Prebiotic enzymatic hydrolysate as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of skin care products, and discloses prebiotic enzymatic hydrolysate as well as a preparation method and application thereof. The preparation method of the prebiotic enzymatic hydrolysate comprises the following steps: (1) inoculating lactobacillus into fermentation liquor for fermentation culture, centrifuging, washing and collecting fermentation thalli, resuspending by using a phosphate buffer solution, and performing high-pressure crushing, homogenizing and cracking treatment to obtain a lactobacillus fermentation lysate product; (2) crushing dried dandelions, adding the crushed dandelions into water, uniformly dispersing, then adding the lactobacillus fermentation lysate obtained in the step (1), carrying out stirring enzymolysis extraction for 1-4 hours under the condition of normal temperature, and filtering to take filtrate, so as to obtain a dandelion enzymolysis extracting solution; and (3) adding the dandelion enzymolysis extracting solution obtained in the step (2), a moisturizing additive and a preservative into water, and uniformly mixing to obtain the prebiotic enzymolysis solution. The prebiotic enzymatic hydrolysate obtained by the invention has the effects of regulating skin micro-ecological distribution, improving skin redness and swelling / skin acne and repairing a skin barrier function.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of skin care products, and particularly relates to a prebiotic enzyme hydrolysate and a preparation method and application thereof. BACKGROUND

[0002] On the skin surface, a micro-ecological barrier is formed by various microorganisms (bacteria, fungi, viruses, mites, etc.), keratinocytes, secretions, dust, etc. The balance of this barrier plays an important protective and regulatory role in the safety of the skin.

[0003] In a healthy skin micro-ecological barrier, there are a large number of resident bacteria that can maintain the skin surface acid mantle by secreting secondary metabolites such as bacteriocins and lactic acid, decompose toxic substances of harmful bacteria and inhibit the growth of harmful bacteria, directly protect the safety of the skin through competition for nutrients and growth space, and indirectly induce the host to increase antibody secretion, activate skin immune regulation, scavenge skin free radicals, and reduce inflammatory factor expression to protect the health of the skin. Once the skin micro-ecology is imbalanced, a series of complex internal and external skin chain reactions will occur, affecting normal skin metabolism and health level. Therefore, developing a skin care product that can regulate the balance of skin microbiota and repair the function of the skin micro-ecological barrier has good market prospects.

[0004] In the previous research results (Chinese patent application No. 2020800796664), we verified that the dandelion extract activated the proliferation of plant lactic acid bacteria RD-02 (preserved number SD1323) as skin lactic acid bacteria, thereby significantly increasing the growth of Staphylococcus epidermidis as a skin beneficial bacteria and significantly reducing the growth of Staphylococcus aureus and Propionibacterium acnes as skin harmful bacteria. However, the efficacy and application of the enzyme hydrolysate obtained by fermenting and lysing the lactic acid bacteria and performing enzyme extraction on Taraxacum officinale have not been disclosed. SUMMARY

[0005] To further develop products containing dandelion extract with better skin care efficacy, the primary purpose of the present application is to provide a preparation method of a prebiotic enzyme hydrolysate.

[0006] Another purpose of the present application is to provide a prebiotic enzyme hydrolysate prepared by the above method.

[0007] Still another purpose of the present application is to provide the application of the above prebiotic enzyme hydrolysate in skin care products.

[0008] The purposes of the present application are achieved by the following technical solutions:

[0009] A preparation method of a prebiotic enzyme solution, comprising the following steps:

[0010] (1) inoculating Lactobacillus into a fermentation broth for fermentation culture, centrifuging, washing, collecting the fermentation bacteria, resuspending with a phosphate buffer, and then performing high-pressure crushing, homogenizing, and lysing treatment to obtain a Lactobacillus fermentation lysate;

[0011] (2) adding dry Taraxacum officinale into water after crushing to disperse uniformly, then adding the Lactobacillus fermentation lysate obtained in step (1), stirring and enzymatic hydrolysis under normal temperature conditions for 1-4 h, filtering to obtain a Taraxacum officinale enzymatic hydrolysis extract;

[0012] (3) mixing the Taraxacum officinale enzymatic hydrolysis extract obtained in step (2) with a moisturizing aid and a preservative in water to obtain the prebiotic enzyme solution.

