Preparation method and application of prebiotic enzyme hydrolysate

Abstract

The application belongs to the technical field of skin care products, and discloses a prebiotic enzyme solution and a preparation method and application thereof. The preparation method of the prebiotic enzyme solution comprises the following steps: (1) inoculating lactobacillus into a fermentation liquid for fermentation culture, centrifuging, washing and collecting the fermentation bacteria, resuspending the fermentation bacteria with a phosphate buffer, and then performing high-pressure disruption homogenization lysis treatment to obtain a lactobacillus fermentation lysate; (2) adding dry dandelion into water after crushing, uniformly dispersing the dandelion, then adding the lactobacillus fermentation lysate obtained in the step (1), and performing stirring enzymolysis extraction under normal temperature conditions for 1-4 hours, filtering to obtain a dandelion enzymolysis extraction liquid; and (3) mixing the dandelion enzymolysis extraction liquid obtained in the step (2), a moisturizing aid and a preservative in water to obtain the prebiotic enzyme solution. The prebiotic enzyme solution obtained by the application has the effects of regulating the skin microecological distribution, improving skin redness / skin acne and repairing the 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) The dry dandelion (TARAXACUM OFFICINALE) is crushed and then added into water in a solid-liquid ratio of 1:10, and then the lactic acid bacteria fermentation lysate obtained in step (1) is added, and the amount of the lactic acid bacteria fermentation lysate added is 3 times the mass of the dandelion. The mixture is stirred for 2 hours at room temperature for enzymatic hydrolysis and extraction, and then filtered through filter cloth to obtain a dandelion enzymatic hydrolysis and extraction liquid.

[0044] (3) 100 parts of the dandelion enzymatic hydrolysis and extraction liquid obtained in step (2) and 15 parts of butanediol, 10 parts of 1,2-hexanediol, 0.2 parts of 1,3-propanediol, and 0.5 parts of p-hydroxyacetophenone are added to 200 parts of deionized water to obtain the prebiotic enzymatic hydrolysis liquid.

[0045] Example 3

[0046] A method for preparing a prebiotic enzymatic hydrolysis liquid, comprising the following steps:

[0047] (1) The plant lactic acid bacteria RD-02 is inoculated into a fermentation broth in an inoculation amount of 1 wt% after being activated and cultured, and the fermentation broth comprises: 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, potassium phosphate dibasic 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 value of the fermentation broth is 6-6.5. The fermentation broth is sterilized at 121°C before use. The temperature for the fermentation culture is controlled at 37°C, and the fermentation culture is performed for 72 hours. The fermentation broth is centrifuged at 4000 r / min for 15 minutes, and the precipitate is washed with deionized water. The collected fermentation bacteria are resuspended in a phosphate buffer to a concentration of 10 wt%, and then added into a high-pressure homogenizer for high-pressure crushing and homogenization. The pressure for the lysis treatment is controlled at 50 MPa, and the temperature is controlled at 4-10°C. A lactic acid bacteria fermentation lysate is obtained.

[0048] (2) The dry dandelion (TARAXACUM OFFICINALE) is crushed and then added into water in a solid-liquid ratio of 1:20, and then the lactic acid bacteria fermentation lysate obtained in step (1) is added, and the amount of the lactic acid bacteria fermentation lysate added is 5 times the mass of the dandelion. The mixture is stirred for 2 hours at room temperature for enzymatic hydrolysis and extraction, and then filtered through filter cloth to obtain a dandelion enzymatic hydrolysis and extraction liquid.

[0049] (3) 100 parts of the dandelion enzymatic hydrolysis and extraction liquid obtained in step (2) and 15 parts of butanediol, 10 parts of 1,2-hexanediol, 0.2 parts of 1,3-propanediol, and 0.5 parts of p-hydroxyacetophenone are added to 200 parts of deionized water to obtain the prebiotic enzymatic hydrolysis liquid.

[0050] Comparative Example 1

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

[0052] (1) The method for preparing the lactobacillus fermentation lysate is the same as in Example 1.

[0053] (2) The dried dandelion (TARAXACUM OFFICINALE) is pulverized and then added to water in a solid-liquid ratio of 1:20 to disperse uniformly, and then stirred for extraction at room temperature for 2 hours. The filter cloth is used to filter and take the filtrate to obtain the dandelion extract.

[0054] (3) In terms of mass parts, 100 parts of the lactobacillus fermentation lysate and the dandelion extract (the amount of the lactobacillus fermentation lysate added is 2 times the mass of the dandelion) are added to 10 parts of butanediol, 6 parts of 1,2-hexanediol, 0.1 part of 1,3-propanediol, and 0.3 parts of p-hydroxyacetophenone in 100 parts of deionized water to mix uniformly to obtain the composition comprising the lactobacillus fermentation lysate and the dandelion extract.

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

[0056] 1. Before using the sample

[0057] One week before the test, the subjects stop using facial cosmetics or external drugs. On the night before the test, the subjects do not apply any skin care products after washing their faces at home. On the morning of the test, the subjects do not apply any skin care products after washing their faces with clean water at home;

[0058] First visit, eligible subjects fill out the informed consent form; the actual number of subjects included is 32, which are randomly divided into 4 groups;

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

[0060] The skin water loss rate is detected using the skin moisture loss test probe TewameterTM300;

[0061] After the subjects receive the samples, they use the test samples according to the instructions at home.

