Water lily polypeptide as well as extraction method and application thereof

Abstract

The invention belongs to the technical field of biological peptide extraction, and particularly relates to a water lily polypeptide as well as an extraction method and application thereof. According to the method, the water lily sample is disinfected, cleaned, aired and crushed in sequence, so that impurities on the surface of the water lily sample can be removed; a crushed sample is mixed with water and then is heated, so that the primary separation of effective components in the water lily is facilitated; the papain is added for enzymolysis, and the ethanol solution is used for analysis, so that water lily polypeptide in a water lily sample can be fully released by penetrating the water lily sample, and relatively high activity is maintained. Therefore, the water lily polypeptide provided by the invention is high in activity and can be used in anti-wrinkle and whitening products.

Description

Technical Field

[0001] The invention belongs to the technical field of biological peptide extraction, and particularly relates to a water lily polypeptide and an extraction method and application thereof. Background Art

[0002] Water lily (Nymphaea L.), a perennial aquatic herb; rhizome is thick. Petiole is cylindrical and slender. Leaves are elliptical, floating on the water surface, with entire margins, heart-shaped leaf base, dark green on the surface, dark purple on the back; seeds are hard, wrapped in gelatinous material, with fleshy cup-shaped aril, small embryo, small endosperm and abundant exosperm. It is often planted as an ornamental plant, rhizome is edible or used for winemaking, and is also used as medicine to treat chronic convulsions in children.

[0003] There are reports on water lily extracts in the prior art, for example, Chinese patent CN 109966347 A extracts flavonoids from water lily petals; another example is Chinese patent CN 109879927 A extracts betulinic acid from water lily rhizomes. It can be seen that although there are reports on water lily extracts in the prior art, there are no reports on how to extract water lily polypeptides. Summary of the invention

[0004] The purpose of the present invention is to provide a water lily polypeptide and an extraction method and application thereof. The method is not only simple and quick, but also the water lily polypeptide extracted thereby has high activity and can be used in anti-wrinkle and whitening products.

[0005] The present invention provides a method for extracting water lily polypeptide, comprising the following steps:

[0006] The water lily samples are sterilized, cleaned, dried and crushed in sequence to obtain crushed samples;

[0007] mixing the crushed sample with water and heating the mixture to obtain a mixture;

[0008] enzymolyze the mixture with papain to obtain an enzymolyzate;

[0009] The enzymatic hydrolysate is mixed with the ethanol solution and then centrifuged to obtain a supernatant;

[0010] The ethanol solvent in the supernatant is removed to obtain the water lily polypeptide.

[0011] Preferably, the water lily sample includes one or more of petals, rhizomes and seeds of the water lily;

[0012] The types of the water lily include yellow water lily, red water lily, blue water lily or white water lily.

[0013] Preferably, the mass ratio of the crushed sample to water is 1:8-10;

[0014] The heating temperature is 38-42° C.; the heating time is 40-60 min.

[0015] Preferably, the amount of papain added is based on the mass of the crushed sample, and the mass ratio of the crushed sample to papain is 1:0.03-0.05;

[0016] The enzymatic hydrolysis temperature is 38-42°C; the enzymatic hydrolysis time is 3-4h;

[0017] The enzymatic activity of the papain is ≧2000 U / g.

[0018] Preferably, the disinfection method comprises: mixing and soaking the water lily sample with a sodium hypochlorite solution to obtain a disinfected water lily sample;

[0019] The soaking time is 1-2 minutes; the mass concentration of the sodium hypochlorite solution is 0.1%.

[0020] Preferably, during the centrifugation, the volume ratio of the enzymatic hydrolysate to the ethanol solution is 1:0.65-0.7;

[0021] The volume concentration of the ethanol solution is 75%-80%;

[0022] The centrifugal speed is 2500-3000 r / min, and the centrifugal time is 10-15 min.

[0023] The present invention provides a water lily polypeptide extracted by the extraction method described in the above technical scheme.

[0024] The present invention provides the use of water lily polypeptides extracted by the extraction method described in the above technical solution in anti-wrinkle and / or whitening products.

[0025] The present invention provides cosmetics, comprising water lily polypeptide extracted by the extraction method described in the above technical scheme, and cosmetically acceptable auxiliary materials.

[0026] Preferably, the auxiliary materials include one or more of a solubilizing agent, a penetration enhancer, a chelating agent and a preservative.