[0013] Further, the Lactobacillus is Lactobacillus plantarum RD-02 (disclosed in Korean patent document KR20190143775A, deposited in Korean Collection for Type Cultures (KCTC), accession number: SD1323).

[0014] Further, the fermentation broth comprises: glucose 15-30 g / L, peptone 5-15 g / L, beef extract 5-15 g / L, yeast extract 2-10 g / L, sodium acetate 2-10 g / L, diammonium hydrogen citrate 1-4 g / L, potassium phosphate dibasic 1-4 g / L, Tween-80 0.5-2 g / L, magnesium sulfate heptahydrate 0.1-0.3 g / L, manganese sulfate heptahydrate 0.01-0.08 g / L; the pH value of the fermentation broth is 6-6.5; and the fermentation broth is sterilized (121℃) before use.

[0015] Further, the fermentation culture temperature is 35-38℃, and the fermentation culture time is 24-72 h.

[0016] Further, the centrifugal speed is 4000-5000 r / min, and the centrifugal time is 10-20 min; and the washing is performed with deionized water.

[0017] Further, the concentration of the bacterial suspension resuspended with the phosphate buffer is 10-20 wt%; and the pressure of the high-pressure crushing, homogenizing, and lysing treatment is 50-80 MPa, and the temperature is 4-10℃.

[0018] Further, the mass ratio of the dry dandelion to water after being crushed and uniformly dispersed is 1:10-30.

[0019] Further, the added amount of the lactic acid bacteria fermentation lysate is 1-5 times of the mass of the dandelion.

[0020] Further, the moisturizing aid is at least one of butanediol, 1,2-hexanediol and 1,3-propanediol, and the preservative is p-hydroxyacetophenone.

[0021] Further, the mass ratio of the dandelion enzymatic extraction liquid, the moisturizing aid and water in step (3) is 100:10-30:100-200.

[0022] The preparation principle of the dandelion enzymatic extraction liquid is that the lactic acid bacteria fermentation lysate containing enzymes and metabolic products is used for enzymatic extraction of the dandelion (not using live bacteria for direct plant fermentation), so that the extraction of water-soluble functional components in the dandelion is maximized, and the prebiotic components of the lactic acid bacteria fermentation lysate are contained, and the obtained prebiotic enzymatic liquid has significantly improved skin care efficacy. The corresponding preparation schematic diagram is shown in Figure 1 .

[0023] A prebiotic enzymatic liquid is prepared by the above method.

[0024] The above prebiotic enzymatic liquid is applied to skin care products.

[0025] Compared with the prior art, the present application has the following advantages:

[0026] (1) The prebiotic enzymatic liquid obtained by the present application has the effects of regulating skin microecological distribution, improving skin redness / acne and repairing skin barrier function.

[0027] (2) The present application uses lactic acid bacteria fermentation lysate to enzymatically extract dandelion, which can maximize the extraction of water-soluble functional components in dandelion, and significantly improve the skin barrier repair efficacy of the obtained prebiotic enzymatic liquid in application. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is a preparation schematic diagram of the dandelion enzymatic extraction liquid of the present application.

[0029] Figure 2 It is a skin microecological efficacy evaluation result diagram of the prebiotic enzymatic liquid obtained in Example 1.

[0030] Figure 3 It is a facial skin improvement efficacy evaluation result diagram of the prebiotic enzymatic liquid obtained in Example 1.

[0031] Figure 4 A skin redness / skin comedones improvement effect evaluation result graph of the prebiotic enzymatic hydrolysate obtained in Example 1. DETAILED DESCRIPTION

[0032] The present application will be further described in detail below with reference to examples and drawings, but the embodiments of the present application are not limited thereto.

