阅读说明：本技术 一种螺旋藻活性肽的制备方法及其在护肤品中的应用 (Preparation method of spirulina active peptide and application of spirulina active peptide in skin care product ) 是由 韦忠明 莫冬海 陈勇 唐申秀 宫猛 于 2020-12-29 设计创作，主要内容包括：本发明公开一种螺旋藻活性肽的制备方法,包括以下步骤：1)取螺旋藻粉处理后,加入蛋白胨、PBS缓冲液配制发酵培养基；2)取葡萄糖、酵母提取物、蛋白胨和PBS缓冲液,配制种子培养基；3)取葡萄汁有孢汉生酵母菌种接种于种子培养基中；然后挑出菌种接种于发酵培养基中,培养得到发酵液；4)将发酵液离心,过截留10KD超滤膜,获得螺旋藻活性肽。本发明还公开了上述螺旋藻活性肽在护肤品中的应用。本发明无需蛋白酶,成本低廉,且多肽水解度高、多肽收率高。(The invention discloses a preparation method of spirulina active peptide, which comprises the following steps: 1) treating spirulina powder, and adding peptone and PBS buffer solution to prepare a fermentation medium; 2) preparing a seed culture medium by taking glucose, a yeast extract, peptone and a PBS buffer solution; 3) inoculating Hanseng yeast strain with grape juice spore in a seed culture medium; then picking out strains and inoculating the strains in a fermentation culture medium to obtain fermentation liquor by culture; 4) and (4) centrifuging the fermentation liquor, and passing through a retained 10KD ultrafiltration membrane to obtain the spirulina active peptide. The invention also discloses application of the spirulina active peptide in skin care products. The invention does not need protease, has low cost, high polypeptide hydrolysis degree and high polypeptide yield.)

1. A preparation method of spirulina active peptide is characterized in that: the method comprises the following steps:

1) mixing Spirulina powder with water, freeze thawing for 3-5 times, and homogenizing to obtain homogeneous solution; adding peptone 1-2% of the homogeneous solution and PBS buffer solution with volume of 10-15% and pH of 7.0 to 1-2mol/L, and sterilizing to obtain fermentation culture medium;

2) mixing glucose, yeast extract and peptone, adding 1-2mol/L PBS buffer solution with pH7.0 to 100ml, sterilizing, and preparing to obtain seed culture medium; the mass concentration of glucose in the seed culture medium is 1-3%, the mass concentration of yeast extract is 1-3%, and the mass concentration of peptone is 1-3%;

3) inoculating Hansenula polymorpha strain of grape juice with spore in seed culture medium, and culturing at 37-40 deg.C for 18-36 hr; selecting strains, inoculating to a fermentation culture medium, and culturing at 37-40 deg.C for 36-48h to obtain fermentation broth;

4) centrifuging the fermentation liquid, taking supernatant, inactivating enzyme, and passing through a 10KD ultrafiltration membrane with the cut-off relative molecular weight to obtain ultrafiltrate with the molecular weight range less than 10000, namely the spirulina active peptide.

2. The method for preparing active spirulina peptide of claim 1, wherein the method comprises the following steps: in the step 1), the adding amount of water is 10-20 times of the weight of the spirulina powder.

3. The method for preparing active spirulina peptide of claim 1, wherein the method comprises the following steps: in the step 1), the freeze thawing is to freeze at-20 ℃ for 8-12h and then unfreeze in water bath at 37 ℃ for 2-3 h.

4. The method for preparing active spirulina peptide of claim 1, wherein the method comprises the following steps: in the step 1), the homogenization is carried out for 5-10min at 5000-10000 rpm.

5. The method for preparing active spirulina peptide of claim 1, wherein the method comprises the following steps: the sterilization temperature in the step 1) and the step 2) is 121 ℃, and the sterilization time is 20-30 minutes.

6. The method for preparing active spirulina peptide of claim 1, wherein the method comprises the following steps: in the step 3), the inoculation weight ratio of the Hansenula botrytis strain of the grape juice in the seed culture medium is 1: 40-50; the inoculation weight ratio of the Hansenula polymorpha strain of the grape juice in the fermentation culture medium is 1: 10-20.

7. The method for preparing active spirulina peptide of claim 1, wherein the method comprises the following steps: in step 3), the shaking culture is performed by shaking table with a shaking frequency of 200-300 rpm.

8. The method for preparing active spirulina peptide of claim 1, wherein the method comprises the following steps: in the step 4), the enzyme inactivation is to inactivate the supernatant in boiling water bath for 10-15 minutes.

