CN105838767A - Method for preparing ACE inhibitory peptide through enzymolysis of konjac fly powder - Google Patents
Method for preparing ACE inhibitory peptide through enzymolysis of konjac fly powder Download PDFInfo
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- CN105838767A CN105838767A CN201610312074.8A CN201610312074A CN105838767A CN 105838767 A CN105838767 A CN 105838767A CN 201610312074 A CN201610312074 A CN 201610312074A CN 105838767 A CN105838767 A CN 105838767A
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- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The invention provides a method for preparing ACE inhibitory peptide through enzymolysis of konjac fly powder. According to the method, konjac fly powder is used as the raw material and subjected to isoelectric precipitation to obtain konjac protein, hydrolysis is conducted with elastinase, inactivation, centrifugation and filtration are conducted to obtain konjac protein enzymatic hydrolysate, the enzymatic hydrolysate is subjected to ultrafiltration and then desalination with macroporous resin, and small molecule protein peptide with ACE inhibition activity is separated out finally and then subjected to concentration and freeze drying to obtain various preparations. The preparing method is simple, bulk production can be achieved, and cost is low. The method has important significance for efficient utilization of konjac fly powder and production of high-value-added products. Processes are simple and easy to implement, operation is easy, adopted equipment is mature, and investment cost is low.
Description
Technical field
The invention belongs to ace inhibitory peptide preparation field, be specifically related to a kind of method that enzymolysis fry starch of konjak prepares ace inhibitory peptide.
Background technology
Angiotensin converting enzyme (Angiotensin I Converting Enzyme, ACE) peptide for inhibiting is a kind of to ACE activity
Inhibited polypeptides matter, is typically made up of 2～20 amino acid residues.Ace inhibitory peptide by emulative with
ACE combines, and has blocked and has had a liter generation for the angiotensinⅡ of blood pressure activity, has simultaneously facilitated and had hypotensive activity
The generation of releive peptide and enkephalin, thus play the effect of blood pressure lowering.Ace inhibitory peptide is generally by protease in a mild condition
Aminosal and obtain, edible safety is high, hyperpietic can be played hypotensive effect and to normotensive without blood pressure lowering
Effect, the most also has the function such as immunomodulating, fat-reducing.At present, in terms of ace inhibitory peptide Source Study, be only from bean cake,
The raw materials such as Semen sojae atricolor, casein, Lac Bovis seu Bubali, wheat germ protein, Concha Ostreae are found that ace inhibitory peptide.And it is relevant with Rhizoma amorphophalli for raw material system
The research of standby ace inhibitory peptide is the fewest.
China is Rhizoma amorphophalli big producing country, and Rhizoma amorphophalli contains the abundantest nutrition, rich in glucomannan, starch, protein, many
Plant vitamin and mineral element.Wherein, protein can obtain having the merits such as defying age, anticancer, blood pressure lowering, immunity through enzymolysis
The biologically active polypeptide of energy, is the very good material producing functional health-care food.Albumen source in Rhizoma amorphophalli is mainly originated from Rhizoma amorphophalli and is flown
Powder.Fry starch of konjak is the light weight descended slowly and lightly in the konjaku powder course of processing, tiny dust, because it has certain egg fishy smell and puckery
Taste and effectively do not developed, major part is directly discarded or is used when animal feed.Testing result finds, fry starch of konjak
Middle protein content is up to 23.8%, can produce branched-chain amino acid oligopeptide, ace inhibitory peptide, high F value oligopeptide, manna after degraded
The Several Active Peptides such as polysaccharide peptide.But the natural resources of this preciousness does not has studied person to pay attention to so far, rarer exploitation and profit
With.
Summary of the invention
It is an object of the invention to provide a kind of method that enzymolysis fry starch of konjak prepares ace inhibitory peptide, the method is by flying Rhizoma amorphophalli
Powder processes, it is possible to obtain ace inhibitory peptide, improves the added value of fry starch of konjak.
