Preparation method of active probiotic freeze-dried powder and its application in medicine

Abstract

The invention relates to a preparation method of active probiotic freeze-dried powder and application thereof in medicines, and the freeze-dried powder contains probiotic freeze-dried powder and antioxidant polypeptide. The probiotic freeze-dried powder is a freeze-dried powder obtained by configuring Lactobacillus rhamnosus ATCC9595 and Lactobacillus paracasei CGMCC No.8903 in a ratio of 2:1, and has strong anti-allergic properties, while the antioxidant polypeptide has good properties of inhibiting skin aging. After the two are used in combination, the effects of allergy and aging caused by skin ultraviolet damage can be effectively prevented, and the application value is high.

Description

Technical Field

[0001] The present application relates to the biological field, and specifically to a method for preparing freeze-dried active probiotic powder and its application in medicine. Background Art

[0002] Probiotics refer to a type of microorganisms that promote the health of the host. They are accepted by consumers worldwide and are widely used in medicine, food, health products, agriculture and other fields. In recent years, many highly functional foods have gradually been applied to the field of cosmetics, and probiotics are no exception, bringing a new development direction to the cosmetics field. They achieve the purpose of maintaining or improving skin health by regulating the balance of skin microecology. At present, probiotic cosmetics can be divided into three categories: topical probiotics: in cosmetics, live probiotics or fermented extracts of probiotics are used. Clinical studies have shown that topical probiotics can improve skin health by affecting the composition of skin microorganisms. Through the external application of live probiotics, the growth of harmful bacteria on the skin surface can be inhibited through metabolites, competition for space, etc.; through the fermentation extracts of topical probiotics, including polysaccharides and amino acids, the skin can be nourished and skin cells can be protected; topical prebiotics: prebiotics refer to specific nutrients that can be absorbed by skin symbiotic bacteria, which can promote the reproduction and growth of skin symbiotic bacteria, facilitate the colonization of topical probiotics on the skin surface, and thus benefit the health of the host skin; probiotic yogurt cosmetics, such as facial masks made with probiotic yogurt, contain a variety of amino acids and vitamins, which play a role in nourishing the skin.

[0003] At present, unlike adding live probiotics to food, most of the probiotic fragments or extracts used in cosmetics play an important physiological function on the skin, inhibiting the growth and reproduction of harmful bacteria on the skin surface and secreting extracellular polysaccharides to protect the skin. Of course, adding "sufficient" and "live" probiotics to cosmetics is more beneficial. However, probiotics are very sensitive to adverse external environments. Oxygen, temperature and humidity have a great impact on the survival of probiotics. Environmental changes can lead to a significant reduction in the number of live bacteria during production, transportation and sales. In addition, when probiotics are introduced to the skin surface, they are easily affected by skin secretions, and it is difficult to keep enough live probiotics colonized on the skin, which is not conducive to the skin care effect. Therefore, it is an important research direction to develop active probiotic freeze-dried powder to provide sufficient probiotics and prebiotics.

[0004] Allergic disease is a specific immune response that occurs when the body is stimulated by the same antigen again after the initial response to certain antigens, mainly characterized by physiological dysfunction or tissue cell damage. It is also called hypersensitivity or allergic reaction. The skin is the most susceptible part to allergies. Applying a probiotic composition to the skin can help treat allergic diseases. It can reduce the probability of the human body producing an allergic reaction to foreign antigens and reduce the risk of allergic diseases.

