CN114948833B

CN114948833B - Day lily fermented product for cosmetics, and preparation method and application thereof

Info

Publication number: CN114948833B
Application number: CN202210772661.0A
Authority: CN
Inventors: 李萌; 王昌涛; 付豪; 张玉芝; 王冬冬; 赵丹
Original assignee: Beijing Technology and Business University
Current assignee: Beijing Technology and Business University
Priority date: 2022-06-30
Filing date: 2022-06-30
Publication date: 2024-04-05
Anticipated expiration: 2042-06-30
Also published as: CN114948833A

Abstract

The present disclosure provides a day lily ferment useful for cosmetics and a preparation method thereof, the preparation method comprising: mixing flos Hemerocallis powder with appropriate amount of water, sterilizing, and cooling to obtain fermentation substrate; inoculating lactobacillus or saccharomycetes to the fermentation substrate for fermentation treatment, and then sterilizing and separating to obtain supernatant fluid to obtain daylily fermentation liquor. The pH=4.3-4.5 of the two day lily fermentation raw pulps provided by the disclosure is suitable for human skin, has high safety to human body, and can be directly used as cosmetics; the two day lily fermented products can be used as natural plant raw materials of cosmetics for cosmetics, and compared with water extracts, the day lily fermented products have strong functions of resisting oxidization, resisting inflammation and whitening skin, and also have good human body safety.

Description

Day lily fermented product for cosmetics, and preparation method and application thereof

Technical Field

The disclosure belongs to the technical field of biological fermentation, and in particular relates to a day lily fermented product for cosmetics, and a preparation method and application thereof.

Background

Daylily (subject name: hemerocallis citrina Baroni) is also known as daylily, day lily, lemon daylily, and amnesia, belonging to the family Liliaceae, and the perennial herb. Day lily has a cultivation history of over 2000 years in China, and is selected naturally and manually for a long time to form rich variety resources.

The daylily not only can be used as ornamental flowers, but also can be used as a plant with medicinal value and edible value, and is one of four precious products in vegetables. Day lily contains abundant carbohydrates, proteins, lipids, fats, carotenes, sugars and abundant trace elements. The daylily can moisten skin, strengthen toughness and elasticity of the skin, make the skin tender, full, smooth and soft, prevent skin aging, remove color spots and keep the skin white, tender and fine. Especially, the Chinese medicinal composition has good effect of relieving chloasma caused by nervous disorder and endocrine disturbance due to mental stress and excessive pressure. The flower bud and root of day lily contains flavonoid, anthraquinone, terpenoid, alkaloid, steroid, saponin and phenolic acid, and has antioxidant, antitumor, antidepressant, sleep improving, tranquilizing, antibacterial, antiinflammatory and liver protecting effects.

At present, the day lily component-containing cosmetics in the market are prepared by adding a certain proportion of day lily flower extract (alias day lily extract) into a basic formula of the cosmetics, wherein the addition amount of the day lily flower extract is low, and the extract is limited by extraction conditions, so that the content of substance components in the extract is limited, and the efficacy of the day lily cannot be fully exerted. At present, a method for fermenting the day lily has been applied to the field of foods, but research for applying the fermented day lily to the field of cosmetics has not been found, and in the prior art, the fermentation period of a day lily fermentation product is longer, the fermentation steps are complicated, so that the energy consumption in the fermentation process is higher, and the product cost is higher. It is necessary to continue to study day lily extract with better effect and preparation method thereof.

Disclosure of Invention

The following presents a simplified summary of the disclosure in order to provide a basic understanding of some aspects of the disclosure. It should be understood that this summary is not an exhaustive overview of the disclosure. It is not intended to identify key or critical elements of the disclosure or to delineate the scope of the disclosure. Its purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is discussed later.

In view of the above-mentioned drawbacks of the prior art, an object of the present disclosure is to provide a day lily fermentation product for cosmetics, a preparation method and application thereof, wherein the day lily fermentation product is prepared by fermenting day lily with lactobacillus or saccharomycetes, all functional components of plants and activities thereof are maintained, loss of active components caused by the conventional extraction method is avoided, and the obtained fermentation product has good safety, and oxidation and inflammation resisting effects.

According to a first aspect of the present disclosure, there is provided a method for preparing a day lily ferment useful for cosmetics, comprising:

mixing flos Hemerocallis powder with appropriate amount of water, sterilizing, and cooling to obtain fermentation substrate;

inoculating lactobacillus or saccharomycetes to the fermentation substrate for fermentation treatment, and then sterilizing and separating to obtain supernatant fluid to obtain daylily fermentation liquor.