For achieving the above object, the present invention taked technical scheme is:
A kind of method that enzymolysis fry starch of konjak prepares ace inhibitory peptide, comprises the following steps:
1) being added to the water by fry starch of konjak after dissolving, regulation pH value, to isoelectric point, IP, is centrifuged to obtain Rhizoma amorphophalli albumen precipitation；To Rhizoma amorphophalli
Adding water in albumen precipitation, obtain Rhizoma amorphophalli protein solution, the pH value of regulation Rhizoma amorphophalli protein solution is 7-9, temperature is 25-50 DEG C,
It is subsequently adding elastoser and carries out enzymolysis, obtain enzymolysis solution；
2) it is centrifuged after regulation pH value is 3.8 after enzymolysis solution enzyme denaturing, collects supernatant；Supernatant is collected after ultrafiltration apparatus
Filtrate, lyophilization, obtain ace inhibitory peptide.
Step 1) in fry starch of konjak when being added to the water, fry starch of konjak is 1:50 with the mass ratio of water.
Step 1) in centrifugal rotating speed be 3000-4000rpm, the time is 20-40min.
Step 1) in pH value be use hydrochloric acid regulation.
Step 1) in the mass concentration of Rhizoma amorphophalli protein solution be 2-10%.
Step 1) in the amount of elastoser that adds in every gram of Rhizoma amorphophalli protein solution be 3000-5000U.
Step 1) in time of enzymolysis be 120-240min.
Step 2) in enzyme denaturing at 90 DEG C, be specifically incubated at least 10min.
Step 2) in centrifugal rotating speed be 8000-10000rpm, the time is 15-30min.
By step 2) obtain ace inhibitory peptide through DA-201 macroporous adsorbent resin desalting and purifying.
Compared with prior art, the invention has the beneficial effects as follows: the present invention, with fry starch of konjak as raw material, is carried out through elastoser
After enzymolysis, obtain enzymolysis solution, obtain ace inhibitory peptide by after enzymolysis solution enzyme denaturing, the suppression of the ace inhibitory peptide that the present invention obtains
Rate is up to more than 50%, illustrates to have prepared ace inhibitory peptide, and activity is higher, can be made into various preparation.The present invention
Utilize fry starch of konjak to be prepared for ace inhibitory peptide, on the one hand the fry starch of konjak of low value is converted into the ACE with higher-value
Peptide for inhibiting, turns waste into wealth, and takes full advantage of resource, has widened the exploitation field of fry starch of konjak, and another is conveniently also widened
The source of ace inhibitory peptide；Preparation method of the present invention is simple, is beneficial to a large amount of production, and low cost.The present invention is to realizing Rhizoma amorphophalli
Fly powder effectively to utilize, produce its high value added product significant.The inventive method process route is simple, operation letter
Single, the equipment used is mature equipment, and cost of investment is relatively low, and the ace inhibitory peptide response rate is high.
Further, the enzymolysis solution obtained is heated to 90 DEG C, is incubated at least 10min, so that elastoser therein is fully gone out
Live.
Further, the ace inhibitory peptide that lyophilization obtains is further across DA-201 macroporous adsorbent resin desalting and purifying, energy
Access highly purified ace inhibitory peptide.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further details.
The mensuration of ACE inhibitory activity of the present invention, concretely comprises the following steps:
The required reagent of preparation, as shown in table 1, joins in test tube by each reagent on request, reacts 30min in 37 DEG C of water-baths,
After reaction terminates, add 1mol/L HCl and terminate reaction (sample C needs to add in advance HCl), in test tube, then add 1.9mL
Ethyl acetate, shake 15s, stand 5min, draw 1mL ethyl acetate layer, be evaporated in the baking oven of 100 DEG C, 40min
Rear taking-up cools down, then is re-dissolved in the deionized water of 3mL, measures absorbance at wavelength 228nm.
Table 1ACE suppression ratio detection method
ACE suppression ratio computing formula is:
In formula:
Aa: be ACE inhibitor and the simultaneous absorbance of ACE in reaction；
Ab: be the absorbance being not added with ACE inhibitor in reaction；
Ac: be the absorbance of ACE and HHL blank reaction.
