Preparation method for micromolecular pearl polypeptide solution containing various active ingredients

Abstract

A preparation method for a micromolecular pearl polypeptide solution containing various active ingredients, comprising the following steps: S1, multi-energy field assisted extraction; S2, centrifugal separation; S3, calcium dissolution; S4, secondary centrifugation; and S5, enzymolysis. A multi-energy field comprises ultrasonic waves, microwaves, a static magnetic field, and an electromagnetic oscillation field. According to the method, readily-available and low-cost narce powder is used as a raw material, an acid hydrolysis method and an enzymatic hydrolysis method are used, the steps are improved, proteins in narce are fully utilized, and the prepared pearl polypeptide solution is rich in various active ingredients, has small peptide molecular weight, has small loss of conchiolin, is easily absorbed by a human body, and has a long shelf life.

Description

Preparation method of small molecule pearl polypeptide liquid containing multiple active ingredients Technical Field

[0001] The present invention relates to the field of pharmaceutical biotechnology, and in particular to a method for preparing a small-molecule pearl polypeptide liquid containing multiple active ingredients. Background Art

[0002] Pearls are formed when foreign matter, such as sand, irritates the oyster's mantle, prompting it to secrete large amounts of nacre, which then envelops the foreign matter layer by layer. Pearls are not only a high-end decorative jewelry item with high market value, but also a valuable traditional Chinese medicine, cosmetic, and nutritional supplement. With the advancement of modern technology, more and more benefits of pearls are being discovered, and their utilization and development has entered a new stage of development.

[0003] Nacre is the innermost layer of the shell, after removing the surface cuticle and the intermediate prismatic layer. It is hard, milky white with a silvery sheen, and composed of calcium carbonate, protein, and organic matter. Nacre and pearls are homogenous and share essentially the same composition, primarily calcium carbonate, followed by keratin. Keratin is a complex protein containing various glycosyl groups. This glycoprotein is a bioactive substance beneficial to the human body, but keratin itself is stable and poorly soluble in water and common solvents, making it difficult for the human body to absorb and utilize. Therefore, it is particularly important to use nacre powder instead of pearl powder to extract pearl peptides, and then hydrolyze the pearl peptides into small, active peptides that are easily absorbed by the human body.

[0004] Currently, two methods for extracting active ingredients from nacre include acid hydrolysis and enzymatic hydrolysis. Acid hydrolysis primarily utilizes chemical reagents such as organic acids to separate and dissolve the calcium carbonate in the nacre, exposing the pearl's organic components to the greatest extent possible. However, acid hydrolysis can easily damage soluble proteins in the nacre during production, denaturing and inactivating them. Traditional enzymatic methods primarily use pancreatic enzymes, but the resulting peptides are unstable and contain a high concentration of amino acids, potentially losing the pearl's original medicinal value. Furthermore, most oligopeptide products are currently presented as powders, which are susceptible to moisture absorption, making them susceptible to bacterial invasion and deterioration. Furthermore, the granulation process can easily destroy the active ingredients in the product.

[0005] To address the above issues, a Chinese invention patent with authorization announcement number CN102018727B discloses a method for preparing a pearl hydrolyzate, which includes the following steps: 1) Place pearl powder in a hydrochloric acid aqueous solution and stir until the reaction is complete. Adjust the pH value of the mixture to neutral with ammonia water, centrifuge, and obtain precipitate A; 2) Dissolve precipitate A in purified water, adjust the pH value to 7.5-8.5 with ammonia water, and centrifuge to obtain precipitate B; 3) Dissolve precipitate B in purified water and add enzyme for enzymatic reaction. The enzymatic hydrolysis solution is centrifuged to obtain a supernatant and precipitate C. The enzyme is a mixture of snail protease and papain, added in an amount of 0.3-1.05% by weight of precipitate B. 4) Precipitate C is dissolved in purified water and enzymatically hydrolyzed again with the enzyme. The enzyme is a mixture of trypsin, papain, and neutral protease, added in an amount of 0.2-0.45% by weight of precipitate B. 5) The two supernatants are combined, boiled, and filtered through a nanofiltration membrane with a pore size of 1-3 nm to obtain the pearl. This method destroys the soluble protein in the pearl, which is discarded and not fully utilized.