In the above preparation method of day lily fermentation product, as a preferred embodiment, the ratio of day lily powder to water is 1:100g/ml to 1:40g/ml (such as 1:90g/ml, 1:80g/ml, 1:70g/ml, 1:60g/ml, 1:50g/ml, etc.). If the concentration of the active ingredient in the fermentation broth prepared with too much water consumption is too low, if the water consumption is too low, the concentration of certain substances such as acids in the aqueous solution before fermentation is too high, which affects the growth of bacteria and is detrimental to fermentation.

In the above method for producing a daylily fermented product, as a preferred embodiment, the lactic acid bacteria are lactobacillus plantarum, and other lactic acid bacteria may be used, but the effect is inferior to lactobacillus plantarum.

In the above method for preparing the daylily fermented product, as a preferred embodiment, the yeast is yellow wine yeast, and other yeasts may also be used, but the effect is inferior to that of yellow wine yeast.

In the above method for preparing the daylily fermented product, as a preferred embodiment, the grain size of the daylily powder is 50 mesh.

In the preparation method of the daylily fermented product, as a preferred embodiment, the lactobacillus solution or the saccharomycete solution for inoculation is obtained by sequentially activating, purifying and amplifying and culturing strains, the OD value of the lactobacillus solution or the saccharomycete solution is 0.5-1.0, and the volume ratio of the lactobacillus solution or the saccharomycete solution to the fermentation substrate is 1:5-1:20 (such as 1:6, 1:8, 1:10, 1:12, 1:15, 1:18 and the like).

In the above method for preparing daylily fermented product, as a preferred embodiment, the temperature of the fermentation treatment of the lactobacillus fermentation broth is 37-45 ℃ (such as 38 ℃, 40 ℃, 42 ℃, 44 ℃ and the like) and the time is 6-16 hours (such as 7 hours, 8 hours, 10 hours, 12 hours, 14 hours, 15 hours and the like), the temperature of the fermentation treatment of the saccharomycete fermentation broth is 25-30 ℃ (such as 26 ℃, 27 ℃, 28 ℃, 29 ℃ and the like) and the time is 36-60 hours (such as 40 hours, 45 hours, 48 hours, 52 hours, 56 hours, 58 hours and the like); more preferably, the fermentation treatment is carried out in a shaker at a rotational speed of 150r/min to 180r/min (e.g. 155r/min, 160r/min, 165r/min, 170r/min, 175 r/min).

In the above method for preparing daylily fermented product, as a preferred embodiment, the sterilization treatment is performed under a pressure of 0.2-0.4Mpa at a temperature of 100-121 ℃ (e.g. 102 ℃, 105 ℃,110 ℃, 115 ℃, 118 ℃ etc.), for 15-30min (e.g. 18min, 20min, 22min, 25min, 28min, etc.).

In the preparation method of the daylily fermented product, as a preferred embodiment, the separation treatment adopts a centrifugal method; more preferably, the centrifugal speed is 4500r/min-5500r/min (such as 4600r/min, 4800r/min, 5000r/min, 5200r/min, 5400r/min, etc.), and the centrifugal time is 20min-40min (such as 25min, 30min, 35min, etc.).

In the preparation method of the daylily ferment, as a preferred embodiment, the preparation method further comprises: drying the daylily fermentation liquor to finally obtain daylily fermentation dry powder; more preferably, the drying treatment may be spray drying, vacuum freeze drying, or the like.

According to a second aspect of the present disclosure, there is also provided a fermented product prepared by the above method, including fermentation broth, fermented dry powder, and the like.

According to a third aspect of the present disclosure, there is also provided the use of the above fermentation product in the preparation of a cosmetic; preferably, the cosmetic may be a mask, an essence, a toner, an emulsion, or the like.

The day lily fermented product prepared by the method has good anti-inflammatory and antioxidant effects, and can be added into a cosmetic formula as an effective component to prepare the day lily fermented product including but not limited to: the face pack, essence, toner, emulsion and other cosmetics.

At present, day lily extracts are available on the market as raw materials of cosmetics, but few extraction methods are disclosed, and the day lily extracts are added into cosmetics, so that the effects of the day lily cannot be fully exerted due to limited substance components. The application field of the daylily fermentation product is mainly concentrated in the food field, and has little research and application in the cosmetic field. In the prior art, the fermentation period of the day lily fermentation product is longer, the fermentation steps are complicated, the energy consumption in the fermentation process is larger, and the product cost is higher.

