CN114796052B

CN114796052B - Moisturizing composition, moisturizing essence and preparation method thereof

Info

Publication number: CN114796052B
Application number: CN202210508245.XA
Authority: CN
Inventors: 罗敏; 吴小端; 陈铭楷; 陈培仲
Original assignee: Baitide Pharmaceutical Biotechnology Guangdong Co ltd
Current assignee: Baitide Pharmaceutical Biotechnology Guangdong Co ltd
Priority date: 2022-05-11
Filing date: 2022-05-11
Publication date: 2023-12-29
Anticipated expiration: 2042-05-11
Also published as: CN114796052A

Abstract

The invention belongs to the field of biotechnology. More particularly, it relates to a moisturizing composition containing roselle extract/fermentation product, a moisturizing essence and a preparation method thereof. The composition comprises at least: roselle extract/fermentation product; the fermentation product is a fermentation supernatant obtained by sequentially inoculating saccharomycetes and bacillus subtilis into a fermentation culture medium containing roselle extract for continuous fermentation. The fermentation supernatant obtained by sequentially adopting the candida albosis and the bacillus subtilis to continuously ferment the roselle extract shows surprising characteristics, and research and test results show that the roselle extract still has the capacities of supernormal moisture retention, water locking and skin cell protection under a low-temperature drying environment.

Description

Moisturizing composition, moisturizing essence and preparation method thereof

Technical Field

The invention belongs to the field of biotechnology. More particularly, it relates to a moisturizing composition containing roselle extract/fermentation product, a moisturizing essence and a preparation method thereof.

Background

The cold environment can lead to reduced air humidity and air drying, which can lead to skin becoming abnormally dry. Skin cells, however, lose luster and elasticity due to the reduction of moisture, even cause a series of skin diseases, and the skin becomes more fragile, which is fatal to skin health in cold environments. At present, moisture retention components such as glycerin and the like on the market are all used for retaining moisture by absorbing moisture in air, but the moisture retention effect is poor under the condition of low air humidity, particularly under the condition of cold. Therefore, it is necessary to provide a product which has a long-lasting moisturizing effect under extreme conditions such as cold drying.

Roselle (Hibiscus sabdariffa) is an annual herb of the genus hibiscus of the family malvaceae; the roselle calyx contains 15-30% of organic acid, mainly oxalic acid, tartaric acid, malic acid, succinic acid and the like, and contains 2% of anthocyanin, mainly delphinidin-3-elderberry diglycoside and cornflower-3-elderberry diglycoside; and flavonoids such as 3,5,7,8,3,4-hexahydroxyflavone, quercetin, roselle red and hibiscus glycoside. Studies have shown that: the 50% alcohol extract of roselle with the concentration of 250 mug/mL has the inhibition rate of 84% on tyrosinase activity, shows that the roselle has better inhibition effect on tyrosinase and strong oxidation resistance, and can be used as skin whitening agent; the inhibition rate of the ethyl acetate extract of roselle calyx of 6.25mg/mL on cyclooxygenase-1 and cyclooxygenase-2 activities is 78.4% and 13.6%, respectively; the rate of promotion of 70% alcohol extract of Hibiscus sabdariffa at 300 μg/mL on neutral lipid hydrolysis is 23%. However, none of these studies disclose the use of roselle for moisturizing, particularly long-lasting moisturizing in cold, dry environments.

Disclosure of Invention

It is an object of the present invention to provide a skin care composition having long lasting moisturization in extreme environments, which possesses the ability to provide moisture to the skin, strengthen the skin barrier and protect cells in specific dry environments.

Another object of the present invention is to provide a moisturizing concentrate with long-lasting moisturizing in extreme environments. The moisture-preserving essence has the capability of providing moisture for skin in a special dry environment, strengthening skin barrier and protecting cells.

It is another object of the present invention to provide a method for improving the water deficit of skin in a specific dry environment. The method improves the phenomenon of skin water deficiency by applying the moisturizing essence on local skin.