[0006] It is clear that it is necessary to seek more effective methods to prepare pearl polypeptide liquids that are rich in multiple active ingredients, have small peptide molecular weights, minimize pearl protein loss, and are easily absorbed by the human body. Summary of the Invention

[0007] The present invention aims to overcome the deficiencies of the prior art by providing a method for preparing a small-molecule pearl polypeptide liquid containing multiple active ingredients. The method utilizes abundant and inexpensive nacre powder as a raw material, employs an acid-enzyme hydrolysis method, and improves the steps to fully utilize the protein in the nacre. The prepared pearl polypeptide liquid is rich in multiple active ingredients, has a small peptide molecular weight, minimizes pearl protein loss, is easily absorbed by the human body, and has a long shelf life.

[0008] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is: a method for preparing a small molecule pearl polypeptide liquid containing multiple active ingredients, comprising the following steps:

[0009] Step S1, multi-energy field-assisted extraction: commercially available nacre powder is ultrafinely ground, dispersed in pure water, heated to 50° C. to 55° C., and subjected to multi-energy field-assisted extraction for 2 h to 4 h;

[0010] Step S2, centrifugation: centrifuging the mixed solution obtained after the multi-energy field-assisted extraction, and collecting the supernatant A and the precipitate B respectively;

[0011] Step S3, dissolving calcium: taking precipitate B, adding organic acid solution, stirring and reacting for 3-5 hours, and after the reaction is completed, adjusting the pH to 6-7 with a NaOH solution with a concentration of 0.8-1.2 mol / L;

[0012] Step S4, secondary centrifugation: the dispersion after dissolving the calcium is subjected to secondary centrifugation to obtain precipitate C, which is insoluble protein;

[0013] Step S5, enzymatic hydrolysis: The supernatant A and the precipitate C are mixed into a homogenate, and a composite plant protease is added. The mixture is hydrolyzed at 50±3°C for 6-8 hours to inactivate the enzyme. The mixture is cooled to 30°C and filtered. The mixture is passed through a 1-3 nm ultrafiltration membrane and an activated carbon fiber membrane in sequence. The mixture is then decolorized and deodorized, sterilized, and canned to obtain a small molecule pearl polypeptide liquid.

[0014] Preferably, the mass ratio of the pearl layer powder to pure water in step S1 is 1:(20-30).

[0015] Preferably, the multi-energy field in step S1 includes ultrasound, microwave, steady magnetic field and electromagnetic oscillation field.

[0016] Preferably, the frequency of the ultrasonic wave is 80-120 kHz, and the power is 150-250 W; the frequency of the microwave treatment is 2.5-3.2 GHz, and the power is 600-1500 W; and the magnetic field strength of the constant magnetic field is 6000-12000 Gs.

[0017] Preferably, the frequency of the electromagnetic oscillation field is 15-30 Hz, and the medium to low duty cycle is 20-30%.

[0018] Preferably, the centrifugal speed in step S2 is 2000-3000 r / min, and the time is 10-20 min.

[0019] Preferably, the organic acid solution in step S3 is any one of citric acid solution, lactic acid solution, and malic acid solution.

[0020] Preferably, the mass percentage concentration of the organic acid solution in step S3 is 20-30 wt %.

[0021] Preferably, the mass ratio of the precipitate B to the organic acid solution in step S3 is 1:(2-4).

[0022] Preferably, the secondary centrifugation in step S4 is performed at a speed of 2000-3000 r / min and for a time of 10-20 min.

[0023] Preferably, the mass ratio of the homogenate to the composite plant protease in step S5 is 100:(1-2).

[0024] Preferably, the composite plant protease in step S5 is a mixture of bromelain, ficin, and papain in a mass ratio of (1-3):2:(5-9).

[0025] Preferably, the enzyme inactivation temperature in step S5 is 95-100° C. and the time is 8-12 min.

