Preparation method of pdrn with soothing anti-wrinkle effect, product and application thereof

Abstract

The application belongs to the technical field of substance extraction, and relates to a preparation method of PDRN with soothing and anti-wrinkle effects, a product and application thereof, and the preparation method comprises the following steps: mixing a strain with a culture medium containing a promoter, and fermenting to obtain a fermentation liquor; centrifuging the fermentation liquor to obtain a bacterial slurry, mixing the bacterial slurry with a lysis solution to lyse, and obtaining a lysis product; mixing the lysis product with a compound enzyme to perform enzymolysis, and obtaining an enzymolysis product; centrifuging the enzymolysis product, mixing the obtained supernatant with a DNA stabilizer, and performing high-temperature denaturation to obtain a denaturation product; centrifuging the denaturation product to obtain supernatant, filtering the supernatant through a filter membrane, and obtaining the PDRN.The preparation method avoids the use of an organic solvent, the addition of the DNA stabilizer can improve the stability of nucleic acids, and the prepared PDRN has excellent soothing and anti-wrinkle effects.

Description

A method for preparing PDRN with soothing and anti-wrinkle effects, its products and applications Technical Field

[0001] This invention belongs to the field of material extraction technology, and relates to a method for preparing PDRN with soothing and anti-wrinkle effects, as well as its products and applications. Background Technology

[0002] The development of polydeoxyribonucleotides (PDRN) stems from the market demand for highly effective repair and anti-aging ingredients and the scientific exploration of natural active substances. In the early days, PDRN was mainly extracted from salmon testes, but this method suffered from pain points such as scarce raw materials, complex extraction, extremely low yield, and high cost. Moreover, traditional extraction methods are constrained by resources and may also pose a risk of biological contamination, making it difficult to meet the needs of large-scale applications.

[0003] Probiotic PDRNs have achieved a core breakthrough through synthetic biology technology, becoming an innovative direction of interest in the industry. Currently, there are only a few types of PDRNs derived from probiotics, and the utilization of probiotics is not yet fully realized. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for preparing PDRN with soothing and anti-wrinkle effects, as well as its products and applications.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for preparing PDRN with soothing and anti-wrinkle effects, the method comprising:

[0007] (1) Mix the strain with a culture medium containing a promoter and ferment to obtain a fermentation broth;

[0008] (2) Centrifuge the fermentation broth to obtain bacterial sludge, mix the bacterial sludge with the lysis solution to obtain lysis products;

[0009] (3) The lysis products are mixed with a complex enzyme for enzymatic hydrolysis to obtain the hydrolysis products;

[0010] (4) Centrifuge the enzymatic hydrolysis product, mix the resulting supernatant with DNA stabilizer, and denature at high temperature to obtain the denatured product;

[0011] (5) Centrifuge the denatured product, collect the supernatant, and filter it through a filter membrane to obtain the product;

[0012] The promoters include D-ribose, 5-phosphate ribose, citric acid, vitamin B1, and white truffle.

[0013] This invention utilizes acid-resistant strains and adds nucleic acid production promoters to the fermentation medium to improve nucleic acid yield. Proteolytic hydrolysis avoids the use of organic reagents (such as isopropanol or chloroform), and the addition of nucleic acid stabilizers improves the stability of nucleic acids between -20℃ and 45℃, while also enhancing efficacy. D-ribose, 5-phosphate ribose, citric acid, vitamin B1, and white truffle have a synergistic effect in promoting strain proliferation and enhancing PDRN efficacy.

[0014] Preferably, the strain includes an acid-resistant strain.

[0015] Preferably, the strain includes at least one of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium animalis, Lactobacillus plantarum, Lactobacillus casei, or Lactobacillus paracasei.

[0016] When using more than one strain for PDRN preparation, the strain is mixed with the culture medium separately and fermented to obtain fermentation broth. After centrifuging the fermentation broth to obtain bacterial sludge, the bacterial sludge obtained from fermentation of different strains is mixed for subsequent operations.

[0017] Preferably, the promoter comprises, by weight parts, 0.01-0.2 parts of D-ribose, 0.01-0.3 parts of 5-phosphate ribose, 0.01-0.1 parts of citric acid, 0.01-0.1 parts of vitamin B1 and 0.5-2 parts of white truffle.

[0018] The mass fractions of D-ribose can be selected from 0.01 parts, 0.02 parts, 0.05 parts, 0.08 parts, 0.1 parts, 0.12 parts, 0.15 parts, 0.18 parts, 0.2 parts, etc., and the mass fractions of 5-phosphate ribose can be selected from 0.01 parts, 0.02 parts, 0.05 parts, 0.08 parts, 0.1 parts, 0.12 parts, 0.15 parts, 0.18 parts, 0.2 parts, 0.22 parts, 0.25 parts, 0.28 parts, 0.3 parts, etc. The mass fractions of citric acid can be selected from 0.01, 0.02, 0.05, 0.08, 0.1, etc.; the mass fractions of vitamin B1 can be selected from 0.01, 0.02, 0.05, 0.08, 0.1, etc.; and the mass fractions of white truffles can be selected from 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, etc. Other specific values ​​within the above ranges can be selected, which will not be elaborated here.