The special drying environment refers to the environment temperature of < -5 ℃ and the relative humidity of 30-50 percent. Although the present invention emphasizes the ability of the composition to retain moisture in a particular dry environment, it does not negate its ability to retain moisture in other, non-particular environments.

The above object of the present invention is achieved by the following technical scheme: a moisturizing composition resistant to cold and dry environments, the composition comprising at least: roselle extract/fermentation product; the fermentation product is a fermentation supernatant obtained by sequentially inoculating saccharomycetes and bacillus subtilis into a fermentation culture medium containing roselle extract for continuous fermentation.

The roselle extract described herein may be an aqueous or alcoholic extract, preferably an alcoholic extract, more preferably an ethanol, methanol extract; more preferably 60 to 85% by volume of ethanol extract. It can be prepared by a conventional technique as long as the soluble components contained in the raw materials can be extracted and dissolved in an extraction solvent. The extraction may be carried out at normal temperature or under reflux heating, removing residues by filtration or the like, and collecting filtrate, which may be further concentrated to obtain the objective extract. In particular, when 60 to 85% ethanol extract is preferred, the preparation method thereof may comprise the steps of:

pulverizing calyx Hibisci Sabdariffae, adding 3-6 times of 60-85% ethanol solution, reflux extracting at 50-65deg.C for 1-3 times, mixing filtrates, removing ethanol under reduced pressure, and concentrating.

In another embodiment, the roselle extract is present in the fermentation medium in an amount of 1 to 20% by weight. The fermentation medium may be a solid medium or a liquid medium, and is usually a liquid medium, and the composition of the medium is not particularly limited as long as it can satisfy the growth and proliferation of yeasts and bacillus subtilis.

The pH of the culture medium is usually 4.0 to 7.5, preferably 5.0 to 7.5, and fermentation is carried out in a preferable pH range to maintain stability between the raw materials; the fermentation temperature is not particularly limited as long as the strain can be proliferated and fermentation is possible; specifically, the fermentation temperature in the invention can be 25-37 ℃, and is preferably 25-32 ℃; for the fermentation time, it is usually 24 to 72 hours, preferably 36 to 72 hours; for the morphology of the fermentation product, although the invention emphasizes the fermentation supernatant, it is not excluded that the fermentation product is present in other morphologies. In theory, the fermentation supernatant obtained by the secondary fermentation usually needs to be subjected to post-treatment such as sterilization, deodorization, decolorization by an ion exchange column or an activated carbon column, and the post-treated fermentation supernatant can be further concentrated or lyophilized.

In another embodiment, the yeast is candida albopictus. The emphasis is placed on the fermentation of the strain of candida utilis because if candida utilis is used it is easier to obtain a composition that has a long lasting moisture retention at low temperature, dry conditions. The present invention emphasizes that the above fermentation product is obtained by continuous fermentation using a specific strain, because the fermentation product obtained by continuous fermentation using candida albosis and bacillus subtilis has more excellent moisture retention under low-temperature, dry conditions than the fermentation product obtained by mixed fermentation or continuous fermentation using a different strain (even the same species of yeast). The inventors have also confirmed by comparative experiments that, although all of the commercial species commonly used for biofermentation are employed in the comparative experiments, the differences in the effects exhibited are unexpected.

The present invention also emphasizes the order of fermentation, as only fermentation with a specific species in a defined order will result in a more desirable product.