The scheme of the invention is that the preparation method of the daylily by respectively fermenting the lactic acid bacteria and the microzyme is adopted, the daylily is adopted as a fermentation substrate, the lactic acid bacteria and the microzyme are adopted as fermentation strains for fermentation, and after the fermentation is finished, sterilization and separation treatment are carried out to obtain supernatant fluid, so that two daylily fermentation liquids are obtained and can be used as raw materials of cosmetics. The method has simple and convenient process and simple operation, and the obtained day lily fermentation product has better anti-inflammatory, antioxidant and whitening capabilities and safety as a raw material of cosmetics.

Compared with the prior art, the method has the following beneficial effects:

(1) The method adopts the fermentation modes of lactic acid bacteria and saccharomycetes respectively, takes day lily as a substrate, carries out sterilization treatment, and is connected with corresponding bacteria to carry out fermentation in a constant temperature and humidity box, so that the method has simple steps in the fermentation process, is simple to operate, has low energy consumption and can save cost.

(2) The fermentation method adopted by the disclosure respectively adopts lactobacillus and saccharomycetes to ferment the day lily, no organic reagent is added in the extraction process of the active ingredients of the day lily, the fermentation temperature and the fermentation pH are mild, the structure of the active ingredients of the plants is not damaged, the natural activity of the plants is improved, and the loss of the active ingredients caused by other extraction methods is avoided.

(3) The pH=4.3-4.5 of the two day lily fermentation raw pulps provided by the disclosure is suitable for human skin, has high safety to human body, and can be directly used as cosmetics.

(4) The two day lily fermented products provided by the disclosure can be used as natural plant raw materials of cosmetics for cosmetics, have strong antioxidation, anti-inflammatory and whitening functions on skin, and simultaneously have good human body safety.

Drawings

FIG. 1 shows the results of the anti-inflammatory performance test of the broccoli fermentation broth of example 3;

FIG. 2 shows the results of the antioxidant efficacy test of the broccoli fermentation broth of example 4;

FIG. 3 shows the results of the whitening efficacy test of the broccoli fermentation broth in example 5;

FIG. 4 shows the results of the chick embryo allantoic test of example 6;

FIG. 5 shows the hemolysis curve in the erythrocyte hemolysis experiment in example 6.

Detailed Description

The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.

The yellow cauliflower powder in the following examples is commercially available sun-dried yellow cauliflower, purchased from Beijing Tongshentang, crushed and sieved (sieved by a 50-mesh sieve) to remove particles with larger size.

The lactobacillus in the following examples is Lactobacillus plantarum, purchased from China center for type culture Collection of microorganisms, and having a strain number of CICC20261; the microzyme is yellow wine microzyme, and is purchased from China industry microbiological culture collection center, and the strain number is CICC21392; the culture medium is MRS culture medium; the culture temperature of the strain activation, purification and expansion culture is 28-37 ℃.

EXAMPLE 1 lactic acid bacteria fermentation preparation of daylily fermented product

In the embodiment, lactobacillus fermentation is adopted to prepare day lily fermented product; the preparation method comprises the following specific steps:

(1) Activating strains: the bacterial colony is picked from the preserved inclined plane and put into a liquid culture medium, and the bacterial colony is put into a shaking table to activate the bacterial strain, so that the higher viability is kept.

(2) And (3) strain purification: activated species were plated in gradient dilutions to obtain single colonies.

(3) And (3) strain expansion culture: the strain to be used is inoculated into a corresponding liquid culture medium, and is cultured in a shaking table at a proper temperature until the OD value of the strain is=0.6, and the strain is in a logarithmic phase, namely the proper inoculation concentration.

(4) Preparing a fermentation substrate: the daylily powder and water are prepared into a daylily powder culture medium, namely a fermentation substrate according to the proportion of 1g to 100 ml.

(5) Preparing lactobacillus fermentation liquor: inoculating bacterial liquid in the culture medium to the day lily powder culture medium, wherein the volume ratio of the bacterial liquid to the fermentation substrate is 1:15, and fermenting in a shaking table at the rotating speed of 180r/min and the fermentation temperature of 37 ℃ for 16 hours. Taking out the material, sterilizing at 110deg.C for 30min, cooling, centrifuging (4800 rpm, 30 min), and sterilizing with ultraviolet irradiation in ultra-clean bench. After centrifugation, the sediment is discarded, and the supernatant is left to obtain lactobacillus daylily fermentation liquor which is put into a sample bottle for standby.

Physicochemical property analysis was performed on the daylily fermentation broth prepared in this example. The appearance of the product is thick liquid and the color is light brownish red; the pH value is 4.4, the viscosity is 160P, the content of soluble solid content is 1.7%, the total colony count is less than 50CFU/ml, and no pathogenic bacteria are detected. According to the cosmetic sanitation standard GB7916-87, the total bacterial count of the cosmetic is not higher than 1000CFU/ml, so that the daylily lactobacillus fermentation liquid meets the cosmetic quality requirement.

