A water lily polypeptide, its extraction method and application

Abstract

This invention belongs to the field of biopeptide extraction technology, specifically relating to a water lily polypeptide, its extraction method, and its application. The invention involves sequentially disinfecting, washing, drying, and pulverizing the water lily sample to remove impurities from its surface. Heating the pulverized sample with water facilitates the initial precipitation of active ingredients from the water lily. Adding papain for enzymatic hydrolysis and then using an ethanol solution for analysis helps to penetrate the water lily sample, fully releasing the internal water lily polypeptides while maintaining high activity. Therefore, the water lily polypeptide provided by this invention has high activity and can be used in anti-wrinkle and whitening products.

Description

Technical Field

[0001] This invention belongs to the field of biopeptide extraction technology, specifically relating to a water lily polypeptide, its extraction method, and its application. Background Technology

[0002] Water lily (Nymphaea L.) is a perennial aquatic herb with a thick rhizome. The petioles are cylindrical and slender. The leaves are elliptical, floating on the water surface, with entire margins, a cordate base, a dark green upper surface, and a dark purple lower surface. The seeds are hard, encased in a gelatinous substance, with a fleshy cup-shaped aril; the embryo is small, with a small amount of endosperm and abundant exosperm. It is commonly cultivated as an ornamental plant; the rhizome is edible or used to make wine, and it is also used medicinally to treat infantile convulsions.

[0003] There are existing reports on water lily extracts, such as Chinese patent CN 109966347 A, which extracted flavonoids from water lily petals; and Chinese patent CN 109879927 A, which extracted betulinic acid from the rhizomes of water lilies. It is evident that while there are existing reports on water lily extracts, there are no reports on how to extract water lily peptides. Summary of the Invention

[0004] The purpose of this invention is to provide a water lily polypeptide, its extraction method, and its application. This method is not only simple and quick, but also yields water lily polypeptides with high activity, which can be used in anti-wrinkle and whitening products.

[0005] This invention provides a method for extracting peptides from water lilies, comprising the following steps:

[0006] The water lily samples were disinfected, washed, dried, and crushed in sequence to obtain the crushed samples.

[0007] The pulverized sample was mixed with water and then heated to obtain a mixture;

[0008] The mixture was enzymatically hydrolyzed using papain to obtain an enzymatic hydrolysate;

[0009] The enzymatic hydrolysate was mixed with an ethanol solution and then centrifuged to obtain the supernatant.

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

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

[0012] The types of water lilies mentioned include yellow water lilies, red water lilies, blue water lilies, or white water lilies.

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

[0014] The heating temperature is 38-42℃; the heating time is 40-60 minutes.

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

[0016] The enzymatic hydrolysis temperature is 38-42℃; the enzymatic hydrolysis time is 3-4 hours.

[0017] The papain activity is ≥2000 U / g.

[0018] Preferably, the disinfection method includes: soaking the water lily sample in a mixture of 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 centrifugation speed is 2500-3000 r / min, and the centrifugation time is 10-15 min.

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

[0024] This invention provides the application of water lily polypeptides extracted by the extraction method described above in anti-wrinkle and / or whitening products.

[0025] This invention provides a cosmetic, comprising water lily polypeptides extracted by the extraction method described above, and cosmetically acceptable excipients.

[0026] Preferably, the excipients include one or more of solvents, penetration enhancers, chelating agents, and preservatives.

[0027] Beneficial effects:

[0028] This invention provides a method for extracting water lily polypeptides. By sequentially disinfecting, washing, drying, and pulverizing the water lily sample, impurities on the surface of the sample are removed. Heating the pulverized sample with water facilitates the initial precipitation of the effective components within the water lily. Adding papain for enzymatic hydrolysis and using ethanol solution for analysis helps to penetrate the water lily sample, allowing the water lily polypeptides inside to be fully released and maintaining high activity.

[0029] Based on the aforementioned technical advantages, this invention also provides water lily peptides and their application in anti-wrinkle and / or whitening products. By blending water lily peptides with other acceptable excipients, the whitening and anti-wrinkle effects of the products are improved. Therefore, the technical solution provided by this invention not only broadens the application scope of water lilies but also realizes the multi-faceted resource transformation of water lilies and related products. Detailed Implementation

[0030] Unless otherwise specified, all reagents and methods used in this invention are conventional.

