CN112390869A - Method for extracting and refining high-purity kidney bean agglutinin - Google Patents
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Abstract
The invention provides a method for extracting and refining high-purity kidney bean agglutinin, which takes kidney beans as raw materials, and the high-purity kidney bean agglutinin is obtained by processing such as ultrasonic-assisted flowing water cleaning, nitrogen-filled ball milling, supercritical extraction and impurity removal, normal-temperature extraction, acid precipitation, activated carbon adsorption, molecular sieve chromatography and the like. The kidney bean agglutinin prepared by the invention has high purity, can be used as a raw material of food and medicine, and has good market prospect.
Description
Technical Field
The invention belongs to the technical field of deep processing of agricultural products, and relates to a method for extracting and refining high-purity kidney bean agglutinin.
Background
Kidney bean, the scientific name kidney bean, commonly known as biquaternary bean or kidney bean, is a plant of the genus phaseolus of the order rosales, an annual, entwined or near-erect herb. Kidney beans are warm in taste and not resistant to frost, are native to America, and are widely cultivated in China. Kidney bean is an edible leguminous plant, and can be used as fresh vegetable for tender pod or seed, or can be processed into canned food, pickled, frozen, and dried. The kidney beans have high medicinal value, are recorded by ancient medical books of China, are sweet and neutral in taste, have the functions of warming middle-jiao and descending qi, benefiting intestines and stomach, stopping hiccup, tonifying kidney and replenishing primordial qi and the like, and are a good nourishing food therapy product; according to modern medical analysis, kidney beans contain various globulins such as agglutinin and the like, and have certain curative effects on various diseases.
Lectin (Lectin) refers to a non-enzymatic protein of non-immunological origin purified from various plants, invertebrates or higher animals, capable of agglutinating cells and precipitating mono-or polysaccharide complexes. Plant lectins are a class of lectins that have important roles in many signaling processes, such as signal transduction, immune response, plant defense, etc., due to their ability to bind specifically to monosaccharides or glycoconjugates; meanwhile, the phytohemagglutinin has various capacities of cell agglutination, virus resistance, fungus resistance, cell apoptosis induction or autophagy induction and the like; therefore, the plant agglutinin has good research value and application prospect in the aspects of life science, medicine and agriculture.
Kidney bean agglutinin (Phytohemagglutinin PHA) is a tetramer glycoprotein extracted from kidney beans, has beneficial biological activities such as sugar binding specificity, alpha-glucosidase inhibition activity, induction of human peripheral blood lymphocytes to generate anticancer lymphokines, anti-HIV virus and the like, and has wide application prospect in the field of medicine and pharmacology.
Disclosure of Invention
The invention aims to provide a method for extracting and refining high-purity kidney bean agglutinin.
In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a method for extracting and refining high-purity kidney bean agglutinin is characterized by comprising the following steps: comprises the following steps:
step 1: putting kidney bean into soaking and cleaning equipment, injecting purified water with temperature of 15-35 deg.C into the soaking and cleaning equipment to submerge kidney bean in the purified water, and treating for 30-45 min; in the cleaning process, water enters from the water inlet at a constant speed, water exits from the water outlet at a constant speed, and the water inlet flow rate is 100 and 200 times of the kidney bean mass per hour; meanwhile, starting ultrasonic treatment once every 5-7min in the cleaning process;
step 2: draining the surface water of the kidney beans obtained in the step 1, and drying for 30-60min at the temperature of 35-40 ℃ and under the vacuum gauge pressure of-0.09 to-0.10 Mpa; then, putting the dried kidney beans into a ball mill, and grinding the kidney beans under the protection of nitrogen until the fineness of the materials is lower than 60 meshes to obtain kidney bean powder;
and step 3: putting the kidney bean powder into an extraction kettle of carbon dioxide supercritical extraction equipment, carrying out extraction and impurity removal treatment, and collecting substances left after extraction;
and 4, step 4: mixing the substance left after extraction obtained in the step 3 with a neutral pH buffer solution according to the mass ratio of 1: 8-12; then stirring the mixed material for 4-6h at 60-120r/min, centrifuging the mixed material at 3000-4000 r/min for 5-10min, and collecting supernatant, namely the crude kidney bean protein extract;
and 5: adding acid liquor into the crude kidney bean protein extract, continuously stirring, adjusting the pH value of the crude kidney bean protein extract to 3.4-3.8, and then standing and precipitating for 30-45 min; centrifuging the solution after standing at the rotation speed of 1000-;
step 6: adding 0.75-1.5% of activated carbon into the supernatant obtained in the step 5, stirring for 90-180s, and standing for 30-45 min; centrifuging the material after standing at the rotation speed of 1000-;
and 7: and (4) performing sephacryls-200 molecular sieve gel filtration column chromatography treatment on the supernatant obtained in the step (6), and collecting the material after chromatography purification, namely the kidney bean agglutinin.