Example 2 yellow wine Yeast fermentation preparation of daylily fermented product

In the embodiment, yellow wine yeast is adopted for fermentation to prepare day lily fermented product; the preparation method comprises the following specific steps:

(3) And (3) strain expansion culture: the strain to be used is inoculated into a corresponding liquid culture medium, and is cultured in a shaking table at a proper temperature until the OD value of the strain is=0.5, and the strain is in a logarithmic phase, namely the proper inoculation concentration.

(5) Preparing a saccharomycete fermentation liquor: inoculating bacterial liquid in the culture medium to the day lily powder culture medium, wherein the volume ratio of the bacterial liquid to the fermentation substrate is 1:15, and fermenting in a shaking table at the rotating speed of 180r/min and the fermentation temperature of 28 ℃ for 48h. Taking out the material, sterilizing at 110deg.C for 30min, cooling, centrifuging (4800 rpm, 30 min), and sterilizing with ultraviolet irradiation in ultra-clean bench. After centrifugation, the sediment is discarded, and the supernatant is left to obtain the yeast daylily fermentation liquor which is put into a sample bottle for standby.

Physicochemical property analysis was performed on the daylily fermentation broth prepared in this example. The appearance of the product is slightly viscous liquid, and the color is light brown; the pH value is 4.3, the viscosity is 160cP, the content of soluble solid content is 1.5 percent, the total number of bacterial colonies is less than 50CFU/ml, and no pathogenic bacteria is detected. According to the cosmetic sanitation standard GB7916-87, the total bacterial count of the cosmetic is not higher than 1000CFU/ml, so that the daylily microzyme fermentation liquor meets the quality requirements of cosmetics.

Example 3 analysis of anti-inflammatory efficacy of daylily fermentation broth

Anti-inflammatory efficacy analysis was performed on the day lily fermentation broth obtained in the above example.

1. The concentration of the inflammatory HaCat cytokines TNF-alpha, IL-6, COX-2 was detected by enzyme-linked immunosorbent assay (ELISA).

Briefly, culture supernatants were collected from 24-well plates using the methods provided by the manufacturer (Nanjing institute of biological engineering, used cartridges model TNF-. Alpha.H 052-1, IL-6H007-1, COX-2H200, COL-1A 005-1-2), including:

1. cell culture supernatant: the secreted components were collected by sterile tube. Centrifuging for 20min (2000-3000 rpm). The supernatant was carefully collected. When detecting the components in the cells, the cell suspension is diluted with PBS (pH 7.2-7.4) to reach a cell concentration of about 100 ten thousand/ml. The freezing and thawing are repeated to destroy cells and release intracellular components. Centrifuging for 20min (2000-3000 rpm). The supernatant was carefully collected. If precipitate is formed during preservation, the solution should be centrifuged again.

2. Dilution and sample addition of standard: setting a standard substance hole 10 holes on an enzyme-labeled coating plate, adding 100 μl of standard substance into the first and second holes respectively, adding 50 μl of standard substance diluent into the first and second holes, and mixing; then 100 mu l of each of the first hole and the second hole is added into a third hole and a fourth hole respectively, 50 mu l of standard substance diluent is added into the third hole and the fourth hole respectively, and the mixture is uniformly mixed; then 50 mu l of each of the third hole and the fourth hole is discarded, 50 mu l of each of the third hole and the fourth hole is added into the fifth hole and the sixth hole respectively, and 50ul of standard substance diluent is added into the fifth hole and the sixth hole respectively and uniformly mixed; mixing, adding 50 μl of each of the fifth and sixth holes into the seventh and eighth holes, adding 50 μl of each of the standard substance diluents into the seventh and eighth holes, mixing, adding 50 μl of each of the seventh and eighth holes into the ninth and tenth holes, adding 50 μl of each of the standard substance diluents into the ninth and tenth holes, mixing, and discarding 50 μl of each of the ninth and tenth holes.

3. Sample adding: blank holes (blank control holes are not added with samples and enzyme-labeled reagents, and the rest steps are the same) and sample holes to be tested are respectively arranged. The sample dilution liquid is added into 40 mu l of the sample to be detected in the hole of the enzyme-labeled coated plate, and then 10 mu l of the sample to be detected is added (the final dilution of the sample is 5 times). The sample is added at the bottom of the ELISA plate hole, the hole wall is not touched as much as possible, and the mixture is gently shaken and uniformly mixed.

4. Incubation: incubate for 30 minutes at 37℃after membrane sealing with a sealing plate (see instructions for specific time).