[0031] In this invention, the water lily species preferably include yellow water lilies, red water lilies, blue water lilies, or white water lilies, and more preferably yellow water lilies. The water lily sample of this invention preferably includes one or more of the water lily petals, rhizomes, and seeds, and more preferably includes water lily petals, rhizomes, or seeds; when the water lily sample comes from multiple parts of the water lily petals, rhizomes, or seeds, there are no special requirements for the amount of each part added, as long as the total amount meets the corresponding sample requirements.

[0032] Example 1

[0033] A method for extracting polypeptides from water lilies, comprising the following steps:

[0034] (1) Soak the seeds of yellow water lily in a sodium chlorate solution with a mass concentration of 0.1% for 1 minute, then rinse them twice with water. After rinsing, dry the seeds and crush them. Then pass them through a 100-mesh sieve to obtain crushed water lily samples.

[0035] (2) The crushed water lily sample from step (1) was mixed with water at a mass ratio of 1:10 and heated at 38°C for 50 minutes to obtain a mixture;

[0036] (3) Add papain to the mixture in step (2). 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 enzyme activity of papain is 2000 U / g. After adding papain, adjust the pH of the mixture to 6.9-7.1. Then, enzymatically hydrolyze the mixture at 38℃ for 4 hours to obtain the enzymatic hydrolysate.

[0037] (4) Mix the enzymatic hydrolysate from step (3) with an ethanol solution with a volume concentration of 78% at a mass ratio of 1:0.7 and centrifuge at a speed of 3000 r / min for 15 min to obtain the supernatant.

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

[0039] Example 2

[0040] A method for extracting polypeptides from water lilies, comprising the following steps:

[0041] (1) Take the petals of yellow water lily and soak them in a sodium chlorate solution with a mass concentration of 0.1% for 1 minute. Then rinse them twice with water. After rinsing, dry the seeds and crush them. Then pass them through a 100-mesh sieve to obtain the crushed water lily sample.

[0042] (2) The crushed water lily sample from step (1) was mixed with water at a mass ratio of 1:10 and heated at 38°C for 50 minutes to obtain a mixture;

[0043] (3) Add papain to the mixture in step (2). 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 enzyme activity of papain is 2000 U / g. After adding papain, adjust the pH of the mixture to 6.9-7.1. Then, enzymatically hydrolyze the mixture at 38℃ for 4 hours to obtain the enzymatic hydrolysate.

[0044] (4) Mix the enzymatic hydrolysate from step (3) with an ethanol solution with a volume concentration of 78% at a mass ratio of 1:0.7 and centrifuge at a speed of 3000 r / min for 15 min to obtain the supernatant.

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

[0046] Example 3

[0047] A method for extracting polypeptides from water lilies, comprising the following steps:

[0048] (1) Soak the rhizomes of yellow water lily in a 0.1% sodium chlorate solution for 1 minute, then rinse twice with water. After rinsing, dry the seeds and crush them. Then pass them through a 100-mesh sieve to obtain crushed water lily samples.

[0049] (2) The crushed water lily sample from step (1) was mixed with water at a mass ratio of 1:10 and heated at 38°C for 50 minutes to obtain a mixture;

[0050] (3) Add papain to the mixture in step (2). 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 enzyme activity of papain is 2000 U / g. After adding papain, adjust the pH of the mixture to 6.9-7.1. Then, enzymatically hydrolyze the mixture at 38℃ for 4 hours to obtain the enzymatic hydrolysate.

[0051] (4) Mix the enzymatic hydrolysate from step (3) with an ethanol solution with a volume concentration of 78% at a mass ratio of 1:0.7 and centrifuge at a speed of 3000 r / min for 15 min to obtain the supernatant.

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

[0053] Example 4

[0054] A method for extracting polypeptides from water lilies, comprising the following steps:

[0055] (1) Soak the seeds of yellow water lily in a sodium chlorate solution with a mass concentration of 0.1% for 1 minute, then rinse them twice with water. After rinsing, dry the seeds and crush them. Then pass them through a 100-mesh sieve to obtain crushed water lily samples.

[0056] (2) The crushed water lily sample from step (1) was mixed with water at a mass ratio of 1:10 and heated at 42°C for 50 minutes to obtain a mixture;

[0057] (3) Add papain to the mixture in step (2). 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 enzyme activity of papain is 2000 U / g. After adding papain, adjust the pH of the mixture to 6.9-7.1. Then, enzymatically hydrolyze the mixture at 38℃ for 4 hours to obtain the enzymatic hydrolysate.