Embodiment 1
1) according to mass ratio 1:50, fry starch of konjak and water being carried out mixed dissolution, (concentration is to drip hydrochloric acid solution while stirring
1.0mol/L), make the pH value of mixed liquor reach 3.8, make Rhizoma amorphophalli albumen precipitate, use low speed centrifuge at 3,000 rpm
After centrifugal 40min, abandoning supernatant, obtain albumen precipitation；
2) gained albumen precipitation is dissolved in water, obtains the Rhizoma amorphophalli protein solution that mass concentration is 2%, use magnetic force heated and stirred
Device is stirred, temperature 38 DEG C, and mixing speed is 800rpm, and dropping hydrochloric acid solution adjusts pH to 8 while stirring, adds elasticity
Protease, enzymolysis 180min, obtain enzymolysis solution；Wherein, the amount of the elastoser added in every gram of Rhizoma amorphophalli protein solution is 5000
U；
3) enzymolysis solution is placed in 90 DEG C of thermostat water baths holding 10min enzyme denaturing live, naturally cools to room temperature afterwards, by enzymolysis
It is 3.8 that liquid is adjusted to pH value, is centrifuged 20min at 10000rpm, collects supernatant；
4) take supernatant liquid filtering, by filtrate lyophilization, i.e. obtain ace inhibitory peptide.Through the mensuration of ACE inhibitory activity, its
Suppression ratio ＞ 50% to ACE activity.Illustrate to have obtained ace inhibitory peptide, and active.
Embodiment 2
1) fry starch of konjak and water are carried out mixed dissolution according to mass ratio 1:50, drip hydrochloric acid solution while stirring, make mixing
The pH value of liquid reaches 3.8, makes Rhizoma amorphophalli albumen precipitate, and uses low speed centrifuge after centrifugal 20min under 4000rpm, abandons
Remove supernatant, obtain albumen precipitation；
2) gained albumen precipitation is dissolved in water, obtains the Rhizoma amorphophalli protein solution that mass concentration is 10%, use magnetic force heated and stirred
Device is stirred, temperature 35 DEG C, and mixing speed is 800rpm, and dropping hydrochloric acid solution adjusts pH to 8.5 while stirring, adds bullet
Property protease, enzymolysis 200min, obtain enzymolysis solution；Wherein, the amount of the elastoser added in every gram of Rhizoma amorphophalli protein solution is
3000U；
3) enzymolysis solution is placed in 90 DEG C of thermostat water baths holding 10min enzyme denaturing live, naturally cools to room temperature afterwards, by enzymolysis
It is 3.8 that liquid is adjusted to pH value, is centrifuged 15min at 10000rpm, collects supernatant；
4) take supernatant liquid filtering, by filtrate lyophilization, i.e. obtain ace inhibitory peptide.Through the mensuration of ACE inhibitory activity, its
Suppression ratio ＞ 45% to ACE activity.
Embodiment 3
1) fry starch of konjak and water are carried out mixed dissolution according to mass ratio 1:50, drip hydrochloric acid solution while stirring, make mixing
The pH value of liquid reaches 3.8, makes Rhizoma amorphophalli albumen precipitate, and uses low speed centrifuge after centrifugal 35min under 3500rpm, abandons
Remove supernatant, obtain albumen precipitation；
2) gained albumen precipitation is dissolved in water, obtains the Rhizoma amorphophalli protein solution that mass concentration is 5%, use magnetic force heated and stirred
Device is stirred, temperature 40 DEG C, and mixing speed is 800rpm, and dropping hydrochloric acid solution adjusts pH to 9 while stirring, adds elasticity
Protease, enzymolysis 240min, obtain enzymolysis solution；Wherein, the amount of the elastoser added in every gram of Rhizoma amorphophalli protein solution is 4000
U；
3) enzymolysis solution is placed in 90 DEG C of thermostat water baths holding 10min enzyme denaturing live, naturally cools to room temperature afterwards, by enzymolysis
It is 3.8 that liquid is adjusted to pH value, is centrifuged 30min at 9000rpm, collects supernatant；
4) take supernatant liquid filtering, by filtrate lyophilization, i.e. obtain ace inhibitory peptide.Through the mensuration of ACE inhibitory activity, its
Suppression ratio ＞ 40% to ACE activity.
