CN101269111B - Method for extracting alpha-diastase restrainer from phaseolus limensis - Google Patents
Method for extracting alpha-diastase restrainer from phaseolus limensis Download PDFInfo
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Abstract
The invention discloses a method of extracting Alpha amylase inhibitor from kidney beans. The method is as follows: the crushed kidney beans are first dispersed in a buffer solution with a definite pH value, and then shifted into an ultrasonic device for ultrasonic extraction, and then conducted enzyme elimination, centrifugalization, dialysis and ethanol sediment, dried and finally the Alpha amylase inhibitor is obtained. The Alpha amylase inhibitor extracted from kidney beans in the invention is a white solid powder with a protein content of 80 to 95 percent and an inhibitory activity of 5,000 to 50,000 U/g. Compared with the prior method of lixiviation and extraction, the method of extracting Alpha amylase inhibitor from kidney beans has the characteristics of high speed, high efficiency, energy conservation and small pollution. The one-time extracted Alpha amylase inhibitor has high content and strong activity, thereby avoiding the fact that circulative extractions consume a large quantity of water and energy. The method of extracting Alpha amylase inhibitor from kidney beans is a novel green environment-friendly technology with a wide range of application prospects.
Description
Technical field
The present invention relates to a kind of method of extracting alpha-amylase inhibitor, particularly a kind of method of from Kidney bean, extracting alpha-amylase inhibitor.
Background technology
A fat big obstacle that has become whole mankind's social progress civilization is a class disease of harm humans health simultaneously.It not only can bring out the arrival in advance of symptoms such as diabetes, hyperlipidemia, but also brings huge mental burden can for the patient.Therefore, prevention and treatment be fat to have become one of problem that the whole mankind merits attention, explores one of emphasis that new prevention and treatment measure are scientific researches and difficult point.
At present, prevention and the fat method of treatment have motion burning fatty method, liposuction method, the method for going on a diet and take health product method etc.The method of movement is a kind of method of at present environmental protection, but because itself otherwise need specialty and expensive sports equipment, or need suitable place, all can bring very big inconvenience to user; Though the liposuction method is a kind of crash diet method of getting instant result, body shaping teacher that it needs specialty goes to operate corresponding apparatus, generally can not oneself finish, and have certain risk; The method of going on a diet is a kind of relatively scientific methods, but because people form some dietary habit for a long time, change in a short time, need pay the hardships that can't weigh, moreover, if there is not the diet program of science also can influence the health of human body; The health product fat-reducing becomes people's prevention and the fat a kind of comparatively ideal method of treatment at present, but because market is raised variety various, and some health product also contains hormone substance, can badly influence the metabolic balance of human body, bring some expendable problems may for the health of human body, so exploitation is green, safety, diet products are prevention and the fat a kind of efficient ways of treatment efficiently.
Alpha-amylase inhibitor is to belong to a kind of in (glycosides) hydrolase inhibitor of sugar, it can suppress the activity of interior saliva of intestinal and pancreatic amylase effectively, hinder the hydrolysis and the digestion of carbohydrate in the food, reduce the picked-up of sugar, reduced blood glucose and lipids contents level, do not produced hyperglycemia after closing food, thereby insulin secretion reduces, lipogenesis reduces, and loses weight.This kind enzyme inhibitor derives from green plants, is a kind of protide health product, without any toxic and side effects, simultaneously, does not have documents and materials to show that this inhibitor has the character of destructive enzyme system to human body.Therefore, the Chinese obese patient that alpha-amylase inhibitor can be used as based on the cereal group food uses, and meets the national conditions of China; Also be a kind of safe medicine of world's diabetics simultaneously, can promote towards the whole world.
Alpha-amylase inhibitor all is a little organic compound, and they and enzyme form enzyme inhibitor complex and make enzyme deactivation.On chemical constitution, they can be protein, polypeptide and carbohydrate.Having α-Dian Fenmei, to suppress active protein distributed more widely, can separate to obtain from plants such as Semen Tritici aestivi, Fructus Hordei Vulgaris, Semen setariae, Sorghum vulgare Pers., corn, Semen arachidis hypogaeae, Semen sojae atricolor, Fructus Colocasiae Esculentae root and some microorganisms, and inhibitor molecules amount size is 9000-63000Da.Compare with other kind according to the alpha-amylase inhibitor in the report Kidney beans such as Leiner and to have height and tire.Because of its extremely external interior many research workers of inhibitor (heat-resisting and thermo-labile) of containing several specific types pay close attention to.According to containing two kinds of isoinhibitors in the black Kidney bean of reports such as Lajolo, some inhibitor are a kind of glycoprotein in the White Kidney Beans, have an identical peptide or different polypeptide, can suppress the diastatic activity of mammal and insecticide.Therefore, at present, Kidney bean is the unique plant source that extracts the efficiency natural alpha-amylase inhibitor.