9. Use of the active spirulina peptide prepared by the method of any one of claims 1 to 8 in skin care products.

10. Use according to claim 9, characterized in that: the addition amount of the spirulina active peptide in the skin care product is 0.01-5%.

Technical Field

The invention belongs to the technical field of microbial fermentation, and particularly relates to a preparation method of spirulina bioactive peptide and application of the spirulina bioactive peptide in skin care products.

Background

Spirulina (Spirulina) belongs to the family Cyanophyta (Cyanophyta) of the order Oscilatoriales (Oscilatoriales) of the genus Spirulina. The appearance of spirulina dates back 35 hundred million years and is one of the oldest organisms on the earth. Spirulina is widely used as natural food supplement worldwide, is a low-fat, low-calorie, cholesterol-free protein source, has effects of improving immunity, resisting virus, resisting oxidation, and the like, can be used for adjuvant treatment of diseases, and has important application value in human health food and beauty treatment.

Bioactive peptides are peptide compounds consisting of amino acids with biological activity. The active peptide with small molecules not only can provide nutrient substances required by the growth and development of organisms, but also has the functions of enhancing immunity, inhibiting bacteria, resisting oxidation and the like. Compared with free amino acids and proteins, the peptides can be absorbed by the body better and faster. Therefore, more and more researchers are focusing on bioactive peptides, and the preparation of small peptides and the biological activities of the small peptides have become hot spots of research. The spirulina contains abundant protein accounting for 60-70% of dry weight, and is a good source of high-quality bioactive peptide. Various functional properties of spirulina protein zymolyte and polypeptide are widely researched, including antioxidant activity, antibacterial activity, antitumor activity, anti-inflammatory activity, blood pressure lowering activity, mineral chelation and the like.

Currently, the preparation method of active peptides mainly comprises a biological enzymolysis method, for example, CN10263384A discloses a method for preparing spirulina polypeptide powder by using living spirulina, filtering and cleaning the living spirulina, and draining algae mud when the pH reaches 8.5 to make the water content of the algae mud less than 90%; loading the treated algae mud into a reaction tank, wherein each tank is 1000 kg, adding 0.5 kg of alkaline protease with the enzyme activity of 40 ten thousand U/g, heating and stirring, wherein the enzymolysis temperature is 45 ℃, and the action time is 70 minutes; adding 0.3 kg of flavourzyme with the enzyme activity of 1.5 ten thousand U/g, heating and stirring, wherein the enzymolysis temperature is 45 ℃, and the action time is 60 minutes; adding 1.5 kg of monascus, heating, stirring, reacting for 180 minutes at 45 ℃, and performing spray drying by using a spray drying tower to obtain the living spirulina polypeptide powder. The method uses a large amount of protease, and has the advantages of high price, extremely high cost and low extraction rate.

Disclosure of Invention

The invention aims to solve the technical problem of providing a preparation method of spirulina active peptide with low cost and high hydrolysis degree and application thereof in skin care products.

The technical scheme provided by the invention is a preparation method of spirulina active peptide, which comprises the following steps:

1) mixing Spirulina powder with water, freeze thawing for 3-5 times, and homogenizing to obtain homogeneous solution; adding peptone 1-2% of the homogeneous solution and PBS buffer solution with volume of 10-15% and pH of 7.0 to 1-2mol/L, and sterilizing to obtain fermentation culture medium;

2) mixing glucose, yeast extract and peptone, adding 1-2mol/L PBS buffer solution with pH7.0 to 100ml, sterilizing, and preparing to obtain seed culture medium; the mass concentration of glucose in the seed culture medium is 1-3%, the mass concentration of yeast extract is 1-3%, and the mass concentration of peptone is 1-3%;

3) inoculating Hansenula polymorpha strain of grape juice with spore in seed culture medium, and culturing at 37-40 deg.C for 18-36 hr; selecting strains, inoculating to a fermentation culture medium, and culturing at 37-40 deg.C for 36-48h to obtain fermentation broth;

4) centrifuging the fermentation liquid, taking supernatant, inactivating enzyme, and passing through a 10KD ultrafiltration membrane with the cut-off relative molecular weight to obtain ultrafiltrate with the molecular weight range less than 10000, namely the spirulina active peptide.

In the step 1), the adding amount of water is 10-20 times of the weight of the spirulina powder.

In the step 1), the freeze thawing is to freeze at-20 ℃ for 8-12h and then unfreeze in water bath at 37 ℃ for 2-3 h.