In another embodiment, the roselle extract is subjected to an enzymatic pretreatment, although the invention emphasizes that the roselle extract needs to be subjected to an enzymatic treatment, but does not exclude the possibility of not being subjected to an enzymatic treatment. The enzymolysis treatment is performed to ensure that the subsequent fermentation effect is more ideal; the enzyme adopted in the enzymolysis process can be one or more of pectase, cellulase and protease, preferably mixed enzyme is adopted for enzymolysis, and when the mixed enzyme is adopted for enzymolysis, the adopted enzyme can be pectase and cellulase, and the two enzymes can be formed in any weight ratio, so long as the enzyme content is in the range of 1-3 wt%. The preferred weight ratio is 1:0.1 to 1, preferably 1:1. The enzymolysis temperature is 35-55 ℃, preferably 40-55 ℃; the pH of enzymolysis is 5.0-6.0, preferably 5.5-6.0; the enzymolysis time is 4-12 h, preferably 4-8 h; the substrate concentration in the enzymolysis process is 3-6wt%, preferably 3-5wt%.

The invention has the following beneficial effects:

the fermentation supernatant obtained by sequentially adopting the candida albosis and the bacillus subtilis to continuously ferment the roselle extract shows novel and surprising characteristics, and research and test results show that the roselle extract still has the capacities of supernormal moisture retention, water locking and skin cell protection under a low-temperature drying environment.

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof.

Example 1 preparation of enzymatic Hibiscus sabdariffa Solanum extract (Re)

Pulverizing calyx Hibisci Sabdariffae, adding 6 times of 80% ethanol solution, reflux-extracting at 60deg.C for 3 times each for 1 hr, mixing filtrates, removing ethanol under reduced pressure, and concentrating to obtain Hibisci Sabdariffae ethanol extract;

adding 5wt% of roselle extract into deionized water, and adding a mixture of pectase and cellulase according to a ratio of 1:1, and carrying out enzymolysis for 8 hours at the temperature of 45 ℃ at the pH of 6.0, and obtaining the enzymatic roselle extract after passivation treatment.

Example 2 preparation of an enzymatic Hibiscus sabdariffa Water extract

Pulverizing calyx Hibisci Sabdariffae, adding distilled water 6 times the weight of calyx Hibisci Sabdariffae, decocting for 3 times each for 1 hr, mixing filtrates, and concentrating to obtain calyx Hibisci Sabdariffae water extract; the enzymolysis method of reference example 1 is used for preparing the enzymolysis roselle water extract for later use.

Example 3 preparation of Hibiscus sabdariffa extract/fermentation product

Adding 12wt% of enzymolysis roselle alcohol extract into a fermentation medium, inoculating 1wt% of candida albosis, and fermenting for 72 hours under the conditions that the temperature is 28 ℃, the pH is 5.0 and the rotation speed of a shaking table is 120 rpm/min; adding fermentation medium, inoculating 1wt% of Bacillus subtilis, fermenting at 35deg.C and pH of 7.5 at rotation speed of 160rpm/min for 48 hr, collecting fermentation supernatant, deodorizing with active carbon, decolorizing, and sterilizing to obtain enzymatic hydrolysis Hibiscus sabdariffa extract/fermentation product (designated Fp 1).

Wherein the fermentation medium comprises the following components: 1L distilled water contains 15g of peptone, 20g of glucose, 0.5g of beef extract, 5g of sodium chloride and 20g of agar.

Comparative example 1: fermentation product Fp2

Adding 12wt% of enzymatic roselle alcohol extract into a fermentation medium, inoculating 1wt% of candida albosis, fermenting for 72 hours at the temperature of 28 ℃ and the pH value of 5.0 and the rotation speed of a shaking table of 120rpm/min, collecting a fermentation supernatant, deodorizing and decoloring by using active carbon, and sterilizing to obtain a fermentation product Fp2, wherein the fermentation medium is the same as that of the example 3.

Comparative example 2: fermentation product Fp3

Adding 12wt% of enzymolysis roselle alcohol extract into a fermentation medium, inoculating 1wt% of bacillus subtilis, fermenting for 48 hours at the temperature of 35 ℃ and the pH of 7.5 and the rotation speed of a shaking table of 160rpm/min, collecting fermentation supernatant, deodorizing and decoloring by using active carbon, and sterilizing to obtain a fermentation product Fp3, wherein the fermentation medium is the same as that of the example 3.