United States Patent (USP) (US 6340699) discloses a kind of method that adopts heat treatment and/or use the method extraction amylase inhibitor of solvent.Yet there are many shortcomings in heat treatment and/or use solvent method.As: some heat-sensitive substances in the easy degradable starch enzyme inhibitor of high temperature extraction make the stability and the active reduction of amylase inhibitor; Solvent method extracts normal because of with an organic solvent causing environmental pollution and product dissolvent residual problem in a large number, has the misgivings of environmental protection and healthy aspect.Chinese patent " purifying starch enzyme inhibitor and be used for obtaining the novel method of this amylase inhibitor " (CN 1665521A) discloses a kind of supercritical carbon dioxide that utilizes and has removed the impurity that grinds bean or pea, is further purified the method for amylase inhibitor.This method is at first ground bean or pea, then under vacuum condition, with supercritical carbon dioxide lixiviate impurity, deionized water with heating repeats to extract the bean or pea of handling 2 times then, each heating 2 hours that continue, and collection extracting solution concentrate with heat-exchanging method at last and lyophilization obtains product.But the supercritical extraction equipment investment is big, and working condition is required harshness, and long-time heating is extracted the activity that also can damage amylase inhibitor.Chinese invention patent (ZL200410041455.4) discloses a kind of two phase aqueous extraction system and has used the method for this system separation and purification amylase inhibitor.This method mainly adds Polyethylene Glycol and fructose-1 in the alpha-amylase inhibitor crude extract; 6-diphosphonic acid slaine; they be biological activity to biogenetic products have stable with protect the main phase reagent that becomes; help the amylase inhibitor emulsion and be separated fast, improve the efficient of membrane separation technique.
The described alpha-amylase inhibitor of above-mentioned open source literature mostly is to utilize traditional heated solvent extraction preparation.Heat in leaching process, cause partial starch generation gelatinizing easily, solution viscosity increases, and brought difficulty for follow-up filtration and centrifugalize operation, and long-time heating is extracted the biological activity that can reduce product; Because the restriction of technology, it is meticulous that raw material can not be pulverized, and causes the degree of fragmentation of cell wall to reduce, and is unfavorable for that the amylase inhibitor in the cell discharges fully, needs to consume to extract solvents and extract for a long time in a large number.Therefore, traditional heated solvent extraction during alpha-amylase inhibitor, has the energy consumption height in preparation, shortcomings such as solvent consumption is big, the production cycle is long, environmental protection pressure is big, product quality is low, production cost height.
Summary of the invention
The objective of the invention is at above-mentioned the deficiencies in the prior art, a kind of method of utilizing the ultrasonic extraction technology to extract alpha-amylase inhibitor from Kidney bean is provided, can be fast, safety, environmental protection ground preparation high-load, highly active medicinal alpha-amylase inhibitor, and save a large amount of extraction solvents (water), simplify production technology, reduce product cost.
For achieving the above object, the technical scheme that the present invention takes is: a kind of method of extracting alpha-amylase inhibitor from Kidney bean is characterized in that may further comprise the steps:
(1) earlier the Kidney bean of pulverizing is joined in the buffer solution that pH is 2-10, after stirring, place ultrasonic equipment to extract 5-60 minute, filter then, obtain filtrate; The percentage by weight of described Kidney bean and buffering solution is 1: 1-20;
(2) enzyme denaturing is handled: the filtrate in the step (1) is adopted conventional heating 30 minutes, or microwave radiation 1-10 minute, make the Kidney bean enzyme deactivation in the filtrate;
(3) solution centrifugal after the enzyme denaturing processing in the step (2) was separated 30 minutes, keep supernatant;
(4) supernatant in the step (3) is sealed with bag filter, place deionized water dialysis 3 hours;
(5) add organic alcohol solvent in the supernatant after dialysis and obtain precipitate; The volume ratio of described supernatant and organic alcohol solvent is 5: 1;
(6) dry sediment promptly obtains alpha-amylase inhibitor.