[0019] Preferably, the basal medium used in the culture medium containing the promoter is MRS medium.

[0020] Preferably, the mass percentage of the promoter in the culture medium containing the promoter is 0.04-2.7%, for example, 0.04%, 0.08%, 0.1%, 0.3%, 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.7%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0021] Preferably, the fermentation method is static fermentation.

[0022] Preferably, the fermentation temperature is 35-38℃ and the time is 10-24 h.

[0023] Temperatures can be selected from 35℃, 36℃, 37℃, 38℃, etc., and fermentation times can be selected from 10 h, 12 h, 15 h, 18 h, 20 h, 22 h, 24 h, etc. Other specific values ​​within the above range can also be selected, which will not be elaborated here.

[0024] Preferably, the centrifugation speed is independently 8000-12000 rpm and the time is independently 4-10 min.

[0025] The speed can be selected from 8000 rpm, 8500 rpm, 9000 rpm, 9500 rpm, 10000 rpm, 10500 rpm, 11000 rpm, 11500 rpm, 12000 rpm, etc., and the time can be selected from 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc. Other specific values ​​within the above range can be selected, which will not be described in detail here.

[0026] Preferably, the fungal mud in step (2) includes single-strain fungal mud or a composite fungal mud made by mixing different single-strain fungal muds.

[0027] Preferably, the compound bacterial mud includes Bifidobacterium animalis bacterial mud and Lactobacillus paracasei bacterial mud.

[0028] Preferably, the ratio of viable bacteria counts of the Bifidobacterium animalis sludge to Lactobacillus paracasei sludge is (1-5):(0.1-2).

[0029] The specific point values ​​in (1-5) can all be selected from 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc., and the specific point values ​​in (0.1-2) can all be selected from 0.1, 0.2, 0.5, 0.8, 1, 1.2, 1.5, 1.8, 2, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0030] Using different types of probiotics in combination can fully utilize their self-produced nucleases, reduce long-fragment nucleic acid residues, and make PDRN distribution more uniform. In addition, the powerful combination of different types of probiotic PDRN can also enhance its soothing and anti-wrinkle effects.

[0031] Preferably, the viable count of the mycelium sludge is 1×10⁻⁶. 11 CFU / g-5×10 11 CFU / g, for example 1×10 11 CFU / g, 1.2×10 11 CFU / g, 1.5×10 11 CFU / g, 1.8×10 11 CFU / g, 2×10 11 CFU / g, 2.5×10 11 CFU / g, 3×10 11 CFU / g, 3.5×10 11 CFU / g, 4×10 11 CFU / g, 4.5×10 11 CFU / g, 5×10 11 CFU / g, etc., other specific point values ​​within the above range can be selected, and will not be elaborated on here.

[0032] Preferably, the ratio of the bacterial sludge to the lysis liquid is (2-10) g:(100-200) mL.

[0033] The specific point values ​​in (2-10) can all be selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., and the specific point values ​​in (100-200) can all be selected from 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0034] Preferably, the pyrolysis temperature is 35-38°C and the time is 1-18 h.

[0035] Temperatures can be selected from 35℃, 36℃, 37℃, 38℃, etc., and time can be selected from 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0036] Preferably, the lysis buffer comprises, in molar concentration, 10-20 mM Tris and 1-5 mM EDTA-disodium, and in mass percentage, 0.1-0.3% SDS and 0.1-0.3% lysozyme, with water as the solvent.

[0037] The molar concentration of Tris can be selected from 10 mM, 12 mM, 15 mM, 18 mM, 20 mM, etc.; the molar concentration of EDTA-disodium can be selected from 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, etc.; the mass percentage of SDS can be selected from 0.1%, 0.12%, 0.15%, 0.18%, 0.2%, 0.22%, 0.25%, 0.28%, 0.3%, etc.; the mass percentage of lysozyme can be selected from 0.1%, 0.12%, 0.15%, 0.18%, 0.2%, 0.22%, 0.25%, 0.28%, 0.3%, etc. Other specific values ​​within the above ranges can be selected, which will not be elaborated here.

[0038] Preferably, the complex enzyme includes papain and bromelain.

[0039] Preferably, the mass ratio of papain to bromelain is (1-2):(1-2).

[0040] The specific point values ​​in (1-2) can all be selected from 1, 1.2, 1.4, 1.6, 1.8, 2, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0041] Preferably, the mass ratio of the lysis product to the complex enzyme is (100-200):(0.1-1).

[0042] The specific point values ​​in (0.1-1) can be selected from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc., and the specific point values ​​in (100-200) can be selected from 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0043] Preferably, the enzymatic hydrolysis is performed at a temperature of 30-60°C for 1-2 hours.