Comparative example 3: fermentation product Fp4

Adding 12wt% of enzymolysis roselle alcohol extract into a fermentation medium, inoculating 1wt% of candida albosis, and fermenting for 72 hours under the conditions that the temperature is 28 ℃, the pH is 5.0 and the rotation speed of a shaking table is 120 rpm/min; supplementing a fermentation medium, continuously inoculating 1wt% of lactobacillus plantarum, fermenting for 72 hours at the temperature of 28 ℃ and the pH value of 4.5 under the condition that the rotation speed of a shaking table is 160rpm/min, collecting fermentation supernatant, deodorizing and decoloring by using active carbon, and sterilizing to obtain a fermentation product Fp4; the fermentation medium is MRS broth.

Comparative example 4: fermentation product Fp5

Adding 12wt% of enzymolysis roselle alcohol extract into a fermentation medium, inoculating 2wt% of mixed bacteria (comprising candida albosis and bacillus subtilis according to the weight ratio of 1:1), and fermenting for 72 hours at the temperature of 30 ℃ and the pH value of 5.0 and the rotation speed of a shaking table of 160 rpm/min; collecting fermentation supernatant, deodorizing and decolorizing with active carbon, and sterilizing to obtain fermentation product Fp5; the fermentation medium was the same as in example 3.

Comparative example 5: fermentation product Fp6

Adding 12wt% of enzymolysis roselle alcohol extract into a fermentation medium, inoculating 1wt% of bacillus subtilis, and fermenting for 48 hours under the conditions that the temperature is 35 ℃, the pH is 7.5 and the rotation speed of a shaking table is 160 rpm/min; adding fermentation medium, inoculating 1wt% of candida albosis, fermenting at 28 deg.c, pH 5.0 and shaking table rotation speed of 120rpm/min for 72 hr, collecting fermentation supernatant, deodorizing with active carbon, decolorizing, and sterilizing to obtain fermentation product Fp6, wherein the fermentation medium is the same as that of example 3.

Comparative example 6: fermentation product Fp7

Adding 12wt% of the enzymolysis roselle water extract prepared in the example 2 into a fermentation medium, inoculating 1wt% of candida alboma, and fermenting for 72 hours at the temperature of 28 ℃ and the pH value of 5.0 and the rotation speed of a shaking table of 120 rpm/min; adding a fermentation medium, continuously inoculating 1wt% of bacillus subtilis, fermenting for 48 hours at the temperature of 35 ℃ and the pH of 7.5 and the rotation speed of a shaking table of 160rpm/min, collecting a fermentation supernatant, deodorizing and decoloring by using active carbon, and sterilizing to obtain a fermentation product Fp7, wherein the fermentation medium is the same as that of the embodiment 3.

Test example one, skin irritation test

The fermentation products Fp1 to Fp7 were tested for skin irritation according to the skin irritation/corrosiveness test in the cosmetic safety technical Specification, and were incorporated into 70 persons of volunteer subjects, and were in accordance with the volunteer selection criteria of the subjects, and the test results are shown in Table 1 below.

Table 1: skin irritation test results

According to the test results of the table, the continuous fermentation or the mixed fermentation of the compound bacteria can reduce the irritation of roselle to the skin and reduce the toxic and side effects of the roselle.

Test example two, test for promoting hyaluronic acid production

Collecting human skin keratinocyte HEKa in logarithmic phase, and regulating cell concentration to 1×10 with DMEM liquid culture medium ⁴ 0.5mL of the cell suspension was inoculated into a 96-well plate at 37℃in 5% CO ₂ Culturing under the condition until the cells grow to be in a nearly fusion state; the medium was discarded, 100. Mu.L of DMEM medium containing 1wt% of Fp1 to Fp7 and Re fermentation product was added, and a negative control group was set, and the mixture was placed at 37℃and 5% CO ₂ Culturing for 24h under the condition, collecting supernatant of each group of cell culture medium, detecting the content of hyaluronic acid in the supernatant by adopting an ELISA method (strictly according to the operation of ELISA kit instructions), recording the OD value of a sample detected by an ELISA reader at the wavelength of 450nm, and expressing the synthesis condition of the hyaluronic acid by using the OD value.