In the step 1), the homogenization is carried out for 5-10min at 5000-10000 rpm.

In the step 1), PBS buffer solution with the molar concentration of 1-2mol/L and the pH value of 7.0 is also added into the homogeneous liquid, and the adding amount is 8-12% of the volume of the homogeneous liquid.

The sterilization in the step 1) and the step 2) is to sterilize the culture medium at the temperature of 121 ℃ for 20-30 minutes.

The Yeast extract in the step 2) is also called Yeast monosodium glutamate, and is named Yeast extract in English, which is called YE for short. YE (Yeast extract) is a brown yellow soluble paste or yellowish powder pure natural product prepared by degrading proteins, nucleic acids and the like in yeast cells by using edible yeast with rich protein content as a raw material and adopting modern biological high and new technologies such as autolysis, enzymolysis, separation, concentration and the like according to the regulations of Chinese pharmacopoeia and refining.

In the step 3), the inoculation weight ratio of the Hansenula botrytis strain of the grape juice in the seed culture medium is 1: 40-50; the inoculation weight ratio of the Hansenula polymorpha strain of the grape juice in the fermentation culture medium is 1: 10-20.

In step 3), the shaking culture is performed by shaking table with a shaking frequency of 200-300 rpm.

In the step 4), the enzyme inactivation is to inactivate the supernatant in boiling water bath for 10-15 minutes.

The invention also provides application of the spirulina active peptide prepared by the preparation method of the spirulina active peptide in skin care products.

The addition amount of the spirulina active peptide in the skin care product is 0.01-5%.

The list names of the spirulina active peptides in the skin care product raw materials are as follows: yeast fermentation product extract.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention utilizes the protease secreted by the Hanseng yeast with botrytis cinerea in the fermentation process to directly carry out enzymolysis on the protein components of the spirulina, does not need the process steps of protease preparation, spirulina crude protein extraction and the like, and has simple process and time saving.

(2) The production process only consumes the spirulina raw material and the culture medium components, does not need expensive raw materials such as protease and the like, and has extremely low cost.

(3) The method has high protein hydrolysis degree of Spirulina and high polypeptide yield.

Detailed Description

The following specific examples further illustrate the invention but are not intended to limit the invention thereto.

Example 1

1) Mixing 50g Spirulina powder (Guangxi agricultural reclamation Lvxian biological health food Co., Ltd.) with 500ml water, freezing at-20 deg.C for 8 hr, thawing in 37 deg.C water bath for 2 hr, repeatedly freezing and thawing for 3 times, homogenizing at 5000rpm for 5min to obtain homogeneous solution; adding peptone (Oxoid, UK) 1% by mass to the homogenized solution, adding PBS buffer solution (pH 7.0) 1 mol/L10% by volume of the homogenized solution, and sterilizing at 121 deg.C for 20 min to obtain fermentation medium;

2) mixing glucose (Biotechnology engineering (Shanghai) Co., Ltd.), yeast extract (Oxoid, UK) and peptone (Oxoid, UK), adding 1mol/L PBS buffer solution with pH of 7.0 to 100ml, sterilizing at 121 deg.C for 20 min, and preparing to obtain seed culture medium; the mass concentration of glucose in the seed culture medium is 1%, the mass concentration of yeast extract is 1%, and the mass concentration of peptone is 1%;

3) thawing a Hansenula polymorpha strain (H.uvarum) of grape juice, and inoculating the strain in a seed culture medium in an inoculation weight ratio of 1: 40, shaking and culturing for 18 hours at 37 ℃ by a shaking table, wherein the shaking frequency is 200 rpm; selecting strains and inoculating the strains in a fermentation medium, wherein the inoculation weight ratio is 1: 10, shaking and culturing for 36 hours at 37 ℃ by a shaking table, wherein the shaking frequency is 200rpm, and obtaining fermentation liquor;

4) centrifuging the fermentation liquid at 5000rpm for 10min, collecting supernatant, inactivating in boiling water bath for 10min, naturally cooling, passing through 10KD ultrafiltration membrane with cut-off relative molecular weight to obtain ultrafiltrate with molecular weight less than 10000, and sterile filtering with 0.22 μm to obtain Spirulina active peptide.