[0044] The enzymatic hydrolysis temperature can be selected from 30℃, 32℃, 35℃, 38℃, 40℃, 42℃, 45℃, 48℃, 50℃, 52℃, 55℃, 58℃, 60℃, etc., and the time can be selected from 1 h, 1.2 h, 1.4 h, 1.6 h, 1.8 h, 2 h, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0045] Preferably, the amount of DNA stabilizer added is 0.1-2% of the mass of the supernatant, such as 0.1%, 0.3%, 0.5%, 0.7%, 0.9%, 1.1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0046] Preferably, the DNA stabilizer comprises any one or a combination of at least two of polyethyleneimine, spermidine, spermine, or chondroitin sulfate.

[0047] Preferably, the DNA stabilizer is spermidine.

[0048] Preferably, the mixing temperature with the DNA stabilizer is 30-80°C, and the mixing time is 30-60 min.

[0049] Temperatures can be selected from 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 65℃, 70℃, 75℃, 80℃, etc., and time can be selected from 30 min, 35 min, 45 min, 45 min, 50 min, 55 min, 60 min, etc. Other specific values ​​within the above range can also be selected, which will not be elaborated here.

[0050] Preferably, the high-temperature denaturation time is 85-95℃ and the time is 30-60 min.

[0051] Temperatures can be selected from 85℃, 88℃, 90℃, 92℃, 95℃, etc., and time can be selected from 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, etc. Other specific values ​​within the above range can also be selected, which will not be elaborated here.

[0052] Secondly, the present invention provides a PDRN prepared according to the preparation method of PDRN with soothing and anti-wrinkle effects described in the first aspect.

[0053] Thirdly, the present invention provides the application of PDRN according to the second aspect in the preparation of products with soothing and anti-wrinkle effects.

[0054] Preferably, the product includes cosmetics or skincare products.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] (1) In terms of fermentation method, the nucleic acid yield can be improved by adding substrates or promoters that promote nucleic acid production.

[0057] (2) In terms of process, protein precipitation is replaced by protease hydrolysis, and nucleic acid stabilizers are added to coordinate and improve the stability of nucleic acids between -20℃ and 45℃.

[0058] (3) In terms of efficacy, the compound lysis of different types of probiotics can make full use of their own nucleases, reduce long-fragment nucleic acid residues, and make PDRN distribution more uniform; the strong combination of different types of probiotic PDRN can also enhance its soothing effect. Attached Figure Description

[0059] Figure 1 is a PDRN agarose gel electrophoresis image.

[0060] Figure 2 is an agarose gel electrophoresis diagram verifying the stability of PDRN.

[0061] Figure 3 shows the test results of the anti-wrinkle efficacy. Detailed Implementation

[0062] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0063] The white truffles were purchased from Yunnan Boshengjia Company and were marketed as quick-frozen white truffles.

[0064] The lysozyme was purchased from Jiahe Xuri Company under the product name "lysozyme";

[0065] Papain was purchased from Jiahe Xuri Company and its product is called Papain.

[0066] The bromelain was purchased from Jiahe Xuri Company under the product name "Bromelain".

[0067] The Lactobacillus plantarum YK-L002 mentioned below is classified as Lactiplantibacillus plantarum, with accession number CGMCC No.29289, accession date of December 11, 2023, and deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0068] The Lactobacillus paracasei mentioned below is classified as Lactobacillus paracasei, with accession number CGMCC No. 29288, accession date of December 11, 2023, and deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0069] The Bifidobacterium animalis YK-L007 mentioned below is classified as Bifidobacterium animalis, with accession number CGMCC No.37117, accession date of December 19, 2025, and deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0070] Preparation Example 1

[0071] This preparation example provides a culture medium containing an accelerator, wherein the culture medium is based on MRS medium and further comprises, by weight percentage, 0.1% D-ribose, 0.1% ribose 5-phosphate, 0.01% citric acid, 0.01% vitamin B1, and 1% white truffle.

[0072] The preparation method is as follows: D-ribose, 5-phosphate ribose, citric acid, vitamin B1, white truffle and MRS medium are mixed and sterilized at 121℃ for 20 min to obtain the product.

[0073] Preparation Example 2

[0074] This preparation example provides a culture medium containing an accelerator, wherein the culture medium is based on MRS medium and further comprises, by weight percentage, 0.05% D-ribose, 0.3% ribose 5-phosphate, 0.1% citric acid, 0.05% vitamin B1, and 0.5% white truffle.

[0075] The preparation method is the same as in Preparation Example 1.

[0076] Preparation Example 3

[0077] This preparation example provides a culture medium containing an accelerator, wherein the culture medium is based on MRS medium and further comprises, by weight percentage, 0.2% D-ribose, 0.05% ribose 5-phosphate, 0.05% citric acid, 0.1% vitamin B1, and 2% white truffle.

[0078] The preparation method is the same as in Preparation Example 1.

[0079] Preparation Example 4

[0080] This preparation example provides a culture medium containing an accelerator, which differs from Preparation Example 1 only in that it does not contain D-ribose, and the reduced mass of D-ribose is proportionally allocated to the mass of 5-phosphoribose, citric acid, vitamin B1, and white truffle, while other components and contents remain unchanged.

[0081] The preparation method is the same as in Preparation Example 1.