[0026] Another object of the present invention is to provide a small molecule pearl polypeptide liquid containing multiple active ingredients prepared by the preparation method of the small molecule pearl polypeptide liquid containing multiple active ingredients.

[0027] Due to the application of the above technical solution, the present invention has the following beneficial effects:

[0028] (1) The method for preparing a small molecule pearl polypeptide liquid containing multiple active ingredients disclosed in the present invention has a simple process, convenient operation and control, low cost, can fully utilize the soluble protein and insoluble protein in the nacre layer, has high preparation efficiency and finished product qualification rate, and has low pearl protein loss. The prepared pearl polypeptide liquid is rich in multiple active ingredients, has a small peptide molecular weight, is easily absorbed by the human body, and has a long shelf life.

[0029] (2) The small molecule pearl polypeptide liquid containing multiple active ingredients disclosed in the present invention adopts multi-energy field-assisted extraction. The nacre powder is ultrafinely crushed before extraction. The cooperation between them can effectively improve the extraction rate. The multi-energy field includes ultrasound, microwave, steady magnetic field and electromagnetic oscillation field. These energy fields act simultaneously, cooperate with each other and work together to achieve high extraction efficiency, less damage to the active ingredients in the nacre, and high extraction rate of pearl protein.

[0030] (3) The small molecule pearl polypeptide liquid containing multiple active ingredients disclosed in the present invention uses nacreous layer powder instead of pearl powder as raw material, which can effectively reduce product preparation costs, enrich the raw material source, and effectively increase its added value. During the preparation process, the soluble protein and insoluble protein are made into a homogenate and enzymatically hydrolyzed together, which avoids the waste of pearl protein, improves its utilization rate and the yield of the final product, and better retains multiple active ingredients in the polypeptide liquid. The active ingredients cooperate with each other to work together, thereby increasing the functional added value of the polypeptide liquid product and making it more effective.

[0031] (4) The small molecule pearl polypeptide liquid containing multiple active ingredients disclosed in the present invention uses a composite plant protease in the enzymatic hydrolysis process. The composite plant protease is a mixture of bromelain, ficin, and papain in a mass ratio of (1-3):2:(5-9). Through the reasonable selection of the composition formula of the protease, the enzymatic hydrolysis effect is more significant and the efficiency is higher, and the small molecule peptide obtained has a smaller molecular weight and is easier to be absorbed by the human body.

[0032] (5) The method for preparing a small molecule pearl polypeptide liquid containing multiple active ingredients disclosed in the present invention is efficient through the reasonable selection of process parameters and the interaction between them, and the method has a high extraction rate for pearl protein without causing damage to it. The prepared polypeptide liquid contains many active ingredients and has high added value of efficacy. DETAILED DESCRIPTION

[0033] The following description is intended to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are merely examples, and those skilled in the art may conceive of other obvious variations.

[0034] Example 1, a method for preparing a small molecule pearl polypeptide solution containing multiple active ingredients, comprising the following steps:

[0035] Step S1, multi-energy field-assisted extraction: commercially available nacre powder is ultrafinely ground, dispersed in pure water, heated to 50° C., and subjected to multi-energy field-assisted extraction for 2 h;

[0036] Step S2, centrifugation: centrifuging the mixed solution obtained after the multi-energy field-assisted extraction, and collecting the supernatant A and the precipitate B respectively;

[0037] Step S3, dissolving calcium: taking precipitate B, adding organic acid solution, stirring and reacting for 3 hours, and after the reaction is completed, adjusting the pH to 6 with a 0.8 mol / L NaOH solution;

[0038] Step S4, secondary centrifugation: the dispersion after dissolving the calcium is subjected to secondary centrifugation to obtain precipitate C, which is insoluble protein;

[0039] Step S5, enzymatic hydrolysis: The supernatant A and the precipitate C are mixed into a homogenate, and a composite plant protease is added. The mixture is hydrolyzed at 50°C for 6 hours to inactivate the enzyme. The mixture is cooled to 30°C and filtered. The mixture is passed through a 1nm ultrafiltration membrane and an activated carbon fiber membrane in sequence. The mixture is then decolorized and deodorized, sterilized, and canned to obtain a small molecule pearl polypeptide liquid.