[0027] Beneficial effects:

[0028] The invention provides a method for extracting water lily polypeptides. The method comprises the steps of sequentially disinfecting, cleaning, air-drying and crushing the water lily samples, thereby facilitating the removal of impurities on the surface of the water lily samples; mixing the crushed samples with water and then heating them, thereby facilitating the preliminary precipitation of effective components in the water lily; and adding papain for enzymolysis and analyzing them with an ethanol solution, thereby facilitating the penetration of the water lily samples so that the water lily polypeptides inside the water lily are fully released and maintain a high activity.

[0029] Based on the above technical advantages, the present invention also provides water lily polypeptides and their use in anti-wrinkle and / or whitening products. By mixing water lily polypeptides with other acceptable excipients, the whitening and anti-wrinkle effects of the products are improved. Therefore, the technical solution provided by the present invention not only broadens the application scope of water lily, but also realizes the multi-faceted resource transformation of water lily and products. DETAILED DESCRIPTION

[0030] In the present invention, unless otherwise specified, the reagents and methods used are conventional reagents and methods.

[0031] In the present invention, the types of water lilies preferably include yellow water lilies, red water lilies, blue water lilies or white water lilies, and more preferably include yellow water lilies. The water lily samples of the present invention preferably include one or more of petals, rhizomes and seeds of water lilies, and more preferably include petals, rhizomes or seeds of water lilies; when the water lily samples are derived from multiple types of petals, rhizomes or seeds of water lilies, there is no special requirement for the amount of each part added, and the total amount can meet the corresponding sample requirements.

[0032] Example 1

[0033] A method for extracting water lily polypeptide, comprising the following steps:

[0034] (1) Soaking the seeds of yellow water lily in a sodium chlorate solution with a mass concentration of 0.1% for 1 min, then rinsing them with clean water twice, drying the rinsed seeds, crushing them, and then passing them through a 100-mesh sieve to obtain a crushed water lily sample;

[0035] (2) mixing the crushed water lily sample in step (1) with water in a mass ratio of 1:10, and heating at 38° C. for 50 min to obtain a mixture;

[0036] (3) adding papain to the mixture of step (2), wherein the amount of papain added is based on the mass of the crushed water lily sample, the mass ratio of the crushed water lily sample to papain is 1:0.03, and the enzymatic activity of papain is 2000 U / g. After adding papain, the pH value of the mixture is adjusted to 6.9-7.1, and then enzymolysis is performed at 38° C. for 4 hours to obtain an enzymolysis solution;

[0037] (4) mixing the enzymatic hydrolysate in step (3) with an ethanol solution having a volume concentration of 78% at a mass ratio of 1:0.7, centrifuging at a speed of 3000 r / min for 15 min to obtain a supernatant;

[0038] (5) The supernatant in step (4) is used to remove the ethanol solvent using a rotary evaporator to obtain a solid substance which is a water lily polypeptide.

[0039] Example 2

[0040] A method for extracting water lily polypeptide, comprising the following steps:

[0041] (1) Soak the petals of yellow water lily in a 0.1% sodium chlorate solution for 1 min, then rinse with clean water twice, dry the rinsed seeds, crush them, and then pass them through a 100-mesh sieve to obtain a crushed water lily sample;

[0042] (2) mixing the crushed water lily sample in step (1) with water in a mass ratio of 1:10, and heating at 38° C. for 50 min to obtain a mixture;

[0043] (3) adding papain to the mixture of step (2), wherein the amount of papain added is based on the mass of the crushed water lily sample, the mass ratio of the crushed water lily sample to papain is 1:0.03, and the enzymatic activity of papain is 2000 U / g. After adding papain, the pH value of the mixture is adjusted to 6.9-7.1, and then enzymolysis is performed at 38° C. for 4 hours to obtain an enzymolysis solution;

[0044] (4) mixing the enzymatic hydrolysate in step (3) with an ethanol solution having a volume concentration of 78% at a mass ratio of 1:0.7, centrifuging at a speed of 3000 r / min for 15 min to obtain a supernatant;

[0045] (5) The supernatant in step (4) is used to remove the ethanol solvent using a rotary evaporator to obtain a solid substance which is a water lily polypeptide.