Kidney bean described in the above-mentioned steps (1) is the ripe seed of one or more plants of pulse family Phaseolus;
Ultrasonic equipment power is 50-5000W described in the above-mentioned steps (1), and frequency is 20-100KHz.
In the oven dry of dry employing normal pressure described in the above-mentioned steps (6), decompression oven dry, spray drying and the lyophilization one or more.
The inventive method is extracted α-Dian Fenmei inhibition method than prior art and had following tangible beneficial effect: (1) adopts steps such as supersound extraction and microwave deactivating enzyme, the production cycle that α-Dian Fenmei suppresses can be shortened to 8-13 hour from 20-28 hour, the once extraction product content that makes α-Dian Fenmei suppress reaches 80-95%, suppressing activity is 5000-50000U/g, has improved production efficiency; (2) α-Dian Fenmei of this method acquisition suppresses the content height, can alleviate the pressure that product is further purified, and production technology is simpler, the extractant demand is little in addition, it is about 50% to economize on water, and reduces production costs greatly, have rapidly and efficiently, energy-conservation, pollute little characteristics; (3) α-Dian Fenmei that the present invention extracted suppresses product has important use to be worth aspect medicine, especially the performance aspect treatment obesity, diabetes, hyperlipidemia good prospects for application and economic worth, for pharmaceuticals industry provides the raw material that enriches.
Description of drawings
Fig. 1 is a process chart of the present invention;
The specific embodiment
The present invention is described in further detail below in conjunction with drawings and Examples.
Embodiment 1
It is in 2.0 the buffer solution that the Kidney bean powder that 100 grams that weigh with scale are pulverized join 100 gram pH, stir, at power is 50W, frequency is to extract in the ultrasonic extraction equipment of 20KHz after 5 minutes, filter to get filtrate, and utilize the heat-exchanging method routine to heat 30 minutes enzyme denaturing, centrifugalize 30 minutes is also got supernatant, dialysis is 3 hours in deionized water, add organic alcohol in the extracting solution after dialysis and obtain precipitate, the volume ratio of described extracting solution and organic alcohol is 5: 1, after the normal pressure oven dry, obtaining protein content is 80.3%, and biological activity is the alpha-amylase inhibitor product of 5000U/g.
Embodiment 2
It is in 3.0 the buffer solution that the Kidney bean powder that 100 grams that weigh with scale are pulverized join 500 gram pH, stir, at power is 100W, frequency is to extract in the ultrasonic extraction equipment of 40KHz after 10 minutes, filter to get filtrate, and utilize the heat-exchanging method routine to heat 30 minutes enzyme denaturing, centrifugalize 30 minutes is also got supernatant, dialysis is 3 hours in deionized water, add organic alcohol in the extracting solution after dialysis and obtain precipitate, the volume ratio of described extracting solution and organic alcohol is 5: 1, after the decompression oven dry, obtaining protein content is 83.6%, and biological activity is the alpha-amylase inhibitor product of 7192U/g.
Embodiment 3
It is in 4.0 the buffer solution that the Kidney bean powder that 100 grams that weigh with scale are pulverized join 1000 gram pH, stir, at power is 200W, frequency is to extract in the ultrasonic extraction equipment of 60KHz after 30 minutes, filter to get filtrate, and utilize the heat-exchanging method routine to heat 30 minutes enzyme denaturing, centrifugalize 30 minutes is also got supernatant, dialysis is 3 hours in deionized water, add organic alcohol in the extracting solution after dialysis and obtain precipitate, the volume ratio of described extracting solution and organic alcohol is 5: 1, after the normal pressure oven dry, obtaining protein content is 89.1%, and biological activity is the alpha-amylase inhibitor product of 8931U/g.