5. Preparing liquid: the 30 (20 times of 48T) concentrated washing solution was diluted with distilled water 30 (20 times of 48T) for use.

6. Washing: carefully removing the sealing plate film, discarding the liquid, spin-drying, filling each hole with the washing liquid, standing for 30 seconds, discarding, repeating the process for 5 times, and beating.

7. Adding enzyme: 50 μl of enzyme-labeled reagent was added to each well, except for blank wells.

8. Incubation: the operation is the same as 3.

9. Washing: the operation is the same as 5.

10. Color development: 50 μl of color reagent A and 50 μl of color reagent B are added into each hole, mixed by gentle shaking, and developed for 15min at 37deg.C in dark place.

11. And (3) terminating: the reaction was stopped by adding 50. Mu.l of stop solution to each well. (at this time the blue color immediately turns yellow)

12. And (3) measuring: the absorbance (OD) of each well was measured sequentially at a wavelength of 450nm with blank air-conditioner zero. The measurement should be performed within 15 minutes after the addition of the stop solution.

13. And (3) calculating: drawing a standard curve on a coordinate paper by taking the concentration of a standard substance as an abscissa and the OD value as an ordinate, and finding out the corresponding concentration from the standard curve according to the OD value of a sample; multiplying by the dilution factor; or calculating a linear regression equation of the standard curve by using the concentration and the OD value of the standard substance, substituting the OD value of the sample into the equation, calculating the concentration of the sample, and multiplying the concentration by the dilution multiple to obtain the actual concentration of the sample.

2. qRT-PCR detects the relative expression level of the inflammatory HaCat cytokines TNF-alpha mRNA and IL-6 mRNA.

1. RNA extraction and reverse transcription experiments were performed as required in the total RNA extraction reagent and reverse transcription kit (Biyun Biotechnology Co., ltd. R0011, D7168L).

2. Specific primers for the target gene (containing housekeeping gene β -actin) were designed using Primer Express software based on the sequence genes published by the national center for biological information (National Center for Biotechnology Information, NCBI) as shown in table 1.

3. Real-time fluorescent quantitative PCR analysis was performed using the reverse transcribed cDNA as a template according to the requirements of the TransStart R Top Green qPCR SuperMix kit (Beijing full gold Biotechnology Co., ltd. AQ 131-01).

As a result, referring to fig. 1, it can be seen that the daylily fermentation broth has a stronger anti-inflammatory ability than the aqueous extract (the aqueous extract is a sample control of the fermentation broth, and the preparation steps are the same as those of the fermentation broth, but no lactobacillus or yellow rice wine yeast is added).

TABLE 1 Real-Time PCR primer sequences

Experimental example 4 analysis of antioxidant efficacy of daylily fermentation broth

The day lily fermentation broth obtained in the above example was subjected to an antioxidant efficacy analysis.

DPPH radical scavenging

DPPH is an early synthetic organic radical, commonly used to evaluate the hydrogen donating ability of antioxidants, which is very stable in organic solvents, purple in color, and has a characteristic absorption peak at 517nm, when a radical scavenger is encountered, the lone pair of electrons of DPPH are paired to fade it, i.e., the absorbance at the maximum absorption wavelength becomes small. Therefore, the effect of the sample on DPPH radical scavenging can be evaluated by measuring the change in absorbance.

All tubes (T, T, C, C0) were supplemented with solvent, water-soluble samples were made up with water, oil-soluble samples were made up with 95% ethanol, 3mL, and mixed well. 1mL of DPPH ethanol solution is added into a sample tube (T) and a DPPH tube (C), the sample background (T0) and the solvent background (C0) are replaced by 95% ethanol, the mixture is gently shaken, and the mixture is kept stand for 5 minutes at room temperature. Each reaction solution was transferred to a 1cm cuvette, and absorbance was measured at 517 nm.

Table 2 sample addition requirements

	T-sample tube	T ₀ Sample background	C-DPPH pipe	C ₀ Solvent background
					Sample solution (mL)	1	1	-	-
Water or 95% ethanol solvent (mL)	2	2	3	3
					DPPH ethanol solution (mL)	1	-	1	-
95% ethanol (mL)	-	1	-	1
					Number of parallel times	3/sample	1/sample	3/test	1/test

DPPH radical scavenging was calculated by the formula: the absorbance value of the T-sample tube, namely the absorbance value of the solution after the reaction of the sample and DPPH; t (T) ₀ -a sample background absorbance value; the average value of the absorbance value of the C-DPPH tube for 3 times is the absorbance value of the DPPH solution when no sample is added;C ₀ solvent background absorbance:

2. radical scavenging of hydroxyl groups

Dissolving 0.5mL of 0.75mmol/L phenanthroline in a test tube, sequentially adding 1mL of 0.15mol/L phosphate buffer solution (PBS, pH=7.4) and 0.5mL of distilled water, mixing thoroughly, adding 0.5mL of 0.75mmol/L ferrous sulfate solution (FeSO) ₄ ) Mixing, adding 0.5mL of 0.01% hydrogen peroxide (H) ₂ O ₂ ) The absorbance at 536nm after 60min in a 37℃water bath was determined as absorbance A of the damaged tube _{Damage to} The absorbance value A of the undamaged tube can be measured by the same operation method that the undamaged tube replaces 0.5mL of 0.01% hydrogen peroxide in the damaged tube with 0.5mL of distilled water _Undamaged The distilled water in the damage tube is replaced by the sample in the sample tube, the operation method is the same as that of the damage tube, and the absorbance A in the sample tube can be measured _{Sample of} The OH clearance of the samples was calculated according to the following formula.

Clearance I (%) =100% (a _{Sample of} -A _{Damage to} )/(A _Undamaged -A _{Damage to} )

TEAC method Total antioxidant Capacity determination

Trolox Equivalent Antioxidant Capacity (TEAC) is one of the methods for measuring the antioxidant capacity of electron transfer type, and is generally a standard method for measuring the antioxidant capacity of fruits and vegetables.

Drawing a standard curve: the standard was diluted with distilled water. 10mM Trolox standard solution was diluted to 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5mM.

Sample measurement: 200 microliters of ABTS working solution is added to each detection well of the 96-well plate, and 10 microliters of distilled water or PBS and other proper solutions are added to the blank control well; 10 microliters of Trolox standard solution with various concentrations is added into a standard curve detection hole; 10 microliters of each sample was added to the sample detection wells. Mix gently. After incubation for 2-6 minutes, absorbance A734 was measured at 734 nm. The results are shown in FIG. 2.

4. Related cytokine detection

The concentration of HaCat cytokine CAT, COL-1 was detected by enzyme-linked immunosorbent assay (ELISA). The procedure is as in example 3 above.

The results are shown in FIG. 2, wherein the first and second graphs are graphs of the change in free radical scavenging rate measured by dilution of stock fermentation broth with water to different concentrations, and the third through fifth graphs are the results of tests conducted with water-diluted fermentation broth (concentration 2%, according to the inventors' previous cytotoxicity test, which concentration fermentation broth was substantially free of cytotoxicity). As can be seen from fig. 2, compared with the aqueous extract, the two day lily fermentation broths have better antioxidant capacity, and in particular, the total antioxidant capacity of the aqueous extract is inferior to that of the yeast fermentation broths and the lactic acid bacteria fermentation broths.

Example 5 day lily fermentation broth whitening efficacy analysis

Whitening efficacy analysis was performed on the day lily fermentation broth obtained in the above examples.

The tyrosinase activity in vitro was determined using the tyrosinase dopa-rate oxidation method.

The amounts and sequences of the materials added to each reaction system are shown in Table 3. After tyrosinase was added, the reaction system was incubated in a 37℃water bath for 10min, then 0.98g/L of L-dopa solution was added, and after 3min of reaction, the absorbance was measured at 475 nm.

Tyrosinase activity inhibition was calculated by the following formula.

I＝[(A－B)－(C－D)]/(A－B)*100％

Wherein, the A-control group has the absorbance value when the tyrosinase system reacts for 3 min;

the absorbance value of the B-control group when the tyrosinase-free system reacts for 3 min;

c-absorbance value of sample group when tyrosinase system is reacted for 3 min;

d-absorbance value of the sample group when the tyrosinase-free system reacts for 3 min; i-tyrosinase activity inhibition (%).

TABLE 3 composition of reaction solution (mL)

Intracellular tyrosinase activity inhibition assay: b16 cells during logarithmic growth were seeded in 6-well cell culture plates at 37 ℃,5% co ₂ Culturing overnight in an incubator. Samples were added at a final concentration of 2% and diluted with DMEM. The non-added sample group served as a cell control group, with 3 duplicate wells per group. After 48h incubation, the supernatant was discarded, washed 1 time with PBS, 100. Mu.L of cell lysate was added to each well, the collected cells were scraped with a spatula, and the supernatant was collected and centrifuged. Taking 50 mu L of supernatant to 96-well plate, adding 1% L-dopa 50 mu L, placing at 37 ℃ and 5% CO ₂ Incubate in incubator for 1h. Absorbance values at 475nm were measured. The relative activity of tyrosinase in the cell reaction system was calculated as follows.