Embodiment 4
1) fry starch of konjak and water are carried out mixed dissolution according to mass ratio 1:50, drip hydrochloric acid solution while stirring, make mixing
The pH value of liquid reaches 3.8, makes Rhizoma amorphophalli albumen precipitate, and after using low speed centrifuge to be centrifuged 40min at 3,000 rpm, abandons
Remove supernatant, obtain albumen precipitation；
2) gained albumen precipitation is dissolved in water, obtains the Rhizoma amorphophalli protein solution that mass concentration is 7%, use magnetic force heated and stirred
Device is stirred, temperature 37 DEG C, and mixing speed is 800rpm, and dropping hydrochloric acid solution adjusts pH to 7 while stirring, adds elasticity
Protease, enzymolysis 200min, obtain enzymolysis solution；Wherein, the amount of the elastoser added in every gram of Rhizoma amorphophalli protein solution is 3500
U；
3) enzymolysis solution is placed in 90 DEG C of thermostat water baths holding 10min enzyme denaturing live, naturally cools to room temperature afterwards, by enzymolysis
It is 3.8 that liquid is adjusted to pH value, is centrifuged 25min at 9500rpm, collects supernatant；
4) take supernatant liquid filtering, by filtrate lyophilization, i.e. obtain ace inhibitory peptide.Through the mensuration of ACE inhibitory activity, its
Suppression ratio ＞ 35% to ACE activity.
Embodiment 5
1) fry starch of konjak and water are carried out mixed dissolution according to mass ratio 1:50, drip hydrochloric acid solution while stirring, make mixing
The pH value of liquid reaches 3.8, makes Rhizoma amorphophalli albumen precipitate, and uses low speed centrifuge after centrifugal 30min under 4000rpm, abandons
Remove supernatant, obtain albumen precipitation；
2) gained albumen precipitation is dissolved in water, obtains the Rhizoma amorphophalli protein solution that mass concentration is 4%, use magnetic force heated and stirred
Device is stirred, temperature 50 C, and mixing speed is 800rpm, and dropping hydrochloric acid solution adjusts pH to 8.1 while stirring, adds bullet
Property protease, enzymolysis 240min, obtain enzymolysis solution；Wherein, the amount of the elastoser added in every gram of Rhizoma amorphophalli protein solution is
4500U；
3) enzymolysis solution is placed in 90 DEG C of thermostat water baths holding 10min enzyme denaturing live, naturally cools to room temperature afterwards, by enzymolysis
It is 3.8 that liquid is adjusted to pH value, is centrifuged 25min at 10000rpm, collects supernatant；
4) take supernatant liquid filtering, by filtrate lyophilization, i.e. obtain ace inhibitory peptide.Through the mensuration of ACE inhibitory activity, its
Suppression ratio ＞ 40% to ACE activity.
Embodiment 6
1) according to mass ratio 1:50, fry starch of konjak and water being carried out mixed dissolution, (concentration is to drip hydrochloric acid solution while stirring
1.0mol/L), make the pH value of mixed liquor reach 3.8, make Rhizoma amorphophalli albumen precipitate, use low speed centrifuge at 3,000 rpm
After centrifugal 40min, abandoning supernatant, obtain albumen precipitation；
2) gained albumen precipitation is dissolved in water, obtains the Rhizoma amorphophalli protein solution that mass concentration is 2%, use magnetic force heated and stirred
Device is stirred, temperature 25 DEG C, and mixing speed is 800rpm, and dropping hydrochloric acid solution adjusts pH to 8 while stirring, adds elasticity
Protease, enzymolysis 120min, obtain enzymolysis solution；Wherein, the amount of the elastoser added in every gram of Rhizoma amorphophalli protein solution is 5000
U；
3) enzymolysis solution is placed in 90 DEG C of thermostat water baths holding 10min enzyme denaturing live, naturally cools to room temperature afterwards, by enzymolysis
It is 3.8 that liquid is adjusted to pH value, is centrifuged 20min at 10000rpm, collects supernatant；
4) take supernatant liquid filtering, by filtrate lyophilization, i.e. obtain ace inhibitory peptide.
The ace inhibitory peptide present invention obtained, through DA-201 macroporous adsorbent resin desalting and purifying, can improve purity.
Claims (10)
1. the method that an enzymolysis fry starch of konjak prepares ace inhibitory peptide, it is characterised in that comprise the following steps:
1) being added to the water by fry starch of konjak after dissolving, regulation pH value, to isoelectric point, IP, is centrifuged to obtain Rhizoma amorphophalli albumen precipitation；To Rhizoma amorphophalli
Adding water in albumen precipitation, obtain Rhizoma amorphophalli protein solution, the pH value of regulation Rhizoma amorphophalli protein solution is 7-9, temperature is 25-50 DEG C,
It is subsequently adding elastoser and carries out enzymolysis, obtain enzymolysis solution；
2) it is centrifuged after regulation pH value is 3.8 after enzymolysis solution enzyme denaturing, collects supernatant；Supernatant is collected after ultrafiltration apparatus
Filtrate, lyophilization, obtain ace inhibitory peptide.