[0040] The mass ratio of the nacre powder to the pure water in step S1 is 1:20; the multi-energy field in step S1 includes ultrasound, microwave, steady magnetic field, and electromagnetic oscillation field; the frequency of the ultrasound is 80 kHz and the power is 150 W; the frequency of the microwave treatment is 2.5 GHz and the power is 600 W; the magnetic field strength of the steady magnetic field is 6000 Gs; the frequency of the electromagnetic oscillation field is 15 Hz, and the medium and low duty cycle is 20%.

[0041] The centrifugal speed in step S2 is 2000 r / min and the time is 10 min; the organic acid solution in step S3 is a citric acid solution; the mass percentage concentration of the organic acid solution in step S3 is 20wt%; the mass ratio of the precipitate B to the organic acid solution in step S3 is 1:2.

[0042] The secondary centrifugation speed in step S4 is 2000 r / min, and the time is 10 minutes; the mass ratio of the homogenate to the composite plant protease in step S5 is 100:1; the composite plant protease in step S5 is a mixture of bromelain, ficin, and papain in a mass ratio of 1:2:5; the enzyme inactivation temperature in step S5 is 95° C., and the time is 8 minutes.

[0043] Example 2, a method for preparing a small molecule pearl polypeptide solution containing multiple active ingredients, comprising the following steps:

[0044] Step S1, multi-energy field-assisted extraction: commercially available nacre powder is ultrafinely ground, dispersed in pure water, heated to 51° C., and subjected to multi-energy field-assisted extraction for 2.5 hours;

[0045] Step S2, centrifugation: centrifuging the mixed solution obtained after the multi-energy field-assisted extraction, and collecting the supernatant A and the precipitate B respectively;

[0046] Step S3, dissolving calcium: taking precipitate B, adding organic acid solution, stirring and reacting for 3.5 hours. After the reaction is completed, using 0.9 mol / L NaOH solution to adjust the pH to 6.3;

[0047] Step S4, secondary centrifugation: the dispersion after dissolving the calcium is subjected to secondary centrifugation to obtain precipitate C, which is insoluble protein;

[0048] Step S5, enzymatic hydrolysis: The supernatant A and the precipitate C are mixed into a homogenate, and a composite plant protease is added. The mixture is hydrolyzed at 48°C for 6.5 hours to inactivate the enzyme. The mixture is cooled to 30°C and filtered. The mixture is passed through a 2nm ultrafiltration membrane and an activated carbon fiber membrane in sequence. The mixture is then decolorized and deodorized, sterilized, and canned to obtain a small molecule pearl polypeptide liquid.

[0049] The mass ratio of the nacre powder to the pure water in step S1 is 1:23; the multi-energy field in step S1 includes ultrasound, microwave, steady-state magnetic field, and electromagnetic oscillation field; the frequency of the ultrasound is 90 kHz and the power is 170 W; the frequency of the microwave treatment is 2.7 GHz and the power is 800 W; the magnetic field strength of the steady-state magnetic field is 8000 Gs; the frequency of the electromagnetic oscillation field is 18 Hz, and the medium and low duty cycle is 23%.

[0050] The centrifugal speed in step S2 is 2300 r / min and the time is 13 min; the organic acid solution in step S3 is a lactic acid solution; the mass percentage concentration of the organic acid solution in step S3 is 23wt%; the mass ratio of the precipitate B to the organic acid solution in step S3 is 1:2.5.

[0051] The secondary centrifugation speed in step S4 is 2200 r / min, and the time is 13 minutes; the mass ratio of the homogenate and the composite plant protease in step S5 is 100:1.3; the composite plant protease in step S5 is a mixture of bromelain, ficin, and papain in a mass ratio of 1.5:2:6; the enzyme inactivation temperature in step S5 is 97° C., and the time is 9 minutes.