[0082] Preparation Example 5

[0083] This preparation example provides a culture medium containing an accelerator, which differs from Preparation Example 1 only in that it does not contain ribose 5-phosphate, and the reduced mass of ribose is proportionally allocated to the mass of D-ribose, citric acid, vitamin B1, and white truffle, while other components and contents remain unchanged.

[0084] The preparation method is the same as in Preparation Example 1.

[0085] Preparation Example 6

[0086] This preparation example provides a culture medium containing an accelerator, which differs from Preparation Example 1 only in that it does not contain citric acid, and the reduced mass of citric acid is proportionally allocated to the mass of D-ribose, 5-phosphate ribose, vitamin B1, and white truffle, while other components and contents remain unchanged.

[0087] The preparation method is the same as in Preparation Example 1.

[0088] Preparation Example 7

[0089] This preparation example provides a culture medium containing an accelerator, which differs from Preparation Example 1 only in that it does not contain vitamin B1. The reduced mass of vitamin B1 is proportionally allocated to the mass of D-ribose, 5-phosphate ribose, citric acid, and white truffle, while other components and contents remain unchanged.

[0090] The preparation method is the same as in Preparation Example 1.

[0091] Preparation Example 8

[0092] This preparation example provides a culture medium containing an accelerator, which differs from Preparation Example 1 only in that it does not contain white truffles, and the reduced mass of white truffles is proportionally allocated to the mass of D-ribose, 5-phosphate ribose, citric acid, and vitamin B1, while other components and contents remain unchanged.

[0093] The preparation method is the same as in Preparation Example 1.

[0094] Example 1

[0095] This embodiment provides a method for preparing PDRN with soothing and anti-wrinkle effects, the preparation method comprising:

[0096] (1) Mix Bifidobacterium animalis YK-L007 with MRS medium and incubate at 37℃ for 15 h to obtain Bifidobacterium animalis YK-L007 seed culture;

[0097] (2) The seed culture of Bifidobacterium animalis YK-L007 (OD) 600 (Controlled at around 1.8) Inoculate into the culture medium described in Preparation Example 1 at an inoculation rate of 3 vt%, and incubate at 37°C for 12 h to obtain fermentation broth;

[0098] (3) Centrifuge the fermentation broth at 10,000 rpm for 10 min to collect the bacterial sludge, and dilute the viable count of the bacterial sludge with purified water to 1×10⁻⁶. 11 The CFU / g was then mixed with the lysis buffer at a feed-to-liquid ratio of 1 g: 50 mL and lysed at 37°C for 12 h to obtain the lysis buffer. The lysis buffer contained Tris 10 mM, EDTA-disodium 2 mM in molar concentration, and included SDS 0.1%, lysozyme 0.2%, and water in mass percentage.

[0099] (4) Centrifuge the lysate at 10,000 rpm for 10 min and collect the supernatant. Mix the supernatant with papain and bromelain. The amount of papain added is 0.2% of the mass of the supernatant, and the amount of bromelain added is 0.2% of the mass of the supernatant. Enzymatically hydrolyze at 45℃ for 1 h to obtain the hydrolysate.

[0100] (5) Mix the enzymatic hydrolysate with spermidine, the amount of spermidine added is 1% of the mass of the enzymatic hydrolysate, react at 30℃ for 45 min, treat at 90℃ for 30 min, centrifuge at 10000rpm for 10 min to collect the filtrate, filter through a 0.1 μm filter membrane to obtain the final product.

[0101] Example 2

[0102] This embodiment provides a method for preparing PDRN with soothing and anti-wrinkle effects, the preparation method comprising:

[0103] (1) Mix Lactobacillus paracasei YK-L003 with MRS medium and incubate at 37℃ for 10 h to obtain Lactobacillus paracasei YK-L003 seed culture;

[0104] (2) The seed culture of Lactobacillus paracasei YK-L003 (OD) 600 (Controlled at around 1.8) Inoculate into the culture medium described in Preparation Example 2 at an inoculation rate of 3 vt%, and incubate at 37°C for 10 h to obtain fermentation broth;

[0105] (3) Centrifuge the fermentation broth at 10,000 rpm for 10 min to collect the bacterial sludge, and dilute the viable count of the bacterial sludge with purified water to 1×10⁻⁶. 11The CFU / g was then mixed with the lysis buffer at a feed-to-liquid ratio of 1 g: 50 mL and lysed at 35°C for 18 h to obtain the lysis buffer. The lysis buffer contained Tris 15 mM, EDTA-disodium 5 mM, and, by mass percentage, SDS 0.2%, lysozyme 0.1%, and water.

[0106] (4) Centrifuge the lysate at 10,000 rpm for 10 min and collect the supernatant. Mix the supernatant with papain and bromelain. The amount of papain added is 0.4% of the mass of the supernatant, and the amount of bromelain added is 0.2% of the mass of the supernatant. Enzymatically hydrolyze at 30℃ for 2 h to obtain the hydrolysate.