[0033] Example 1

[0034] A prebiotic enzymatic hydrolysate preparation method, comprising the following steps:

[0035] (1) After the plant lactic acid bacteria RD-02 is activated and cultured, it is inoculated into a fermentation broth at an inoculation amount of 1.5 wt%, and the fermentation broth composition includes: glucose 20 g / L, peptone 10 g / L, beef extract 8 g / L, yeast extract 6 g / L, sodium acetate 5 g / L, diammonium hydrogen citrate 2 g / L, potassium phosphate dibasic 1.5 g / L, Tween-80 1 g / L, magnesium sulfate heptahydrate 0.2 g / L, and manganese sulfate heptahydrate 0.04 g / L; the pH value of the fermentation broth is 6-6.5; the fermentation broth is sterilized at 121℃ before use. The fermentation culture temperature is controlled at 37℃, and the fermentation culture time is 48 h. After centrifugation at 4500 r / min for 15 min, the precipitate is washed with deionized water, the fermentation bacteria are collected and resuspended in phosphate buffer to a concentration of 15 wt%, and then added to a high-pressure homogenizer for high-pressure crushing and homogenization lysis treatment. The lysis treatment pressure is controlled at 60 MPa, and the temperature is controlled at 4-10℃. The lactic acid bacteria fermentation lysate product is obtained.

[0036] (2) The dried dandelion (TARAXACUM OFFICINALE) is crushed and then added to water at a solid-liquid mass ratio of 1:20 to disperse uniformly, and then the lactic acid bacteria fermentation lysate obtained in step (1) is added, and the addition amount of the lactic acid bacteria fermentation lysate is 2 times the mass of the dandelion. Stirring enzymatic hydrolysis extraction is carried out at room temperature for 2 h, the filter cloth is filtered to obtain the dandelion enzymatic hydrolysis extract.

[0037] (3) 100 parts of the dandelion enzymatic hydrolysis extract obtained in step (2) and 10 parts of butanediol, 6 parts of 1,2-hexanediol, 0.1 part of 1,3-propanediol, and 0.3 parts of p-hydroxyacetophenone are added to 100 parts of deionized water to mix uniformly to obtain the prebiotic enzymatic hydrolysate.

[0038] The skin microecological effect of the prebiotic enzymatic hydrolysate obtained in this example is evaluated. According to the cosmetic use specification, the human face effect evaluation experiment is carried out, and the sample (diluted at a concentration of 3 wt%) is applied before and after 4 weeks, and the distribution of beneficial bacteria and harmful bacteria (the corresponding bacteria are detected by DNA) is collected and detected from the face of the experimental object, respectively. The results are as follows: Figure 2As shown. By Figure 2 The results showed that 27 types of bacteria were collected from the faces of the experimental subjects. Before application, 80% of harmful bacteria (including Propionibacterium acnes) and 20% of beneficial bacteria (including lactic acid bacteria) were detected. After 4 weeks of application, the results showed that 55% of harmful bacteria (including Propionibacterium acnes) and 45% of beneficial bacteria (including lactic acid bacteria) were detected, with harmful bacteria decreasing by 31% and beneficial bacteria increasing by 123%. This indicates that the prebiotic enzymatic hydrolysate obtained in this invention has a significant effect on regulating the skin microecology.

[0039] The facial skin improvement efficacy and skin redness / acne improvement efficacy of the prebiotic enzymatic hydrolysate obtained in this embodiment were evaluated. Human facial efficacy evaluation experiments were conducted according to cosmetic usage guidelines. The sample (diluted at 3wt%) was applied to the face twice a day for 4 weeks. The degree of facial skin improvement was assessed using Soft Plus, and skin redness and acne were assessed using OBSERV. The test results are as follows: Figure 3 and Figure 4 As shown. By Figure 3 and Figure 4 The results showed that the dry or oily skin of the experimental subjects improved to a healthy skin state (hydration increased by 10%, and sebum decreased by 20%). The content of porphyrin, one of the causes of skin redness and acne, decreased by 25%. This indicates that the prebiotic enzymatic hydrolysate obtained in this invention has significant effects on improving facial skin and reducing skin redness / acne.