[0062] 2. After using the sample for 1 day

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

[0064] The skin transepidermal water loss rate was detected using a skin moisture loss test probe Tewameter™ 300.

[0065] The test was completed and the subjects left the venue.

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

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

[0068] The skin transepidermal water loss rate was detected using a skin moisture loss test probe Tewameter™ 300.

[0069] The test was completed and the subjects left the venue.

[0070] 4. Data processing

[0071] The transepidermal water loss rate test results of the subjects before using the sample, after using the sample for 1 day, and after using the sample for 28 days were recorded, and the average improvement rate was calculated The calculation formula is as follows:

[0072]

[0073] In the formula, T0 is the skin parameter value before the test area uses the product;

[0074] T t is the skin parameter value after the test area uses the product;

[0075] N is the number of effective subjects.

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

[0077] Table 1 Skin barrier repair efficacy test results of Examples 1-3 and Comparative Example 1

[0078]

[0079] As can be seen from the results in Table 1, the dandelion is subjected to synergistic enzymatic extraction using lactobacillus fermentation lysate, which can significantly improve the skin barrier repair efficacy of the obtained composition.

[0080] II. The skin soothing effect of the prebiotic enzymatic hydrolysate obtained in Examples 1-3 and the composition obtained in Comparative Example 1 was tested (experimental principle and basis: hyaluronidase is a hydrolase that degrades hyaluronic acid, which is the main component of the extracellular matrix of connective tissue cells, and is related to most IgE-mediated type I and T cell-mediated type IV allergic reactions. Therefore, the in vitro inhibition experiment of hyaluronidase can reflect the correlation of the corresponding product with the skin soothing, anti-allergic, anti-inflammatory and other effects to a certain extent. Hyaluronic acid is a high-molecular-weight polysaccharide composed of D-glucuronic acid and N-acetylglucosamine, and can undergo color reaction according to the Elson-morgan reaction. The inhibition rate of hyaluronidase in the sample can be determined by this reaction. In this experiment, the inhibition rate of hyaluronidase in the sample was determined according to the detection method of laboratory method (enzyme inhibition rate method) QTTZ-XZ-35 "Verification and detection of soothing effect of cosmetics", and by comparing with the negative control, it was determined whether the sample had soothing effect). The test procedure is as follows:

[0081] 1. Accurately weigh the sample and extract and pretreat the sample;

[0082] 2. The pretreated sample was subjected to modified Elson-morgan reaction, and a negative control was also set up;

[0083] 3. Measure the OD value according to the operation of the detection kit;

[0084] 4. Calculate the inhibition rate of hyaluronidase;

[0085]

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

[0087] C is the hyaluronic acid content of the negative control.

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

[0089] Table 2 Skin soothing effect test results of 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 use of lactobacillus fermentation lysate for synergistic enzymatic extraction of dandelion can significantly improve the skin soothing effect of the obtained composition.

[0092] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A method for preparing a prebiotic enzymatic hydrolysate, characterized in that, It comprises the following steps: (1) inoculating lactobacillus RD-02 into a fermentation broth for fermentation culture, centrifuging, washing, collecting the fermentation bacteria, resuspending with a phosphate buffer, high-pressure crushing, homogenizing and lysing to obtain a lactobacillus fermentation lysate; (2) adding dry dandelion into water for uniform dispersion, then adding the lactobacillus fermentation lysate obtained in step (1), stirring and enzymatic hydrolysis at room temperature for 1-4 hours, filtering to obtain a dandelion enzymatic hydrolysate; (3) mixing the dandelion enzymatic hydrolysate obtained in step (2), a moisturizing aid and a preservative in water to obtain the prebiotic enzymatic hydrolysate; 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 centrifugal speed is 4000-5000 r / min, and the centrifugal time is 10-20 min; the washing is performed with deionized water; The concentration of the bacterial suspension resuspended with the phosphate buffer is 10-20 wt%; The mass ratio of the dandelion to water is 1:10-30; the addition amount of the lactobacillus fermentation lysate is 1-5 times the mass of the dandelion; The moisturizing aid is at least one of butanediol, 1,2-hexanediol and 1,3-propanediol, and the preservative is p-hydroxyacetophenone; The mass ratio of the dandelion enzymatic hydrolysate, the moisturizing aid and water is 100:10-30:100-200.

2. The method for preparing a prebiotic enzymatic hydrolysate according to claim 1, characterized in that, The pH value of the fermentation broth is 6-6.5, and the fermentation broth is sterilized at high temperature and high pressure before use; the fermentation culture temperature is 35-38℃, and the fermentation culture time is 24-72 h.

3. The method for preparing a prebiotic enzymatic hydrolysate according to claim 1, characterized in that, The pressure of the high-pressure crushing, homogenizing and lysing treatment is 50-80 MPa, and the temperature is 4-10℃.

4. A prebiotic enzymatic hydrolysate, characterized in that, Prepared by the method of any one of claims 1-3.

5. Use of the prebiotic enzymatic hydrolysate of claim 4 in the preparation of skin care products.

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