[0058] (4) Mix the enzymatic hydrolysate from step (3) with an ethanol solution with a volume concentration of 78% at a mass ratio of 1:0.7 and centrifuge at a speed of 3000 r / min for 15 min to obtain the supernatant.

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

[0060] Comparative Example 1

[0061] A method for extracting polypeptides from water lilies, comprising the following steps:

[0062] (1) Soak the seeds of yellow water lily in a sodium chlorate solution with a mass concentration of 0.1% for 1 minute, then rinse them twice with water. After rinsing, dry the seeds and crush them. Then pass them through a 100-mesh sieve to obtain crushed water lily samples.

[0063] (2) The crushed water lily sample from step (1) was mixed with water at a mass ratio of 1:10 and heated at 38°C for 50 minutes to obtain a mixture;

[0064] (3) Add trypsin to the mixture in step (2). 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 2000U / g. After adding trypsin, adjust the pH of the mixture to 6.9-7.1. Then, enzymatically hydrolyze at 38℃ for 4 hours to obtain the enzymatic hydrolysate.

[0065] (4) Mix the enzymatic hydrolysate from step (3) with an ethanol solution with a volume concentration of 78% at a mass ratio of 1:0.7 and centrifuge at a speed of 3000 r / min for 15 min to obtain the supernatant.

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

[0067] Comparative Example 2

[0068] A method for extracting polypeptides from water lilies, comprising the following steps:

[0069] (1) Soak the seeds of yellow water lily in a sodium chlorate solution with a mass concentration of 0.1% for 1 minute, then rinse them twice with water. After rinsing, dry the seeds and crush them. Then pass them through a 100-mesh sieve to obtain crushed water lily samples.

[0070] (2) The crushed water lily sample from step (1) was mixed with water at a mass ratio of 1:10 and heated at 38°C for 50 minutes to obtain a mixture;

[0071] (3) Add papain to the mixture in step (2). 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 enzyme activity of papain is 2000 U / g. After adding papain, adjust the pH of the mixture to 6.9-7.1. Then, enzymatically hydrolyze the mixture at 38℃ for 4 hours to obtain the enzymatic hydrolysate.

[0072] (4) Mix the enzymatic hydrolysate from step (3) with an 85% ethanol solution at a mass ratio of 1:0.7 and centrifuge at 3000 r / min for 15 min to obtain the supernatant.

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

[0074] Comparative Example 3

[0075] A method for extracting polypeptides from water lilies, comprising the following steps:

[0076] (1) Soak the seeds of yellow water lily in a sodium chlorate solution with a mass concentration of 0.1% for 1 minute, then rinse them twice with water. After rinsing, dry the seeds and crush them. Then pass them through a 100-mesh sieve to obtain crushed water lily samples.

[0077] (2) The crushed water lily sample from step (1) was mixed with water at a mass ratio of 1:10 and heated at 38°C for 50 minutes to obtain a mixture;

[0078] (3) Add papain to the mixture in step (2). 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 enzyme activity of papain is 2000 U / g. After adding papain, adjust the pH of the mixture to 6.9-7.1. Then, enzymatically hydrolyze the mixture at 38℃ for 4 hours to obtain the enzymatic hydrolysate.

[0079] (4) Mix the enzymatic hydrolysate from step (3) with a 70% ethanol solution at a mass ratio of 1:0.7 and centrifuge at 3000 r / min for 15 min to obtain the supernatant.

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

[0081] Comparative Example 4

[0082] A method for extracting polypeptides from water lilies, comprising the following steps:

[0083] (1) Soak the seeds of yellow water lily in a sodium chlorate solution with a mass concentration of 0.1% for 1 minute, then rinse them twice with water. After rinsing, dry the seeds and crush them. Then pass them through a 100-mesh sieve to obtain crushed water lily samples.

[0084] (2) The crushed water lily sample from step (1) was mixed with water at a mass ratio of 1:10 and heated at 38°C for 70 minutes to obtain a mixture;

[0085] (3) Add papain to the mixture in step (2). 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 enzyme activity of papain is 2000 U / g. After adding papain, adjust the pH of the mixture to 6.9-7.1. Then, enzymatically hydrolyze the mixture at 38℃ for 4 hours to obtain the enzymatic hydrolysate.