[0040] Example 2

[0041] A method for preparing a prebiotic enzymatic hydrolysate includes the following steps:

[0042] (1) Plant lactic acid bacteria RD-02 was activated and then inoculated into the fermentation broth at a 2wt% inoculum for fermentation. The fermentation broth consisted of: glucose 30g / L, peptone 15g / L, beef extract 10g / L, yeast extract 10g / L, sodium acetate 8g / L, diammonium citrate 3g / L, dipotassium hydrogen phosphate 2g / L, Tween-80 1g / L, magnesium sulfate heptahydrate 0.2g / L, and manganese sulfate heptahydrate 0.04g / L. The pH of the fermentation broth was 6-6.5. The fermentation broth was sterilized at 121℃ under high pressure before use. The fermentation temperature was controlled at 37℃, and the fermentation time was 24h. Centrifuge at 5000 r / min for 15 min, take the precipitate and wash it with deionized water. Collect the fermentation cells and resuspend them in phosphate buffer to a concentration of 20 wt%. Add them to a high-pressure homogenizer for high-pressure homogenization and lysis treatment. Control the lysis treatment pressure at 70 MPa and the temperature at 4-10℃ to obtain the lactobacillus fermentation lysate.

[0043] (2) After pulverizing the dried dandelion (TARAXACUM OFFICINALE), add it to water at a material-to-liquid mass ratio of 1:10 and disperse it evenly. Then add the lactobacillus fermentation lysate obtained in step (1). The amount of lactobacillus fermentation lysate added is 3 times the mass of dandelion. Stir and enzymatically extract at room temperature for 2 hours. Filter the filtrate through a filter cloth to obtain dandelion enzymatic extract.

[0044] (3) By mass, take 100 parts of the dandelion enzymatic hydrolysate obtained in step (2) and add 15 parts of butanediol, 10 parts of 1,2-hexanediol, 0.2 parts of 1,3-propanediol and 0.5 parts of p-hydroxyacetophenone to 200 parts of deionized water and mix evenly to obtain the prebiotic enzymatic hydrolysate.

[0045] Example 3

[0046] A method for preparing a prebiotic enzymatic hydrolysate includes the following steps:

[0047] (1) Plant lactic acid bacteria RD-02 was activated and then inoculated into the fermentation broth at a 1 wt% inoculum for fermentation. The fermentation broth consisted of: glucose 15 g / L, peptone 6 g / L, beef extract 5 g / L, yeast extract 3 g / L, sodium acetate 2 g / L, diammonium hydrogen citrate 1 g / L, dipotassium hydrogen phosphate 1 g / L, Tween-80 1 g / L, magnesium sulfate heptahydrate 0.2 g / L, and manganese sulfate heptahydrate 0.04 g / L. The pH of the fermentation broth was 6-6.5. The fermentation broth was sterilized at 121℃ under high pressure before use. The fermentation temperature was controlled at 37℃, and the fermentation time was 72 h to complete the culture. Centrifuge at 4000 r / min for 15 min, take the precipitate and wash it with deionized water. Collect the fermentation cells and resuspend them in phosphate buffer to a concentration of 10 wt%. Add them to a high-pressure homogenizer for high-pressure homogenization and lysis treatment. Control the lysis treatment pressure at 50 MPa and the temperature at 4-10℃ to obtain the Lactobacillus fermentation lysate.

[0048] (2) After pulverizing the dried dandelion (TARAXACUM OFFICINALE), add it to water at a material-to-liquid mass ratio of 1:20 and disperse it evenly. Then add the lactobacillus fermentation lysate obtained in step (1). The amount of lactobacillus fermentation lysate added is 5 times the mass of dandelion. Stir and enzymatically extract at room temperature for 2 hours. Filter the filtrate through a filter cloth to obtain dandelion enzymatic extract.

[0049] (3) By weight, take 100 parts of the dandelion enzymatic hydrolysate obtained in step (2) and add 12 parts of butanediol, 8 parts of 1,2-hexanediol, 0.15 parts of 1,3-propanediol and 0.4 parts of p-hydroxyacetophenone to 150 parts of deionized water and mix evenly to obtain the prebiotic enzymatic hydrolysate.

[0050] Comparative Example 1

[0051] A method for preparing a composition comprising lactobacillus fermentation lysate and dandelion extract, comprising the following steps:

[0052] (1) The preparation method of the fermentation lysate of Lactobacillus is the same as that in Example 1.

[0053] (2) After pulverizing dried dandelion (TARAXACUM OFFICINALE), add it to water at a mass ratio of 1:20 and disperse it evenly. Stir and extract at room temperature for 2 hours. Filter the solution through a filter cloth to obtain dandelion extract.

