CN114796052A

CN114796052A - Moisturizing composition, moisturizing essence and preparation method thereof

Info

Publication number: CN114796052A
Application number: CN202210508245.XA
Authority: CN
Inventors: 罗敏; 吴小端; 陈铭楷; 陈培仲
Original assignee: Guangzhou Qingsongquan Biotechnology Co ltd
Current assignee: Baitide Pharmaceutical Biotechnology Guangdong Co ltd
Priority date: 2022-05-11
Filing date: 2022-05-11
Publication date: 2022-07-29
Anticipated expiration: 2042-05-11
Also published as: CN114796052B

Abstract

The invention belongs to the field of biotechnology. More particularly, to a moisturizing composition and a moisturizing essence containing a roselle extract/fermentation product, 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 a roselle extract and carrying out continuous fermentation. The fermentation supernatant obtained by continuously fermenting the roselle extractive by adopting the eremothecium ashbyi and the bacillus subtilis in sequence shows surprising characteristics, and research and test results show that the roselle extractive still has the capabilities of preserving moisture, locking water and protecting skin cells in a low-temperature dry environment.

Description

Moisturizing composition, moisturizing essence and preparation method thereof

Technical Field

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

Background

Cold environments can result in reduced air humidity and air dryness, which can cause the skin to become abnormally dry. The skin cells lose their luster and elasticity due to the reduction of moisture, even cause a series of skin diseases, and become more fragile, which is fatal to the health of the skin in cold environments. Currently, moisturizing ingredients such as glycerin on the market absorb moisture in the air to moisturize the air, but the moisturizing effect is poor in the case of low air humidity, particularly in the case of cold. Therefore, there is a need to provide a product that has long-lasting moisturization under extreme conditions such as cold drying.

Hibiscus sabdariffa (Hibiscus sabdariffa) is an annual herb of Hibiscus of Malvaceae; the content of organic acid in the roselle calyx is up to 15-30%, the organic acid mainly comprises oxalic acid, tartaric acid, malic acid, succinic acid and the like, the content of anthocyanin is up to 2%, and the anthocyanin mainly comprises delphinidin-3-elderberry diglycoside and cyanidin-3-elderberry diglycoside; further contains flavonoids such as 3,5,7,8,3, 4-hexahydroxyflavone, quercetin, hibiscus red and hibiscus glycoside. The study showed that: the inhibition rate of 250 mu g/mL roselle 50% alcohol extract on tyrosinase activity is 84%, which shows that the roselle 50% alcohol extract has better inhibition effect on tyrosinase and strong inoxidizability, and can be used as a skin whitening agent; the inhibition rates of the roselle ethyl acetate extract with the concentration of 6.25mg/mL on the activity of the cyclooxygenase-1 and the cyclooxygenase-2 are respectively 78.4 percent and 13.6 percent; the 300. mu.g/mL 70% alcohol extract of Hibiscus sabdariffa showed a 23% promotion of neutral lipid hydrolysis. 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 has the ability to provide moisture to the skin, strengthen the skin barrier, and protect cells in particularly dry environments.

The invention also aims to provide the moisturizing essence with long-acting moisturizing effect in extreme environments. The moisturizing essence has the capability of providing moisture for skin in a special dry environment, strengthening skin barrier and protecting cells.

Another object of the present invention is to provide a method for improving the lack of water in the skin in a particularly dry environment. The method improves skin hydration by topically applying the moisturizing essence to the skin.

The special drying environment is that the ambient temperature is less than-5 ℃ and the relative humidity is 30-50%. Although the present invention emphasizes the ability of the composition to retain moisture in a particular dry environment, it does not negate the ability to retain moisture in other, non-particular environments.

The above purpose of the invention is realized by the following technical scheme: a moisturizing composition for combating cold and dry environments, said composition comprising: 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 a roselle extract and carrying out 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 60-85% volume fraction ethanol extract. It can be prepared by a conventional technique as long as it can extract soluble components contained in the raw material and dissolve into the extraction solvent. The extraction can be carried out at room temperature or under reflux heating, and the residue can be removed by filtration or the like, and the filtrate can be collected and further concentrated to obtain the desired extract. Particularly, when the 60-85% ethanol extract is preferred, the preparation method thereof can comprise the following steps:

taking roselle calyx, crushing, adding 60-85% volume fraction ethanol solution with the weight being 3-6 times of that of the roselle calyx, heating and refluxing for 1-3 times at 50-65 ℃, combining filtrates, removing ethanol under reduced pressure, and concentrating to obtain the roselle calyx.