The preferable technical scheme is as follows: in step 1, the ultrasonic frequency is 25-35 KHz, and the power density is 0.25-0.50W/cm2At each time of treatmentThe time interval is 1.0-1.5 min.
The preferable technical scheme is as follows: the extraction and impurity removal treatment comprises 2 times of continuous supercritical extraction operation, and the technological parameters of the first extraction are as follows: extracting under 25-30Mpa at 37.0 + -0.3 deg.C for 15-30 min; the technological parameters of the second extraction are as follows: the extraction pressure is 28-33Mpa, the extraction temperature is 45.0 +/-0.3 ℃, and the extraction time is 15-30 min.
The preferable technical scheme is as follows: the neutral pH buffer solution is a PBS buffer solution with pH 7.2 or a Tris-HCl buffer solution.
The preferable technical scheme is as follows: the ionic strength of the neutral pH buffer solution is 10-20 mM.
The preferable technical scheme is as follows: the acid solution is at least one of citric acid, acetic acid and phosphoric acid.
The preferable technical scheme is as follows: the activated carbon is dried food activated carbon, and has a specific surface area of 10-20m2/g。
The preferable technical scheme is as follows: the operation of the sephacryls-200 molecular sieve gel filtration column chromatography treatment is as follows: and (3) balancing the gel chromatographic column by a pH 7.2 PBS buffer solution system, loading a sample with the volume not more than 15% of the column volume, continuously eluting by using the pH 7.2 PBS buffer solution with the flow rate of 0.8ml/min, collecting a first peak area sample, and freeze-drying to obtain the high-purity kidney bean agglutinin.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
1. according to the invention, the kidney beans are treated by ultrasonic-assisted running water cleaning and nitrogen-filled ball milling, so that impurities on the surfaces of the kidney beans are effectively removed, oxidation in crushing is avoided, and a clean and non-oxidized kidney bean powder raw material is provided for lectin extraction.
2. The invention adopts 2 times of supercritical carbon dioxide extraction treatment, removes fat and fat-soluble substances in the kidney bean by extraction, and is beneficial to improving the extraction efficiency and the extraction purity of the kidney bean agglutinin.
3. The high-purity kidney bean agglutinin is obtained by refining and purifying the acid precipitation, the activated carbon adsorption, the molecular sieve chromatography and the like.
Drawings
FIG. 1 is a liquid phase chromatogram of a kidney bean agglutinin sample.
FIG. 2 is a liquid phase chromatogram of the supernatant.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
Please refer to fig. 1-2. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention. In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.
Example 1: method for extracting and refining high-purity kidney bean agglutinin
A method for extracting and refining high-purity kidney bean agglutinin comprises the following technical steps.
(1) Kidney bean ultrasonic-assisted running water cleaning
The kidney bean material is one of white kidney bean, red kidney bean and black kidney bean with moderate maturity, dryness, no insect pest and no mildew.
Putting kidney beans into soaking and cleaning equipment with an ultrasonic generating device, and injecting food-grade purified water into the equipment to submerge the kidney beans in the water; the top of the equipment soaking and cleaning tank is provided with a water inlet, the bottom of the equipment soaking and cleaning tank is provided with a water outlet with a filter screen, in the cleaning process, water enters from the water inlet at a constant speed, water exits from the water outlet at a constant speed, the water temperature is 20 ℃,the water inlet flow is 150 times of the kidney bean mass per hour, and a filter screen at the water outlet can intercept the kidney beans; meanwhile, the cleaning equipment starts ultrasonic treatment once every 6min, the ultrasonic frequency is 30 KHz, and the power density is 0.35/cm2Each treatment time is 1.2 min. Ultrasonic wave-assisted running water cleaning kidney beans, wherein the treatment time is about 40 min.