A _Cells ＝(OD _{Measuring hole} －OD _{Blank control} )/(OD _{Cell control group} －OD _{Blank control} )*100％

Wherein A is _Cells -tyrosinase relative activity,%;

OD _{sample of} -absorbance of a reaction system containing a sample to be tested;

OD _{blank control} -empty plate absorbance without any substance;

OD _{cell control} Absorbance of the reaction system without the test sample.

As a result, referring to fig. 3, it can be seen that the two day lily fermentation broths have better whitening effect compared to the aqueous extract.

Experimental example 6 safety efficacy analysis of daylily fermentation liquor

Safety efficacy analysis was performed on the day lily fermentation broth obtained in the above example.

1. Chick embryo chorioallantoic membrane assay (HET-CAM)

The chick embryo chorioallantoic membrane test (HET-CAM) is a classical in vitro evaluation method of ocular irritation and is suitable for evaluating cosmetic products or materials. Chorioallantoic membrane (CAM) is the respiratory membrane located around the chick embryo. The test utilizes the characteristics of complete, clear and transparent CAM vascular membrane system in the middle hatching period, a certain amount of test substances are directly contacted with CAM, and after a period of action, the change of chorioallantoic membrane toxicity effect indexes (such as bleeding, coagulation and vascular thawing) is observed, and a score is obtained by combining, so that the eye irritation of the test substances is evaluated. These indices reflect changes in morphology, structure, color and permeability of blood vessels and vascular networks, and changes in chorioallantoic membrane protein denaturation and the like, and the degree of damage thereof.

An end point score (ES) should be calculated for the test performed using the end point evaluation method. According to the degree of bleeding, coagulation and vascular thawing observed with chick embryos; six sets of parallel experiments were performed for each sample and the vascular response scores were summed, with the highest vascular response score in the total score being the final score and the highest scoring value in the set being the response ES value. The eye irritation of the test subjects was classified according to the ES values as shown in Table 4.

TABLE 4 eye irritation prediction model for HET-CAM endpoint evaluation

The test substance includes: negative control (0.9% sodium chloride), positive control (0.1 mol/L sodium hydroxide) and two daylily fermentation broths (stock solution, 6 replicates were set). The results of the chick embryo chorioallantoic membrane test are shown in fig. 4, and as can be seen from the figure, the sample has no bleeding phenomenon before and after the sample acts, and no eye irritation of the day lily fermentation liquor can be known according to the score.

Morphological observations and scoring of CAM before and after addition of negative control (0.9% sodium chloride), positive control (0.1 mol/L sodium hydroxide) and day lily fermentation broth gave the following Table 5, where numbers 1, 2, 3, 4, 5, 6 are parallel to the samples, respectively.

Table 5 day lily fermentation broth chick embryo chorioallantoic membrane test record

2. Erythrocyte hemolysis experiment

The erythrocyte hemolysis test (Red blood cell test, RBC) is one of the alternatives to the rabbit eye stimulation test (Draize test). Erythrocytes are considered to be the best biological source for studying the biofilm effect, and are highly operable and homogenous. The basic principle of RBC experiments is to evaluate the irritation of chemicals to ocular tissues by detecting the leakage of hemoglobin and the degree of protein denaturation in red blood cells. RBC experiments are also widely used internationally for eye irritation studies of chemicals such as cosmetic products and raw materials.

The specific steps of the experiment are as follows:

pretreatment of RBC

(1) Blood collection and transport

Fresh rabbit blood is taken from slaughterhouses and contained in polyethylene plastic containers according to the following ratio of 1: and 9, adding an anticoagulant citric acid buffer solution, and uniformly mixing. Immediately mixing the blood sample, and keeping the temperature in an incubator at 21-22 ℃. The sample is transported to the laboratory within 30 minutes and the time can be extended to 1 hour if the sample is not contaminated.

(2) Separation of RBC

1) Split charging and dilution: fresh blood (treated with citric acid anticoagulant) was collected and diluted with PBS solution (blood: pbs=4:10 volume ratio);

2) And (5) centrifuging to remove impurities: the above dilution was centrifuged at 1500 Xg for 10min at room temperature, the supernatant after centrifugation and the pale yellow leucocyte layer were carefully aspirated off, and the above washing centrifugation steps were repeated 2-3 times with PBS (the microplate reader was turned on for 15min at the 2 nd centrifugation).

3) RBC suspension preparation: after the last centrifugation, the pelleted cells were diluted with PBS to a concentration of approximately 2% red blood cell suspension (approximately 2mL pellet +98mL PBS) and gently shaken well.