Embodiment 4
It is in 6.0 the buffer solution that the Kidney bean powder that 100 grams that weigh with scale are pulverized join 1500 gram pH, stir, at power is 1000W, frequency is to extract in the ultrasonic extraction equipment of 40KHz after 40 minutes, filter to get filtrate, and utilize the heat-exchanging method routine to heat 30 minutes enzyme denaturing, centrifugalize 30 minutes is also got supernatant, dialysis is 3 hours in deionized water, add organic alcohol in the extracting solution after dialysis and obtain precipitate, the volume ratio of described extracting solution and organic alcohol is 5: 1, after the normal pressure oven dry, obtaining protein content is 92.4%, and biological activity is the alpha-amylase inhibitor product of 25390U/g.
Embodiment 5
It is in 8.0 the buffer solution that the Kidney bean powder that 100 grams that weigh with scale are pulverized join 1000 gram pH, stir, at power is 3000W, frequency is to extract in the ultrasonic extraction equipment of 80KHz after 35 minutes, filter to get filtrate, and utilize the heat-exchanging method routine to heat 30 minutes enzyme denaturing, centrifugalize 30 minutes is also got supernatant, dialysis is 3 hours in deionized water, add organic alcohol in the extracting solution after dialysis and obtain precipitate, the volume ratio of described extracting solution and organic alcohol is 5: 1, and obtaining protein content is 82.9%, and biological activity is the alpha-amylase inhibitor product of 10843U/g.
Embodiment 6
It is in 9.0 the buffer solution that the Kidney bean powder that 100 grams that weigh with scale are pulverized join 400 gram pH, stir, at power is 4000W, frequency is to extract in the ultrasonic extraction equipment of 20KHz after 60 minutes, filter to get filtrate, and utilize the heat-exchanging method routine to heat 30 minutes enzyme denaturing, centrifugalize 30 minutes is also got supernatant, dialysis is 3 hours in deionized water, add organic alcohol in the extracting solution after dialysis and obtain precipitate, the volume ratio of described extracting solution and organic alcohol is 5: 1, after the decompression oven dry, obtaining protein content is 80%, and biological activity is the alpha-amylase inhibitor product of 5124U/g.
Embodiment 7
It is in 10.0 the buffer solution that the Kidney bean powder that 100 grams that weigh with scale are pulverized join 2000 gram pH, stir, at power is 5000W, frequency is to extract in the ultrasonic extraction equipment of 20KHz after 45 minutes, filter to get filtrate, and utilize the heat-exchanging method routine to heat 30 minutes enzyme denaturing, centrifugalize 30 minutes is also got supernatant, dialysis is 3 hours in deionized water, add organic alcohol in the extracting solution after dialysis and obtain precipitate, the volume ratio of described extracting solution and organic alcohol is 5: 1, after the normal pressure oven dry, obtaining protein content is 93.8%, and biological activity is the alpha-amylase inhibitor product of 43819U/g.
Embodiment 8
It is in 5.0 the buffer solution that the Kidney bean powder that 100 grams that weigh with scale are pulverized join 100 gram pH, stir, at power is 3000W, frequency is to extract in the ultrasonic extraction equipment of 60KHz after 60 minutes, filter to get filtrate, and utilize 1 minute enzyme denaturing of microwave radiation, centrifugalize 30 minutes is also got supernatant, dialysis is 3 hours in deionized water, add organic alcohol in the extracting solution after dialysis and obtain precipitate, the volume ratio of described extracting solution and organic alcohol is 5: 1, after the decompression oven dry, obtaining protein content is 81.7%, and biological activity is the alpha-amylase inhibitor product of 6528U/g.
Embodiment 9
It is in 7.0 the buffer solution that the Kidney bean powder that 100 grams that weigh with scale are pulverized join 500 gram pH, stir, at power is 100W, frequency is to extract in the ultrasonic extraction equipment of 100KHz after 15 minutes, filter to get filtrate, and utilize 4 minutes enzyme denaturing of microwave radiation, centrifugalize 30 minutes is also got supernatant, dialysis is 3 hours in deionized water, add organic alcohol in the extracting solution after dialysis and obtain precipitate, the volume ratio of described extracting solution and organic alcohol is 5: 1, after the decompression oven dry, obtaining protein content is 86.9%, and biological activity is the alpha-amylase inhibitor product of 42839U/g.