(2) Ball milling by filling nitrogen
Draining the cleaned kidney beans to remove surface water, and drying under vacuum at 35-40 deg.C under-0.09-0.10 Mpa for 30-60 min.
And putting the kidney beans subjected to vacuum low-temperature drying into food sanitation closed ball milling equipment, exhausting air, filling nitrogen, starting the ball milling equipment, and fully milling until the material fineness is lower than 60 meshes to obtain the kidney bean powder for later use.
(3) Supercritical impurity removal treatment
And (3) putting the kidney bean powder in the step (2) into an extraction kettle of carbon dioxide supercritical extraction equipment, carrying out extraction and impurity removal treatment, and collecting substances left after extraction for later use. The extraction and impurity removal treatment adopts 2 times of continuous supercritical extraction operation, and the technological parameters of the first extraction are as follows: extracting at 28Mpa and 37.0 deg.C for 20 min; the technological parameters of the second extraction are as follows: the extraction pressure is 30Mpa, the extraction temperature is 45.0 ℃, and the extraction time is 20 min.
(4) Leaching at normal temperature
Mixing the extracted and impurity-removed substance of the kidney bean powder in the step (3) and a neutral pH buffer solution according to a mass ratio of 1: 10; keeping the mixed material at normal temperature and stirring speed of 80 r/min; after leaching for 5h, centrifuging the mixed material at the rotation speed of 3500 r/min for 8min, and collecting supernatant, namely the crude kidney bean protein extract.
The neutral pH buffer solution is a PBS buffer solution with the pH value of 7.2 or a Tris-HCl buffer solution, and preferably, the ionic strength of the buffer solution is 15 mM. This example specifically selects a PBS buffer solution with a pH of 7.2.
(5) Acid precipitation treatment
Slowly adding acid liquor into the crude kidney bean protein extract in the step (4) and continuously stirring, adjusting the pH value of the crude kidney bean protein extract to 3.6, and then standing and precipitating for 35 min; and centrifuging the solution after standing at the rotating speed of 1500 r/min for 7min, and collecting supernatant for later use.
Preferably, the acid solution is one or more of citric acid, acetic acid and phosphoric acid. Citric acid was specifically chosen in this example.
(6) Constant temperature adsorption impurity removal by activated carbon
Adding 1% of activated carbon into the supernatant in the step (5), fully stirring for 150s, and then standing for 35 min; and centrifuging the material after standing at the rotating speed of 1500 r/min for 8min, and collecting supernatant for later use. The liquid phase spectrum of the supernatant is shown in figure 2.
The activated carbon is dried food activated carbon, and has a specific surface area of 10-20m2/g。
(7) Purifying by molecular sieve chromatography
And (4) performing gel filtration column chromatography treatment on the supernatant in the step (6) by using sephacryls-200 molecular sieves, and collecting a material after chromatography purification, namely the kidney bean agglutinin.
The sephacryls-200 molecular sieve gel filtration column chromatography comprises the following specific operations: and (3) balancing the gel chromatographic column by using a pH 7.2 buffer solution system, loading a sample with the volume not more than 15% of the column volume, continuously eluting by using the pH 7.2 buffer solution at the flow rate of 0.8ml/min, collecting a first peak area sample (a sample between the starting point and the end point of the first peak), and freeze-drying to obtain the high-purity kidney bean agglutinin.
Detecting the supernatant in the step (6) prepared by the method by adopting a liquid phase method, and taking a sigma kidney bean agglutinin sample; comparing the liquid phase spectrum of the kidney bean agglutinin sample of sigma with the liquid phase spectrum of the supernatant in step (6), the first peak value can be found, and the high-purity kidney bean agglutinin can be obtained.
The method for analyzing the liquid phase comprises the following steps: gel chromatographic column, adopting imported Japan Tosoh TSK G3000 SWXL (globulin molecular weight 1W-50W Da), analyzing liquid phase system, mobile phase pH 7.220 mmol/L PBS buffer solution containing 0.05% sodium azide, flow rate 0.8ml/min, sample loading amount 5-20 μ L, detector ultraviolet 278 nm.