4) RBC concentration calibration: 0.5mL of the cell suspension is taken in a 10mL EP tube, distilled water is added to dilute to 5mL, the mixture is uniformly mixed and reacted for 1 minute, the reaction is measured at 541nm by an enzyme label instrument (PBS is used as a blank control, absorbance is regulated by red cell suspension), and the ideal absorbance value is 0.5 (+ -5%). The treated RBC suspension was sealed and stored at 4 ℃.

2. Determination of the haemolysis Curve

(1) Diluting daylily fermentation liquor PBS into sample solutions with volume concentrations of 10%, 20%, 40%, 60%, 80% and 100%, and then mixing each concentration gradient sample solution with RBC suspension according to a ratio of 3:1 (750. Mu.L sample+250. Mu.L RBC) were added and mixed.

(2) The subject RBC mix was incubated on a shaker at room temperature for 60min.

(3) Each EP tube was placed in a centrifuge and centrifuged at 10000rpm/min for 1 min, and incubation was terminated.

(4) The supernatant was taken and its absorbance at 540nm was measured. Three replicates were made for each concentration and the results averaged.

(5) Control groups were assayed simultaneously:

negative control (zero hemolysis): 750 μl PBS+250 μl RBC, with a hemolysis rate of 0% for the negative control;

positive control: 750 μl 0.1% SDS water+250 μl RBC;

complete hemolysis control: 750 μl of water+250 μl of RBC, the hemolysis rate of the complete hemolysis control was set to 100%.

3. Data processing

The absorbance OD values measured at 560nm wavelength and the concentration gradient curve of each group were taken as regression lines, and the difference between the negative control OD values and the positive control OD values at 560nm was taken into the regression line equation to obtain H50 (expressed as the concentration in the test system).

The calculation formula is as follows:

the results of the erythrocyte hemolysis experiment are as follows.

The hemolysis curve is shown in fig. 5, and it can be seen from fig. 5 that the hemolysis rate of the daylily fermentation broth prepared by the steps is lower than that of the water extract.

Finally it is pointed out that in the present disclosure relational terms such as first and second, and the like, are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

While the disclosure has been disclosed by the foregoing description of specific embodiments thereof, it will be understood that various modifications, improvements, or equivalents may be devised by those skilled in the art that will fall within the spirit and scope of the appended claims. Such modifications, improvements, or equivalents are intended to be included within the scope of this disclosure.

Claims

1. A method for preparing a day lily ferment for cosmetics, comprising:

inoculating lactobacillus to the fermentation substrate for fermentation treatment, and then sterilizing and separating to obtain supernatant fluid to obtain day lily fermentation liquor;

the lactobacillus is Lactobacillus plantarumLactiplantibacillus plantarum) Purchased from China center for type culture collection of Industrial microorganisms, with a strain number of CICC20261;

the dosage ratio of the day lily powder to the water is 1:100g/ml-1:40 g/ml; the temperature of the lactobacillus fermentation treatment is 37-45 ℃ and the time is 6-16h.

2. The method for preparing a daylily ferment according to claim 1, wherein the grain size of the daylily powder is 50 mesh.

3. The method for preparing daylily fermented product according to claim 1 or 2, wherein the lactic acid bacteria liquid for inoculation is obtained by sequentially activating, purifying and amplifying strains, the OD value of the lactic acid bacteria liquid is 0.5-1.0, and the volume ratio of the lactic acid bacteria liquid to the fermentation substrate is 1:5-1:20.

4. The method for preparing a fermented product of day lily according to claim 1, wherein the fermentation treatment is performed in a shaker at a rotation speed of 150r/min-180 r/min.

5. The method for preparing a fermented daylily product according to any one of claims 1, 2 and 4, wherein the sterilizing treatment is performed under a pressure of 0.2-0.4Mpa at a temperature of 100-121 ℃ for 15-30min.

6. The method for producing a fermented product of daylily according to any one of claims 1, 2 and 4, wherein the separation treatment is performed by a centrifugation method.

7. The method of claim 6, wherein the separation is performed at a centrifugal speed of 4500r/min-5500r/min for 20min-40min.

8. The method for producing a daylily fermented product according to any one of claims 1, 2, 4, and 7, further comprising: and (3) drying the daylily fermentation liquid to finally obtain the daylily fermentation dry powder.

9. The method for preparing a daylily ferment according to claim 8, wherein the drying treatment is spray drying or vacuum freeze drying.

10. A day lily ferment for cosmetics, characterized in that the ferment produced by the method according to any one of claims 1 to 9 is used.

11. A cosmetic comprising the daylily ferment of claim 10.