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 growth and proliferation of yeast and bacillus subtilis.

The pH value of the culture medium is usually 4.0-7.5, preferably 5.0-7.5, and the fermentation can be carried out in a preferable pH range to maintain the stability among raw materials; the temperature for fermentation is not particularly limited as long as it satisfies the growth of the strain and enables fermentation; specifically, the fermentation temperature in the invention can be 25-37 ℃, and preferably 25-32 ℃; the fermentation time is usually 24-72 h, preferably 36-72 h; with respect to the morphology of the fermentation product, although the present invention is directed to the fermentation supernatant, it is not excluded that the fermentation product exists in other forms. Theoretically, the fermentation supernatant obtained by the secondary fermentation usually needs to be subjected to post-treatment such as sterilization, deodorization, decoloration and the like by using an ion exchange column and an activated carbon column, and the post-treated fermentation supernatant can be further concentrated or lyophilized.

In another embodiment, the yeast is Eremothecium ashbyii. The emphasis is placed on the fermentation of the species Eremothecium ashbyii, since it is easier to obtain compositions with long-lasting moisturization under low temperature, dry conditions if Eremothecium ashbyii is used. The present invention emphasizes that the above-mentioned fermentation product is obtained by continuous fermentation using a specific bacterial species, because the fermentation product obtained by continuous fermentation using eremothecium and bacillus subtilis has a superior moisturizing ability under low-temperature, dry conditions, as compared to mixed fermentation or continuous fermentation using different bacterial species (even the same yeast). The inventor also proves that the difference of the effect is unexpected although the comparative experiment adopts industrial strains which are commonly used for biological fermentation.

Meanwhile, the invention emphasizes the fermentation sequence because a more ideal product can be obtained only by adopting specific strains for fermentation under the limited sequence.

In another embodiment, the roselle extract is pre-treated by enzymatic hydrolysis, although the present invention emphasizes that roselle extract needs to be treated by enzymatic hydrolysis, but does not exclude the possibility that it is not treated by enzymatic hydrolysis. The enzymolysis treatment is carried out for the purpose that the subsequent fermentation effect is more ideal; the enzyme adopted in the enzymolysis process can be one or more of pectinase, cellulase and protease, preferably mixed enzyme is adopted for enzymolysis, and when the mixed enzymolysis is adopted for enzymolysis, the adopted enzyme can be pectinase and cellulase, and the two enzymes can be composed in any weight ratio as long as the enzyme content is within the range of 1-3 wt%. The weight ratio is preferably 1:0.1 to 1, and preferably 1: 1. The enzymolysis temperature is 35-55 ℃, and preferably 40-55 ℃; the pH value of enzymolysis is 5.0-6.0, preferably 5.5-6.0; the enzymolysis time is 4-12 h, preferably 4-8 h; the concentration of the substrate in the enzymolysis process is 3-6 wt%, and preferably 3-5 wt%.

The invention has the following beneficial effects:

the fermentation supernatant obtained by continuously fermenting the roselle extractive by adopting the eremothecium ashbyi and the bacillus subtilis in sequence shows a surprising new characteristic, and research and test results show that the roselle extractive still has the capabilities of preserving moisture, locking water and protecting skin cells in a low-temperature dry 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. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.

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

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

adding 5 wt% of Hibiscus sabdariffa extract to deionized water, and adding pectinase and cellulase at a ratio of 1:1, carrying out enzymolysis for 8 hours at the pH of 6.0 and the temperature of 45 ℃, and carrying out passivation treatment to obtain an enzymolysis roselle alcohol extract for later use.

Example 2 enzymatic hydrolysis of aqueous extract of Hibiscus sabdariffa

Pulverizing calyx Hibisci Sabdariffae, adding 6 times of distilled water, decocting for 3 times (1 hr each time), mixing filtrates, and concentrating to obtain calyx Hibisci Sabdariffae water extract; reference example 1 the enzymatic hydrolysis method was used to prepare an enzymatic hydrolyzed roselle aqueous extract for use.

