A method for preparing a small molecule pearl polypeptide liquid containing multiple active ingredients

Abstract

The present invention discloses a method for preparing a small molecule pearl polypeptide liquid containing multiple active ingredients, which relates to the field of pharmaceutical biotechnology and includes the following steps: step S1, multi-energy field assisted extraction; step S2, centrifugal separation; step S3, calcium dissolution; step S4, secondary centrifugation; step S5, enzymatic hydrolysis; the multi-energy field includes ultrasound, microwave, steady magnetic field and electromagnetic oscillation field. The method uses the abundant and cheap pearl layer powder as raw material, uses the acid-enzyme hydrolysis method, improves the steps, and makes full use of the protein in the pearl layer. The prepared pearl polypeptide liquid is rich in multiple active ingredients, has a small peptide molecular weight, has little pearl protein loss, is easily absorbed by the human body, and has a long shelf life.

Description

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 stimulates the mantle of the oyster, prompting it to secrete a large amount of nacre, which then wraps up the foreign matter layer by layer. Pearls are not only a high-end jewelry decoration with high market value, but also a precious Chinese herbal medicine, cosmetics and nutritional health product. With the advancement of modern science and technology, more and more benefits of pearls have been discovered, and the utilization and development of pearls has entered a new stage of development.

[0003] The nacre is the innermost layer of the shell after removing the surface cuticle and the middle prism layer. It is hard, milky white, and silvery. It is composed of calcium carbonate, protein, and organic matter. The nacre and pearl grains are homogeneous and have basically the same composition. The main component is calcium carbonate, followed by shell keratin. Shell keratin is a complex protein containing different sugar groups. This glycoprotein is a biologically active substance that is beneficial to the human body. However, shell keratin itself is stable in nature and is not easily soluble in water and general 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 molecule active peptides that are easily absorbed by the human body.

[0004] At present, there are two methods for extracting effective ingredients from nacre: acid hydrolysis and enzymatic hydrolysis. Acid hydrolysis mainly uses chemical reagents such as organic acids to separate and dissolve the calcium carbonate in the nacre, so that the organic components of the pearls can be exposed to the maximum extent. However, acid hydrolysis can easily destroy the soluble proteins in the nacre during the production process, causing them to denature and lose their activity. The traditional enzymatic method mainly uses pancreatic enzymes. The peptides after degradation are unstable and contain many amino acids, which may lose the original medicinal value of the pearls. In addition, most oligopeptide products are currently presented in powder form, which is easy to absorb moisture, easily lead to bacterial invasion, and cause qualitative changes. In addition, the active substances in the product are easily destroyed during the granulation process.

[0005] In order to solve the above problems, the Chinese invention patent with the authorization announcement number CN102018727B discloses a method for preparing a pearl hydrolyzate, and the steps are as follows: 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 to obtain precipitate A; 2) Dissolve precipitate A in pure water, adjust the pH value to 7.5-8.5 with ammonia water, centrifuge to obtain precipitate B; 3) Dissolve precipitate B in pure water, add enzyme for enzyme reaction The enzyme solution is centrifuged to obtain a supernatant and a precipitate C; the enzyme is a mixture of snail protease and papain, and the amount added is 0.3-1.05% of the weight of the precipitate B; 4) the precipitate C is dissolved in pure water and the enzyme is added for secondary enzymolysis, centrifuged, and the supernatant is collected; the enzyme is a mixture of trypsin, papain and neutral protease, and the amount added is 0.2-0.45% of the weight of the precipitate B; 5) the two supernatants are combined, boiled, and filtered with a nanofiltration membrane with a pore size of 1-3nm to obtain. This method destroys the soluble protein in the pearl, and it is discarded and not fully utilized.

[0006] It can be seen that it is necessary to seek a more effective method to prepare a pearl polypeptide liquid that is rich in multiple active ingredients, has a small peptide molecular weight, has little pearl protein loss, and is easily absorbed by the human body. Summary of the invention

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

[0008] To achieve the above object, 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: take commercially available pearl layer powder, perform ultrafine grinding, disperse it in pure water, heat it to 50°C to 55°C, and perform multi-energy field assisted extraction for 2h to 4h;

[0010] Step S2, centrifugal separation: 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, after the reaction is completed, using NaOH solution with a concentration of 0.8-1.2 mol / L to adjust the pH to 6-7;

[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 enzyme is inactivated after hydrolysis at 50±3°C for 6-8h, and the mixture is cooled to 30°C and filtered. The mixture is passed through a 1-3nm ultrafiltration membrane and an activated carbon fiber membrane in sequence, and then decolorized, deodorized, sterilized and canned to obtain a small molecule pearl polypeptide liquid.