[0054] (3) By weight, take 100 parts of Lactobacillus fermentation lysate and dandelion extract (the amount of Lactobacillus fermentation lysate added is twice the mass of dandelion) and 10 parts of butanediol, 6 parts of 1,2-hexanediol, 0.1 parts of 1,3-propanediol and 0.3 parts of p-hydroxyacetophenone and add them to 100 parts of deionized water and mix evenly to obtain a composition containing Lactobacillus fermentation lysate and dandelion extract.

[0055] I. The skin barrier repair efficacy of the prebiotic enzymatic hydrolysate obtained in Examples 1-3 and the composition obtained in Comparative Example 1 was tested. The test procedure is as follows:

[0056] 1. Before using the sample

[0057] One week prior to the test, participants discontinued the use of facial cosmetics or topical medications. The night before the test, participants washed their face at home and did not apply any skincare products. On the morning of the test, participants washed their face with water at home and did not apply any skincare products.

[0058] Upon initial visit, qualified participants completed informed consent forms; the actual number of qualified participants was 32, who were randomly divided into 4 groups.

[0059] After cleaning their faces with water, the subjects rested for 30 minutes in a constant temperature and humidity room (temperature (21±1)℃, relative humidity (50±10)%RH).

[0060] The skin's transepidermal water loss rate was measured using the Tewameter™ 300 skin moisture loss test probe.

[0061] After receiving the sample, the subject went home and used the test sample according to the instructions.

[0062] 2. One day after using the sample

[0063] After arriving at the venue, the subjects cleaned their faces with water and rested for 30 minutes in a constant temperature and humidity room (temperature (21±1)℃, relative humidity (50±10)%RH).

[0064] The Tewameter™ 300 skin moisture loss test probe was used to detect the transepidermal moisture loss rate of the skin.

[0065] After completing the test, the participants left the venue.

[0066] 3. After using the sample for 28 days

[0067] After arriving at the venue, the subjects cleaned their faces with water and rested for 30 minutes in a constant temperature and humidity room (temperature (21±1)℃, relative humidity (50±10)%RH).

[0068] The Tewameter™ 300 skin moisture loss test probe was used to detect the transepidermal moisture loss rate of the skin.

[0069] After completing the test, the participants left the venue.

[0070] 4. Data Processing

[0071] Record the transdermal water loss rate test results of subjects before sample use, 1 day after use, and 28 days after use, and calculate the average improvement rate. The calculation formula is as follows:

[0072]

[0073] In the formula, T0 represents the skin parameter values ​​in the test area before the product was used;

[0074] T t -Skin parameter values ​​after product application to the test area;

[0075] N - Number of valid subjects.

[0076] The test results are shown in Table 1 below:

[0077] Table 1. Results of skin barrier repair efficacy tests in Examples 1-3 and Comparative Example 1

[0078]

[0079] As can be seen from the results in Table 1, the present invention uses lactobacillus fermentation lysate to synergistically extract dandelion, which can significantly improve the skin barrier repair efficacy of the obtained composition.

[0080] II. The skin-soothing efficacy of the prebiotic enzymatic hydrolysates obtained in Examples 1-3 and the composition obtained in Comparative Example 1 was tested (Experimental principle and basis: Hyaluronidase is a hydrolytic enzyme that degrades hyaluronic acid. It is a major component of the extracellular matrix of connective tissue cells and is associated with most type I and type IV allergic reactions mediated by IgE and T cells. Therefore, in vitro inhibition experiments of hyaluronidase can, to a certain extent, reflect the correlation between the corresponding product's skin-soothing, anti-allergic, and anti-inflammatory effects. Hyaluronic acid is a high-grade polysaccharide composed of D-glucuronic acid and N-acetylglucosamine, and can undergo a colorimetric reaction according to the Elson-Morgan reaction. This reaction can be used to determine the inhibition rate of hyaluronidase in the sample. In this experiment, the inhibition rate of hyaluronidase in the sample was determined according to the laboratory method (enzyme inhibition rate method) QTTZ-XZ-35 "Verification and Testing of Soothing Efficacy of Cosmetics". By comparing with a negative control, the soothing efficacy of the sample was determined). The test procedure is as follows:

[0081] 1. Accurately weigh the sample and perform extraction pretreatment on the sample;

[0082] 2. The pretreated samples were processed according to the modified Elson-Morgan reaction procedure, with a negative control included.

