CN110343145B - Polypeptide derived from grub enzymolysis three-stage isolate and preparation method thereof - Google Patents
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Abstract
The invention belongs to the technical field of agricultural biology, and particularly relates to a preparation method of polypeptide derived from grub enzymolysis tertiary isolate. According to the method, the gel chromatography and the reversed-phase high-performance liquid chromatography are combined to separate the grub enzymatic hydrolysate to obtain a third-level isolate, and the fraction obtained by the method has high oxidation resistance and high specific activity.
Description
Technical Field
The invention belongs to the technical field of agricultural biology, and particularly relates to a preparation method of polypeptide derived from grub enzymolysis tertiary isolate.
Background
The grub is a general name of larva of the Tortoise plastron general (Scarabaeoedea), belongs to the order Coleoptera, and is also called walnut worm, cutworm and tussah silkworm. The grub contains various amino acids and inorganic elements required by human beings, and has been clinically used for promoting blood circulation for hundreds of years, and modern pharmacological studies show that the grub has various medicinal activities of resisting tumors, protecting the liver, treating aphtha, infantile asthma, stroke, schistosomiasis liver cirrhosis and the like, and is a medicinal material with great research and development values. Researches find that the crude extract of the grubs has anticoagulant activity, and several fibrinolytic agents (proto) in the crude extract have antithrombotic effect; the ethanol and petroleum ether extracts of the grubs have excellent antifungal activity.
More and more protein hydrolysates are found to have antioxidant capacity and new antioxidant peptides are continuously being developed. The antioxidant peptide has the capacity of resisting lipid peroxidation and chelating metal ions, and can also eliminate excessive free radicals in organisms. The rise of the resource insect breeding leads the research of insect antioxidant peptide to increase. In the prior art, different types of antioxidant peptides are released according to hydrolysates of animal and vegetable proteins from different sources, and the antioxidant peptides are generally prepared by an extraction method, a chemical hydrolysis method, an enzymatic hydrolysis method, a fermentation method, a chemical synthesis method, a gene recombination method and the like. The current enzymolysis method is the most main production method, the hydrolysis is easy to control, the specific peptide can be produced in a positioning way, the product safety is extremely high, the cost is low, and the method becomes the main mode for preparing the food-borne antioxidant peptide. Although the components with relatively concentrated molecular mass can be obtained by optimizing the enzymolysis process, the great variety of peptides in the enzymolysis product, the molecular weight of the peptides being relatively close to each other, and the sensitivity of the active components, which causes the difficulty of peptide separation and purification to a great extent, have become the main factors that restrict further research on the structure and function of the active peptides.
Disclosure of Invention
The invention aims to provide a polypeptide derived from a grub enzymolysis tertiary isolate.
It is still another object of the present invention to provide a method for producing the above polypeptide.
According to the specific embodiment of the invention, the polypeptide derived from the grub enzymolysis tertiary isolate has an amino acid sequence shown in SEQ ID No. 1:
EHTLLLVQPTK
according to a specific embodiment of the present invention, the polypeptide derived from a grub enzymatic hydrolysis three-stage isolate is prepared by a method comprising the steps of:
(1) separating the grub enzymolysis liquid by using sephadex column and ultrapure water as mobile phase under the conditions of flow rate of 14-16mL/min, column temperature of 27-33 ℃ and wavelength of 215-225nm,
collecting the fraction for 2-4 min to obtain primary isolate;
(2) octadecylsilane chemically bonded silica is used as a stationary phase, and the volume ratio of the octadecylsilane chemically bonded silica to the stationary phase is 95:5 0.1% trifluoroacetic acid: water is used as a mobile phase, the primary separated substance is separated under the conditions of the flow rate of 1mL/min, the column temperature of 27-33 ℃ and the wavelength of 215-225nm,
collecting 15-16 min fraction to obtain secondary isolate;
(3) octadecylsilane chemically bonded silica is used as a stationary phase, and the volume ratio is 95:5 0.1% trifluoroacetic acid: water is used as a mobile phase, the secondary separated substance is separated under the conditions of the flow rate of 1mL/min, the column temperature of 27-33 ℃ and the wavelength of 215-225nm,
collecting the fraction for 2-3 min.
According to the polypeptide derived from the grub enzymolysis tertiary isolate in the embodiment of the invention, the sephadex column selected in the step (1) is sephadex column G-15.
