CN109438557A - A kind of Agroeybe cylindracea mycelia anti-oxidation peptide and application thereof - Google Patents
A kind of Agroeybe cylindracea mycelia anti-oxidation peptide and application thereof Download PDFInfo
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- CN109438557A CN109438557A CN201811090002.9A CN201811090002A CN109438557A CN 109438557 A CN109438557 A CN 109438557A CN 201811090002 A CN201811090002 A CN 201811090002A CN 109438557 A CN109438557 A CN 109438557A
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- Prior art keywords
- oxidation peptide
- cylindracea mycelia
- agroeybe cylindracea
- peptide
- agroeybe
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
Abstract
The invention discloses a kind of Agroeybe cylindracea mycelia anti-oxidation peptide, which contains the peptide fragment that amino acid sequence is PAQPPPPPNR.The invention also discloses the preparation methods of the anti-oxidation peptide.A kind of present invention isolated biologically active peptide from Agroeybe cylindracea mycelia for the first time, and its antioxidant activity is demonstrated, it can be used as additive and be used to prepare health care product, food, cosmetics and daily chemical product.The anti-oxidation peptide has many advantages, such as that purity is high, yield are big, realizes the deep processing and utilization of Agroeybe cylindracea mycelia, improves added value of product.
Description
Technical field
The invention belongs to biological field, it is related to a kind of Agroeybe cylindracea mycelia anti-oxidation peptide and application thereof.
Background technique
Currently, people separate from animal, plant and microorganism, Several Active Peptides have been prepared, these activity
There are many physiological functions, such as anti-oxidant, reducing blood lipid, inhibition virus for peptide.Wherein inoxidizability polypeptide is with specific amino acids
The peptide fragment of sequence is hidden in the inside of protein molecule from some originals and releases, and passes through scavenging activated oxygen, hydroxy radical
Etc. oxidation resistant effect is reached, compares and synthetized oxidation preventive agent, safety are higher, it can not only be prevented in food system
Oxidation, and the harm of people's interior free yl can be prevented, various degenerative disorders caused by delaying senescence, such as cancer, coronary disease
Disease, diabetes etc..
Agroeybe cylindracea mycelia (Agrocybe aegerita) also known as agrocybe belong to Agaricales, Fen Xiu umbrella section, field mushroom category, are foods
With the appreciable variety in bacterium.Its delicious flavour, nutritive value is high, there is decompression, anti-ephritis, antioedematous and other effects, there is " China's mind
The good reputation of mushroom ".It is mainly cultivated in Fujian Province, China, Guangdong Province and Guangxi province, year output is up to 1,000,000 tons.
China is world's big country of mushroom producing, but mainly based on primary processing agricultural product, intensive processing is less.
CN102224879A discloses a kind of preparation method of champignon polypeptide, has and adjusts blood lipid and antifatigue effect；
CN101709320A discloses a kind of preparation method of champignon polypeptide powder, is mainly used for health care product and food processing field, these
Document is all to carry out enzymatic hydrolysis using mushroom amyloid proteins to prepare active peptide.Reported in literature about Agroeybe cylindracea mycelia anti-oxidation peptide at present
It is less.
Summary of the invention
The object of the present invention is to provide a kind of Agroeybe cylindracea mycelia anti-oxidation peptides.
Agroeybe cylindracea mycelia anti-oxidation peptide provided by the present invention is to be prepared as follows:
1. weighing Agroeybe cylindracea mycelia fructification powder, adds water temperature extraction to take, saturated ammonium sulfate is added after centrifugation into leaching liquor
It saltouts, the precipitating after saltouing is dissolved with water, then dialyse through the bag filter that molecular cut off is 3000-4000Da, it will
Precipitating drying after dialysis, obtains Agroeybe cylindracea mycelia crude protein；
2. the water of 20-50 times of weight is added into crude protein, pH to 7.5-8.5 is adjusted, according still further to the 1- of crude protein weight
5% be added neutral proteinase, 50-60 DEG C enzymatic hydrolysis 5-10 hour, then boil inactivate after be centrifuged, collection filtrate；
3. ultrafiltration then is carried out with the ultrafiltration membrane that molecular cut off is 3000-4000Da by filtrate first through micro-filtrate membrane filtration,
Collect ultrafiltrate；
4. by ultrafiltrate through the further separation component of preparation scale high performance liquid chromatograph, using PRC-ODS chromatographic column, with
The mixed solution of 0.05-0.5% (percent by volume) trifluoroacetic acid and acetonitrile is mobile phase, carries out gradient elution, examines under 215nm
It surveys, the second component is collected according to peak position out, is concentrated, after drying to obtain the final product.