[0005] In addition, at present, active biological peptides have the advantages of safety, stability, easy solubility in water, easy absorption, and small molecular weight. At the same time, they have multiple biological activities such as scavenging free radicals, anti-oxidation, and anti-aging. They can not only promote the proliferation of skin cells, but also provide nutrition for the skin, delay skin aging, and promote skin wound repair. Therefore, they are widely used in skin cosmetics and anti-aging cosmetics. Nowadays, due to the increasing aging of society and the continuous pursuit of youth and beauty by consumers, the proportion of anti-aging skin care products in the cosmetics market is also increasing year by year. Consumers' demand for the safety and effectiveness of anti-aging cosmetics has also promoted the continuous improvement and perfection of the research and development technology of anti-aging cosmetics. According to the survey of anti-aging products currently on the market, many internationally renowned brands have launched peptide anti-aging cosmetics, such as: Olay, Avon, Estee Lauder, Lancome, L'Oreal, Chanel, Dior, SK-Ⅱ and other international first-line well-known brands, which can be said to have set off a wave of peptide beauty and skin care products. For example, the brand series under Canada's DECIEM is a multi-peptide high-concentration anti-aging full-effect repair essence. Its active ingredient, peptide 3000, as an amino acid peptide complex, can reduce fine lines, wrinkles, and restore skin elasticity; amino acid peptide complex can reduce fine lines, wrinkles and facial defects, and snake venom protein peptide complex can prevent the formation of deep lines and restore skin elasticity. Amino acid peptide complex can stimulate collagen regeneration and restore skin firmness and elasticity. Tianjin University of Traditional Chinese Medicine cited the concept of Swiss enzyme-decomposable peptides. After years of research and development, it has developed a small polypeptide cosmetic with a molecular weight of less than 800Dr. It is specifically for pregnant women and has highly significant anti-aging, beauty and skin care effects.

[0006] However, currently, there are not enough studies on compositions or cosmetics using active peptides and probiotics for treating skin anti-aging and anti-allergy. Summary of the invention

[0007] The invention provides a medicine with dual effects of anti-aging and anti-allergy.

[0008] Specifically, the medicine contains active probiotic freeze-dried powder and anti-aging polypeptides.

[0009] Furthermore, the active probiotic freeze-dried powder is obtained by configuring Lactobacillus rhamnosus ATCC9595 and Lactobacillus paracasei CGMCC No.8903 in a ratio of 2:1.

[0010] Specifically, the anti-aging polypeptide is GSO-2, and its amino acid sequence is shown in SEQ ID NO: 1.

[0011] The anti-aging polypeptide is obtained from fresh wolfberry fruit through screening and identification.

[0012] The present invention also relates to variants of polypeptides having an amino acid sequence of SEQ ID NO: 1 comprising substitutions, deletions and / or insertions of one or more amino acids. The amino acid sequence of the variant polypeptide may differ from the amino acid sequence of SEQ ID NO: 1 by insertion or deletion of one or more amino acid residues and / or substitution of one or more amino acid residues with different amino acid residues. Preferred are conservative amino acid substitutions where the amino acid changes are not important for properties, i.e., do not significantly affect the folding and / or activity of the protein; small deletions, usually of 1 to about 4 amino acids; small amino or carboxyl terminal extensions, such as an amino terminal methionine residue; or small extensions that facilitate purification by altering the net charge or other functions, such as polyhistidine sequences. Examples of conservative substitutions are within the following groups: basic amino acid group (arginine, lysine and histidine), acidic amino acid group (glutamic acid and aspartic acid), polar amino acid group (glutamine and asparagine), hydrophobic amino acid group (leucine, isoleucine and valine), aromatic amino acid group (phenylalanine, tryptophan and tyrosine) and small amino acid group (glycine, alanine, serine, threonine and methionine). Thus, for example, the present invention relates to a polypeptide having or comprising a sequence as set forth in SEQ ID NO: 1 or an active fragment thereof, wherein conservative amino acid substitutions are contained in basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (alanine, leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine, threonine and methionine), replacing one with another; or any combination thereof.

[0013] The present invention also provides a medicine containing a therapeutically effective amount of the polypeptide of the present invention. As used herein, the "effective amount" refers to an amount that can produce functions or activities on humans and / or animals and can be accepted by humans and / or animals. For example, for liquid preparations or compositions, the concentration of the polypeptide can be above 20 ng / ml, such as above 50 ng / ml, above 80 ng / ml, above 100 ng / ml or higher. The medicine may contain a pharmaceutically acceptable carrier. Herein, "pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not necessary active ingredients themselves and are not excessively toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. In the composition, the pharmaceutically acceptable carrier may contain a liquid, such as water, saline, buffer. In addition, auxiliary substances such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffer substances, etc. may also be present in these carriers. The pharmaceutical composition of the present invention can be administered to a subject in various conventional and appropriate ways in the art, including but not limited to: oral administration, subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, sustained-release administration, and the like.