Example 2: method for extracting and refining high-purity kidney bean agglutinin
A method for extracting and refining high-purity kidney bean agglutinin is characterized by comprising the following steps: comprises the following steps:
step 1: putting kidney beans into soaking and cleaning equipment, injecting purified water with the temperature of 15 ℃ into the soaking and cleaning equipment to submerge the kidney beans in the purified water, and treating for 30 min; in the cleaning process, water enters from the water inlet at a constant speed, water exits from the water outlet at a constant speed, and the water inlet flow is 100 times of the mass of the kidney beans per hour; meanwhile, starting ultrasonic treatment once every 5min in the cleaning process;
step 2: draining the kidney beans obtained in the step 1 to remove surface moisture, and drying for 30min at the vacuum gauge pressure of-0.09 Mpa and the temperature of 35 ℃; then, putting the dried kidney beans into a ball mill, and grinding the kidney beans under the protection of nitrogen until the fineness of the materials is lower than 60 meshes to obtain kidney bean powder;
and step 3: putting the kidney bean powder into an extraction kettle of carbon dioxide supercritical extraction equipment, carrying out extraction and impurity removal treatment, and collecting substances left after extraction;
and 4, step 4: mixing the substance left after extraction obtained in the step 3 with a neutral pH buffer solution according to a mass ratio of 1: 8; then stirring the mixed material at a speed of 60r/min for 4h, centrifuging the mixed material at a rotating speed of 3000r/min for 5min, and collecting supernatant, namely the crude kidney bean protein extract;
and 5: adding acid liquor into the crude kidney bean protein extracting solution, continuously stirring, adjusting the pH value of the crude kidney bean protein extracting solution to 3.4, and then standing and precipitating for 30 min; centrifuging the solution after standing at the rotating speed of 1000r/min for 5min, and collecting supernatant;
step 6: adding active carbon with the mass of 0.75% of the supernatant obtained in the step 5 into the supernatant, stirring the mixture for 90 seconds, and standing the mixture for 30 min; centrifuging the material after standing at the rotating speed of 1000r/min for 5min, and collecting supernatant;
and 7: and (4) performing sephacryls-200 molecular sieve gel filtration column chromatography treatment on the supernatant obtained in the step (6), and collecting the material after chromatography purification, namely the kidney bean agglutinin.
The preferred embodiment is: in step 1, the ultrasonic frequency is 25KHz, and the power density is 0.25W/cm2Time of each treatmentIt is 1.0 min.
The preferred embodiment is: the extraction and impurity removal treatment comprises 2 times of continuous supercritical extraction operation, and the technological parameters of the first extraction are as follows: extracting at 25Mpa at 36.7 deg.C for 15 min; the technological parameters of the second extraction are as follows: the extraction pressure is 28Mpa, the extraction temperature is 44.7 ℃, and the extraction time is 15 min.
The preferred embodiment is: the neutral pH buffer solution is Tris-HCl buffer solution.
The preferred embodiment is: the neutral pH buffer solution had an ionic strength of 10 mM.
The preferred embodiment is: the acid solution is acetic acid.
The preferred embodiment is: the activated carbon is dried food activated carbon, and has a specific surface area of 10-20m2/g。
The preferred embodiment is: the operation of the sephacryls-200 molecular sieve gel filtration column chromatography treatment is as follows: and (3) balancing the gel chromatographic column by a pH 7.2 PBS buffer solution system, loading a sample with the volume not more than 15% of the column volume, continuously eluting by using the pH 7.2 PBS buffer solution with the flow rate of 0.8ml/min, collecting a first peak area sample, and freeze-drying to obtain the high-purity kidney bean agglutinin.