Preferably, the temperature that the temperature extraction takes is 50-80 DEG C, and the time is 2-8 hours.
Preferably, the saturation degree of the ammonium sulfate is 80%.
Preferably, the aperture of the microfiltration membranes is 0.22 μm.
Preferably, the molecular cut off of the ultrafiltration membrane is 3500Da.
According to one embodiment of present invention, optimal preparation process is:
1) Agroeybe cylindracea mycelia fructification powder is weighed, the water of 10 times of weight is added, 60 DEG C of temperature extractions take 4 hours, and centrifugation is backward
Saturated ammonium sulfate is added in leaching liquor to saltout, until the saturation degree of ammonium sulfate is 80%, the precipitating after saltouing is dissolved with water,
Then it dialyses through the bag filter that molecular cut off is 3000-4000Da, the precipitating after dialysis is dried, it is thick to obtain Agroeybe cylindracea mycelia
Albumen；
2) water of 35 times of weight is added into crude protein, adjusts pH to 8.0, according still further in 3% addition of crude protein weight
Property protease, 50 DEG C digest 6 hours, then boil inactivation after be centrifuged, collect filtrate；
3) then the micro-filtrate membrane filtration for being 0.22 μm by filtrate elder generation via hole diameter is the super of 3500 dalton with molecular cut off
Filter membrane carries out ultrafiltration, collects ultrafiltrate；
4) by ultrafiltrate through the further separation component of preparation scale high performance liquid chromatograph, using PRC-ODS chromatographic column, with
The mixed solution of 0.1% trifluoroacetic acid and acetonitrile is mobile phase, carries out gradient elution (elution process is shown in Table 1), flow velocity 1mL/
It is detected under min, 215nm, the second component is collected according to peak position out, is concentrated, after drying to obtain the final product.
1 gradient elution process of table
| Elution time | 0.1% trifluoroacetic acid phase | Acetonitrile phase |
| 0~5min | 100 → 95% | 0 → 5% |
| 5~25min | 95 → 85% | 5 → 15% |
| 25~40min | 85 → 80% | 15 → 20% |
| 40~45min | 80 → 70% | 20 → 30% |
| 45~50min | 70 → 60% | 30 → 40% |
Through detecting, gained Agroeybe cylindracea mycelia anti-oxidation peptide contains the peptide fragment that amino acid sequence is PAQPPPPPNR.
The beneficial effects of the present invention are:
1) isolated a kind of biologically active peptide from Agroeybe cylindracea mycelia for the first time of the invention, and demonstrate its anti-oxidant work
Property, it can be used as additive and be used to prepare health care product, food, cosmetics and daily chemical product.
2) Agroeybe cylindracea mycelia anti-oxidation peptide provided by the invention has many advantages, such as that purity is high, yield are big, realizes column field
The deep processing and utilization of head mushroom, improve added value of product.
Detailed description of the invention
Fig. 1 is half preparative high-performance liquid chromatographic figure of Agroeybe cylindracea mycelia anti-oxidation peptide, is total in figure there are three separation component, this
Invention is the second component (appearance time was at the 32nd minute).
Fig. 2 is PAQPPPPPNR peptide fragment mass spectrogram contained by Agroeybe cylindracea mycelia anti-oxidation peptide.
Specific embodiment
The present invention is described in detail below by specific embodiment.
Embodiment 1
1) Agroeybe cylindracea mycelia fructification powder 1kg is weighed, the water of 10 times of weight is added, 60 DEG C extract 4 hours, to leaching after centrifugation
Saturated ammonium sulfate is added in extract to saltout, the saturation degree of ammonium sulfate final concentration is 80%.By the precipitating after saltouing with water-soluble
Then solution dialyses through the bag filter that molecular cut off is 4000Da, the precipitating after dialysis is dried, the thick egg of Agroeybe cylindracea mycelia is obtained
It is white；
2) water of 35 times of weight is added into crude protein, adjusts pH to 8.0, then 3% neutral proteinase is added by weight
(Rate activity is 2.0 million international units), 50 DEG C digest 6 hours, are centrifuged after then boiling inactivation, collect filtrate；
3) by filtrate first through 0.22 μm of filter membrane micro-filtration, ultrafiltration then is carried out with the ultrafiltration membrane of 3500 dalton, is collected super
Filtrate；
4) ultrafiltrate is further isolated and purified through preparation scale high performance liquid chromatograph (HPLC), using PRC-ODS column
(250mm × 20mm, 5 μm, SHIMADZU, Japan), the mixed solution of 0.1% trifluoroacetic acid and acetonitrile are mobile phase, carry out ladder
Degree elution (elutes process and is shown in Table 1), detects under flow velocity 1mL/min, 215nm, collects the second component (result according to peak position out
See Fig. 1, through detecting, the antioxidant activity of the second component is apparently higher than other two components), it is concentrated, it is dry, obtain column the edge of a field
Mushroom anti-oxidation peptide 15.7g, purity 91.8% (reference standard: GB18186-2000).