[0014] Preferably, the mass ratio of the pearl layer powder to the 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 80-120kHz, and the power is 150-250W; the frequency of the microwave treatment is 2.5-3.2GHz, and the power is 600-1500W; and the magnetic field strength of the steady magnetic field is 6000-12000Gs.

[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 20-30wt%.

[0021] Preferably, the mass ratio of the precipitate B and 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 to 3000 r / min for 10 to 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, 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 pearl layer 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 pearl layer, 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 pearl layer powder instead of pearl powder as raw material, which can effectively reduce the product preparation cost, has a richer source of raw materials, and can effectively improve its added value. In the preparation process, the soluble protein and the 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, better retains multiple active ingredients in the polypeptide liquid, and the active ingredients cooperate with each other to work together, thereby improving the functional added value of the polypeptide liquid product and making its efficacy richer.

[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, wherein 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 obtained small molecule peptide 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 has high efficiency and a high extraction rate of pearl protein without causing damage to the pearl protein through the reasonable selection of process parameters and the cooperation between them. The prepared polypeptide liquid has more active ingredients and high added value of efficacy. DETAILED DESCRIPTION

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

[0034] Example 1

[0035] A method for preparing a small molecule pearl polypeptide liquid containing multiple active ingredients comprises the following steps:

[0036] Step S1, multi-energy field assisted extraction: take commercially available pearl layer powder, grind it into ultrafine powder, disperse it in pure water, heat it to 50° C., and perform multi-energy field assisted extraction for 2 hours;

[0037] Step S2, centrifugal separation: centrifuging the mixed solution obtained after the multi-energy field-assisted extraction, and collecting the supernatant A and the precipitate B respectively;

[0038] Step S3, dissolving calcium: taking precipitate B, adding organic acid solution, stirring and reacting for 3 hours, and after the reaction, adjusting the pH to 6 with a NaOH solution with a concentration of 0.8 mol / L;

[0039] Step S4, secondary centrifugation: the dispersion after dissolving the calcium is subjected to secondary centrifugation to obtain precipitate C, which is insoluble protein;

[0040] Step S5, enzymatic hydrolysis: the supernatant A and the precipitate C are mixed into a homogenate, and the composite plant protease is added, and the enzyme is inactivated after hydrolysis at 50°C for 6 hours, and the solution is filtered after cooling to 30°C, and then passed through a 1nm ultrafiltration membrane and an activated carbon fiber membrane in sequence, and then decolorized, deodorized, sterilized and canned to obtain a small molecule pearl polypeptide liquid.

[0041] The mass ratio of the pearl layer powder and 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 80kHz and the power is 150W; the frequency of the microwave treatment is 2.5GHz and the power is 600W; the magnetic field intensity of the steady magnetic field is 6000Gs; the frequency of the electromagnetic oscillation field is 15HZ, and the medium and low duty cycle is 20%.

[0042] The centrifugal speed in step S2 is 2000r / min, and the time is 10min; the organic acid solution in step S3 is 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.

[0043] The speed of the secondary centrifugation in step S4 is 2000r / min, and the time is 10min; the mass ratio of the homogenate and 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 temperature of the enzyme inactivation in step S5 is 95°C, and the time is 8min.

[0044] Example 2

[0045] A method for preparing a small molecule pearl polypeptide liquid containing multiple active ingredients comprises the following steps:

[0046] Step S1, multi-energy field assisted extraction: take commercially available pearl layer powder, perform ultrafine grinding, disperse it in pure water, heat it to 51° C., and perform multi-energy field assisted extraction for 2.5 hours;

[0047] Step S2, centrifugal separation: centrifuging the mixed solution obtained after the multi-energy field-assisted extraction, and collecting the supernatant A and the precipitate B respectively;

[0048] 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;

[0049] Step S4, secondary centrifugation: the dispersion after dissolving the calcium is subjected to secondary centrifugation to obtain precipitate C, which is insoluble protein;

[0050] Step S5, enzymatic hydrolysis: the supernatant A and the precipitate C are mixed into a homogenate, and a composite plant protease is added. The enzyme is inactivated after hydrolysis at 48°C for 6.5 hours, and 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, and then decolorized, deodorized, sterilized and canned to obtain a small molecule pearl polypeptide liquid.