[0107] (5) Mix the enzymatic hydrolysate with spermidine. The amount of spermidine added is 0.8% of the mass of the enzymatic hydrolysate. After reacting at 50°C for 60 min, treat at 85°C for 30 min, centrifuge at 10000 rpm for 10 min to collect the filtrate, and filter it through a 0.1 μm filter membrane to obtain the final product.

[0108] Example 3

[0109] This embodiment provides a method for preparing PDRN with soothing and anti-wrinkle effects, the preparation method comprising:

[0110] (1) Mix Lactobacillus plantarum YK-L002 with MRS medium and incubate at 37℃ for 24 h to obtain Lactobacillus plantarum YK-L002 seed culture;

[0111] (2) The seed culture of Lactobacillus plantarum YK-L002 (OD) 600 (Controlled at around 1.8) Inoculate into the culture medium described in Preparation Example 3 at an inoculation rate of 3 vt%, and incubate at 37°C for 24 h to obtain fermentation broth;

[0112] (3) Centrifuge the fermentation broth at 10,000 rpm for 10 min to collect the bacterial sludge, and dilute the viable count of the bacterial sludge with purified water to 1×10⁻⁶. 11 The CFU / g was then mixed with the lysis buffer at a feed-to-liquid ratio of 1 g: 50 mL and lysed at 38°C for 2 h to obtain the lysis buffer. The lysis buffer contained Tris 20 mM, EDTA-disodium 1 mM, and, by mass percentage, SDS 0.3%, lysozyme 0.3%, and water.

[0113] (4) Centrifuge the lysate at 10,000 rpm for 10 min and collect the supernatant. Mix the supernatant with papain and bromelain. The amount of papain added is 0.1% of the mass of the supernatant, and the amount of bromelain added is 0.2% of the mass of the supernatant. Enzymatically hydrolyze at 60℃ for 1 h to obtain the hydrolysate.

[0114] (5) Mix the enzymatic hydrolysate with spermidine, the amount of spermidine added is 2% of the mass of the enzymatic hydrolysate, react at 80℃ for 30 min, treat at 95℃ for 30 min, centrifuge at 10000 rpm for 10 min to collect the filtrate, filter through a 0.1 μm filter membrane to obtain the final product.

[0115] Example 4

[0116] This embodiment provides a method for preparing PDRN with soothing and anti-wrinkle effects. The only difference between this method and Example 1 is that step (2) is "to prepare Bifidobacterium animalis YK-L007 seed liquid (OD)". 600 (Controlled at around 1.8%) Inoculate into the culture medium described in Preparation Example 4 at an inoculation rate of 3 vt%, and incubate at 37°C for 12 h to obtain fermentation broth. Other operations remain unchanged.

[0117] Example 5

[0118] This embodiment provides a method for preparing PDRN with soothing and anti-wrinkle effects. The only difference between this method and Example 1 is that step (2) is "to prepare Bifidobacterium animalis YK-L007 seed liquid (OD)". 600 (Controlled at around 1.8%) Inoculate into the culture medium described in Preparation Example 5 at an inoculation rate of 3 vt%, and incubate at 37°C for 12 h to obtain fermentation broth. Other operations remain unchanged.

[0119] Example 6

[0120] This embodiment provides a method for preparing PDRN with soothing and anti-wrinkle effects. The only difference between this method and Example 1 is that step (2) is "to prepare Bifidobacterium animalis YK-L007 seed liquid (OD)". 600 (Controlled at around 1.8%) Inoculate into the culture medium described in Preparation Example 6 at an inoculation rate of 3 vt%, and incubate at 37°C for 12 h to obtain fermentation broth. Other operations remain unchanged.

[0121] Example 7

[0122] This embodiment provides a method for preparing PDRN with soothing and anti-wrinkle effects. The only difference between this method and Example 1 is that step (2) is "to prepare Bifidobacterium animalis YK-L007 seed liquid (OD)". 600 (Controlled at around 1.8%) Inoculate into the culture medium described in Preparation Example 7 at an inoculation rate of 3 vt%, and incubate at 37°C for 12 h to obtain fermentation broth. Other operations remain unchanged.

[0123] Example 8

[0124] This embodiment provides a method for preparing PDRN with soothing and anti-wrinkle effects. The only difference between this method and Example 1 is that step (2) is "to prepare Bifidobacterium animalis YK-L007 seed liquid (OD)".600 (Controlled at around 1.8%) Inoculate into the culture medium described in Example 8 at an inoculation rate of 3 vt%, and incubate at 37°C for 12 h to obtain fermentation broth. Other operations remain unchanged.

[0125] Example 9

[0126] This embodiment provides a method for preparing PDRN with soothing and anti-wrinkle effects. The only difference between this embodiment and Example 1 is that step (4) is to "centrifuge the lysate at 10,000 rpm for 10 min to collect the supernatant, mix the supernatant with papain, add papain at 0.4% of the mass of the supernatant, and enzymatically hydrolyze at 45°C for 1 h to obtain the hydrolysate". Other operations remain unchanged.