The method that a kind of enzymolysis fry starch of konjak the most according to claim 1 prepares ace inhibitory peptide, it is characterised in that step
1), when in, fry starch of konjak is added to the water, fry starch of konjak is 1:50 with the mass ratio of water.
The method that a kind of enzymolysis fry starch of konjak the most according to claim 1 prepares ace inhibitory peptide, it is characterised in that step
1) rotating speed centrifugal in is 3000-4000rpm, and the time is 20-40min.
The method that a kind of enzymolysis fry starch of konjak the most according to claim 1 prepares ace inhibitory peptide, it is characterised in that step
1) in, pH value is to use hydrochloric acid regulation.
The method that a kind of enzymolysis fry starch of konjak the most according to claim 1 prepares ace inhibitory peptide, it is characterised in that step
1) in, the mass concentration of Rhizoma amorphophalli protein solution is 2-10%.
The method that a kind of enzymolysis fry starch of konjak the most according to claim 5 prepares ace inhibitory peptide, it is characterised in that step
1) amount of the elastoser added in every gram of Rhizoma amorphophalli protein solution in is 3000-5000U.
The method that a kind of enzymolysis fry starch of konjak the most according to claim 1 prepares ace inhibitory peptide, it is characterised in that step
1) in, the time of enzymolysis is 120-240min.
The method that a kind of enzymolysis fry starch of konjak the most according to claim 1 prepares ace inhibitory peptide, it is characterised in that step
2) in, enzyme denaturing is specifically incubated at least 10min at 90 DEG C.
The method that a kind of enzymolysis fry starch of konjak the most according to claim 1 prepares ace inhibitory peptide, it is characterised in that step
2) rotating speed centrifugal in is 8000-10000rpm, and the time is 15-30min.
The method that a kind of enzymolysis fry starch of konjak the most according to claim 1 prepares ace inhibitory peptide, it is characterised in that will
Step 2) obtain ace inhibitory peptide through DA-201 macroporous adsorbent resin desalting and purifying.
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| CN201610312074.8A CN105838767A (en) | 2016-05-11 | 2016-05-11 | Method for preparing ACE inhibitory peptide through enzymolysis of konjac fly powder |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755244A (en) * | 2016-12-29 | 2017-05-31 | 陕西科技大学 | A kind of method that flavor protease enzymolysis prepares ace inhibitory peptide |
| CN108949873A (en) * | 2018-04-19 | 2018-12-07 | 金华市景和科技有限公司 | The method of polypeptide active substance is extracted from konjaku |
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|---|---|---|---|---|
| CN1557474A (en) * | 2004-02-04 | 2004-12-29 | 高春平 | Blood pressure reducing natural polypeptide nutrient |
| WO2007080894A1 (en) * | 2006-01-11 | 2007-07-19 | Seiya Sakurai | Fluid konjac material, method of producing the same and use thereof |
| CN103436582A (en) * | 2013-07-10 | 2013-12-11 | 马杰坤 | Konjac polypeptide extract, as well as preparation method and application thereof |
-
2016
- 2016-05-11 CN CN201610312074.8A patent/CN105838767A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1557474A (en) * | 2004-02-04 | 2004-12-29 | 高春平 | Blood pressure reducing natural polypeptide nutrient |
| WO2007080894A1 (en) * | 2006-01-11 | 2007-07-19 | Seiya Sakurai | Fluid konjac material, method of producing the same and use thereof |
| CN103436582A (en) * | 2013-07-10 | 2013-12-11 | 马杰坤 | Konjac polypeptide extract, as well as preparation method and application thereof |
Non-Patent Citations (1)
| Title |
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| 徐怀德等: "酶解魔芋飞粉蛋白制备ACE 抑制肽", 《食品科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755244A (en) * | 2016-12-29 | 2017-05-31 | 陕西科技大学 | A kind of method that flavor protease enzymolysis prepares ace inhibitory peptide |
| CN108949873A (en) * | 2018-04-19 | 2018-12-07 | 金华市景和科技有限公司 | The method of polypeptide active substance is extracted from konjaku |
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Application publication date: 20160810 |