Embodiment 2
1) Agroeybe cylindracea mycelia fructification powder 1kg is weighed, the water of 20 times of weight is added, stirs evenly, 80 DEG C extract 3 hours,
Saturated ammonium sulfate is added after centrifugation into leaching liquor to saltout, the saturation degree of ammonium sulfate final concentration is 80%.After saltouing
Precipitating is dissolved with water, then dialyses through the bag filter that molecular cut off is 3000Da, the precipitating after dialysis is dried, column is obtained
Ricetulus rodents crude protein；
2) water of 25 times of weight is added into crude protein, adjusts pH to 7.5, then 4% neutral proteinase is added by weight,
60 DEG C digest 5 hours, are centrifuged after then boiling inactivation, collect filtrate；
3) by filtrate first through 0.22 μm of filter membrane micro-filtration, ultrafiltration then is carried out with the ultrafiltration membrane of 3500 dalton, is collected super
Filtrate；
4) ultrafiltrate is further isolated and purified through preparation scale high performance liquid chromatograph, using PRC-ODS column, 0.05% 3
The mixed solution of fluoroacetic acid and acetonitrile is mobile phase, carries out gradient elution (elution process is same as Example 1), flow velocity 1mL/
It is detected under min, 215nm, the second component is collected according to peak position out, is concentrated, it is dry, obtain Agroeybe cylindracea mycelia anti-oxidation peptide
14.6g, purity 90.5%.
Embodiment 3
1) Agroeybe cylindracea mycelia fructification powder 1kg is weighed, the water of 6 times of weight is added, stirs evenly, 65 DEG C extract 5 hours, from
Saturated ammonium sulfate is added after the heart into leaching liquor to saltout, the saturation degree of ammonium sulfate final concentration is 80%.It is heavy after saltouing
Shallow lake is dissolved with water, then dialyses through the bag filter that molecular cut off is 4000Da, the precipitating after dialysis is dried, column field is obtained
Head mushroom crude protein；
2) water of 50 times of weight is added into crude protein, adjusts pH to 8.5, then 2% neutral proteinase is added by weight,
55 DEG C digest 10 hours, are centrifuged after then boiling inactivation, collect filtrate；
3) by filtrate first through 0.22 μm of filter membrane micro-filtration, ultrafiltration then is carried out with the ultrafiltration membrane of 3500 dalton, is collected super
Filtrate；
4) ultrafiltrate is further isolated and purified through preparation scale high performance liquid chromatograph, using PRC-ODS column, 0.5% 3
The mixed solution of fluoroacetic acid and acetonitrile is mobile phase, carries out gradient elution (elution process is same as Example 1), flow velocity 1mL/
It is detected under min, 215nm, the second component is collected according to peak position out, is concentrated, it is dry, obtain Agroeybe cylindracea mycelia anti-oxidation peptide
15.2g, purity 91.1%.
Test 1: antioxidant activity detection
Using total antioxidant capacity detection kit, using the free radical scavenging ability of ABTS method measurement anti-oxidation peptide.It surveys
Anti-oxidation peptide obtained by embodiment 1-3 antioxidant activity be respectively 1.62umol TE/mg, 1.55umol TE/mg,
1.58umol TE/mg.From the above results, Agroeybe cylindracea mycelia antioxidation polypeptide prepared by the present invention has very high antioxygen
Change activity.