[0127] Example 10

[0128] This embodiment provides a method for preparing PDRN with soothing and anti-wrinkle effects. The only difference between this embodiment and Example 1 is that step (4) is to "centrifuge the lysate at 10,000 rpm for 10 min to collect the supernatant, mix the supernatant with bromelain, the amount of bromelain added is 0.4% of the mass of the supernatant, and enzymatically hydrolyze at 45°C for 1 h to obtain the enzymatic hydrolysate". Other operations remain unchanged.

[0129] Example 11

[0130] This embodiment provides a method for preparing PDRN with soothing and anti-wrinkle effects, the preparation method comprising:

[0131] (1) Bifidobacterium animalis YK-L007 and Lactobacillus paracasei YK-L003 were each independently mixed with MRS medium and cultured at 37℃ for 15 h to obtain seed culture of Bifidobacterium animalis YK-L007 and seed culture of Lactobacillus paracasei YK-L003.

[0132] (2) The seed culture of Bifidobacterium animalis YK-L007 (OD) 600 (Controlled at around 1.8) and Lactobacillus paracasei seed culture YK-L003 (OD) 600 (Controlled at around 1.8%) Each was independently inoculated into the culture medium described in Preparation Example 1 at an inoculation rate of 3 vt%, and cultured statically at 37°C for 12 h to obtain fermentation broth of Bifidobacterium animalis YK-L007 and fermentation broth of Lactobacillus paracasei YK-L003.

[0133] (3) Collect the bacterial sludge by centrifuging Bifidobacterium animalis YK-L007 fermentation broth and Lactobacillus paracasei YK-L003 fermentation broth at 10000 rpm for 10 min respectively. Mix the Bifidobacterium animalis YK-L007 bacterial sludge and Lactobacillus paracasei YK-L003 bacterial sludge at a viable cell ratio of 4:1. Dilute the compound bacterial sludge with purified water to 1×10⁻⁶.11 The CFU / g was then mixed with the lysis buffer at a feed-to-liquid ratio of 1 g: 50 mL and lysed at 37°C for 12 h to obtain the lysis buffer. The lysis buffer contained Tris 10 mM, EDTA-disodium 2 mM in molar concentration, and included SDS 0.1%, lysozyme 0.2%, and water in mass percentage.

[0134] (4) Centrifuge the lysate at 10,000 rpm for 10 min and collect the supernatant. Mix the supernatant with papain and bromelain. The amount of papain added is 0.2% of the mass of the supernatant, and the amount of bromelain added is 0.2% of the mass of the supernatant. Enzymatically hydrolyze at 45℃ for 1 h to obtain the hydrolysate.

[0135] (5) Mix the enzymatic hydrolysate with spermidine, the amount of spermidine added is 1% of the mass of the enzymatic hydrolysate, react at 30℃ for 45 min, treat at 90℃ for 30 min, centrifuge at 10000rpm for 10 min to collect the filtrate, filter through a 0.1 μm filter membrane to obtain the final product.

[0136] Example 12

[0137] This embodiment provides a method for preparing PDRN with soothing and anti-wrinkle effects. The only difference between this embodiment and Example 1 is that Bifidobacterium animalis YK-L007 is replaced with Lactobacillus paracasei YK-L003, while other operations remain unchanged.

[0138] Figure 1 shows the gel electrophoresis results of PDRN prepared from Lactobacillus paracasei YK-L003 (Example 12), PDRN prepared from Bifidobacterium animalis YK-L007 (Example 1), and a composite PDRN (Example 11). Both the PDRN prepared in Example 12 and Example 1 showed long nucleic acid fragments (long bands in the electrophoresis results), while the composite PDRN prepared in Example 11 did not show any long bands. This indicates that the combined lysis of different types of probiotics can result in a more uniform distribution of PDRN.

[0139] Example 13

[0140] This embodiment provides a method for preparing PDRN with soothing and anti-wrinkle effects, the preparation method comprising:

[0141] (1) Bifidobacterium animalis YK-L007 and Lactobacillus paracasei YK-L003 were each independently mixed with MRS medium and cultured at 37℃ for 15 h to obtain seed culture of Bifidobacterium animalis YK-L007 and seed culture of Lactobacillus paracasei YK-L003.

[0142] (2) The seed culture of Bifidobacterium animalis YK-L007 (OD)600 (Controlled at around 1.8) and Lactobacillus paracasei seed culture YK-L003 (OD) 600 Each of the two bacteria (controlled at around 1.8%) was independently inoculated into the culture medium described in Preparation Example 1 at an inoculation rate of 3 vt%, and cultured statically at 37°C for 15 h to obtain fermentation broth of Bifidobacterium animalis YK-L007 and fermentation broth of Lactobacillus paracasei YK-L003.

[0143] (3) The fermentation broths of Bifidobacterium animalis YK-L007 and Lactobacillus paracasei YK-L003 were centrifuged at 10,000 rpm for 10 min to collect bacterial sludge. The bacterial sludge of Bifidobacterium animalis YK-L007 and Lactobacillus paracasei YK-L003 were diluted with purified water to a concentration of 2×10⁻⁶. 11 The CFU / g was then mixed with the lysis buffer at a ratio of 1 g:50 mL and lysed at 37°C for 12 h to obtain Bifidobacterium animalis YK-L007 lysis buffer and Lactobacillus paracasei YK-L003 lysis buffer, respectively. The lysis buffer contained Tris 10 mM, EDTA-disodium 2 mM, and, by mass percentage, SDS 0.1%, lysozyme 0.2%, and water.