Example 3: method for extracting and refining high-purity kidney bean agglutinin
A method for extracting and refining high-purity kidney bean agglutinin is characterized by comprising the following steps: comprises the following steps:
step 1: putting kidney beans into soaking and cleaning equipment, injecting purified water with the temperature of 35 ℃ into the soaking and cleaning equipment to submerge the kidney beans in the purified water, and treating for 45 min; in the cleaning process, water enters from the water inlet at a constant speed, water exits from the water outlet at a constant speed, and the water inlet flow is 200 times of the mass of kidney beans per hour; meanwhile, starting ultrasonic treatment once every 7min in the cleaning process;
step 2: draining the kidney beans obtained in the step 1 to remove surface moisture, and drying for 60min at the temperature of 40 ℃ and under the vacuum gauge pressure of-0.10 Mpa; then, putting the dried kidney beans into a ball mill, and grinding the kidney beans under the protection of nitrogen until the fineness of the materials is lower than 60 meshes to obtain kidney bean powder;
and step 3: putting the kidney bean powder into an extraction kettle of carbon dioxide supercritical extraction equipment, carrying out extraction and impurity removal treatment, and collecting substances left after extraction;
and 4, step 4: mixing the substance left after extraction obtained in the step 3 with a neutral pH buffer solution according to the mass ratio of 1: 12; then stirring the mixed material at 120r/min for 6h, centrifuging the mixed material at the rotating speed of 4000 r/min for 10min, and collecting supernatant, namely the crude kidney bean protein extract;
and 5: adding acid liquor into the crude kidney bean protein extracting solution, continuously stirring, adjusting the pH value of the crude kidney bean protein extracting solution to 3.8, and then standing and precipitating for 45 min; centrifuging the solution after standing at a rotating speed of 2000 r/min for 10min, and collecting supernatant;
step 6: adding activated carbon accounting for 1.5% of the mass of the supernatant obtained in the step 5 into the supernatant, stirring the mixture for 180 seconds, and standing the mixture for 45 min; centrifuging the material after standing at a rotating speed of 2000 r/min for 10min, and collecting supernatant;
and 7: and (4) performing sephacryls-200 molecular sieve gel filtration column chromatography treatment on the supernatant obtained in the step (6), and collecting the material after chromatography purification, namely the kidney bean agglutinin.
The preferred embodiment is: in step 1, the ultrasonic frequency is 35 KHz, and the power density is 0.50W/cm2The treatment time was 1.5min each time.
The preferred embodiment is: the extraction and impurity removal treatment comprises 2 times of continuous supercritical extraction operation, and the technological parameters of the first extraction are as follows: the extraction pressure is 30Mpa, the extraction temperature is 37.3 ℃, and the extraction time is 30 min; the technological parameters of the second extraction are as follows: the extraction pressure is 33Mpa, the extraction temperature is 45.3 ℃, and the extraction time is 30 min.
The preferred embodiment is: the neutral pH buffer solution is a pH 7.2 PBS buffer solution.
The preferred embodiment is: the neutral pH buffer solution had an ionic strength of 20 mM.
The preferred embodiment is: the acid liquor is phosphoric acid.
The preferred embodiment is: the active carbon is dryDried food-use activated carbon with a specific surface area of 10-20m2/g。
The preferred embodiment is: the operation of the sephacryls-200 molecular sieve gel filtration column chromatography treatment is as follows: and (3) balancing the gel chromatographic column by a pH 7.2 PBS buffer solution system, loading a sample with the volume not more than 15% of the column volume, continuously eluting by using the pH 7.2 PBS buffer solution with the flow rate of 0.8ml/min, collecting a first peak area sample, and freeze-drying to obtain the high-purity kidney bean agglutinin.
The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.