Example 3 Hibiscus sabdariffa extract/fermentation product preparation

Adding 12 wt% of enzymolysis roselle alcohol extract into a fermentation culture medium, inoculating 1 wt% of eremothecium alniforme, and fermenting for 72h under the conditions that the temperature is 28 ℃, the pH value is 5.0 and the rotating speed of a shaking table is 120 rpm/min; supplementing a fermentation culture medium, continuously inoculating 1 wt% of bacillus subtilis, fermenting for 48h at the temperature of 35 ℃, the pH value of 7.5 and the rotating speed of a shaking table of 160rpm/min, collecting fermentation supernatant, deodorizing and decolorizing by using activated carbon, and sterilizing to obtain an enzymolysis roselle alcohol extract/fermentation product (named as Fp 1).

Wherein the fermentation medium comprises the following components: the 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 12 wt% of enzymolysis roselle alcohol extract into a fermentation medium, inoculating 1 wt% of eremothecium alniforme, fermenting for 72h at the temperature of 28 ℃, the pH value of 5.0 and the rotating speed of a shaking table of 120rpm/min, collecting fermentation supernatant, deodorizing and decolorizing by active carbon, and sterilizing to obtain a fermentation product Fp2, wherein the fermentation medium is the same as that in example 3.

Comparative example 2: fermentation product Fp3

Adding 12 wt% of enzymolysis roselle alcohol extract into a fermentation culture medium, inoculating 1 wt% of bacillus subtilis, fermenting for 48h under the conditions that the temperature is 35 ℃, the pH value is 7.5 and the rotating speed of a shaking table is 160rpm/min, collecting fermentation supernatant, deodorizing and decolorizing by using activated carbon, and sterilizing to obtain a fermentation product Fp3, wherein the fermentation culture medium is the same as that in example 3.

Comparative example 3: fermentation product Fp4

Adding 12 wt% of enzymolysis roselle alcohol extract into a fermentation culture medium, inoculating 1 wt% of eremothecium alniforme, and fermenting for 72h under the conditions that the temperature is 28 ℃, the pH value is 5.0 and the rotating speed of a shaking table is 120 rpm/min; supplementing a fermentation culture medium, continuously inoculating 1 wt% of lactobacillus plantarum, fermenting for 72 hours at the temperature of 28 ℃, the pH value of 4.5 and the rotation speed of a shaking table of 160rpm/min, collecting fermentation supernatant, deodorizing and decolorizing with active carbon, and sterilizing to obtain a fermentation product Fp 4; the fermentation medium is MRS broth.

Comparative example 4: fermentation product Fp5

Adding 12 wt% of enzymolysis roselle alcohol extract into a fermentation culture medium, inoculating 2 wt% of mixed bacteria (composed of eremothecium alniformis and bacillus subtilis according to the weight ratio of 1: 1), and fermenting for 72h under the conditions that the temperature is 30 ℃, the pH is 5.0 and the rotating speed of a shaking table is 160 rpm/min; collecting fermentation supernatant, deodorizing and decolorizing with active carbon, and sterilizing to obtain fermentation product Fp 5; the fermentation medium was the same as in example 3.

Comparative example 5: fermentation product Fp6

Adding 12 wt% of enzymolysis roselle alcohol extract into a fermentation culture medium, inoculating 1 wt% of bacillus subtilis, and fermenting for 48h under the conditions that the temperature is 35 ℃, the pH value is 7.5 and the rotating speed of a shaking table is 160 rpm/min; supplementing a fermentation culture medium, continuously inoculating 1 wt% of eremothecium alniforme, fermenting for 72 hours at the temperature of 28 ℃, the pH value of 5.0 and the rotation speed of a shaking table of 120rpm/min, collecting fermentation supernatant, deodorizing and decolorizing by using activated carbon, and sterilizing to obtain a fermentation product Fp6, wherein the fermentation culture medium is the same as that in example 3.

Comparative example 6: fermentation product Fp7

Adding 12 wt% of the enzymolyzed roselle aqueous extract prepared in example 2 into a fermentation medium, inoculating 1 wt% of eremothecium alniforme, and fermenting for 72h at the temperature of 28 ℃, the pH of 5.0 and the rotating speed of a shaker of 120 rpm/min; supplementing a fermentation culture medium, continuously inoculating 1 wt% of bacillus subtilis, fermenting for 48 hours at the temperature of 35 ℃, the pH value of 7.5 and the rotating speed of a shaking table of 160rpm/min, collecting fermentation supernatant, deodorizing and decolorizing by using activated carbon, and sterilizing to obtain a fermentation product Fp7, wherein the fermentation culture medium is the same as that in example 3.

