~ 5 repeats above-mentioned steps, and when protein concentration is 2.0mg/mL, the inhibiting rate of embodiment 2 is 77%, and the inhibiting rate of embodiment 3 is 78%, and the inhibiting rate of embodiment 4 is 74%, and the inhibiting rate of embodiment 5 is 75%.
Above-mentioned protein solution rice natural bioactive albumen (dry powder) being mixed with 2mg/mL with sterile distilled water, namely can be considered a kind of oral liquid.Certain oral liquid can also adopt other concentration and formula, adds other ancillary component obtained by limited number of time optimum experimental through those skilled in the art according to this area knowledge.
Result is known by experiment, and rice natural bioactive albumen has the activity suppressing pancreaticα-amylase in vitro.Alpha-amylase inhibitor can suppress the activity of saliva in gi tract, pancreatic amylase, obstruction or delaying human body absorb the decomposition of starchy carbohydrate material in food, thus play hypoglycemic activity, suppress the rising of blood sugar concentration, thus be conducive to dietetic treatment (Arai M & Nishimura T.Newproteinaceous α-amylase inhibitor (T-76) the from Streptomyces nitrosporeus.Journalof Fermentation and Bioengineering of diabetic subject, 1989,68:56-57; Wei Pengjuan etc.The progress of α-amylase protein inhibitor.Food science, 2011,32(9): 312-318).Moreover, product on current foreign market "
carb Controller " healing activity composition be exactly the alpha-amylase inhibitor deriving from North America Semen Phaseoli Vulgaris, many cases research prove
carb Controller also can reduce diabetics and normal people's level of postprandial blood sugar by the clear and definite effect of weight reducing of its alpha-amylase inhibitor activation plays, and with food, after beverage mix, its biological activity is constant, the outward appearance of corresponding food is not changed yet, matter structure and local flavor, have no side effect (Barrett M L & Udani J K.A proprietaryalpha-amylase inhibitor form white bean (Phaseolus vulgaris): A review ofclinical studies on weight loss and glycemic control.Nutritional Journal, 2011, 10:24).The rice natural bioactive albumen adopting the present invention to prepare has the activity suppressing pancreaticα-amylase in vitro, illustrates that this rice natural bioactive albumen can be used in preparing hypoglycemic drug or blood sugar reducing food.
Claims (3)
1. utilize rice natural bioactive albumen to prepare a method for hypoglycemic drug or blood sugar reducing food, it is characterized in that, comprise the following steps:
1) by brown rice or/and rice bran obtains degreasing ground rice through skimming treatment;
2) by step 1) in degreasing ground rice mix with sodium chloride aqueous solution, through mechanical shaking extraction, the first separated and collected supernatant liquor after mixing, be total salting-in protein solution;
The concentration of described sodium chloride aqueous solution is 0.5mol/L ~ 1.5mol/L;
The quality of described degreasing ground rice and the volume ratio of sodium chloride aqueous solution are 1:5 ~ 12g/mL;
The condition of described mechanical shaking extraction was: 20 ~ 40 DEG C of mechanical shaking extractions 2 ~ 4 hours;
Described first condition be separated is: at the centrifugal 10 ~ 30min of 5000 ~ 8000rpm;
3) to step 2) in total salting-in protein solution add ethanol, mix, through leave standstill, second be separated after remove sediment fraction, obtain solutions of biologically active proteins;
Described total salting-in protein solution and the volume ratio of ethanol are 1:1 ~ 3;
The described condition left standstill is: 1 ~ 10 DEG C leaves standstill 0.5 ~ 1.5h;
Described second condition be separated is: at the centrifugal 10 ~ 30min of 7000 ~ 9000rpm;
4) by step 3) in solutions of biologically active proteins through aftertreatment except ethanol, except obtaining rice natural bioactive albumen after salinity and drying;
5) using step 4) in rice natural bioactive albumen as activeconstituents, prepare hypoglycemic drug or blood sugar reducing food.
2. the method utilizing rice natural bioactive albumen to prepare hypoglycemic drug or blood sugar reducing food according to claim 1, it is characterized in that, step 1) in, described skimming treatment comprises: by brown rice or/and rice bran crosses 30 ~ 80 mesh sieves after pulverizing obtain powder material, in powder material, add acetone degreasing, after vibration, separation, drying, obtain degreasing ground rice.
3. the method utilizing rice natural bioactive albumen to prepare hypoglycemic drug or blood sugar reducing food according to claim 2, it is characterized in that, the quality of described powder material and the volume ratio of acetone are 1:2 ~ 4g/mL.
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| The amino acid sequence of a 20kD bifunctional subtilisin/α-amylase inhibitor from grain of rice (Oryza sativa L.) seeds;OHTSUBO K et al;《FEBS Letters》;19920831;第309卷(第1期);68-72 * |
| 米糠与米糠蛋白质的开发与利用;姚惠源等;《无锡轻工大学学报》;20020531;第21卷(第3期);312-316 * |
| 米糠中功能性成分的研究现状与发展趋势;王永斌;《中国食物与营养》;20061231(第5期);17-20 * |
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