[0046] Example 3

[0047] A method for extracting water lily polypeptide, comprising the following steps:

[0048] (1) Soak the rhizomes of yellow water lilies in a 0.1% sodium chlorate solution for 1 min, then rinse them twice with clean water, dry the rinsed seeds, crush them, and then pass them through a 100-mesh sieve to obtain a crushed water lily sample;

[0049] (2) mixing the crushed water lily sample in step (1) with water in a mass ratio of 1:10, and heating at 38° C. for 50 min to obtain a mixture;

[0050] (3) adding papain to the mixture of step (2), wherein the amount of papain added is based on the mass of the crushed water lily sample, the mass ratio of the crushed water lily sample to papain is 1:0.03, and the enzymatic activity of papain is 2000 U / g. After adding papain, the pH value of the mixture is adjusted to 6.9-7.1, and then enzymolysis is performed at 38° C. for 4 hours to obtain an enzymolysis solution;

[0051] (4) mixing the enzymatic hydrolysate in step (3) with an ethanol solution having a volume concentration of 78% at a mass ratio of 1:0.7, centrifuging at a speed of 3000 r / min for 15 min to obtain a supernatant;

[0052] (5) The supernatant in step (4) is used to remove the ethanol solvent using a rotary evaporator to obtain a solid substance which is a water lily polypeptide.

[0053] Example 4

[0054] A method for extracting water lily polypeptide, comprising the following steps:

[0055] (1) Soaking the seeds of yellow water lily in a sodium chlorate solution with a mass concentration of 0.1% for 1 min, then rinsing them with clean water twice, drying the rinsed seeds, crushing them, and then passing them through a 100-mesh sieve to obtain a crushed water lily sample;

[0056] (2) mixing the crushed water lily sample in step (1) with water in a mass ratio of 1:10, and heating at 42° C. for 50 min to obtain a mixture;

[0057] (3) adding papain to the mixture of step (2), wherein the amount of papain added is based on the mass of the crushed water lily sample, the mass ratio of the crushed water lily sample to papain is 1:0.03, and the enzymatic activity of papain is 2000 U / g. After adding papain, the pH value of the mixture is adjusted to 6.9-7.1, and then enzymolysis is performed at 38° C. for 4 hours to obtain an enzymolysis solution;

[0058] (4) mixing the enzymatic hydrolysate in step (3) with an ethanol solution having a volume concentration of 78% at a mass ratio of 1:0.7, centrifuging at a speed of 3000 r / min for 15 min to obtain a supernatant;

[0059] (5) The supernatant in step (4) is used to remove the ethanol solvent using a rotary evaporator to obtain a solid substance which is a water lily polypeptide.

[0060] Comparative Example 1

[0061] A method for extracting water lily polypeptide, comprising the following steps:

[0062] (1) Soaking the seeds of yellow water lily in a sodium chlorate solution with a mass concentration of 0.1% for 1 min, then rinsing them with clean water twice, drying the rinsed seeds, crushing them, and then passing them through a 100-mesh sieve to obtain a crushed water lily sample;

[0063] (2) mixing the crushed water lily sample in step (1) with water in a mass ratio of 1:10, and heating at 38° C. for 50 min to obtain a mixture;

[0064] (3) adding trypsin to the mixture of step (2), wherein the amount of trypsin added is based on the mass of the crushed water lily sample, the mass ratio of the crushed water lily sample to trypsin is 1:0.03, and the enzyme activity of trypsin is 2000 U / g, and after adding trypsin, the pH value of the mixture is adjusted to 6.9-7.1, and then enzymolysis is performed at 38° C. for 4 hours to obtain an enzymolysis solution;

[0065] (4) mixing the enzymatic hydrolysate in step (3) with an ethanol solution having a volume concentration of 78% at a mass ratio of 1:0.7, centrifuging at a speed of 3000 r / min for 15 min to obtain a supernatant;

[0066] (5) The supernatant in step (4) is used to remove the ethanol solvent using a rotary evaporator to obtain a solid substance which is a water lily polypeptide.