Claims (8)
1. A method for extracting and refining high-purity kidney bean agglutinin is characterized by comprising the following steps: comprises the following steps:
step 1: putting kidney bean into soaking and cleaning equipment, injecting purified water with temperature of 15-35 deg.C into the soaking and cleaning equipment to submerge kidney bean in the purified water, and treating for 30-45 min; in the cleaning process, water enters from the water inlet at a constant speed, water exits from the water outlet at a constant speed, and the water inlet flow rate is 100 and 200 times of the kidney bean mass per hour; meanwhile, starting ultrasonic treatment once every 5-7min in the cleaning process;
step 2: draining the surface water of the kidney beans obtained in the step 1, and drying for 30-60min at the temperature of 35-40 ℃ and under the vacuum gauge pressure of-0.09 to-0.10 Mpa; then, putting the dried kidney beans into a ball mill, and grinding the kidney beans under the protection of nitrogen until the fineness of the materials is lower than 60 meshes to obtain kidney bean powder;
and step 3: putting the kidney bean powder into an extraction kettle of carbon dioxide supercritical extraction equipment, carrying out extraction and impurity removal treatment, and collecting substances left after extraction;
and 4, step 4: mixing the substance left after extraction obtained in the step 3 with a neutral pH buffer solution according to the mass ratio of 1: 8-12; then stirring the mixed material for 4-6h at 60-120r/min, centrifuging the mixed material at 3000-4000 r/min for 5-10min, and collecting supernatant, namely the crude kidney bean protein extract;
and 5: adding acid liquor into the crude kidney bean protein extract, continuously stirring, adjusting the pH value of the crude kidney bean protein extract to 3.4-3.8, and then standing and precipitating for 30-45 min; centrifuging the solution after standing at the rotation speed of 1000-;
step 6: adding 0.75-1.5% of activated carbon into the supernatant obtained in the step 5, stirring for 90-180s, and standing for 30-45 min; centrifuging the material after standing at the rotation speed of 1000-;
and 7: and (4) performing sephacryls-200 molecular sieve gel filtration column chromatography treatment on the supernatant obtained in the step (6), and collecting the material after chromatography purification, namely the kidney bean agglutinin.
2. The method for extracting and refining high-purity kidney bean agglutinin according to claim 1, wherein the method comprises the following steps: in step 1, the ultrasonic frequency is 25-35 KHz, and the power density is 0.25-0.50W/cm2The treatment time is 1.0-1.5min each time.
3. The method for extracting and refining high-purity kidney bean agglutinin according to claim 1, wherein the method comprises the following steps: the extraction and impurity removal treatment comprises 2 times of continuous supercritical extraction operation, and the technological parameters of the first extraction are as follows: extracting under 25-30Mpa at 37.0 + -0.3 deg.C for 15-30 min; the technological parameters of the second extraction are as follows: the extraction pressure is 28-33Mpa, the extraction temperature is 45.0 +/-0.3 ℃, and the extraction time is 15-30 min.
4. The method for extracting and refining high-purity kidney bean agglutinin according to claim 1, wherein the method comprises the following steps: the neutral pH buffer solution is a PBS buffer solution with pH 7.2 or a Tris-HCl buffer solution.
5. The method for extracting and refining high-purity kidney bean agglutinin according to claim 4, wherein the method comprises the following steps: the ionic strength of the neutral pH buffer solution is 10-20 mM.
6. The method for extracting and refining high-purity kidney bean agglutinin according to claim 1, wherein the method comprises the following steps: the acid solution is at least one of citric acid, acetic acid and phosphoric acid.
7. The method for extracting and refining high-purity kidney bean agglutinin according to claim 1, wherein the method comprises the following steps: the activated carbon is dried food activated carbon, and has a specific surface area of 10-20m2/g。
8. The method for extracting and refining high-purity kidney bean agglutinin according to claim 1, wherein the method comprises the following steps: the operation of the sephacryls-200 molecular sieve gel filtration column chromatography treatment is as follows: and (3) balancing the gel chromatographic column by a pH 7.2 PBS buffer solution system, loading a sample with the volume not more than 15% of the column volume, continuously eluting by using the pH 7.2 PBS buffer solution with the flow rate of 0.8ml/min, collecting a first peak area sample, and freeze-drying to obtain the high-purity kidney bean agglutinin.
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| CN103073627A (en) * | 2013-02-22 | 2013-05-01 | 哈尔滨工业大学 | Method for microwave-assisted reverse micelle separation and purification of kidney bean agglutinin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103073627A (en) * | 2013-02-22 | 2013-05-01 | 哈尔滨工业大学 | Method for microwave-assisted reverse micelle separation and purification of kidney bean agglutinin |
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| JINLONG ZHAO等: "Low-pH induced structural changes, allergenicity and in vitro digestibility of lectin from black turtle bean (Phaseolus vulgaris L.)", 《FOOD CHEMISTRY》 * |
| 吴梧桐主编: "《生物制药工艺学》", 30 April 2013 * |
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