[0051] The mass ratio of the pearl layer powder and the pure water in step S1 is 1:23; the multi-energy field in step S1 includes ultrasound, microwave, steady magnetic field and electromagnetic oscillation field; the frequency of the ultrasound is 90kHz and the power is 170W; the frequency of the microwave treatment is 2.7GHz and the power is 800W; the magnetic field intensity of the steady magnetic field is 8000Gs; the frequency of the electromagnetic oscillation field is 18HZ, and the medium and low duty cycle is 23%.

[0052] The centrifugal speed in step S2 is 2300r / min, and the time is 13min; 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.

[0053] The speed of the secondary centrifugation in step S4 is 2200r / min, and the time is 13min; 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 temperature of the enzyme inactivation in step S5 is 97°C, and the time is 9min.

[0054] Example 3

[0055] A method for preparing a small molecule pearl polypeptide liquid containing multiple active ingredients comprises the following steps:

[0056] Step S1, multi-energy field assisted extraction: take commercially available pearl layer powder, grind it into ultrafine powder, disperse it in pure water, heat it to 53° C., and perform multi-energy field assisted extraction for 3 hours;

[0057] Step S2, centrifugal separation: centrifuging the mixed solution obtained after the multi-energy field-assisted extraction, and collecting the supernatant A and the precipitate B respectively;

[0058] Step S3, dissolving calcium: taking precipitate B, adding organic acid solution, stirring and reacting for 4 hours, and after the reaction, adjusting the pH to 6.5 with 1 mol / L NaOH solution;

[0059] Step S4, secondary centrifugation: the dispersion after dissolving the calcium is subjected to secondary centrifugation to obtain precipitate C, which is insoluble protein;

[0060] Step S5, enzymatic hydrolysis: the supernatant A and the precipitate C are mixed into a homogenate, and a composite plant protease is added. The enzyme is inactivated after hydrolysis at 51°C for 7 hours, and the mixture is filtered after cooling to 30°C, and then passed through a 2nm ultrafiltration membrane and an activated carbon fiber membrane in sequence. After decolorization and deodorization, sterilization and canning, a small molecule pearl polypeptide liquid is obtained.

[0061] The mass ratio of the pearl layer powder and 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 100kHz and the power is 200W; the frequency of the microwave treatment is 2.9GHz and the power is 1200W; the magnetic field strength of the steady magnetic field is 9500Gs; the frequency of the electromagnetic oscillation field is 22HZ, and the medium and low duty cycle is 25% at this time; the centrifugal speed in step S2 is 2500r / min and the time is 15min; the organic acid solution in step S3 is malic acid solution; the mass percentage concentration of the organic acid solution in step S3 is 25wt%; the mass ratio of the precipitate B and the organic acid solution in step S3 is 1:3.

[0062] The speed of the secondary centrifugation in step S4 is 2500r / min, and the time is 15min; 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 temperature of the enzyme inactivation in step S5 is 98°C, and the time is 10min.

[0063] Example 4

[0064] A method for preparing a small molecule pearl polypeptide liquid containing multiple active ingredients comprises the following steps:

[0065] Step S1, multi-energy field assisted extraction: take commercially available pearl layer powder, perform ultrafine grinding, disperse it in pure water, heat it to 54° C., and perform multi-energy field assisted extraction for 3.5 hours;

[0066] Step S2, centrifugal separation: centrifuging the mixed solution obtained after the multi-energy field-assisted extraction, and collecting the supernatant A and the precipitate B respectively;

[0067] Step S3, dissolving calcium: taking precipitate B, adding organic acid solution, stirring and reacting for 4.5 hours, after the reaction is completed, using 1.1 mol / L NaOH solution to adjust the pH to 6.8;

[0068] Step S4, secondary centrifugation: the dispersion after dissolving the calcium is subjected to secondary centrifugation to obtain precipitate C, which is insoluble protein;

[0069] Step S5, enzymatic hydrolysis: the supernatant A and the precipitate C are mixed into a homogenate, and a composite plant protease is added. The enzyme is inactivated after hydrolysis at 52°C for 7.5 hours, and 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, and then decolorized, deodorized, sterilized and canned to obtain a small molecule pearl polypeptide liquid.