[0067] Comparative Example 2

[0068] A method for extracting water lily polypeptide, comprising the following steps:

[0069] (1) Soaking the seeds of yellow water lily in a sodium chlorate solution with a mass concentration of 0.1% for 1 min, then rinsing them with clean water twice, drying the rinsed seeds, crushing them, and then passing them through a 100-mesh sieve to obtain a crushed water lily sample;

[0070] (2) mixing the crushed water lily sample in step (1) with water in a mass ratio of 1:10, and heating at 38° C. for 50 min to obtain a mixture;

[0071] (3) adding papain to the mixture of step (2), wherein the amount of papain added is based on the mass of the crushed water lily sample, the mass ratio of the crushed water lily sample to papain is 1:0.03, and the enzymatic activity of papain is 2000 U / g. After adding papain, the pH value of the mixture is adjusted to 6.9-7.1, and then enzymolysis is performed at 38° C. for 4 hours to obtain an enzymolysis solution;

[0072] (4) mixing the enzymatic hydrolysate in step (3) with an ethanol solution having a volume concentration of 85% at a mass ratio of 1:0.7, centrifuging at a speed of 3000 r / min for 15 min to obtain a supernatant;

[0073] (5) The supernatant in step (4) is used to remove the ethanol solvent using a rotary evaporator to obtain a solid substance which is a water lily polypeptide.

[0074] Comparative Example 3

[0075] A method for extracting water lily polypeptide, comprising the following steps:

[0076] (1) Soaking the seeds of yellow water lily in a sodium chlorate solution with a mass concentration of 0.1% for 1 min, then rinsing them with clean water twice, drying the rinsed seeds, crushing them, and then passing them through a 100-mesh sieve to obtain a crushed water lily sample;

[0077] (2) mixing the crushed water lily sample in step (1) with water in a mass ratio of 1:10, and heating at 38° C. for 50 min to obtain a mixture;

[0078] (3) adding papain to the mixture of step (2), wherein the amount of papain added is based on the mass of the crushed water lily sample, the mass ratio of the crushed water lily sample to papain is 1:0.03, and the enzymatic activity of papain is 2000 U / g. After adding papain, the pH value of the mixture is adjusted to 6.9-7.1, and then enzymolysis is performed at 38° C. for 4 hours to obtain an enzymolysis solution;

[0079] (4) mixing the enzymatic hydrolysate in step (3) with an ethanol solution having a volume concentration of 70% at a mass ratio of 1:0.7, and centrifuging at a speed of 3000 r / min for 15 min to obtain a supernatant;

[0080] (5) The supernatant in step (4) is used to remove the ethanol solvent using a rotary evaporator to obtain a solid substance which is a water lily polypeptide.

[0081] Comparative Example 4

[0082] A method for extracting water lily polypeptide, comprising the following steps:

[0083] (1) Soaking the seeds of yellow water lily in a sodium chlorate solution with a mass concentration of 0.1% for 1 min, then rinsing them with clean water twice, drying the rinsed seeds, crushing them, and then passing them through a 100-mesh sieve to obtain a crushed water lily sample;

[0084] (2) mixing the crushed water lily sample in step (1) with water in a mass ratio of 1:10, and heating at 38° C. for 70 min to obtain a mixture;

[0085] (3) adding papain to the mixture of step (2), wherein the amount of papain added is based on the mass of the crushed water lily sample, the mass ratio of the crushed water lily sample to papain is 1:0.03, and the enzymatic activity of papain is 2000 U / g. After adding papain, the pH value of the mixture is adjusted to 6.9-7.1, and then enzymolysis is performed at 38° C. for 4 hours to obtain an enzymolysis solution;

[0086] (4) mixing the enzymatic hydrolysate in step (3) with an ethanol solution having a volume concentration of 78% at a mass ratio of 1:0.7, centrifuging at a speed of 3000 r / min for 15 min to obtain a supernatant;

[0087] (5) The supernatant in step (4) is used to remove the ethanol solvent using a rotary evaporator to obtain a solid substance which is a water lily polypeptide.

[0088] Comparative Example 5

[0089] A method for extracting water lily polypeptide, comprising the following steps:

[0090] (1) Soaking the seeds of yellow water lily in a sodium chlorate solution with a mass concentration of 0.1% for 1 min, then rinsing them with clean water twice, drying the rinsed seeds, crushing them, and then passing them through a 100-mesh sieve to obtain a crushed water lily sample;

[0091] (2) mixing the crushed water lily sample in step (1) with water in a mass ratio of 1:10, and heating at 45° C. for 50 min to obtain a mixture;

[0092] (3) adding papain to the mixture of step (2), wherein the amount of papain added is based on the mass of the crushed water lily sample, the mass ratio of the crushed water lily sample to papain is 1:0.03, and the enzymatic activity of papain is 2000 U / g. After adding papain, the pH value of the mixture is adjusted to 6.9-7.1, and then enzymolysis is performed at 38° C. for 4 hours to obtain an enzymolysis solution;

[0093] (4) mixing the enzymatic hydrolysate in step (3) with an ethanol solution having a volume concentration of 78% at a mass ratio of 1:0.7, centrifuging at a speed of 3000 r / min for 15 min to obtain a supernatant;

[0094] (5) The supernatant in step (4) is used to remove the ethanol solvent using a rotary evaporator to obtain a solid substance which is a water lily polypeptide.