Test example I, skin irritation test

The skin irritation of the fermentation products Fp 1-Fp 7 was tested according to the skin irritation/corrosion test in the technical Specification for cosmetic safety, and the fermentation products were included in 70 volunteers to meet the volunteer selection criteria, and the test results are shown in Table 1 below.

Table 1: results of skin irritation test

According to the test results in the table, the irritation of the roselle to the skin can be reduced and the toxic and side effects can be reduced by adopting the compound bacteria for continuous fermentation or mixed fermentation.

Test example II hyaluronic acid production promoting test

Taking human skin keratinocyte HEKa in logarithmic growth phase, and adjusting cell concentration to 1 × 10 with DMEM liquid culture medium ⁴ Perml, 0.5mL of the cell suspension was inoculated into a 96-well plate at 37 ℃ with 5% CO ₂ Culturing under the condition until the cell growth is in a near-fusion state; discarding the culture medium, adding 100 μ L DMEM medium containing 1 wt% Fp 1-Fp 7 and Re fermentation product, setting negative control group, placing at 37 deg.C 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 an ELISA method (strictly according to the operation of an ELISA kit instruction), recording the OD value of a detection sample of an enzyme-labeling instrument under the wavelength of 450nm, and expressing the synthesis condition of hyaluronic acid by the OD value.

Table 2: promotion of hyaluronic acid Synthesis

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

the results in table 2 show that the fermentation product (Fp1) obtained by sequential continuous fermentation of eremothecium ashbyii and bacillus subtilis has the effect of remarkably promoting the synthesis of hyaluronic acid by HEKa cells, and has very remarkable difference (P < 0.01) compared with single-strain fermentation; however, the fermentation product Fp4 obtained by continuous fermentation of the eremothecium ashbyii and other strains, the fermentation product Fp5 obtained by mixed fermentation of the eremothecium ashbyii and other strains, and the fermentation product Fp6 obtained by exchanging the fermentation sequence of the strains have obvious difference (P is less than 0.05) compared with Fp1, which fully proves that the HA synthesis promoting property of the roselle fermentation product HAs obvious relationship with the type and the fermentation mode of the fermentation strains thereof.

Test example III promotion of expression of filaggrin

Taking human skin keratinocyte HEKa in logarithmic growth phase, and adjusting cell concentration to 1 × 10 with DMEM liquid culture medium ⁴ Perml, inoculated in a 96-well plate at 37 ℃ with 5% CO ₂ Culturing for 24h under the condition, adding 100 mu L DMEM medium containing 1 wt% Fp 1-Fp 7 fermentation products and Re, and continuing culturing for 24 h. Cell RNA was extracted using Trizol reagent, RT-PCR analysis was performed using one-step reverse transcription PCR kit, and expression levels of each group were compared using filaggrin primers and β -actin as an internal control group.

Table 3: promoting expression of filaggrin

Note: the blank control group was set to 100%.

As can be seen from Table 3, in general, the synthesis of filaggrin by HEKa cells can be significantly promoted by using mixed bacteria in either a continuous fermentation mode or a mixed fermentation mode, wherein the fermentation product (Fp1) obtained by sequentially carrying out continuous fermentation on the Eremothecium ashbyii and the Bacillus subtilis has the strongest effect of promoting the synthesis of filaggrin by HEKa cells, and the mixed fermentation is carried out secondly by using the Eremothecium ashbyii and the Bacillus subtilis, and the worst is the fermentation product obtained by using a single fermentation.

Test example four skin moisture content test under different environments

160 subjects were selected and randomized into 8 groups of 20 subjects each. The test area was washed before each test and was set aside in a constant temperature and humidity environment for 30 minutes before each test. Smearing the same mass of tested sample on the tested area until the sample is completely absorbed, respectively testing the moisture content of the skin of the tested area by a skin moisture tester CM825 for 4h, 8h and 12h before and after the use of the sample, measuring for 3 times, averaging, recording the result, and calculating the moisture content increase rate of the skin.

In the formula, MMV ₀ MMV on skin before application; MMV _t MMV of skin t time period after application.