[0144] (4) Centrifuge the lysate of Bifidobacterium animalis YK-L007 and the lysate of Lactobacillus paracasei YK-L003 at 10000 rpm for 10 min and collect the supernatant. Mix the supernatant with papain and bromelain respectively. The amount of papain added is 0.2% of the mass of the supernatant and the amount of bromelain added is 0.2% of the mass of the supernatant. Enzymatic hydrolysis is carried out at 45℃ for 1 h to obtain the enzymatic hydrolysate of Bifidobacterium animalis YK-L007 and the enzymatic hydrolysate of Lactobacillus paracasei YK-L003.

[0145] (5) Mix the enzymatic hydrolysate of Bifidobacterium animalis YK-L007 and Lactobacillus paracasei YK-L003 with spermidine respectively. The amount of spermidine added is 1% of the mass of the enzymatic hydrolysate. After reacting at 30℃ for 45 min, treat at 90℃ for 30 min, centrifuge at 10000 rpm for 10 min to collect the filtrate, filter through a 0.1 μm filter membrane, and mix the filtrate of Bifidobacterium animalis YK-L007 with the filtrate of Lactobacillus paracasei YK-L003 in equal volumes to obtain the final product.

[0146] Comparative Example 1

[0147] This comparative example provides a method for preparing PDRN with soothing and anti-wrinkle effects. The only difference between this method and Example 1 is that step (5) is to "treat the enzymatic hydrolysate at 90°C for 30 min, centrifuge at 10000 rpm for 10 min to collect the filtrate, filter it through a 0.1 μm filter membrane, and freeze-dry it to obtain the product". Other operations remain unchanged.

[0148] Comparative Example 2

[0149] This comparative example provides a method for preparing PDRN with soothing and anti-wrinkle effects. The only difference between this method and Example 1 is that step (2) is "to prepare Bifidobacterium animalis YK-L007 seed liquid (OD)". 600 (Controlled at around 1.8%) Inoculate into MRS medium at a rate of 3 vt%, and incubate at 37°C for 15 h to obtain fermentation broth. Other operations remain unchanged.

[0150] Test Example 1

[0151] Nucleic acid content detection

[0152] Test method: Turn on the NanoDrop ultra-micro spectrophotometer and preheat for 15 minutes. Select the nucleic acid detection mode, use the lysis buffer as a blank control for baseline calibration, take 1~2 μL of PDRN stock solution into the instrument detection platform, start the detection, and the nucleic acid content will be automatically output.

[0153] Table 1

[0154]

[0155] The nucleic acid content detection results show that, compared with Comparative Example 2 without the addition of promoters, the nucleic acid content of Examples 1 and Examples 4-8 with the addition of different promoters (D-ribose, 5-phosphate ribose, citric acid, vitamin B1, white truffle) is relatively high. This indicates that the addition of promoters is beneficial to promoting nucleic acid generation and thus increasing the concentration and yield of nucleic acid.

[0156] Test Example 2

[0157] Stability test

[0158] The stability results of nucleic acid content after being placed at room temperature for 28 days are shown in Table 2.

[0159] Table 2

[0160]

[0161] After being placed at room temperature for 28 days, the nucleic acid degradation rate of the samples with added nucleic acid stabilizers (Examples 1-3, Examples 11-13) was significantly lower than that of the samples without added nucleic acid stabilizers (Comparative Example 1).

[0162] In addition, the Lactobacillus plantarum YK-L002PDRN prepared in Example 3 was detected by gel electrophoresis. The PDRN was treated at high temperature (45℃), room temperature, and frozen (-20℃) for 28 days and then detected by gel electrophoresis. The results are shown in Figure 2.

[0163] As shown in Figure 2, the PDRN prepared by this invention has strong stability.

[0164] Test Example 3

[0165] Soothing efficacy test

[0166] Test method:

[0167] Hyaluronidase was dissolved in 0.4% BSA solution to prepare a 0.5 U / mL enzyme working solution. Hyaluronic acid (HA) substrate was diluted with purified water to 0.5 mg / mL. 50 μL of the test sample, 50 μL of the hyaluronidase working solution, and 100 μL of the hyaluronic acid (HA) substrate were added to establish a sample group, a blank control group, and a positive control group, with three replicates for each group. After pre-incubation at 37℃ for 30 min, 50 μL of 0.1 mol / L NaOH solution was added to terminate the reaction. 50 μL of 0.2 mol / L acetylacetone was added and the mixture was boiled in a water bath for 15 min. After cooling, 150 μL of DMAB (p-dimethylaminobenzaldehyde) chromogenic reagent was added, and the mixture was shaken well. The mixture was then incubated at 37℃ for 20 min for color development. The absorbance was measured at 528 nm using a microplate reader. The inhibition rate of hyaluronidase was calculated using the following formula.

