CN106565839A - Method for extracting trypsin inhibitor from pitaya seeds - Google Patents
Method for extracting trypsin inhibitor from pitaya seeds Download PDFInfo
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- CN106565839A CN106565839A CN201610893580.0A CN201610893580A CN106565839A CN 106565839 A CN106565839 A CN 106565839A CN 201610893580 A CN201610893580 A CN 201610893580A CN 106565839 A CN106565839 A CN 106565839A
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- nacl
- trypsin inhibitor
- dragon fruit
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- seeds
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
Abstract
The invention relates to a method for extracting a trypsin inhibitor from pitaya seeds. The method comprises the following steps: freezing fresh pitaya in -30 - -10 DEG C for 45-50 h, unfreezing the frozen fresh pitaya, and removing peels; filtering out obtained liquid, washing obtained solid with clear water to remove pulp residual on the surfaces of the seeds, drying the washed seeds, and crushing the dried seeds to form powder; adding the powder to a citric acid-sodium citrate buffering solution, heating the obtained solution to 60-70 DDEG C, and keeping the temperature unchanged for 1.5-2.5 h; centrifuging the heated solution under 9000-11000 rpm, taking obtained supernatant, and carrying out suction filtration on the supernatant with quantitative filter paper to obtain a filtrate; carrying out adsorption elution on the filtrate in an anion exchange fiber column by using a NaCl-Tris-HCl buffering solution; carrying out gradient elution on the anion-exchange fiber column by using a NaCl-Tris-HCl buffering solution containing 0-1 M of NaCl; and detecting the obtained solution at wavelengths of 230 nm and 280 nm, collecting the eluate obtained by using 0-1 M of NaCl, carrying out concentration desalination by using a 3.5 KD tangential flow ultrafilter membrane, and carrying out vacuum freeze drying to obtain the trypsin inhibitor.
Description
Technical field
The present invention relates to the extracting method of trypsin inhibitor, refers specifically to one kind and Trypsin is extracted from dragon fruit seed
The method of enzyme inhibitor.
Background technology
Hylocereus undatuss, also known as Hylocereus undatus, Herba Passiflorae Foetidae, Pitaya fruit, Yu Longguo, are Cactaceae, Hylocereus undatus platymiscium.Fruit is in
Ellipse, 10～12 centimetres of diameter, outward appearance peel are red or yellow, have the thalluves of green rounded triangle, and sarcocarp has white
Color, redness or yellow point, with melanospermous fruit.Hylocereus undatuss fruits nutrition is abundant, function is unique, containing general plant
Rare vegetable albumin, betanin, abundant vitamin and water soluble dietary fiber etc..In its natural state, fruit in
Autumn in summer is ripe, flat flavor, succulence.One Hylocereus undatuss black seeds rich content, contains a large amount of unsaturated fatty acidss in dragon fruit seed
Acid and protein.But in dragon fruit seed, protein is not fully used at present.
The content of the invention
The technical problem to be solved is to provide that a kind of inhibition is good, low cost for the present situation of prior art
And the few method that trypsin inhibitor is extracted from dragon fruit seed of side reaction.
The present invention solve the technical scheme that adopted of above-mentioned technical problem for：Trypsin suppression should be extracted from dragon fruit seed
The method of preparation, it is characterised in that comprise the steps：
Fresh Hylocereus undatuss are freezed into 45～50h at -30～-10 DEG C, epidermis after defrosting, is taken out；Liquid is filtered out, is obtained
Solidss with clear water clean seed surface residual sarcocarp after, be dried 24-48h at 45～55 DEG C, be ground into dragon fruit seed powder；
Dragon fruit seed powder will be obtained to be added in citric acid-sodium citrate buffer, 60-70 DEG C will be heated to, constant temperature
1.5～2.5h；Then it is centrifuged under 9000～11000rpm, takes supernatant, supernatant carries out sucking filtration using qualitative filter paper, obtains
Filtrate；
In the citric acid-sodium citrate buffer, the concentration of citric acid and sodium citrate is 90～110mmol/
Ml, pH are 5-8；The ratio of the dragon fruit seed powder and citric acid-sodium citrate buffer is 1:10-20g/ml,
The filtrate for obtaining is carried out into absorb-elute, the buffering used by eluting in DEAE-52 anion-exchange fibre posts
System is NaCl-Tris-HCl buffer, and pH is 7.0-7.2；
With containing 0～1M of NaCl NaCl-Tris-HCl buffer Gradient elution DEAE-52 anion-exchange fibre posts,
Flow velocity is 1mL/min；It is to detect under 230nm and 280nm in wavelength, collects the eluent under 0～1M NaCl, cut in 3.5KD
It is to stream ultrafilter membrane lower film concentrating and desalinating to 1/5th of original volume, then true at a temperature of 120～140MPa, -90～-70 DEG C
Vacuum freecing-dry 46-50h, that is, obtain trypsin inhibitor.