[0014] Further, "pharmaceutically acceptable carrier" broadly refers to any component other than the active therapeutic ingredient (also referred to in the art as a drug substance or active pharmaceutical ingredient). An excipient may be an inert substance in the sense that it does not have substantially any therapeutic and / or prophylactic effect by itself. An excipient may be used for a variety of different purposes, for example, as an enhancer, absorption enhancer, vehicle, filler (also referred to as a diluent), binder, lubricant, glidant, disintegrant, crystallization retardant, acidifier, alkalizer, preservative, antioxidant, buffer, chelating agent, complexing agent, surfactant, emulsifier and / or solubilizer, sweetener, wetting agent, stabilizer, colorant, flavoring agent, and / or to improve the administration and / or absorption of the active substance. In the present invention, the term "lubricant" refers to stearic acid, magnesium stearate, calcium stearate or other metal stearates, talc, wax, glyceride, light mineral oil, behenic acid glyceride, hydrogenated vegetable oil, sodium stearyl fumarate, polyethylene glycol, alkyl sulfate or sodium benzoate. In the present invention, the term "filler" refers to lactose (e.g., spray-dried lactose, α-lactose, β-lactose, various grades), microcrystalline cellulose (various grades), other cellulose derivatives, sucrose, sorbitol, mannitol, dextrin, dextran, maltodextrin, dextrose, fructose, kaolin, mannitol, sorbitol, sucrose, sugar, starch or modified starch (including potato starch, corn starch and rice starch), calcium phosphate (e.g., basic calcium phosphate, calcium hydrogen phosphate, hydrated dicalcium phosphate), calcium sulfate, calcium carbonate or sodium alginate. In the present invention, the term "binder" includes lactose (e.g., spray-dried lactose, α-lactose, β-lactose, various grades), microcrystalline cellulose (various grades), hydroxypropyl cellulose, L-hydroxypropyl cellulose (low substituted), hydroxypropyl methylcellulose (HPMC, such as Methocel E, F and K, Metolose SH of Shin-Etsu, Ltd, such as 4,000 cps grade Methocel E and Metolose 60 SH, 4,000 cps grade Methocel F and Metolose 65 SH, 4,000, 15,000 and 100,000 cps grade Methocel K; and 4,000, 15,000, 39,000 and 100,000 grades of Metolose 90 SH), methylcellulose polymers (e.g., Methocel A, Methocel A4C, Methocel A15C, Methocel A5C, Methocel A60C, Methocel A70C, Methocel A80C, Methocel A90C, Methocel A110C, Methocel A120C, Methocel A130C, Methocel A140C, Methocel A150C, Methocel A150C, Methocel A13 ... A4M), hydroxyethyl cellulose, ethyl cellulose, sodium carboxymethyl cellulose, other cellulose derivatives, sucrose, dextrin, maltodextrin, starch or modified starch (including potato starch, corn starch and rice starch), calcium lactate, calcium carbonate, gum arabic, sodium alginate, agar, carrageenan, gelatin, guar gum, pectin, PEG or povidone.

[0015] Furthermore, the medicine also contains active probiotic freeze-dried powder.

[0016] Furthermore, the active probiotic freeze-dried powder is obtained by configuring Lactobacillus rhamnosus ATCC9595 and Lactobacillus paracasei CGMCC No.8903 in a ratio of 2:1.

[0017] The additives may also include scavengers, antioxidants, vitamins such as vitamin C and its derivatives, such as ascorbyl acetate, ascorbyl phosphate and ascorbyl palmitate; vitamin A and its derivatives; folic acid and its derivatives; vitamin E and its derivatives, such as tocopherol acetate; flavonoids and flavonoids; amino acids, such as histidine, glycine, tyrosine, tryptophan and their derivatives; carotenoids and carotenes, such as α-carotene, β-carotene; uric acid and its derivatives; α-hydroxy acids, such as citric acid, lactic acid, and maleic acid.