[0086] (4) Mix the enzymatic hydrolysate from step (3) with an ethanol solution with a volume concentration of 78% at a mass ratio of 1:0.7 and centrifuge at a speed of 3000 r / min for 15 min to obtain the supernatant.

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

[0088] Comparative Example 5

[0089] A method for extracting polypeptides from water lilies, comprising the following steps:

[0090] (1) Soak the seeds of yellow water lily in a sodium chlorate solution with a mass concentration of 0.1% for 1 minute, then rinse them twice with water. After rinsing, dry the seeds and crush them. Then pass them through a 100-mesh sieve to obtain crushed water lily samples.

[0091] (2) The crushed water lily sample from step (1) was mixed with water at a mass ratio of 1:10 and heated at 45°C for 50 minutes to obtain a mixture;

[0092] (3) Add papain to the mixture in step (2). 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 enzyme activity of papain is 2000 U / g. After adding papain, adjust the pH of the mixture to 6.9-7.1. Then, enzymatically hydrolyze the mixture at 38℃ for 4 hours to obtain the enzymatic hydrolysate.

[0093] (4) Mix the enzymatic hydrolysate from step (3) with an ethanol solution with a volume concentration of 78% at a mass ratio of 1:0.7 and centrifuge at a speed of 3000 r / min for 15 min to obtain the supernatant.

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

[0095] Antioxidant performance test

[0096] Prepare a 0.1 mM DPPH solution and store it away from light for later use;

[0097] The water lily polypeptide samples extracted in Examples 1-4 and Comparative Examples 1-5 were diluted with water to obtain 0.1 mM test samples.

[0098] 100 μL of different test samples and 100 μL of DPPH alcohol solution from the examples and comparative examples were added to 96-well plates and incubated in the dark. 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 in 3 parallel replicates.

[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 for calculating the DPPH scavenging rate for each concentration of sample is as follows:

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

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

[0103] Table 1 DPPH removal rate in different treatments

[0104]

[0105]

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

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

[0108] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for extracting a Nelumbo nucifera polypeptide, characterized by, The method comprises the following steps: Disinfect, clean, dry and crush the sample of Nymphaea, to obtain the crushed sample; Mix the crushed sample with water and heat to obtain a mixture; Carry out enzymolysis on the mixture by using papain to obtain an enzymolysis solution; Mix the enzymolysis solution with an ethanol solution and centrifuge to obtain a supernatant; Remove the ethanol solvent from the supernatant to obtain the Nymphaea polypeptide; The sample of Nymphaea is one or more of petals, rhizomes and seeds of Nymphaea; The species of Nymphaea is Nymphaea alba, Nymphaea hybrid, Nymphaea colorata or Nymphaea tetragona; The papain is added in an amount of 0.03 times the mass of the pulverized sample 0.05； The temperature of the enzymatic hydrolysis is 38 42°C; the time of the enzymatic hydrolysis is 3 4h; The enzyme activity of the papain is ≧2000 U / g; The volume ratio of the enzymatic hydrolysate to the ethanol solution is 1 : 0.65 when the centrifugation is performed 0.7; The volume concentration of the ethanol solution is 75% 80% The speed of centrifugation was 2500 3000 r / min, and the time of centrifugation was 10 15 min.

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

3. The extraction method of claim 1, wherein, The disinfection method comprises the following steps: mixing the sample of Nymphaea with a sodium hypochlorite solution and soaking to obtain the disinfected sample of Nymphaea; The soaking time is 1 2 min; the mass concentration of the sodium hypochlorite solution is 0.1%.

4. The method of claim 1 3. The polypeptide of Nymphaea tetragona extracted by the extraction method of any one of claims 1-2.

5. The method of claim 1 3. Use of the polypeptide extracted by the extraction method of any one of claims 1-2 in anti-wrinkle and / or whitening products.

6. A cosmetic product, characterized by, The method of claim 1 3 The lotus peptide extracted by the extraction method according to any one of claims 1-2, and a cosmetically acceptable adjuvant.

7. The cosmetic product according to claim 6, characterized in that, The auxiliary material is one or more of dissolving agents, penetration enhancers, chelating agents and preservatives.

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