[0070] The mass ratio of the pearl layer powder and the 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 110kHz and the power is 230W; the frequency of the microwave treatment is 3.1GHz and the power is 1400W; the magnetic field strength of the steady magnetic field is 11000Gs; the frequency of the electromagnetic oscillation field is 27HZ, and the medium and low duty cycle is 28% at this time; the centrifugal speed in step S2 is 2900r / min and the time is 18min; the organic acid solution in step S3 is citric acid solution; the mass percentage concentration of the organic acid solution in step S3 is 28wt%; the mass ratio of the precipitate B and the organic acid solution in step S3 is 1:3.5.

[0071] The speed of the secondary centrifugation in step S4 is 2900r / min, and the time is 18min; 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 temperature of the enzyme inactivation in step S5 is 99°C, and the time is 11min.

[0072] Example 5

[0073] A method for preparing a small molecule pearl polypeptide liquid containing multiple active ingredients comprises the following steps:

[0074] Step S1, multi-energy field assisted extraction: take commercially available pearl layer powder, grind it into ultrafine powder, disperse it in pure water, heat it to 55° C., and perform multi-energy field assisted extraction for 4 hours;

[0075] Step S2, centrifugal separation: centrifuging the mixed solution obtained after the multi-energy field-assisted extraction, and collecting the supernatant A and the precipitate B respectively;

[0076] Step S3, dissolving calcium: taking precipitate B, adding organic acid solution, stirring and reacting for 5 hours, and after the reaction, adjusting the pH to 7 with a NaOH solution with a concentration of 1.2 mol / L;

[0077] Step S4, secondary centrifugation: the dispersion after dissolving the calcium is subjected to secondary centrifugation to obtain precipitate C, which is insoluble protein;

[0078] Step S5, enzymatic hydrolysis: the supernatant A and the precipitate C are mixed into a homogenate, and the composite plant protease is added, and the enzyme is inactivated after hydrolysis at 53°C for 8 hours, and the solution is filtered after cooling to 30°C, and then passed through a 3nm ultrafiltration membrane and an activated carbon fiber membrane in sequence, and then decolorized, deodorized, sterilized and canned to obtain a small molecule pearl polypeptide liquid.

[0079] The mass ratio of the pearl layer powder and the 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 120kHz and the power is 250W; the frequency of the microwave treatment is 3.2GHz and the power is 1500W; the magnetic field strength of the steady magnetic field is 12000Gs; the frequency of the electromagnetic oscillation field is 30HZ, and the medium and low duty cycle is 30% at this time; the centrifugal speed in step S2 is 3000r / min and the time is 20min; the organic acid solution in step S3 is malic acid solution; the mass percentage concentration of the organic acid solution in step S3 is 30wt%; the mass ratio of the precipitate B and the organic acid solution in step S3 is 1:4.

[0080] The speed of the secondary centrifugation in step S4 is 3000r / min, and the time is 20min; the mass ratio of the homogenate and 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 temperature of the enzyme inactivation in step S5 is 100°C, and the time is 12min.

[0081] Comparative Example 1

[0082] 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.

[0083] Comparative Example 2

[0084] A method for preparing a small molecule pearl polypeptide liquid containing multiple active ingredients is basically the same as that of Example 1, except that the composite plant protease is a mixture of bromelain and papain in a mass ratio of 1:5.

[0085] Experimental Example 1

[0086] 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 respectively, repeated three times, and the average value was taken. The results are shown in Tables 1 and 2 below.

[0087] Table 1 Active ingredient content

[0088]

[0089] Table 2 Peptide molecular weight distribution

[0090]

[0091] The results of active ingredient content in Table 1 show that the SOD superoxide dismutase content in the pearl polypeptide liquid obtained by the method in Examples 1 and 2 is relatively high, and this method can extract taurine substances in the pearl layer, fully retaining the active ingredients in the pearl. The molecular weight distribution in Table 2 shows that the pearl polypeptide liquid obtained by the method in Examples 1 and 2 has a molecular weight of most peptides less than 180, which is smaller than the molecular weight of pearl peptides currently published. Modern pharmacological studies have shown that small molecule peptides with a molecular weight of less than 500 are more conducive to rapid absorption by the human body.

[0092] 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 the absorption and utilization by the human body and can be used as a raw material for health food and cosmetics.

[0093] Experimental Example 2 Detection of Peptide Content in Pearl Peptide Liquid

[0094] The peptide contents of Example 1 and Example 2 were tested according to GB / T 22729-2008, and the test results were as follows:

[0095] Table 3 Peptide content in pearl polypeptide liquid

[0096]

[0097] In the prior art, the peptides extracted from pearls are 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, which greatly improves the extraction rate of pearl polypeptides.