Example 2

1) Mixing 50g Spirulina powder (Guangxi agricultural reclamation Lvxian biological health food Co., Ltd.) with 1000ml water, freezing at-20 deg.C for 12 hr, thawing in 37 deg.C water bath for 3 hr, repeatedly freezing and thawing for 5 times, homogenizing at 10000rpm for 10min to obtain homogeneous solution; adding peptone (Oxoid, UK) 2% of the homogenized solution, adding PBS buffer solution (pH 7.0) 2 mol/L15% of the homogenized solution volume, and sterilizing at 121 deg.C for 30min to obtain fermentation medium;

2) mixing glucose (Biotechnology engineering (Shanghai) Co., Ltd.), yeast extract (Oxoid, UK) and peptone (Oxoid, UK), adding 2mol/L PBS buffer solution with pH of 7.0 to 100ml, sterilizing at 121 deg.C for 30min, and preparing to obtain seed culture medium; the mass concentration of glucose in the seed culture medium is 3%, the mass concentration of yeast extract is 3%, and the mass concentration of peptone is 3%;

3) thawing a Hansenula polymorpha strain (H.uvarum) of grape juice, and inoculating the strain in a seed culture medium in an inoculation weight ratio of 1: 50, shaking and culturing for 36 hours at 40 ℃ by a shaking table, wherein the shaking frequency is 300 rpm; selecting strains and inoculating the strains in a fermentation medium, wherein the inoculation weight ratio is 1: 20, shaking and culturing for 48 hours at 40 ℃ by a shaking table, wherein the shaking frequency is 300rpm, and obtaining fermentation liquor;

4) centrifuging the fermentation liquid at 5000rpm for 10min, placing the supernatant in boiling water bath for inactivating for 15 min, naturally cooling, passing through 10KD ultrafiltration membrane with cut-off relative molecular weight to obtain ultrafiltrate with molecular weight less than 10000, and sterile filtering with 0.22 μm to obtain the filtrate which is Spirulina active peptide.

Example 3

1) Mixing spirulina powder 50g (Guangxi agricultural reclamation Lvxian biological health food Co., Ltd.) with water 750ml, freezing at-20 deg.C for 10 hr, thawing in water bath at 37 deg.C for 2.5 hr, repeatedly freezing and thawing for 4 times, homogenizing at 8000rpm for 7min to obtain homogeneous solution; adding peptone 1.5% of the homogeneous solution and PBS buffer solution (1.5 mol/L and pH7.0) 12% of the homogeneous solution, and sterilizing at 121 deg.C for 25 min to obtain fermentation medium;

2) mixing glucose (Biotechnology engineering (Shanghai) Co., Ltd.), yeast extract (Oxoid, UK) and peptone (Oxoid, UK), adding 1.5mol/L PBS buffer solution with pH of 7.0 to 100ml, sterilizing at 121 deg.C for 25 min, and preparing to obtain seed culture medium; the mass concentration of glucose in the seed culture medium is 2%, the mass concentration of yeast extract is 2%, and the mass concentration of peptone is 2%;

3) thawing a Hansenula polymorpha strain (H.uvarum) of grape juice, and inoculating the strain in a seed culture medium in an inoculation weight ratio of 1: 45, shaking and culturing for 24 hours at 38 ℃ by a shaking table, wherein the shaking frequency is 270 rpm; selecting strains and inoculating the strains in a fermentation medium, wherein the inoculation weight ratio is 1: 15, shaking and culturing for 42 hours at 38 ℃ by a shaking table, wherein the shaking frequency is 280rpm, and obtaining fermentation liquor;

4) centrifuging the fermentation liquid at 5000rpm for 10min, placing the supernatant in boiling water bath for inactivating for 12 min, naturally cooling, passing through 10KD ultrafiltration membrane with cut-off relative molecular weight to obtain ultrafiltrate with molecular weight less than 10000, and sterile filtering with 0.22 μm to obtain the filtrate which is Spirulina active peptide.

EXAMPLE 1 determination of hydrolysis degree and polypeptide yield of Spirulina active peptide

Method for measuring degree of hydrolysis: the Degree of Hydrolysis (DH) is expressed as the amount of alpha-amino nitrogen in the sample as a percentage of the total nitrogen content. Using the spirulina species bioactive peptides prepared in examples 1 to 3 as samples, the amount of alpha-amino nitrogen in the samples was measured by the indetrione method, the total nitrogen content in the samples was measured by the Kjeldahl method, and the degree of hydrolysis was calculated according to the following formula.