[0052] Example 3, a method for preparing a small molecule pearl polypeptide solution containing multiple active ingredients, comprising the following steps:

[0053] Step S1, multi-energy field-assisted extraction: commercially available nacre powder is ultrafinely ground, dispersed in pure water, heated to 53° C., and subjected to multi-energy field-assisted extraction for 3 h;

[0054] Step S2, centrifugation: centrifuging the mixed solution obtained after the multi-energy field-assisted extraction, and collecting the supernatant A and the precipitate B respectively;

[0055] Step S3, dissolving calcium: taking precipitate B, adding organic acid solution, stirring and reacting for 4 hours, and after the reaction is completed, adjusting the pH to 6.5 with a 1 mol / L NaOH solution;

[0056] Step S4, secondary centrifugation: the dispersion after dissolving the calcium is subjected to secondary centrifugation to obtain precipitate C, which is insoluble protein;

[0057] Step S5, enzymatic hydrolysis: The supernatant A and the precipitate C are mixed into a homogenate, and a composite plant protease is added. The mixture is hydrolyzed at 51°C for 7 hours to inactivate the enzyme. The mixture is cooled to 30°C and filtered. The mixture is passed through a 2nm ultrafiltration membrane and an activated carbon fiber membrane in sequence. The mixture is then decolorized and deodorized, sterilized, and canned to obtain a small molecule pearl polypeptide liquid.

[0058] The mass ratio of the nacre powder to the pure water in step S1 is 1:25; the multi-energy field in step S1 includes ultrasound, microwave, steady magnetic field, and electromagnetic oscillation field; the frequency of the ultrasound is 100 kHz and the power is 200 W; the frequency of the microwave treatment is 2.9 GHz and the power is 1200 W; the magnetic field strength of the steady magnetic field is 9500 Gs; the frequency of the electromagnetic oscillation field is 22 Hz, and the medium and low duty cycle is 25% at this time; the centrifugal speed in step S2 is 2500 r / min and the time is 15 minutes; the organic acid solution in step S3 is a malic acid solution; the mass percentage concentration of the organic acid solution in step S3 is 25 wt%; and the mass ratio of the precipitate B to the organic acid solution in step S3 is 1:3.

[0059] The secondary centrifugation speed in step S4 is 2500 r / min, and the time is 15 min; the mass ratio of the homogenate and the composite plant protease in step S5 is 100:1.5; the composite plant protease in step S5 is a mixture of bromelain, ficin, and papain in a mass ratio of 2:2:7; the enzyme inactivation temperature in step S5 is 98° C., and the time is 10 min.

[0060] Example 4, a method for preparing a small molecule pearl polypeptide solution containing multiple active ingredients, comprising the following steps:

[0061] Step S1, multi-energy field-assisted extraction: commercially available nacre powder is ultrafinely ground, dispersed in pure water, heated to 54° C., and subjected to multi-energy field-assisted extraction for 3.5 hours;

[0062] Step S2, centrifugation: centrifuging the mixed solution obtained after the multi-energy field-assisted extraction, and collecting the supernatant A and the precipitate B respectively;

[0063] Step S3, dissolving calcium: taking precipitate B, adding organic acid solution, stirring and reacting for 4.5 hours. After the reaction is completed, adjusting the pH to 6.8 with a 1.1 mol / L NaOH solution;

[0064] Step S4, secondary centrifugation: the dispersion after dissolving the calcium is subjected to secondary centrifugation to obtain precipitate C, which is insoluble protein;

[0065] Step S5, enzymatic hydrolysis: The supernatant A and the precipitate C are mixed into a homogenate, and a composite plant protease is added. The mixture is hydrolyzed at 52°C for 7.5 hours to inactivate the enzyme. The mixture is cooled to 30°C and filtered. The mixture is passed through a 3nm ultrafiltration membrane and an activated carbon fiber membrane in sequence. The mixture is then decolorized and deodorized, sterilized, and canned to obtain a small molecule pearl polypeptide liquid.