[0098] Experimental Example 3: Antioxidant Activity Test of Pearl Peptide Liquid in Vitro

[0099] 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 to calculate the scavenging rate of DPPH free radicals. Take 5mL of the small molecule pearl polypeptide solution in each case and place it in a test tube. Add Tris-Hcl buffer to each test tube. After constant temperature, add preheated pyrogallol solution and measure its absorbance. Read the absorbance every 30s for 5 minutes. Draw a standard curve with absorbance and time. Take VC as the control group to calculate the superoxide anion (O2 － ) free radical scavenging rate. Take 0.2mL of the small molecule pearl polypeptide solution in each case, add it to each test tube with ABTS solution, and measure its absorbance after standing reaction. Take VC as the control group and calculate the ABTS scavenging rate. Take 5mL of the small molecule pearl polypeptide solution in each case, place it in a test tube, add salicylic acid-ethanol solution, ferrous sulfate solution and hydrogen peroxide solution respectively, and measure its absorbance after reaction. Take VC as the control group and measure its hydroxyl (OH － )Free radical scavenging rate.

[0100] Table 4 Antioxidant test results of pearl peptide hydrolyzate

[0101]

[0102] As can be seen from Table 4, the small molecule pearl polypeptide solution prepared by the present invention has a significant effect on DPPH free radicals and O2 － It has a strong scavenging effect, but its scavenging ability for ABTS free radicals and hydroxyls is relatively weak. The products made from each embodiment of the present invention have higher antioxidant activity than the products of the comparative examples, and the combined use of multi-energy field assistance and ficin is beneficial to improving the above performance.

[0103] Experimental Example 4: Experimental verification of the effect of small molecule pearl polypeptide liquid in delaying aging

[0104] 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.

[0105] Experimental materials: Drosophila melanogaster, culture medium: 8.0 g corn flour, 7.5 g glucose, 0.7 g agar, 2.0 g yeast powder, 0.2 mL 15% benzoic acid and 0.2 mL propionic acid. The small molecule pearl polypeptide solution in Example 1 was added to the culture medium in proportion.

[0106] Experimental process: Drosophila melanogaster within 10 hours of eclosion were collected and grouped according to gender. After ether anesthesia, 100 male and female fruit flies were collected and divided into two groups. The fruit flies were placed in Φ2.3×12cm test tubes, 10wt% small molecule pearl polypeptide liquid was added to the experimental group, and only distilled water was added to the control group. Then the test tubes were placed in an environment of 25±1℃ and 60%-70% humidity for incubation. The culture medium was changed every 3 days, and the number of dead fruit flies was recorded every day.

[0107] Experimental results: Compared with the control group, the small molecule pearl polypeptide liquid can significantly increase the average lifespan of fruit flies, and the effect is more obvious in female fruit flies than in male fruit flies. The average lifespan of female fruit flies increased from 30.1±3.2 days to 41.5±2.3 days, and the average lifespan of male fruit flies increased from 29.9±1 days to 38.3±6 days.

[0108] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the contents of the present invention and implement them accordingly. They cannot be used to limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a small molecule pearl polypeptide liquid containing multiple active ingredients, characterized in that: The steps include: Step S1, multi-energy field assisted extraction: take commercially available pearl layer powder, perform ultrafine grinding, disperse it in pure water, heat it to 50°C to 55°C, and perform multi-energy field assisted extraction for 2h to 4h; the multi-energy field includes ultrasound, microwave, steady magnetic field and electromagnetic oscillation field; the frequency of the ultrasound is 80-120kHz, and the power is 150-250W; the frequency of the microwave treatment is 2.5-3.2GHz, and the power is 600-1500W; the magnetic field strength of the steady magnetic field is 6000-12000Gs; The frequency of the electromagnetic oscillation field is 15-30HZ, and the medium and low duty cycle is 20-30%; Step S2, centrifugal separation: 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, after the reaction is completed, using NaOH solution with a concentration of 0.8-1.2 mol / L to adjust the pH to 6-7; 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 enzyme is inactivated after hydrolysis at 50±3°C for 6-8h, and the mixture is cooled to 30°C and filtered, and the mixture is passed through a 1-3nm ultrafiltration membrane and an activated carbon fiber membrane in sequence, and then decolorized, deodorized, sterilized and canned to obtain a small molecule pearl polypeptide liquid; 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 liquid 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 liquid 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 liquid 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 liquid containing multiple active ingredients according to claim 1, characterized in that: The mass ratio of the precipitate B and 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 liquid 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 liquid 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 min.

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