According to the polypeptide derived from the grub enzymolysis tertiary isolate, the grub enzymolysis liquid is prepared by the following steps:
(a) adding the grubs into a buffer system with the pH value of 6.8-7.2, and homogenizing at a high speed to obtain worm slurry;
(b) adding lipase into the insect slurry, and carrying out enzymolysis at 38-42 ℃;
(c) inactivating the liquid obtained in the step (b), standing for layering, centrifuging, and taking supernatant to obtain grub enzymatic hydrolysate.
According to the polypeptide derived from the grub enzymatic hydrolysis three-stage isolate according to the embodiment of the present invention, in the step (a), the grub is added into the buffer system so that the concentration of the grub is 5%.
According to the polypeptide derived from the grub enzymatic hydrolysis three-stage isolate according to the embodiment of the present invention, in the step (b), after adding the lipase, the ratio of the lipase to the grub is 0.6%.
According to the polypeptide derived from the grub enzymatic hydrolysis of the tertiary isolate according to the embodiment of the present invention, in the step (b), the enzymatic hydrolysis time is 3 hours.
According to the polypeptide derived from the grub enzymolysis tertiary isolate in the embodiment of the invention, in the step (c), the liquid is inactivated at 70 ℃ for 30min, kept stand at 4 ℃ overnight, centrifuged at 4000r/min at 4 ℃ for 15min, and the supernatant is taken.
A method for preparing a polypeptide derived from a grub enzymatic hydrolysis tertiary isolate according to a specific embodiment of the present invention, the method comprising the steps of:
(1) separating the grub enzymolysis liquid by using sephadex column and ultrapure water as mobile phase under the conditions of flow rate of 14-16mL/min, column temperature of 27-33 ℃ and wavelength of 215-225nm,
collecting the fraction for 2-4 min to obtain primary isolate;
(2) octadecylsilane chemically bonded silica is used as a stationary phase, and the volume ratio of the octadecylsilane chemically bonded silica to the stationary phase is 95:5 0.1% trifluoroacetic acid: water is used as a mobile phase, the primary separated substance is separated under the conditions of the flow rate of 1mL/min, the column temperature of 27-33 ℃ and the wavelength of 215-225nm,
collecting 15-16 min fraction to obtain secondary isolate;
(3) octadecylsilane chemically bonded silica is used as a stationary phase, and the volume ratio is 95:5 0.1% trifluoroacetic acid: water is used as a mobile phase, the secondary separated substance is separated under the conditions of the flow rate of 1mL/min, the column temperature of 27-33 ℃ and the wavelength of 215-225nm,
collecting the fraction for 2-3 min.
According to the preparation method of the polypeptide derived from the grub enzymolysis tertiary isolate, which is a specific embodiment of the invention, the sephadex column selected in the step (1) is sephadex column G-15. .
The invention has the beneficial effects that:
the sequence of the grub enzymolysis tertiary isolate is identified by adopting an LC-MS/MS method, the sequence is determined to be EHTLLLVQPTK according to mass spectrum information, the relative molecular weight is 1278.49Da, and the anti-oxidation activity is stronger.
Drawings
Fig. 1 shows HPLC separation of grub extracts;
figure 2 shows the polypeptide content of each fraction of the grub extract,
FIG. 3 shows the antioxidant capacity profile of the primary fraction;
FIG. 4 shows the specific activity profile of the primary fraction;
FIG. 5 shows the antioxidant peptide content profile of the secondary fraction;
FIG. 6 shows the total antioxidant capacity profile of the secondary fraction;
FIG. 7 shows the specific activity profile of the secondary fraction;
FIG. 8 shows HPLC fractionation of the secondary fraction PB 2-16;
FIG. 9 shows the secondary mass spectrum of PB 2-16-1.
Detailed Description
Materials and instruments:
grub: larvae of Protaetia brevitarsis of the family Scarabaeidae;
lipase (more than or equal to 3000.0U/g), trypsin (more than or equal to 50000U/g), acid protease (more than or equal to 50000U/g) and neutral protease (more than or equal to 60000U/g).
Example 1 preparation of grub enzymatic hydrolysate
Taking several cleaned grubs, putting into 100 ℃ boiled water for about 40s, immediately taking out, wiping the water, putting into a refrigerator at-20 ℃ and storing for later use.