The Hylocereus undatuss are preferably red meat Hylocereus undatuss.
Compared with prior art, the extracting method of trypsin inhibitor provided by the present invention, overcomes technology prejudice, adopts
It is raw material with dragon fruit seed, coordinates specific extraction process, using citric acid-sodium citrate buffer heating from Hylocereus undatuss
Trypsin inhibitor is extracted in seed, method is easy to operate, the trypsin inhibitor activity for obtaining is high, and trypsin is had
Fine inhibitory action.And prior art is typically to extract from the crops such as Semen sojae atricolor, Rhizoma Solani tuber osi, more bean, Semen phaseoli radiati, Radix Et Rhizoma Fagopyri Tatarici, method is multiple
Miscellaneous, the trypsin inhibitor activity for obtaining is low.
Description of the drawings
The relation that Fig. 1 suppresses to tryptic activity for addition extract 1, extract 2 in the embodiment of the present invention.
Specific embodiment
The present invention is described in further detail below in conjunction with accompanying drawing embodiment.
The method that trypsin inhibitor is extracted from dragon fruit seed is specific as follows：
October harvesting is selected, ripe fresh, the red meat Hylocereus undatuss of 300-500g/ are a collection of.It is put into -20 DEG C of refrigerator freezings
48h, thaws after peeling, falls liquid with filtered through gauze, cleans the sarcocarp of seed surface residual with clear water, is then placed in baking oven at 50 DEG C
Lower drying 36 hours, with crusher for Chinese herbal medicine crush, obtain dragon fruit seed powder, loaded on hermetic bag in, be placed in -20 DEG C of refrigerators standby
With.
To in dragon fruit seed powder add pH be 6.25 citric acid-sodium citrate buffer, dragon fruit seed powder with
The proportioning of citric acid-sodium citrate buffer is 1:10g/ml；60 DEG C are heated to, constant temperature 2h is centrifuged under 10000r/min
10min, carries out sucking filtration using qualitative filter paper, removes lubricant component in solution, obtains filtrate.
The filtrate for obtaining is carried out into absorb-elute in DEAE-52 anion-exchange fibres post, elution requirement is：Buffer system
For the NaCl-Tris-HCl buffer of pH7.0, the NaCl-Tris-HCl buffer isocratic elution DEAE- containing 0～1M NaCl are used
52 anion-exchange fibre posts, flow velocity is 1mL/min, and in being to monitor eluent liquid under 230nm and 280nm in wavelength, extract contains
Amount, is shown in that peak is collected.
The eluent under 0M NaCl, i.e. extract 1 are collected respectively；Eluent under 1M NaCl is extract 2.Respectively
In 3.5KD cross-flow ultrafiltrations film lower film concentrating and desalinating to 1/5th of original volume；It is at -80 DEG C in vacuum 130Pa, temperature
Vacuum lyophilization 48h；Trypsin inhibitor is obtained.
The inhibitory action of trypsin inhibitor obtained in checking：
Reagent：Porcine trypsin, is purchased from Yuan Ye biotech firms；N- Benzoyl-L-arginine ethyl esters (BAEE), is purchased from me
Fourth biotech firm；Sodium dihydrogen phosphate, disodium hydrogen phosphate, are purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Distilled water is by laboratory
There is provided.