Table 4: comparison of skin moisture content growth rates at different time points

As can be seen from Table 4, the fermentation product (Fp1) obtained by sequential continuous fermentation of Eremothecium ashbyi and Bacillus subtilis still has ideal moisturizing effect in cold and low-humidity environments, reaches the optimal moisturizing effect within 4h after smearing and then slightly decreases, but still keeps a higher water content increase rate within 12h after smearing, which indicates that the moisturizing effect is good and has stronger lasting moisturizing capability. However, even if the continuous fermentation is carried out by using the composite bacteria, the moisture-retaining effects of Fp4 and Fp6 are obvious compared with Fp1, especially under the environment of low temperature and low humidity.

Test example five, measurement of percutaneous moisture loss value

70 tested volunteers are randomly divided into 7 groups, the face of each test subject is cleaned before testing, the test subjects sit still for 30 minutes in a constant temperature and humidity environment, then the faces are respectively coated with Fp 1-Fp 7 fermentation products and Re, and the test subjects are gently massaged until absorption. Cheek skin water dispersion amount [ TEWL, g/(h · m2) was measured using an epidermal water loss meter TM300 before and 12h after use; the lower the TEWL value, the less water loss, the better the skin barrier function ], the left and right cheeks were measured 5 times each, averaged, and the values recorded to calculate the percent reduction in skin water loss through the epidermis.

The percentage of decrease in skin moisture through epidermal dissipation was ═ [ (amount of moisture loss through epidermis before use-amount of moisture loss through epidermis after use)/amount of moisture loss through epidermis before use ] × 100%

Table 5: the loss of the skin through epidermis after being smeared for 12 hours is reduced by percentage

According to the results of the above table 5, it can be known that, besides the single-strain fermentation, the fermentation products obtained by the mixed-strain fermentation have strong effects of repairing skin barrier and locking water.

Test example six protective action against cells

The assay was performed using the MTT method: human skin fibroblasts were arranged at 1X 10 ⁴ Inoculating the cells in a 96-well plate at a density of/mL, adding a DMEM medium to culture the cells until the cells reach a fused state, respectively adding 1% of Fp 1-Fp 7 fermentation products and Re, setting a negative control group, and continuously culturing for 12 hours; the medium was removed, the culture was continued for 4 hours under a dry environment (25 ℃, 40%), the mitochondrial activity in the cells was measured, and the cell survival rate (%) was calculated.

Table 6: has cell protecting effect

Note: compared with the negative control group, the test results show that, ^* P＜0.05， ^** P＜0.01。

as is clear from the results in Table 6, the cell survival rate was improved and the effect of protecting cells was shown by the intervening culture of the fermentation product (Fp1) obtained by sequential continuous fermentation of Eremothecium ashbyii and Bacillus subtilis. The cell survival rate of the fermentation product Fp2 obtained by fermenting the eremothecium alfoschi alone after intervening culture is slightly lower than that of a negative control group, which shows that the fermentation product Fp2 possibly has a certain toxic effect on human skin fibroblasts, and the result is consistent with the result of the irritation test.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A moisturizing composition resistant to cold and dry environments, said composition comprising: 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 a roselle extract and carrying out continuous fermentation.

2. The moisturizing composition of claim 1, wherein the yeast is eremothecium ashbyii.

3. A moisturizing composition according to claim 1 or 2, characterized in that the roselle extract is present in the fermentation medium in a quantity of 1 to 20% by weight.

4. A moisturizing composition according to any of claims 1 to 3, characterized in that the fermentation conditions are: pH4.0-7.5; the fermentation time is 24-72 h; the fermentation temperature is 25-37 ℃.

5. The moisturizing composition as claimed in claim 4, wherein the Hibiscus sabdariffa extract is obtained by extracting Hibiscus sabdariffa calyx with 60-85% volume fraction ethanol solution.

6. The moisturizing composition of claim 5, wherein the Hibiscus sabdariffa extract is pretreated by enzymatic hydrolysis, wherein the enzymes used in the enzymatic hydrolysis are one or more of pectinase, cellulase and protease.

7. The moisturizing composition of claim 6, 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 h; the concentration of the substrate is 3-6 wt%; the final concentration of the enzyme is 1-3 wt%.

8. Use of the moisturizing composition as claimed in any one of claims 1 to 7 in the preparation of a skin care product with long-lasting moisturizing effect in normal and special dry environments.

9. The use according to claim 8, wherein the special drying environment is an environment temperature of less than-5 ℃ and a relative humidity of 30-50%.

10. A long-lasting moisturizing essence characterized by comprising the moisturizing composition as claimed in any one of claims 1 to 7.