[0018] The carrier also includes a surfactant such as an anionic, amphoteric, nonionic or cationic surfactant or a mixture thereof. Cationic polymers or mixtures of anionic and amphoteric surfactants are preferred surfactants. Non-limiting examples of anionic foaming surfactants include surfactants selected from the group consisting of alkyl sulfates, alkyl ether sulfates, sulphated mono-glycerides, sulfonated paraffins, alkyl aryl sulfonates, primary or secondary alkyl sulfonates, alkyl sulfosuccinates, acyl taurates, acyl isothionates, alkyl glyceryl ether sulfonates, methyl ester sulfonates, sulphonated fatty acids, alkyl phosphates, acyl glutamates, acyl sarcosinates, alkyl sulfoacetates, acylated peptides, alkyl ether carboxylates, acyl lactylates, fluorine-containing anionic surfactants and mixtures thereof. Mixtures of anionic surfactants can be effectively used in the present invention. Suitable nonionic surfactants include, but are not limited to, coconut acyl monoethanolamide or coconut acyl diethanolamide, alkyl polysaccharides, lacto-bionamide, glycol esters, glycerol monoethers, polyhydroxy amides (glucamide), ethoxylates of primary and secondary alcohols, especially ethoxylates of C8-20 fatty alcohols having an average of 1 to 20 moles of ethylene oxide per mole of alcohol. Mixtures of the aforementioned surfactants may also be used. Beneficial Effects

[0019] The invention provides a medicine with good anti-allergy and anti-oxidation effects, which contains probiotic freeze-dried powder and antioxidant polypeptide. The probiotic freeze-dried powder is a freeze-dried powder obtained by configuring Lactobacillus rhamnosus ATCC9595 and Lactobacillus paracasei CGMCC No.8903 in a ratio of 2:1, which has strong anti-allergy properties, and the antioxidant polypeptide has good properties of inhibiting skin aging. After the two are used in combination, the effects of allergy and aging caused by skin ultraviolet damage can be effectively prevented, and the application value is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Identification results of the anti-allergic properties of probiotic freeze-dried powder

[0021] Figure 2 Effects of each group on the histamine content in mouse skin DETAILED DESCRIPTION

[0022] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention. The methods, equipment, and materials in the following implementation cases, if not specifically stated, are all conventional methods, equipment, and materials in the art, and can be purchased from the market.

[0023] Example 1 Isolation and identification of anti-aging peptides

[0024] Take 200g of fresh Ningxia wolfberry, crush and pulp it, centrifuge it at 3000r / min for 10min, take the supernatant and perform trypsin hydrolysis; the trypsin conditions are as follows: temperature 37℃, pH 8.5, enzyme amount 2500U / g protein, material-liquid ratio 1:12, hydrolysis time 4h, inactivate the enzyme after hydrolysis, microfilter it, and remove macromolecular impurities by ultrafiltration with a pore size of 5000u. Glucose gel G50 gel was used for peptide sorting, and a total of 4 main elution peaks were obtained, among which the hydroxyl radical scavenging rate of the third elution peak reached the highest 95.46%. The third elution peak was used for mass spectrometry analysis, and the chromatographic column used was Waters Acquity UPLC BEHC18 colum (50mm×2.1mm,1.7μm). The elution conditions were as follows: mobile phase A: 0.1% (v / v) formic acid aqueous solution; mobile phase B: acetonitrile. Mobile phase B changed from 0 to 40% within 40min, and from 40 to 42min, mobile phase B changed back to 0% from 40%; the flow rate was 0.3mL / min; and the injection was 5μL. The mass spectrometry conditions were: electrospray ionization source, positive ion mode, scanning molecular weight 500-3000Da. The mass spectrometry parameters were: capillary voltage 3000V, cone voltage 40V, spray 35psi, degassing temperature 350℃, ion source temperature 120℃, N2 flow rate 900L / h. Four peptides were obtained through analysis and identification, namely GSO-1, GSO-2, GSO-3, and GSO-4. The above four peptides were commissioned for artificial synthesis.