Degree of hydrolysis ═ 100% (amount of α -amino nitrogen of sample/total nitrogen content of sample) ×

The method for measuring the polypeptide yield comprises the following steps: high-molecular proteins are easily precipitated under acidic conditions, and protein hydrolysates (acid-soluble proteins) having relatively small molecular weights are soluble in an acidic solution. Respectively acidifying the supernatants prepared in the step 4) of the embodiments 1-3, filtering out precipitates, and determining the content of the polypeptide by adopting a Kjeldahl method; determining the protein content of the spirulina powder by adopting a Kjeldahl method (GB/T6432-2018), and calculating the polypeptide yield:

the yield of the polypeptide is the total amount of the polypeptide in the spirulina active peptide/the total amount of the spirulina powder protein is multiplied by 100 percent

The results of the measurements are given in the following table:

group of	Degree of hydrolysis	Yield of polypeptide
			Example 1	46.2％	74.7％
Example 2	48.3％	73.2％
			Example 3	47.9％	78.5％

EXAMPLE 2 research on antioxidant Activity of Spirulina-active peptides

1. Ability to scavenge DPPH free radicals: 2mL of the spirulina active peptides obtained in examples 1 to 3 were mixed well with 2mL of 0.2mmol/L DPPH 95% ethanol solution, and then left at room temperature for 30min, and the absorbance at 517nm was measured. DPPH.removing Capacity is calculated as follows.

DPPH.Clearance (1- (A1-A2)/A3). times.100%

In the formula: a1 is the absorbance value measured by DPPH solution and spirulina active peptide; a2 is the absorbance value measured by replacing DPPH solution with 95% ethanol and spirulina active peptide; a3 is the absorbance value measured by using distilled water instead of spirulina active peptide and DPPH solution.

2. Ability to scavenge hydroxyl radicals: using the spirulina active peptides of examples 1-3 as samples, 8.8mmol/LH was added to 1mL of the samples₂O₂、9mmol/LFeSO₄The solution, 9mmol/L salicylic acid-70% ethanol solution each 1ml, and finally H₂O₂Starting the reaction, reacting for 0.5h at 37 ℃, taking distilled water as a reference, and measuring the absorbance of a sample at 510nm and recording as AX; a blank control was also run with distilled water instead of the sample and the absorbance measured was recorded as A0. Considering the absorbance of the sample itself, 1ml of 9mmol/L FeSO₄The background absorbance of the solution, 1ml of a 9mmol/L salicylic acid-70% ethanol solution, 1ml of the sample and 1ml of distilled water as a sample solution was designated as AX 0. OH scavenging ability was calculated as follows.

OH clearance (A0-AX + AX 0)/A0X 100%

In the formula: a0 is the absorbance of the blank control solution; AX is the absorbance after the sample is added; AX0 Absorbance of sample background

The detection result of the antioxidant activity of the spirulina bioactive peptide is as follows:

group of	DPPH.Rate of removal (%)	OH Rate (%)
			Example 1	70.2	88.6
Example 2	69.7	86.4
			Example 3	71.3	87.9

EXAMPLE 3 study of cell growth-promoting Activity of Spirulina-active peptide

The research on the cell growth promoting activity of the spirulina bioactive peptide adopts a cell proliferation method/MTT colorimetric method.

Reagent:

(1) RPMI 1640 culture solution: dissolving RPMI 1640 culture medium powder in 1 bag (1L), adding water, diluting to 1000ml, adding penicillin 105IU and streptomycin 105IU, adding sodium bicarbonate 2.1g, dissolving, mixing, sterilizing, filtering, and storing at 4 deg.C.

(2) Maintaining the culture solution: measuring 4ml of newborn bovine serum, and adding RPMI 1640 culture solution to 1000 ml.

(3) Complete culture solution: 100ml of newborn bovine serum is measured and RPMI 1640 culture solution is added to 1000 ml.

(4) PBS: weighing 8g of sodium chloride, 0.2g of potassium chloride, 1.44g of disodium hydrogen phosphate and 0.24g of potassium dihydrogen phosphate, adding water to dissolve and dilute the mixture to 1000ml, and sterilizing the mixture at 121 ℃ for 15 minutes.

(5) Thiazole blue (MTT) solution: 0.10g of MTT powder was weighed, dissolved in 20ml of PBS, and sterilized by filtration through a 0.22 μm filter. Storing at 4 ℃ in the dark.

Standard solution: taking the recombinant human epidermal growth factor standard substance, re-dissolving according to the instruction, and diluting with a maintenance culture solution until each 1ml contains 50 IU. In 96-well cell culture plates, 4-fold serial dilutions were made for 8 dilutions, 2 wells for each concentration. The above operations are carried out under aseptic conditions.