Embodiment 10
It is in 6.0 the buffer solution that the Kidney bean powder that 100 grams that weigh with scale are pulverized join 1000 gram pH, stir, at power is 2000W, frequency is to extract in the ultrasonic extraction equipment of 80KHz after 5 minutes, filter to get filtrate, and utilize 5 minutes enzyme denaturing of microwave radiation, centrifugalize 30 minutes is also got supernatant, dialysis is 3 hours in deionized water, add organic alcohol in the extracting solution after dialysis and obtain precipitate, the volume ratio of described extracting solution and organic alcohol is 5: 1, after the normal pressure oven dry, obtaining protein content is 92.7%, and biological activity is the alpha-amylase inhibitor product of 24381U/g.
Embodiment 11
It is in 8.0 the buffer solution that the Kidney bean powder that 100 grams that weigh with scale are pulverized join 600 gram pH, stir, at power is 200W, frequency is to extract in the ultrasonic extraction equipment of 100KHz after 50 minutes, filter to get filtrate, and utilize 10 minutes enzyme denaturing of microwave radiation, centrifugalize 30 minutes is also got supernatant, dialysis is 3 hours in deionized water, add organic alcohol in the extracting solution after dialysis and obtain precipitate, the volume ratio of described extracting solution and organic alcohol is 5: 1, spray-dried after, obtaining protein content is 90.2%, and biological activity is the alpha-amylase inhibitor product of 17391U/g.
Embodiment 12
It is in 2.0 the buffer solution that the Kidney bean powder that 100 grams that weigh with scale are pulverized join 1300 gram pH, stir, at power is 4000W, frequency is to extract in the ultrasonic extraction equipment of 40KHz after 55 minutes, filter to get filtrate, and utilize 8 minutes enzyme denaturing of microwave radiation, centrifugalize 30 minutes is also got supernatant, dialysis is 3 hours in deionized water, add organic alcohol in the extracting solution after dialysis and obtain precipitate, the volume ratio of described extracting solution and organic alcohol is 5: 1, spray-dried after, obtaining protein content is 85.1%, and biological activity is the alpha-amylase inhibitor product of 5627U/g.
Embodiment 13
It is in 4.0 the buffer solution that the Kidney bean powder that 100 grams that weigh with scale are pulverized join 2000 gram pH, stir, at power is 5000W, frequency is to extract in the ultrasonic extraction equipment of 20KHz after 15 minutes, filter to get filtrate, and utilize 8 minutes enzyme denaturing of microwave radiation, centrifugalize 30 minutes is also got supernatant, dialysis is 3 hours in deionized water, add organic alcohol in the extracting solution after dialysis and obtain precipitate, the volume ratio of described extracting solution and organic alcohol is 5: 1, after lyophilization, obtaining protein content is 91.9%, and biological activity is the alpha-amylase inhibitor product of 50000U/g.
Embodiment 14
It is in 10.0 the buffer solution that the Kidney bean powder that 100 grams that weigh with scale are pulverized join 1900 gram pH, stir, at power is 2000W, frequency is to extract in the ultrasonic extraction equipment of 100KHz after 60 minutes, filter to get filtrate, and utilize 10 minutes enzyme denaturing of microwave radiation, centrifugalize 30 minutes is also got supernatant, dialysis is 3 hours in deionized water, add organic alcohol in the extracting solution after dialysis and obtain precipitate, the volume ratio of described extracting solution and organic alcohol is 5: 1, after lyophilization, obtaining protein content is 95.0%, and biological activity is the alpha-amylase inhibitor product of 8219U/g.
Embodiment 15
It is in 6.0 the buffer solution that the Kidney bean powder that 100 grams that weigh with scale are pulverized join 100 gram pH, stir, at power is 4000W, frequency is to extract in the ultrasonic extraction equipment of 40KHz after 55 minutes, filter to get filtrate, and utilize the traditional hot exchange to heat 30 minutes enzyme denaturing, centrifugalize 30 minutes is also got supernatant, dialysis is 3 hours in deionized water, add organic alcohol in the extracting solution after dialysis and obtain precipitate, the volume ratio of described extracting solution and organic alcohol is 5: 1, after lyophilization, obtaining protein content is 94.2%, and biological activity is the alpha-amylase inhibitor product of 36184U/g.
The used ultrasonic device of the foregoing description can be in ultrasonic cleaning machine, probe type ultrasonic pulverizer and the circulating ultrasonic extractor any one.