[0025] Take 2mL of the above polypeptide sample (adjust the concentration to 10μg / mL, and use the same concentration of glutathione as a control) and oscillate and mix with 2mL of 0.1mmol.L-1 DPPH solution, react at room temperature in the dark for 30min, adjust to zero with 95% ethanol, and measure the absorbance value A1 at a wavelength of 517nm. At the same time, measure the absorbance value A2 of 2mL sample mixed with 2mL 95% ethanol solution in the dark for 30min and the absorbance value A3 of 2mL sample mixed with 2mL deionized water in the dark for 30min. Calculate the DPPH free radical scavenging rate according to the formula: DPPH free radical scavenging rate = (1-(A1-A2) / A3) × 100%. The results are shown in Table 1.

[0026] Table 1 DPPH free radical scavenging rate of each peptide

[0027] Components DPPH free radical scavenging rate (%) Glutathione 67.53±1.24 GSO-1 46.57±0.67 GSO-2 89.67±1.84 GSO-3 70.14±1.03 GSO-4 52.47±0.89

[0028] As can be seen from Table 1, among the four peptides, GSO-2 peptide has the highest DPPH free radical scavenging rate and is better than the control glutathione.

[0029] Example 2 Preparation of freeze-dried powder of probiotics with anti-allergic activity

[0030] The inventors obtained through screening and identification that the combination of Lactobacillus rhamnosus ATCC9595 and Lactobacillus paracasei CGMCCNo.8903 has good anti-allergic properties, and the specific operation is as follows.

[0031] A single colony of Lactobacillus rhamnosus strain ATCC9595 was picked and inoculated into MRS liquid culture medium, and cultured at 37° C. and 150 rpm for 24 h to obtain probiotic culture solution 1.

[0032] Similarly, a single colony of Lactobacillus paracasei CGMCC No. 8903 was selected and inoculated into MRS liquid culture medium, and cultured at 37° C. and 150 rpm for 24 h to obtain probiotic culture solution 2.

[0033] The culture solution 1 and the culture solution 2 were mixed at a bacterial concentration ratio of 5:1, 2:1, 1:1, 1:2, and 1:5.

[0034] Then, the mixture is mixed with a freeze-drying protective agent, wherein the protective agent comprises the following components in parts by weight: 20-30 parts of maltodextrin, 20-30 parts of trehalose, 19-29 parts of sodium L-glutamate, 15-25 parts of glycerol, and 3-9 parts of galactomannan.

[0035] The weight ratio of the bacterial liquid to the lyophilization protective agent is 10:1. The lyophilization protective agent and the bacterial liquid are fully mixed to obtain a mixed solution; then pre-freezing treatment is performed: the mixed solution is placed in a refrigerator at a temperature of 4°C and kept at a constant temperature for 12 hours to obtain a pre-freezing treatment solution; the pre-freezing treatment solution is added to a vacuum freeze dryer for freeze drying, and the temperature is reduced to -35°C to -40°C at a cooling rate of 3°C / min, and maintained for 20 minutes to obtain freeze-dried bacterial bodies. The freeze-dried bacterial bodies obtained after freeze drying are crushed, and the crushed product is sieved through a 40-mesh sieve for standby use.

[0036] Example 3 Identification of the anti-allergic properties of probiotic freeze-dried powder

[0037] Take 500 μL of rat skin mast cell suspension (article number: C12087, Hewu Biological), incubate at 37°C for 5 minutes, and add 4 μL of calcium chloride solution (250 mM). Continue to culture in a 37°C incubator for 5 minutes, add 5 μL of each group of freeze-dried powder test samples prepared in Example 2 (5 μg / μL), and add 5 μL of Compound 48 / 80 solution (0.05 mg / mL) after 10 minutes to stimulate the cells. After 10 minutes of reaction, cool and terminate. Centrifuge at 6000 rpm for 3 minutes at 4°C, and collect the supernatant. Take 400 μL of supernatant and add 20 μL of 60wt% perchloric acid to remove protein. After standing at room temperature for 20 minutes, add 400 μL of normal saline, then centrifuge at 13000 rpm for 3 minutes, and collect the supernatant. The histamine content in the supernatant was determined using a kit and calculated using the following formula: Histamine release inhibition rate (%) = (1-(sample-blank)) / (Comp48 / 80-blank) × 100%. Figure 1 shown.