Table 2: promotion of hyaluronic acid Synthesis

Note that: in comparison with the negative control group, ^* P＜0.05， ^** p is less than 0.01; in comparison with the ratio of Fp1, ^# P＜0.05， ^## P＜0.01。

as can be seen from the results of Table 2, the fermentation product (Fp 1) obtained by sequentially carrying out continuous fermentation of Candida albopictus and Bacillus subtilis has a remarkable effect of promoting synthesis of hyaluronic acid by HEKa cells, and has a very remarkable difference (P < 0.01) compared with single strain fermentation; however, the fermentation products Fp4 obtained by continuous fermentation of the same strain with other strains, the fermentation products Fp5 obtained by mixed fermentation of the same strain and the fermentation products Fp6 obtained by changing the fermentation sequence of the strain are significantly different from those of Fp1 (P < 0.05), which fully shows that the HA synthesis promoting characteristics of the roselle fermentation products are obviously related to the types and fermentation modes of the fermentation strains thereof.

Test example III, promotion of filaggrin expression

Taking human skin cutin in logarithmic growth phaseForming cell HEKa, regulating cell concentration to 1×10 with DMEM liquid culture medium ⁴ Inoculating into 96-well plate at 37deg.C with 5% CO ₂ Culturing for 24 hours under the condition, adding 100 mu L of DMEM medium containing 1wt% of Fp 1-Fp 7 fermentation products and Re, and continuing culturing for 24 hours. Cellular RNA was extracted using Trizol reagent, RT-PCR analysis was performed using a one-step reverse transcription PCR kit, using a filaggrin primer, and the expression levels of the respective groups were compared using beta-actin as an internal control group.

Table 3: promoting filaggrin expression

Note that: the blank was set to 100%.

As is clear from Table 3, in general, the use of the mixed bacteria significantly promoted the synthesis of silk fibroin by HEKa cells, either in a continuous fermentation or in a mixed fermentation mode, wherein the fermentation product (Fp 1) obtained by sequentially carrying out continuous fermentation by Candida albopicta and Bacillus subtilis promoted the synthesis of silk fibroin by HEKa cells to be the strongest, and the mixed fermentation by Candida albopicta and Bacillus subtilis was the worst fermentation product obtained by single fermentation.

Test example four, skin moisture content test under different environments

160 subjects were selected and randomized into 8 groups of 20 persons each. The test areas were cleaned before each group of subjects was tested, and after sitting still for 30 minutes in a constant temperature and humidity environment. Smearing the same quality of the tested sample on the tested area until the sample is completely absorbed, respectively testing the skin moisture content of the tested area by a skin moisture content tester Corneometer CM825 before using and after using the sample for 4 hours, 8 hours and 12 hours, measuring for 3 times, taking an average value, recording the result, and calculating the skin moisture content increase rate.

In MMV ₀ MMV for pre-application skin; MMV (MMV) _t Skin MMV for t period after application.

Table 4: comparison of skin moisture increase Rate at different time points

According to Table 4, the fermentation product (Fp 1) obtained by sequentially and continuously fermenting the candida utilis and the bacillus subtilis still has ideal moisturizing effect in cold and low-humidity environments, the optimal moisturizing effect is achieved within 4 hours after the application, and then the moisturizing effect is slightly reduced, but the water content increase rate is still higher after 12 hours after the application, which indicates that the moisturizing effect is good and the lasting moisturizing capability is strong. However, although the continuous fermentation is carried out by adopting the composite bacteria, the moisturizing effect of Fp4 and Fp6 is obviously different from that of Fp1, and the difference is obvious especially in low-temperature and low-humidity environments.