Accurately weighing 10g of grub, adding a buffer system with pH of 7.0 (or 6.8, 6.9, 7.1 and 7.2) according to 5% substrate concentration (g/mL), and homogenizing by a high-speed dispersion machine at 8000r/min to obtain worm slurry. Adding lipase according to the ratio of enzyme to insect of 0.6%, placing in a water bath constant temperature oscillator, and performing enzymolysis for 3h at 150r/min (or 100r/min, 200r/min) and 40 deg.C (or 38 deg.C, 39 deg.C, 41 deg.C, 42 deg.C). Inactivating in a water bath constant temperature oscillator at 70 deg.C for 30min, and standing at 4 deg.C overnight. Centrifuging at 4000r/min at 4 ℃ for 15min, taking supernatant, and freeze-drying to obtain grub enzymatic hydrolysate.
The measurement of the total protein content of the grub is detected according to a Kjeldahl method GBl2309-90, and the measurement of the polypeptide content in the grub hydrolysate is detected according to a Folin-phenol method. The calculation formula of the polypeptide extraction rate is as follows:
the extraction rate (%). is grub polypeptide content (g)/grub total protein mass (g) × 100;
the total antioxidant capacity was measured according to the method of the total antioxidant capacity kit (FRAP method). Diluting the enzymolysis solution by a certain time, centrifuging for 15min at 4000r/min, sequentially adding 5 mu L of sample to be detected, 150 mu L of TPTZ diluent, 15 mu L of TPTZ solution and 15 mu L of detection buffer solution into a 96-well plate, and lightly mixing uniformly. After incubation at 37 ℃ for 3-5 min, the OD value of each well is read by an enzyme-linked immunosorbent assay at 593 nm.
By calculation, the grub polypeptide extraction rate is 14.21%, and the total antioxidant capacity is 0.752 mmol/L.
Example 2 isolation and purification of Tabanus antioxidant peptides
2.1 separation of the first fraction
Fractionating the grub hydrolysate by SephadexG-15(5 μm,300 × 21.2mm) by HPLC preparative chromatography, and preparing chromatographic conditions for the grub antioxidant peptide extract: the mobile phase ultrapure water has a flow rate of 15mL/min (or 14mL/min, 16mL/min), a column temperature of 30 deg.C (or 27 deg.C, 28 deg.C, 29 deg.C, 31 deg.C, 32 deg.C, 33 deg.C), and a wavelength of 220nm (or 215nm, 225 nm). Gradient elution procedure was 16 min. A grub enzymatic hydrolysate solution with a concentration of 10mg/mL was prepared with ultrapure water, and the sample loading was 2 mL. One tube was collected every 2min according to the separation time of the grub extract in the preparative chromatography.
As shown in fig. 1, 8 fractions PB1(Protaetia brevitarsis larvae) were obtained from the grub extract, PB2, PB3, PB4, PB5, PB6, PB7 and PB8, the collected samples were subjected to rotary evaporation at 45 ℃ and 85r/min, and the antioxidant peptide content, total antioxidant capacity and specific activity of the first fraction were measured, respectively, and the results are shown in table 1 and fig. 2 to 4:
TABLE 1 antioxidant peptide content, Total antioxidant Capacity and specific Activity data of the first fraction
The results show that the specific activities of PB1, PB2, PB3, PB4, PB5, PB6, PB7 and PB8 are 0.0000, 0.0178, 0.0055, 0.0026, 0.0033, 0.0042, 0.0039 and 0.0033mmoL/mg respectively, wherein the specific activity of PB2 is 3.42 times that of PB crude extract (0.0052 mmoL/mg).
2.2 separation of the second fraction
The fraction PB2 with the highest specific activity was further fractionated by C18(5 μm, 4.6X 150mm) in an analytical chromatograph under the following conditions: the mobile phase was a 0.1% trifluoroacetic acid-water (a) -acetonitrile (B) system (VA: VB 95:5), flow rate 1mL/min, column temperature 30 ℃ (alternatively 27 ℃, 28 ℃, 29 ℃, 31 ℃, 32 ℃, 33 ℃), wavelength 220nm (alternatively 215nm, 225nm), collection time 20 min.
The grub extract was fractionated according to the polarity on the C18 column, and PB2 was divided into 20 fractions of different polarity in the order of PB2 flowing out of the C18 column. Collecting one tube every 1min, collecting 20 tubes, and fractionating the components into 20 segments, namely PB2-1, PB2-2, PB2-3, PB2-4, PB2-5, PB2-6, PB2-7, PB2-8, PB2-9, PB2-10, PB2-11, PB2-12, PB2-13, PB2-14, PB2-15, PB2-16, PB2-17, PB2-18, PB2-19 and PB 2-20.
And (3) carrying out rotary evaporation on the fractions at 45 ℃ and 85r/min, and detecting the antioxidant peptide content and the total antioxidant capacity of the fractions, wherein the specific results are shown in table 2 and fig. 5-7.