[0066] The mass ratio of the nacre powder to pure water in step S1 is 1:28; the multi-energy field in step S1 includes ultrasound, microwave, steady magnetic field, and electromagnetic oscillation field; the frequency of the ultrasound is 110 kHz and the power is 230 W; the frequency of the microwave treatment is 3.1 GHz and the power is 1400 W; the magnetic field strength of the steady magnetic field is 11000 Gs; the frequency of the electromagnetic oscillation field is 27 Hz, and the medium-low duty cycle is 28%; the centrifugal speed in step S2 is 2900 r / min and the time is 18 minutes; the organic acid solution in step S3 is a citric acid solution; the mass percentage concentration of the organic acid solution in step S3 is 28 wt%; and the mass ratio of the precipitate B to the organic acid solution in step S3 is 1:3.5.

[0067] The secondary centrifugation speed in step S4 is 2900 r / min, and the time is 18 minutes; the mass ratio of the homogenate and the composite plant protease in step S5 is 100:1.8; the composite plant protease in step S5 is a mixture of bromelain, ficin, and papain in a mass ratio of 2.5:2:8.5; the enzyme inactivation temperature in step S5 is 99° C., and the time is 11 minutes.

[0068] Example 5, a method for preparing a small molecule pearl polypeptide solution containing multiple active ingredients, comprising the following steps:

[0069] Step S1, multi-energy field-assisted extraction: commercially available nacre powder is ultrafinely ground, dispersed in pure water, heated to 55° C., and subjected to multi-energy field-assisted extraction for 4 hours;

[0070] Step S2, centrifugation: centrifuging the mixed solution obtained after the multi-energy field-assisted extraction, and collecting the supernatant A and the precipitate B respectively;

[0071] Step S3, dissolving calcium: taking precipitate B, adding organic acid solution, stirring and reacting for 5 hours, and after the reaction is completed, adjusting the pH to 7 with a 1.2 mol / L NaOH solution;

[0072] Step S4, secondary centrifugation: the dispersion after dissolving the calcium is subjected to secondary centrifugation to obtain precipitate C, which is insoluble protein;

[0073] Step S5, enzymatic hydrolysis: The supernatant A and the precipitate C are mixed into a homogenate, and a composite plant protease is added. The mixture is hydrolyzed at 53°C for 8 hours to inactivate the enzyme. The mixture is cooled to 30°C and filtered. The mixture is passed through a 3nm ultrafiltration membrane and an activated carbon fiber membrane in sequence. After decolorization and deodorization, the mixture is sterilized and canned to obtain a small molecule pearl polypeptide liquid.

[0074] The mass ratio of the nacre powder to pure water in step S1 is 1:30; the multi-energy field in step S1 includes ultrasound, microwave, steady magnetic field, and electromagnetic oscillation field; the frequency of the ultrasound is 120 kHz and the power is 250 W; the frequency of the microwave treatment is 3.2 GHz and the power is 1500 W; the magnetic field strength of the steady magnetic field is 12000 Gs; the frequency of the electromagnetic oscillation field is 30 Hz, and the medium and low duty cycle is 30% at this time; the centrifugal speed in step S2 is 3000 r / min and the time is 20 min; the organic acid solution in step S3 is a malic acid solution; the mass percentage concentration of the organic acid solution in step S3 is 30 wt%; and the mass ratio of the precipitate B to the organic acid solution in step S3 is 1:4.

[0075] The secondary centrifugation speed in step S4 is 3000 r / min, and the time is 20 min; the mass ratio of the homogenate to the composite plant protease in step S5 is 100:2; the composite plant protease in step S5 is a mixture of bromelain, ficin, and papain in a mass ratio of 3:2:9; the enzyme inactivation temperature in step S5 is 100° C., and the time is 12 min.

[0076] Comparative Example 1, a method for preparing a small molecule pearl polypeptide liquid containing multiple active ingredients, is basically the same as Example 1, except that ultrasonic assistance is used instead of multi-energy field assistance; the frequency of the ultrasonic wave is 80kHz and the power is 150W.

[0077] Comparative Example 2, a method for preparing a small molecule pearl polypeptide liquid containing multiple active ingredients, is basically the same as Example 1, except that the composite plant protease is a mixture of bromelain and papain in a mass ratio of 1:5.