Test example five, percutaneous moisture loss test

70 tested volunteers are randomly divided into 7 groups, the faces of each group of subjects are cleaned before the test, the subjects sit still for 30 minutes under the environment of constant temperature and humidity, and then Fp 1-Fp 7 fermentation products and Re are respectively smeared on the faces, and the faces are gently massaged until the faces are absorbed. The cheek skin moisture loss [ TEWL, g/(h·m2) was measured using an epidermal moisture loss meter TM300 before and 12h after use; the lower the TEWL value, the less water loss and the better the skin barrier function, the 5 measurements were taken on each of the left and right cheeks, the average was taken, the values were recorded, and the percent reduction in skin water loss through the epidermis was calculated.

Percentage decrease in skin moisture transepidermal loss = [ (amount of moisture transepidermal before use-amount of moisture transepidermal after use)/amount of moisture transepidermal before use ] ×100%

Table 5: the percentage of the skin loss through the epidermis is reduced after 12 hours of application

As shown in the results of Table 5, the fermentation products obtained by fermenting with mixed strains have strong effects of repairing skin barrier and locking water, except for fermenting with single strain.

Test example six, cytoprotective effect

The measurement was performed by the MTT method: human skin fibroblasts were cultured at 1X 10 ⁴ Inoculating the density of/mL into a 96-well plate, adding a DMEM culture medium to culture until cells reach a fusion state, respectively adding 1% of Fp 1-Fp 7 fermentation products and Re, setting a negative control group, and continuing to culture for 12 hours; the medium was removed and the culture was continued for 4 hours in a dry environment (25 ℃, 40%) to determine the activity of mitochondria in the cells and calculate the cell survival (%).

Table 6: protective action on cells

Note that: in comparison with the negative control group, ^* P＜0.05， ^** P＜0.01。

as is clear from the results shown in Table 6, the cell viability was improved after the intervention culture of the fermentation product (Fp 1) obtained by sequentially subjecting the Candida albopictus and the Bacillus subtilis to continuous fermentation, and the effect of the fermentation product on the cells was shown to be protective. And the survival rate of the cells after the fermentation product Fp2 obtained by fermenting the candida albosis alone is slightly lower than that of a negative control group after the fermentation product Fp2 is subjected to the intervention culture, which shows that the cells possibly have a certain toxic effect on human skin fibroblasts, and the result is consistent with the result of the stimulus test.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. A moisturizing composition resistant to cold and dry environments, said composition comprising at least: roselle extract/fermentation product; the fermentation product is a fermentation supernatant obtained by sequentially inoculating candida arvensis and bacillus subtilis into a fermentation culture medium containing roselle extract for continuous fermentation; the roselle extract is prepared by taking roselle calyx as a raw material and extracting the roselle calyx with 60-85% ethanol solution by volume fraction; the roselle extract is subjected to enzymolysis pretreatment, and the enzyme adopted in the enzymolysis process is one or more of pectase, cellulase and protease.

2. The moisturizing composition of claim 1, wherein the roselle extract is present in the fermentation medium in an amount of 1-20% by weight.

3. The moisturizing composition of claim 1 or 2, wherein the fermentation conditions are: the pH is 4.0 to 7.5; the fermentation time is 24-72 h; the fermentation temperature is 25-37 ℃.

4. The moisturizing composition of claim 1, wherein the enzymatic treatment is performed under the following conditions: the pH is 5.0-6.0, and the temperature is 35-55 ℃; the enzymolysis time is 4-12 hours; the concentration of the substrate is 3-6wt%; the final concentration of the enzyme is 1-3wt%.

5. Use of the moisturizing composition according to any one of claims 1 to 4 for preparing a skin care product having long-lasting moisturizing effect under normal and special dry environments.

6. The method according to claim 5, wherein the special drying environment is an environment temperature of < -5 ℃ and a relative humidity of 30-50%.

7. A long-lasting moisturizing concentrate comprising the moisturizing composition of any one of claims 1-4.