Instrument：Japanese Shimadzu UV-2600 visible spectrophotometer；TLE104E electronic balances, prunus mume (sieb.) sieb.et zucc. Teller-support benefit instrument
Device (Shanghai) Co., Ltd.；Haier's BCD-215DC refrigerators, Qingdao HaiEr Co., Ltd；RC-200 mesolow tomographic systems,
Guangzhou Rui Bai instruments Science and Technology Ltd..
Determination of tryptic activity：
Trypsin can be catalyzed and be hydrolyzed to H- Benzoyl-L-arginines to N- Benzoyl-L-arginine ethyl esters (BAEE)
(BA).BA has maximum absorption band at 253nm wavelength.Therefore by determining the suction that system is determined under enzymic catalytic reaction system A253
Light value and the relation of time, map and obtain a straight line, and cut-off line head and the tail numerical value substitutes into formula (1) and calculates enzymatic activity size.
P=(A2- A1)/0.003TW (1)
In formula, P is tryptic amount (U/mg) in every 1mg test samples；
A1Light absorption value during to start on straight line；
A2Light absorption value during to terminate on straight line；
T is A1To A2The time of reading, min；
W is the tryptic amount containing test sample in measure liquid, mg；
0.003 is light absorption value change value per minute, i.e., equivalent to 1 trypsin unit.
Specifically assay method is：Substrate B AEE0.086g is weighed, the phosphorus of the 0.067mol/L that 1000mL pH are 6.8 is dissolved in
In acid buffer, BAEE solution is obtained；With water as blank, adjusting substrate B AEE solution with phosphate buffer makes its A253nm exist
Between 0.575～0.585.Take substrate solution 3.0mL, plus 100uL distilled water, add after mixing 200uL concentration be 0.020～
0.030mg/mL trypsin aqueous solutions, it is rapid to mix and timing immediately, A253nm light absorption values are determined at ambient temperature.It is another with
The mixed liquor of BAEE solution 3mL and distilled water 300uL makees blank, and A253nm values are read per 1min, 6min is read altogether, to inhale
Light value is vertical coordinate, and the time is abscissa mapping.
In the every 1min of control, between 0.015-0.018, the linear time must not be less than for the change of light absorption value
3min.Head and the tail numerical value on straight line is substituted in formula (1) and is calculated enzymatic activity.Figure line is less with x-axis angle, shows to suppress
Rate is higher.
The measure of extract manufactured in the present embodiment 1 and extract 2 to trypsin inhibitory action：
The extract solution that concentration is 1mg/mL is configured to by the extract 1 and extract 2 of preparation soluble in water respectively.
Make blank with the mixed liquor of BAEE 3mL, distilled water 200uL and extract solution 100uL.BAEE solution 3mL are taken separately
After mixing with each component protein extract 100uL, 200uL concentration is added for 0.020～0.030mg/mL trypsin enzyme water
Mix in solution, 1s and be put into timing immediately in spectrophotometer, determine A253nm light absorption values at ambient temperature, to be added
Tryptic activity after inhibitor.
Calculated according to formula (1) and add tryptic activity after each extract, each extract pair is calculated according to measurement result
Trypsin suppression ratio.
Tryptic activity/tryptic activity after suppression ratio=each extract of tryptic activity-addition
Tryptic activity and each extract tryptic activity such as Fig. 1 of addition.
By Fig. 1 it is known that extract 1 reaches 90% to trypsin suppression ratio, extract 2 is to trypsin suppression ratio
Reach 99%.Illustrate that the inhibitor prepared by the present invention has extraordinary inhibitory action to trypsin, can be used as a kind of new
Trypsin inhibitor.