[0095] Antioxidant performance test

[0096] Prepare 0.1 mM DPPH solution and store in dark place for later use;

[0097] Take the water lily polypeptide samples extracted in Examples 1-4 and Comparative Examples 1-5, respectively, and dilute them with water to obtain 0.1 mM samples to be tested;

[0098] 100 μL of different samples to be tested and 100 μL of DPPH alcohol solution in the examples and comparative examples were added to a 96-well plate for dark culture, and 100 μL of DPPH alcohol solution and 100 μL of water were used as blank controls; each example, comparative example or blank control was a treatment, and each treatment was repeated 3 times in parallel;

[0099] After the culture was completed, the absorbance at 517 nm was measured and the average value was taken. The results are shown in Table 1.

[0100] The formula used to calculate the DPPH clearance of samples at each concentration was as follows:

[0101] (1-(Asample-Ablank) / Acontrol)×100%;

[0102] Among them, Asample is the absorbance value of the experimental group, Ablank is the absorbance value of the blank group, and Acontrol is the absorbance value of the control group.

[0103] Table 1 DPPH removal rate in different treatments

[0104]

[0105]

[0106] It can be seen from Table 1 that the DPPH scavenging rate of the water lily polypeptide extracted by adopting the technical solution provided by the present invention can reach up to 86.5%.

[0107] In summary, the water lily polypeptide provided by the present invention has high activity and can be used in cosmetics to have anti-wrinkle, whitening and other effects.

[0108] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for extracting water lily polypeptide, characterized in that: The steps include: The water lily samples are sterilized, cleaned, dried and crushed in sequence to obtain crushed samples; mixing the crushed sample with water and heating the mixture to obtain a mixture; enzymolyze the mixture with papain to obtain an enzymolyzate; The enzymatic hydrolysate is mixed with the ethanol solution and then centrifuged to obtain a supernatant; The ethanol solvent in the supernatant is removed to obtain the water lily polypeptide.

2. The extraction method according to claim 1, characterized in that The water lily sample includes one or more of petals, rhizomes and seeds of the water lily; The types of the water lily include yellow water lily, red water lily, blue water lily or white water lily.

3. The extraction method according to claim 1 or 2, characterized in that The mass ratio of the crushed sample to water is 1:8-10; The heating temperature is 38-42° C.; the heating time is 40-60 min.

4. The extraction method according to claim 1, characterized in that The amount of papain added is based on the mass of the crushed sample, and the mass ratio of the crushed sample to papain is 1:0.03-0.05; The enzymatic hydrolysis temperature is 38-42°C; the enzymatic hydrolysis time is 3-4h; The enzymatic activity of the papain is ≧2000 U / g.

5. The extraction method according to claim 1, characterized in that The disinfection method comprises: mixing and soaking the water lily sample with a sodium hypochlorite solution to obtain a disinfected water lily sample; The soaking time is 1-2 minutes; the mass concentration of the sodium hypochlorite solution is 0.1%.

6. The extraction method according to claim 1, characterized in that During the centrifugation, the volume ratio of the enzymatic hydrolysate to the ethanol solution is 1:0.65-0.7; The volume concentration of the ethanol solution is 75%-80% The centrifugal speed is 2500-3000 r / min, and the centrifugal time is 10-15 min.

7. The water lily polypeptide obtained by the extraction method according to any one of claims 1 to 6.

8. Use of the water lily polypeptide extracted by the extraction method according to any one of claims 1 to 6 in anti-wrinkle and / or whitening products.

9. A cosmetic, characterized in that The invention comprises water lily polypeptide extracted by the extraction method according to any one of claims 1 to 6, and auxiliary materials acceptable to cosmetics.

10. The cosmetic according to claim 9, characterized in that: The auxiliary materials include one or more of a solubilizing agent, a permeation enhancer, a chelating agent and a preservative.

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