Test 2: amino acid sequence analysis
The above-mentioned component isolated and purified is anti-oxidant by Liquid Chromatography-Mass Spectrometry (HPLC-MS/MS) identification
The amino acid sequence of peptide, and by identifying the sequence of peptide fragment to Agroeybe cylindracea mycelia protein data library searching.The Agroeybe cylindracea mycelia
Anti-oxidation peptide contains the feature peptide fragment that amino acid sequence is PAQPPPPPNR, and molecular weight is that (mass spectral results are shown in figure to 1069.5g/mol
2)。
4 vegetable protein beverage of embodiment
By following weight proportion at being grouped as:
Walnut kernel, 6%, major ingredient；
Pea protein, 2%, increase protein content, increases stability；
Agroeybe cylindracea mycelia anti-oxidation peptide, 0.5%, antioxidant obtained in embodiment 1, slow down unsaturated fat in walnut
The oxidation of acid；
Sucrose, 6%, sweetening material；
Glycerin monostearate, 0.16%, emulsifier；
Sucrose fatty ester, 0.012%, emulsifier；
Soybean lecithin, 4%, emulsifier；
Sodium carboxymethylcellulose, 0.04%, stabilizer；
Sodium tripolyphosphate, 0.06%, stabilizer；
Agar, 0.15%, thickener；
Sodium bicarbonate, 0.03%, adjust pH；
Pure water, surplus.
Preparation method: being made lotion according to a conventional method, sterilizing, it is filling to obtain the final product.
Agroeybe cylindracea mycelia proteolysis peptide, can increase the anti-oxidation protection to unsaturated fatty acid in vegetable protein beverage
Function, and the protein content of product is improved, increase emulsibility.It can be used as green, safe and healthy multifunctional food is matched
Material.
5 toner of embodiment
By following weight proportion at being grouped as:
Preparation method: above each ingredient is mixed, sterilizing to get.
Anti-oxidation peptide can be absorbed rapidly by skin, mainly play effect of scavenging radical.
6 pet food of embodiment
By following weight proportion at being grouped as:
Preparation method: above each ingredient is mixed, sterilizing, moulding to obtain the final product.
Agroeybe cylindracea mycelia proteolysis peptide can play removing as natural as feed nutritional supplement
Interior free yl effect, active peptide itself can also supplement rapidly animal protein nutrition, promote growth, enhance animal immunizing power.
7 shower cream of embodiment
By following weight proportion at being grouped as:
Preparation method: it adds, stirs evenly according to the above ratio to obtain the final product.
Anti-oxidation peptide can be absorbed rapidly by skin, play effect of scavenging radical.
Claims (7)
1. a kind of Agroeybe cylindracea mycelia anti-oxidation peptide, it is characterised in that: it is PAQPPPPPNR that the anti-oxidation peptide, which contains amino acid sequence,
Peptide fragment.
2. Agroeybe cylindracea mycelia anti-oxidation peptide as described in claim 1, it is characterised in that be to be prepared as follows:
1. weighing Agroeybe cylindracea mycelia fructification powder, water temperature extraction is added to take, saturated ammonium sulfate is added after centrifugation into leaching liquor and carries out
It saltouts, the precipitating after saltouing is dissolved with water, the bag filter that molecular cut off is 3000-4000Da is then crossed, after dialysis
Precipitating drying, obtains Agroeybe cylindracea mycelia crude protein；
2. the water of 20-50 times of weight is added into crude protein, pH to 7.5-8.5 is adjusted, is added according still further to the 1-5% of crude protein weight
Enter neutral proteinase, 50-60 DEG C enzymatic hydrolysis 5-10 hours, be centrifuged after then boiling inactivation, collect filtrate；
3. then carrying out ultrafiltration with the ultrafiltration membrane that molecular cut off is 3000-4000Da by filtrate first through micro-filtrate membrane filtration, collect
Ultrafiltrate；
4. by ultrafiltrate through the further separation component of preparation scale high performance liquid chromatograph, using PRC-ODS chromatographic column, with 0.05-
The mixed solution of 0.5% trifluoroacetic acid and acetonitrile is mobile phase, carries out gradient elution, detects under 215nm, is received according to peak position out
Collect the second component, be concentrated, after drying to obtain the final product.
3. Agroeybe cylindracea mycelia anti-oxidation peptide as claimed in claim 2, it is characterised in that: the temperature that the temperature extraction takes is 50-
80 DEG C, the time is 2-8 hours.
4. Agroeybe cylindracea mycelia anti-oxidation peptide as claimed in claim 2, it is characterised in that: the saturation degree of the ammonium sulfate is
80%.
5. Agroeybe cylindracea mycelia anti-oxidation peptide as claimed in claim 2, it is characterised in that: the aperture of the microfiltration membranes is 0.22 μ
m。
6. Agroeybe cylindracea mycelia anti-oxidation peptide as claimed in claim 2, it is characterised in that: the molecular cut off of the ultrafiltration membrane is
3500Da。
7. Agroeybe cylindracea mycelia anti-oxidation peptide described in claim 1-6 any one is as antioxidant, for health care product, food
The purposes of product, cosmetics or daily chemical product.
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