Claims (2)
1. it is a kind of from dragon fruit seed extract trypsin inhibitor method, it is characterised in that comprise the steps：
Fresh Hylocereus undatuss are freezed into 45～50h at -30～-10 DEG C, epidermis after defrosting, is taken out；Liquid is filtered out, what is obtained consolidates
After body thing cleans the sarcocarp of seed surface residual with clear water, 24-48h is dried at 45～55 DEG C, is ground into dragon fruit seed powder；
Dragon fruit seed powder will be obtained to be added in citric acid-sodium citrate buffer, 60-70 DEG C will be heated to, constant temperature 1.5～
2.5h；Then it is centrifuged under 9000～11000rpm, takes supernatant, supernatant carries out sucking filtration using qualitative filter paper, obtains filtrate；
In the citric acid-sodium citrate buffer, the concentration of citric acid and sodium citrate is 90～110mmol/ml, pH
For 5-8；The ratio of the dragon fruit seed powder and citric acid-sodium citrate buffer is 1:10-20g/ml；
The filtrate for obtaining is carried out into absorb-elute, the buffer system used by eluting in DEAE-52 anion-exchange fibre posts
For NaCl-Tris-HCl buffer, pH is 7.0-7.2；
With the NaCl-Tris-HCl buffer Gradient elution DEAE-52 anion-exchange fibre posts containing 0～1M of NaCl, flow velocity
For 1mL/min；It is to detect under 230nm and 280nm in wavelength, collects the eluent under 0～1M NaCl, in 3.5KD slipstreams
, to 1/5th of original volume, then at a temperature of 120～140MPa, -90～-70 DEG C, vacuum is cold for ultrafilter membrane lower film concentrating and desalinating
The dry 46-50h of lyophilizing, that is, obtain trypsin inhibitor.
2. it is according to claim 1 from dragon fruit seed extract trypsin inhibitor method, it is characterised in that it is described
Hylocereus undatuss are red meat Hylocereus undatuss.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610893580.0A CN106565839B (en) | 2016-10-13 | 2016-10-13 | Method for extracting trypsin inhibitor from pitaya seeds |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610893580.0A CN106565839B (en) | 2016-10-13 | 2016-10-13 | Method for extracting trypsin inhibitor from pitaya seeds |
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| CN106565839A true CN106565839A (en) | 2017-04-19 |
| CN106565839B CN106565839B (en) | 2020-01-03 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101759801A (en) * | 2008-12-11 | 2010-06-30 | 湖州来色生物基因工程有限公司 | Separation and purification method of novel soybean trypsin inhibitor |
| CN102295700A (en) * | 2011-07-29 | 2011-12-28 | 集美大学 | Separation and purification method of mung bean trypsin inhibitor (MBTI) and application of MBTI in production of surimi products |
| CN104558158A (en) * | 2014-12-17 | 2015-04-29 | 江西省林业科学院 | Method for extracting trypsin inhibitor from bamboo shoots |
| CN105153099A (en) * | 2015-10-17 | 2015-12-16 | 重庆都好生物科技有限公司 | Method for extracting procyanidins (OPC) from pitaya peels |
-
2016
- 2016-10-13 CN CN201610893580.0A patent/CN106565839B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101759801A (en) * | 2008-12-11 | 2010-06-30 | 湖州来色生物基因工程有限公司 | Separation and purification method of novel soybean trypsin inhibitor |
| CN102295700A (en) * | 2011-07-29 | 2011-12-28 | 集美大学 | Separation and purification method of mung bean trypsin inhibitor (MBTI) and application of MBTI in production of surimi products |
| CN104558158A (en) * | 2014-12-17 | 2015-04-29 | 江西省林业科学院 | Method for extracting trypsin inhibitor from bamboo shoots |
| CN105153099A (en) * | 2015-10-17 | 2015-12-16 | 重庆都好生物科技有限公司 | Method for extracting procyanidins (OPC) from pitaya peels |
Non-Patent Citations (2)
| Title |
|---|
| MEHRNOUSH AMID ET AL.,: "Purification and Characterization of Alkaline-Thermostable Protease Enzyme from Pitaya(Hylocereus polyrhizus) Waste: A potential Low Cost of the Enzyme", 《JOURNAL OF BIOMEDICINE AND BIOTECHNOLOGY》 * |
| 陈丽娜等: "红肉火龙果与白肉火龙果品质分析", 《中国南方果树》 * |
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