[0083] 3. Follow the instructions on the test kit to measure the OD value;

[0084] 4. Calculate the hyaluronidase inhibition rate;

[0085]

[0086] In the formula, T represents the hyaluronic acid content of the sample solution;

[0087] C - Negative control hyaluronic acid content.

[0088] The test results are shown in Table 2 below:

[0089] Table 2. Results of skin soothing efficacy tests in Examples 1-3 and Comparative Example 1

[0090] Test Example Inhibition rate (%) Example 1 40.53±0.90 Example 2 41.89±1.12 Example 3 38.26±0.79 Comparative Example 1 29.77±0.85

[0091] As can be seen from the results in Table 2, the present invention uses Lactobacillus fermentation lysate to synergistically extract dandelion, which can significantly improve the skin-soothing effect of the obtained composition.

[0092] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a prebiotic enzymatic hydrolysate, characterized in that, Includes the following steps: (1) Lactobacillus was inoculated into the fermentation broth for fermentation culture. The fermentation cells were collected by centrifugation and washing. After resuspending in phosphate buffer, the cells were homogenized by high pressure to obtain the fermentation lysate of Lactobacillus. (2) After the dried dandelion is crushed, it is added to water and dispersed evenly. Then, the lactobacillus fermentation lysate obtained in step (1) is added. The mixture is stirred and enzymatically extracted at room temperature for 1-4 hours. The filtrate is filtered to obtain dandelion enzymatic extract. (3) Add the dandelion enzymatic hydrolysate obtained in step (2) to water and mix evenly to obtain the prebiotic enzymatic hydrolysate.

2. The method for preparing a prebiotic enzymatic hydrolysate according to claim 1, characterized in that, The lactobacillus used is plant-based lactobacillus RD-02.

3. The method for preparing a prebiotic enzymatic hydrolysate according to claim 1, characterized in that, The fermentation broth comprises: glucose 15-30 g / L, peptone 5-15 g / L, beef extract 5-15 g / L, yeast extract 2-10 g / L, sodium acetate 2-10 g / L, diammonium hydrogen citrate 1-4 g / L, dipotassium hydrogen phosphate 1-4 g / L, Tween-80 0.5-2 g / L, magnesium sulfate heptahydrate 0.1-0.3 g / L, and manganese sulfate heptahydrate 0.01-0.08 g / L; the pH of the fermentation broth is 6-6.5; the fermentation broth is sterilized by high temperature and high pressure before use; the fermentation temperature is 35-38℃, and the fermentation time is 24-72 h.

4. The method for preparing a prebiotic enzymatic hydrolysate according to claim 1, characterized in that, The centrifugation speed is 4000-5000 r / min, and the centrifugation time is 10-20 min; The washing process uses deionized water.

5. The method for preparing a prebiotic enzymatic hydrolysate according to claim 1, characterized in that, The concentration of the bacterial suspension resuspended in phosphate buffer is 10-20 wt%; the pressure of the high-pressure crushing and homogenization pyrolysis treatment is 50-80 MPa, and the temperature is 4-10℃.

6. The method for preparing a prebiotic enzymatic hydrolysate according to claim 1, characterized in that, The mass ratio of the dried dandelion powdered and then evenly dispersed in water is 1:10-30; the amount of lactobacillus fermentation lysate added is 1-5 times the mass of the dandelion.

7. The method for preparing a prebiotic enzymatic hydrolysate according to claim 1, characterized in that, The moisturizing agent is at least one of butylene glycol, 1,2-hexanediol, and 1,3-propanediol, and the preservative is p-hydroxyacetophenone.

8. The method for preparing a prebiotic enzymatic hydrolysate according to claim 1, characterized in that, The mass ratio of the dandelion enzymatic hydrolysis extract, moisturizing agent and water in step (3) is 100:10-30:100-200.

9. A prebiotic enzymatic hydrolysate, characterized in that, It is prepared by the method described in any one of claims 1 to 8.

10. The application of the prebiotic enzymatic hydrolysate as described in claim 9 in skin care products.

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