[0168] Inhibition rate (%) = [1 - (Absorbance of sample group / Absorbance of control group)] × 100%

[0169] Sample group: Dilute with pure water to a sample concentration of 3%;

[0170] Positive control (epigallocatechin gallate, purity >98%): diluted with water to a positive control concentration of 1 mg / mL;

[0171] Negative control: pure water.

[0172] This method directly assesses the potential for soothing and repairing efficacy by quantifying the sample's ability to inhibit HA decomposition. The test results are shown in Table 3.

[0173] Table 3

[0174]

[0175] The higher the hyaluronidase inhibition rate, the stronger the soothing effect of PDRN. In this invention, the soothing effect of PDRN from strong to weak is: Bifidobacterium animalis and Lactobacillus paracasei PDRN > Bifidobacterium animalis PDRN > Lactobacillus paracasei PDRN.

[0176] Test Example 4

[0177] Anti-wrinkle efficacy test

[0178] This invention uses human efficacy evaluation to test the anti-wrinkle effect of PDRN.

[0179] Experimental group: Apply face cream containing 3% YK double bacteria PDRN (Example 11) (0.2g / side) after cleansing every morning and evening. The face cream formula is shown in Table 4.

[0180] Control group: Apply face cream (0.2g / side) without YK double bacteria PDRN after cleansing every morning and evening. The face cream (the formula does not contain YK double bacteria PDRN, and other ingredients are the same as the experimental group)

[0181] After 14 days of use, standard photos of the crow's feet area were taken using the VISIA-CR skin image analyzer to analyze the wrinkles. The results are shown in Figure 3.

[0182] Table 4

[0183]

[0184] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for preparing PDRN with soothing and anti-wrinkle effects, characterized in that, The preparation method includes: (1) mixing the strain with a culture medium containing a promoter and fermenting to obtain a fermentation broth; (2) centrifuging the fermentation broth to obtain bacterial sludge, mixing the bacterial sludge with a lysis buffer to obtain a lysis product; (3) mixing the lysis product with a complex enzyme for enzymatic hydrolysis to obtain an enzymatic hydrolysis product; (4) centrifuging the enzymatic hydrolysis product, mixing the obtained supernatant with a DNA stabilizer, and denaturing at high temperature to obtain a denatured product; (5) centrifuging the denatured product and filtering the supernatant through a filter membrane to obtain the product; the promoter includes D-ribose, 5-phosphate ribose, citric acid, vitamin B1 and white truffle; the strain is Bifidobacterium animalis strain YK-L007 with accession number CGMCC No.37117 or Bifidobacterium animalis strain YK-L007 with accession number CGMCC No.37117 and Lactobacillus paracasei strain YK-L003 with accession number CGMCC No.29288; the complex enzyme is papain and bromelain.

2. The method for preparing PDRN with soothing and anti-wrinkle effects according to claim 1, characterized in that, The promoter comprises, by weight parts, 0.01-0.2 parts of D-ribose, 0.01-0.3 parts of 5-phosphate ribose, 0.01-0.1 parts of citric acid, 0.01-0.1 parts of vitamin B1, and 0.5-2 parts of white truffle; the promoter content in the culture medium containing the promoter is 0.8-2.7% by weight.

3. The method for preparing PDRN with soothing and anti-wrinkle effects according to claim 1, characterized in that, The fungal mud in step (2) includes single-strain fungal mud or a composite fungal mud made by mixing different single-strain fungal muds.

4. The method for preparing PDRN with soothing and anti-wrinkle effects according to claim 3, characterized in that, The composite microbial mud includes Bifidobacterium animalis microbial mud and Lactobacillus paracasei microbial mud; the ratio of the number of viable bacteria in the Bifidobacterium animalis microbial mud and Lactobacillus paracasei microbial mud is (1-5):(0.1-2).

5. The method for preparing PDRN with soothing and anti-wrinkle effects according to claim 1, characterized in that, The lysis buffer comprises, by molar concentration, 10-20 mM Tris and 1-5 mM EDTA-disodium, by mass percentage, 0.1-0.3% SDS and 0.1-0.3% lysozyme, and the solvent is water.

6. The method for preparing PDRN with soothing and anti-wrinkle effects according to claim 1, characterized in that, The mass ratio of papain to bromelain is (1-2):(1-2).

7. The method for preparing PDRN with soothing and anti-wrinkle effects according to claim 1, characterized in that, The amount of DNA stabilizer added is 0.1-2% of the mass of the supernatant.

8. The method for preparing PDRN with soothing and anti-wrinkle effects according to claim 1, characterized in that, The DNA stabilizer includes any one or a combination of at least two of polyethyleneimine, spermidine, spermine, or chondroitin sulfate.

9. PDRN prepared by the method for preparing PDRN with soothing and anti-wrinkle effects according to any one of claims 1-8.

10. The use of PDRN according to claim 9 in the preparation of products with soothing and anti-wrinkle effects.

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