TABLE 2 antioxidant peptide content, Total antioxidant capacity and specific Activity data of the second fraction
The results show that the specific activities of PB2-1, PB2-2, PB2-3, PB2-2-4, PB2-5, PB2-6, PB2-7, PB2-8, PB2-9, PB2-10, PB2-11, PB2-12, PB2-13, PB2-2-14, PB2-15, PB2-16, PB2-17, PB2-18, PB2-19 and PB2-20 are 7.63, 55.62, 2.04, 7.68, 14.67, 16.30, 10.32, 31.80, 45.30, 8.27, 5.40, 1.90, 8.74, 11.61, 19.92, 84.85, 13.60, 11.76, 28.26 and 9.65/g respectively, wherein the maximum specific activity of the fraction is 8653.53 g/8653 g/g.
2.3 separation of the third fraction
The secondary fraction PB2-16 obtained was subjected to the next fractionation in analytical chromatography using C18(3.5 μm, 4.6X 150mm), analytical chromatographic conditions: the mobile phase was a 0.1% trifluoroacetic acid-water (a) -acetonitrile (B) system (VA: VB 95:5), flow rate 1mL/min, column temperature 30 ℃ (alternatively 27 ℃, 28 ℃, 29 ℃, 31 ℃, 32 ℃, 33 ℃) and wavelength 220nm (alternatively 215nm, 225 nm).
As shown in FIG. 8, the sample, PB2-16-1, was collected according to the highest chromatographic peak of the active ingredient in the analytical chromatogram. The specific activity of fraction PB2-16-1 was 65.10 mmol/g.
PB2-16-1 is obtained by distillation concentration and freeze drying, and a PB2-16-1 sequence is identified by an LC-MS/MS method, as shown in figure 9, and is determined to be EHTLLLVQPTK according to mass spectrum information, and the relative molecular weight is 1278.49 Da.
Sequence listing
<110> institute of feed of Chinese academy of agricultural sciences
<120> polypeptide derived from grub enzymolysis tertiary isolate and preparation method thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 11
<212> PRT
<213> Tabanus (Holotrichia diomophila Bates)
<400> 1
Glu His Thr Leu Leu Leu Val Gln Pro Thr Lys
1 5 10
Claims (4)
1. The polypeptide derived from the grub enzymolysis tertiary isolate is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID No. 1.
2. The polypeptide derived from a grub enzymatic hydrolysis tertiary isolate according to claim 1, wherein the polypeptide is prepared by a process comprising the steps of:
(1) separating the grub enzymatic hydrolysate by using a sephadex column G-15 and ultrapure water as a mobile phase under the conditions that the flow rate is 14-16mL/min, the column temperature is 27-33 ℃, and the wavelength is 215-225nm, performing a gradient elution procedure for 16min, preparing a grub enzymatic hydrolysate solution with the concentration of 10mg/mL by using the ultrapure water, wherein the sample loading amount is 2mL, collecting one tube every 2min according to the separation time of the grub extract in the preparation chromatogram, collecting fractions for 2-4 min to obtain a primary isolate,
the grub enzymolysis solution is prepared by the following steps:
(a) adding the grub into a buffer system with pH of 6.8-7.2, and homogenizing at high speed to obtain a grub liquid, wherein the concentration of the grub is 5%;
(b) adding lipase into the worm pulp, wherein the ratio of the lipase to the grubs is 0.6%, and carrying out enzymolysis for 3 hours at 38-42 ℃;
(c) inactivating the liquid obtained in the step (b), standing for layering, centrifuging, and taking supernatant to obtain grub enzymatic hydrolysate;
(2) using octadecylsilane chemically bonded silica as a stationary phase, using a 0.1% trifluoroacetic acid-water-acetonitrile system as a mobile phase, wherein the volume ratio of trifluoroacetic acid-water to acetonitrile is 95:5, separating the primary separator under the conditions of the flow rate of 1mL/min, the column temperature of 27-33 ℃ and the wavelength of 215-225nm, and collecting 15-16 min fractions to obtain a secondary separator;
(3) and (2) taking octadecylsilane chemically bonded silica as a stationary phase, taking a mobile phase of a 0.1% trifluoroacetic acid-water-acetonitrile system, wherein the volume ratio of trifluoroacetic acid-water to acetonitrile is 95:5, separating the secondary separator under the conditions of the flow rate of 1mL/min, the column temperature of 27-33 ℃ and the wavelength of 215-225nm, and collecting the fraction for 2-3 min.