Test solution: the spirulina active peptides prepared in examples 1 to 3 were diluted to contain about 1. mu.g of the peptides per 1ml with the maintenance medium. In 96-well cell culture plates, 4-fold serial dilutions were made for 8 dilutions, 2 wells for each concentration.

The determination method comprises the following steps: mouse embryo fibroblast (BALB/c 3T3 cell) was cultured in whole culture medium at 37 deg.C under 5% carbon dioxide with cell concentration controlled1.0X 10 per 1mL⁵-5.0×10⁵Removing culture medium from culture flask 24-36 hr after passage, digesting and collecting cells, and preparing with complete culture medium to obtain a culture medium containing 5.0 × 10/1 mL⁴-8.0×10⁴The cell suspension of each cell was seeded in a 96-well cell culture plate at 100. mu.L/well and cultured at 37 ℃ under 5% carbon dioxide. After 24 hours, the culture medium was changed to the maintenance medium and incubated at 37 ℃ under 5% carbon dioxide for 24 hours. The prepared cell culture plate is discarded the maintenance solution, and 100 μ l of standard solution and test solution are added into each well, and cultured for 64-72 hours at 37 ℃ under 5% carbon dioxide. Mu.l of MTT solution was added to each well, and the mixture was incubated at 37 ℃ under 5% carbon dioxide for 5 hours. After discarding the liquid in the plate, 100. mu.l of dimethyl sulfoxide was added to each well, and after mixing, absorbance was measured at a wavelength of 570mn on a microplate reader with 630nm as a reference wavelength. The cell growth promoting activity of the spirulina active peptide is calculated according to the following formula:

activity (IU/ml) ═ Pr XDs XEs/(Dr XEr)

In the formula: pr is the biological activity of a standard substance, IU/ml

Ds is the pre-dilution multiple of the test sample

Dr is pre-dilution multiple of standard substance

Es is the dilution multiple of the half effective amount of the standard substance equivalent to the test substance

Er is the dilution multiple of half effective amount of the standard product

Cell growth promoting activity of spirulina active peptide

Group of	Activity (IU/ml)
		Example 1	2.45×106
Example 2	2.68×106
		Example 3	2.65×106

EXAMPLE 4 safety study of Spirulina active peptide into skin Care products

The spirulina bioactive peptides obtained in the embodiments 1 to 3 are respectively prepared into facial mask liquid or essence liquid samples for researching the skin irritation and the anaphylaxis of related products of the spirulina bioactive peptides.

The formula of the mask liquid is as follows: 0.3 percent of sodium hyaluronate, 5 percent of butanediol, 0.1 percent of ethylparaben, 5 percent of spirulina active peptide and 89.6 percent of water.

The formula of the essence liquid is as follows: 2% of glycerin, 0.1% of sodium hyaluronate, 2% of betaine, 0.5% of trehalose, 5% of methyl propylene glycol, 0.15% of polyacrylic acid, 0.15% of arginine, 0.05% of disodium EDTA (ethylene diamine tetraacetic acid), 0.4% of p-hydroxyacetophenone, 0.8% of 1, 2-pentanediol, 0.01% of spirulina active peptide and 88.84% of water.

The research on the safety of related products of spirulina active peptides conforms to the related regulations of technical Specification for cosmetic safety in 2015 edition.

Skin irritation test the irritation of test samples to intact skin and damaged skin was investigated using the back skin of a Japanese big-ear white rabbit. A Single dose Study (SD) is applied to animal skin once, and after 24hr, the sample is washed off and observed for 1h, 24h, 48h and 72 h. Multiple dosing research (MD) animal skin is smeared with a test sample once a day, continuously smeared for 14 days, smeared for 24 hours a day and then washed off for 1 hour for observation; and finally, smearing the test sample for 24h, washing off the test sample, and observing for 1h, 24h, 48h and 72 h.

Skin allergy test guinea pig skin application test article was used, and the administration was performed 4 times in total. Sensitizing on the 1 st, 7 th and 14 th days, and smearing the test sample on the left skin of the animal; the test article was applied to the right skin of the right side of the animal on day 28 of challenge. Allergic symptoms were observed 6h after challenge and skin allergic symptoms were again observed at 24h, 48h and 72 h.

As a result:

skin irritation study: the facial mask solutions and essence solutions prepared from the spirulina bioactive peptides of examples 1-3 have no irritation to the intact skin and damaged skin of the white rabbits.

Skin allergy study: the spirulina active peptide facial mask solutions and essences of examples 1-3 were not allergenic to guinea pig skin.