[0078] Experimental Example 1: Taking Example 1 and Example 2 as examples, the pearl polypeptide liquid was tested for SOD superoxide dismutase activity, taurine content, and peptide molecular weight distribution. The results were repeated three times and the average values ​​were taken. The results are shown in Tables 1 and 2 below.

[0079]

[0080]

[0081] The active ingredient content results in Table 1 indicate that the pearl polypeptide liquid obtained using the methods of Examples 1 and 2 contains a high level of SOD superoxide dismutase. Furthermore, this method is capable of extracting taurine from the nacre, fully preserving the active ingredients in the pearls. The molecular weight distribution in Table 2 shows that the majority of the peptides in the pearl polypeptide liquid obtained using the methods of Examples 1 and 2 have molecular weights less than 180, which is lower than the molecular weights of all currently published pearl peptides. Modern pharmacological research indicates that small peptides with molecular weights less than 500 are more readily absorbed by the human body.

[0082] The above results show that the methods involved in the embodiments of the present invention retain more active ingredients of pearls and are completely hydrolyzed, which is beneficial to human absorption and utilization, and can be used as raw materials for health foods and cosmetics.

[0083] Experimental Example 2: Detection of Peptide Content in Pearl Polypeptide Liquid

[0084] The peptide contents of Examples 1 and 2 were tested according to GB / T 22729-2008. The test results are as follows:

[0085]

[0086] In the prior art, the peptide content extracted from pearls is basically 5 ppm (5 parts per million). The peptide content in the pearl polypeptide liquid prepared by the methods of the embodiments of the present invention is increased by about 40 times, greatly improving the extraction rate of pearl polypeptides.

[0087] Experimental Example 3: In vitro antioxidant activity test of pearl polypeptide liquid

[0088] Take 2mL of the small molecule pearl polypeptide solution in each case, add DPPH solution, and measure its absorbance after reaction. Take VC as the control group and calculate the DPPH free radical scavenging rate. Take 5mL of the small molecule pearl polypeptide solution in each case and place it in a test tube. Add Tris-HCl buffer and add preheated pyrogallol solution after constant temperature. Measure its absorbance. Read the absorbance value every 30s for 5 minutes. Draw a standard curve with absorbance and time. Take VC as the control group and calculate the superoxide anion (O2 － ) free radical scavenging rate. Take 0.2mL of the small molecule pearl polypeptide solution in each case and add it to the ABTS solution in each test tube. After standing for reaction, measure the absorbance value. Take VC as the control group and calculate the ABTS scavenging rate. Take 5mL of the small molecule pearl polypeptide solution in each case and place it in a test tube. Add salicylic acid-ethanol solution, ferrous sulfate solution and hydrogen peroxide solution respectively. After reaction, measure the absorbance value. Take VC as the control group and measure the hydroxyl (OH － ) Free radical scavenging rate.

[0089]

[0090] From Table 4, we can see that the small molecule pearl polypeptide solution prepared by the present invention has the following effects on DPPH free radicals and O2 － It has a strong scavenging effect, but its scavenging ability for ABTS free radicals and hydroxyl radicals is relatively weak. The products made from each embodiment of the present invention have higher antioxidant activity than the products of the comparative examples. The combined use of multi-energy field assistance and ficin is beneficial to improving the above performance.

[0091] Experimental Example 4: Experimental verification of the anti-aging effect of small molecule pearl polypeptide solution

[0092] The small molecule pearl polypeptide solution in Example 1 was used to culture Drosophila melanogaster to verify the anti-aging effect of the present invention.

[0093] Experimental Materials: Drosophila melanogaster, Culture Medium: 8.0g cornstarch, 7.5g glucose, 0.7g agar, 2.0g yeast powder, 0.2mL 15% benzoic acid, and 0.2mL propionic acid. The small molecule pearl polypeptide solution from Example 1 was added to the culture medium in the appropriate proportions.