3. The polypeptide derived from a grub enzymatic hydrolysis three-stage isolate according to claim 2, wherein in step (c), the liquid is inactivated at 70 ℃ for 30min, left overnight at 4 ℃, centrifuged at 4000r/min at 4 ℃ for 15min, and the supernatant is taken.
4. A preparation method of polypeptide derived from a grub enzymolysis tertiary isolate is characterized in that the amino acid sequence of the polypeptide derived from the grub enzymolysis tertiary isolate is shown as SEQ ID No.1, and the method comprises the following steps:
(1) separating the grub enzymatic hydrolysate by using a sephadex column G-15 and ultrapure water as a mobile phase under the conditions that the flow rate is 14-16mL/min, the column temperature is 27-33 ℃, and the wavelength is 215-225nm, performing a gradient elution procedure for 16min, preparing a grub enzymatic hydrolysate solution with the concentration of 10mg/mL by using the ultrapure water, wherein the sample loading amount is 2mL, collecting one tube every 2min according to the separation time of the grub extract in the preparation chromatogram, collecting fractions for 2-4 min to obtain a primary isolate,
the grub enzymolysis solution is prepared by the following steps:
(a) adding the grub into a buffer system with pH of 6.8-7.2, and homogenizing at high speed to obtain a grub liquid, wherein the concentration of the grub is 5%;
(b) adding lipase into the worm pulp, wherein the ratio of the lipase to the grubs is 0.6%, and carrying out enzymolysis for 3 hours at 38-42 ℃;
(c) inactivating the liquid obtained in the step (b), standing for layering, centrifuging, and taking supernatant to obtain grub enzymatic hydrolysate;
(2) using octadecylsilane chemically bonded silica as a stationary phase, using a 0.1% trifluoroacetic acid-water-acetonitrile system as a mobile phase, wherein the volume ratio of trifluoroacetic acid-water to acetonitrile is 95:5, separating the primary separator under the conditions of the flow rate of 1mL/min, the column temperature of 27-33 ℃ and the wavelength of 215-225nm, and collecting 15-16 min fractions to obtain a secondary separator;
(3) and (2) taking octadecylsilane chemically bonded silica as a stationary phase, taking a mobile phase of a 0.1% trifluoroacetic acid-water-acetonitrile system, wherein the volume ratio of trifluoroacetic acid-water to acetonitrile is 95:5, separating the secondary separator under the conditions of the flow rate of 1mL/min, the column temperature of 27-33 ℃ and the wavelength of 215-225nm, and collecting the fraction for 2-3 min.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100050141A (en) * | 2008-11-05 | 2010-05-13 | 대한민국(농촌진흥청장) | An anti-fungal peptide gene isolated from beetle, protaetia brevitarsis, larvae ,said anti-fungal peptide and its analogue |
| CN106084007A (en) * | 2016-07-26 | 2016-11-09 | 青岛农业大学 | The isolation identification of a kind of tenebrio molitor natural anti-oxidation octapeptide and functional analysis |
| CN108220378A (en) * | 2018-03-08 | 2018-06-29 | 佛山汇沐化学科技有限公司 | A kind of extracting method of grub albumen and its application |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100050141A (en) * | 2008-11-05 | 2010-05-13 | 대한민국(농촌진흥청장) | An anti-fungal peptide gene isolated from beetle, protaetia brevitarsis, larvae ,said anti-fungal peptide and its analogue |
| CN106084007A (en) * | 2016-07-26 | 2016-11-09 | 青岛农业大学 | The isolation identification of a kind of tenebrio molitor natural anti-oxidation octapeptide and functional analysis |
| CN108220378A (en) * | 2018-03-08 | 2018-06-29 | 佛山汇沐化学科技有限公司 | A kind of extracting method of grub albumen and its application |
Non-Patent Citations (3)
| Title |
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| Enzymatic Preparation and Antioxidant Activities of Protein;Hyo-Seon Lee,et al.;《J Korean Soc Food Sci Nutr》;20071001;第46卷(第10期);第1164-1170页 * |
| 白星花金龟( Protaetia brevitarsis) 幼虫抗菌物质的分离纯化;徐明旭等;《生命科学研究》;20080331;第12卷(第1期);第53-56页 * |
| 蛴螬酶法制备抗氧化活性肽;张海英等;《食品研究与开发》;20190430;第40卷(第8期);摘要，第153页左栏第2段，第153页1.2-第158页结论 * |
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| CN110343145A (en) | 2019-10-18 |
| CN110372771B (en) | 2021-03-26 |
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