The quality determining method of alpha-amylase inhibitor product:
1, protein content detects the Lowry method that adopts;
2, the Bernfeld method is adopted in the inhibition determination of activity of alpha-amylase inhibitor:
Utilize the reducing sugar and 3 in the starch hydrolyzate, the 5-DNS color combining changes, and reducing sugar is in finite concentration, in the linear characteristics of amount of the light absorption value and the reducing sugar at 540nm place, the difference of the reducing sugar amount that the α-Dian Fenmei starch-splitting produces before and after adding according to amylase inhibitor can calculate the activity of inhibitor.
Get an amount of α-Dian Fenmei storing solution with 0.12mol/L phosphate buffer (pH 6.10) preparation, 37 ℃ of insulation 15min, adding 100uL concentration is the soluble starch of 10g/L, accurate response 5min.Add 500uL 3 after reaction is finished, 5-DNS, and boiling water bath heating 10min take out reactant liquor ice bath cooling 15min, and measure absorbance (A at the 540nm place
540Nm).Concentration or the consumption of adjusting inhibitor solution and α-Dian Fenmei solution make A
540The value of nm is in 0.4～0.7 scope is advisable.
The active definition of alpha-amylase inhibitor:
1 is suppressed unit of activity (U) under these conditions, and suppression ratio is the consumption of 50% o'clock inhibitor;
α-Dian Fenmei suppression ratio (%)=(AE
540-AI
540)/AE
540
Wherein: AE
540When only adding amylase, hydrolyzate is at the absorbance at 540nm place;
AI
540After adding inhibitor, hydrolyzate is at the absorbance at 540nm place.
Claims (4)
1. method of extracting alpha-amylase inhibitor from Kidney bean is characterized in that may further comprise the steps:
(1) earlier the Kidney bean of pulverizing is joined in the buffer solution that pH is 2-10, after stirring, place ultrasonic equipment to extract 5-60 minute, filter then, obtain filtrate; The weight ratio of described Kidney bean and buffering solution is 1: 1-20;
(2) enzyme denaturing is handled: the filtrate in the step (1) is adopted conventional heating 30 minutes, or microwave radiation 1-10 minute, make the Kidney bean enzyme deactivation in the filtrate;
(3) solution centrifugal after the enzyme denaturing processing in the step (2) was separated 30 minutes, keep supernatant;
(4) supernatant in the step (3) is sealed with bag filter, place deionized water dialysis 3 hours;
(5) add organic alcohol solvent in the supernatant after dialysis and obtain precipitate; The volume ratio of described supernatant and organic alcohol solvent is 5: 1;
(6) dry sediment promptly obtains alpha-amylase inhibitor.
2. a kind of method of extracting alpha-amylase inhibitor from Kidney bean according to claim 1 is characterized in that Kidney bean described in the step (1) is the ripe seed of one or more plants of pulse family Phaseolus.
3. a kind of method of extracting alpha-amylase inhibitor from Kidney bean according to claim 1 is characterized in that ultrasonic equipment power is 50-5000W described in the step (1), and frequency is 20-100KHz.
4. a kind of method of extracting alpha-amylase inhibitor from Kidney bean according to claim 1 is characterized in that drying described in the step (6) adopts one or more in normal pressure oven dry, decompression oven dry, spray drying and the lyophilization.
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| CN102552376A (en) * | 2010-12-31 | 2012-07-11 | 西安天一生物技术有限公司 | White kidney bean extract extracted by microwave as well as preparation method and application for same |
| CN103638089B (en) * | 2013-11-29 | 2016-01-27 | 广东省食品工业研究所 | A kind of method utilizing microwave-compound enzyme coupled method to extract alpha-amylase inhibitor from Semen Phaseoli Vulgaris |
| CN113318143A (en) * | 2021-05-28 | 2021-08-31 | 德州职业技术学院 | Preparation method of alpha-amylase inhibitor by using beans as raw materials |
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Non-Patent Citations (3)
| Title |
|---|
| 吕凤霞等.α-淀粉酶抑制剂的研究进展.《食品科学》.2002,第23卷(第3期),152-155. * |
| 谭胜兵.超声波技术在天然产物提取中的应用.《枣庄学院学报》.2007,第24卷(第2期),84-86. * |
| 贾光锋.α-淀粉酶抑制剂的制备及检测.《食品与药品》.2007,第9卷(第02A期),34-36. * |
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