[0094] Experimental Procedure: Drosophila melanogaster within 10 hours of eclosion were collected and grouped by sex. After ether anesthesia, 100 male and 100 female fruit flies were collected and divided equally into two groups. The fruit flies were placed in a 2.3 x 12 cm test tube. The experimental group was treated with a 10 wt% small molecule pearl peptide solution, while the control group was treated with distilled water alone. The test tubes were then incubated at 25 ± 1°C and 60%-70% humidity. The culture medium was changed every three days, and the number of fruit flies that died was recorded daily.

[0095] Experimental results: Compared to the control group, the small molecule pearl peptide solution significantly increased the average lifespan of fruit flies, with the effect being more pronounced in female fruit flies than in males. The average lifespan of female fruit flies increased from 30.1±3.2 days to 41.5±2.3 days, while the average lifespan of male fruit flies increased from 29.9±1 days to 38.3±6 days.

[0096] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a small molecule pearl polypeptide solution containing multiple active ingredients, characterized in that: The steps include: Step S1, multi-energy field-assisted extraction: Commercially available nacre powder is ultrafinely ground, dispersed in pure water, heated to 50°C to 55°C, and subjected to multi-energy field-assisted extraction for 2 to 4 hours; the multi-energy field includes ultrasound, microwave, steady magnetic field, and electromagnetic oscillation field; the ultrasound has a frequency of 80-120kHz and a power of 150-250W; the microwave treatment has a frequency of 2.5-3.2GHz and a power of 600-1500W; and the steady magnetic field has a magnetic field strength of 6000-12000Gs; The frequency of the electromagnetic oscillation field is 15-30 Hz, and the medium to low duty cycle is 20-30%; Step S2, centrifugation: centrifuging the mixed solution obtained after the multi-energy field-assisted extraction, and collecting the supernatant A and the precipitate B respectively; Step S3, dissolving calcium: taking precipitate B, adding organic acid solution, stirring and reacting for 3-5 hours, and after the reaction is completed, adjusting the pH to 6-7 with a NaOH solution with a concentration of 0.8-1.2 mol / L; Step S4, secondary centrifugation: the dispersion after dissolving the calcium is subjected to secondary centrifugation to obtain precipitate C, which is insoluble protein; Step S5, enzymatic hydrolysis: The supernatant A and the precipitate C are mixed into a homogenate, a composite plant protease is added, and the mixture is hydrolyzed at 50±3°C for 6-8 hours to inactivate the enzyme. The mixture is cooled to 30°C and filtered, and sequentially passed through a 1-3 nm ultrafiltration membrane and an activated carbon fiber membrane. After decolorization and deodorization, sterilization and canning, a small molecule pearl polypeptide liquid is obtained; the composite plant protease is a mixture of bromelain, ficin, and papain in a mass ratio of (1-3):2:(5-9).

2. The method for preparing a small molecule pearl polypeptide solution containing multiple active ingredients according to claim 1, characterized in that: The mass ratio of the pearl layer powder to the pure water in step S1 is 1:(20-30).

3. The method for preparing a small molecule pearl polypeptide solution containing multiple active ingredients according to claim 1, characterized in that: The centrifugal speed in step S2 is 2000-3000 r / min, and the time is 10-20 min.

4. The method for preparing a small molecule pearl polypeptide solution containing multiple active ingredients according to claim 1, characterized in that: The organic acid solution in step S3 is any one of citric acid solution, lactic acid solution, and malic acid solution; the mass percentage concentration of the organic acid solution is 20-30wt%.

5. The method for preparing a small molecule pearl polypeptide solution containing multiple active ingredients according to claim 1, characterized in that: The mass ratio of the precipitate B to the organic acid solution in step S3 is 1:(2-4); the speed of the secondary centrifugation in step S4 is 2000-3000 r / min, and the time is 10-20 min.

6. The method for preparing a small molecule pearl polypeptide solution containing multiple active ingredients according to claim 1, characterized in that: The mass ratio of the homogenate to the composite plant protease in step S5 is 100:(1-2).

7. The method for preparing a small molecule pearl polypeptide solution containing multiple active ingredients according to claim 1, characterized in that: The enzyme inactivation temperature in step S5 is 95-100° C. and the time is 8-12 minutes.

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