[0038] according to Figure 1 The results show that the probiotic freeze-dried powder prepared by culture medium 1 and culture medium 2 in a ratio of 2:1 has the highest anti-allergic effect, with the histamine release inhibition rate reaching (86.4±2.03)%, while the anti-allergic effect of freeze-dried powder in other ratios is significantly weakened, which indicates that a specific bacterial solution ratio has a synergistic anti-allergic effect.

[0039] Example 4 Animal Experiment Verification

[0040] After the mice were adaptively fed for 1 week, the hair on their backs was shaved with a depilatory knife, leaving a bare area of ​​3cm×3cm on the back. New hair was shaved off daily to keep the back skin always exposed. LED ultraviolet lamps [ultraviolet intensity: UVA (365nm) 360μW·cm−2 and UVB (310nm) 30μW·cm−2] were used to irradiate the back skin of the mice daily for 8 weeks, with an average total irradiation dose of 4500mJ·cm−2. 100mg of crude extract of dust mites was injected intradermally in the right ear of the mice twice a week for a total of 4 injections. At the beginning of irradiation, the mice were dosed according to the following groups:

[0041] The model group and the blank group were not given any medication, the model group was irradiated with UV light, and the blank group was not irradiated;

[0042] Peptide group: daily oral administration of GSO-2 peptide 10 mg / kg;

[0043] Freeze-dried powder group: Daily application of freeze-dried powder of Lactobacillus rhamnosus ATCC9595 and Lactobacillus paracasei CGMCC No.8903 prepared in a ratio of 2:1 1 mg / cm2

[0044] Peptide and lyophilized powder combined treatment group: daily oral administration of GSO-2 peptide 10mg / kg; daily application of lyophilized powder 1mg / cm2 prepared by Lactobacillus rhamnosus ATCC9595 and Lactobacillus paracasei CGMCC No.8903 in a ratio of 2:1

[0045] Positive control group: Retinoic acid tablets (Shandong Liangfu Pharmaceutical Co., Ltd.), 10 mg / kg per day by intragastric administration;

[0046] At week 9, skin grade was scored: After the mice were killed by carbon dioxide anesthesia, the back skin was photographed and the skin grade was scored. The specific scoring criteria are: 0 points: no wrinkles or sagging, no thickening, longitudinal fine lines running through the back, appearing or disappearing with movement; 1 point: most of the skin is longitudinal fine lines, a small number of transverse fine lines, which can appear or disappear with movement; 2 points: there are still a small number of longitudinal fine lines, most of the transverse fine lines, appear or disappear with movement, and the skin is slightly thickened; 3 points: all longitudinal fine lines disappear, a small number of transverse shallow wrinkles appear, do not appear or disappear with movement, and the skin is slightly thickened; 4 points: there are continuous transverse deep wrinkles and sagging, the skin is red, and the skin is obviously thickened; 5 points: there are continuous large numbers of transverse rough deep wrinkles and sagging, small erythema, and obvious thickening of the skin; 6 points: severe wrinkles, large areas of erythema, development of tumors or lesions, and abnormal skin hyperplasia. Determination of total skin thickness: after long-term UV irradiation, the skin tissue will produce hyperplasia and thickening. After treatment, the thickness will decrease. The results are shown in Table 2.

[0047] Table 2 Skin characterization evaluation results

[0048] Group Skin score Total skin thickness (mm) Blank group 0±0 0.38±0.02* Model Group 5.69±0.39 1.05±0.07 Peptide Group 1.89±0.17# 0.60±0.05* Freeze-dried powder group 3.52±0.22# 0.73±0.04* Peptide and lyophilized powder combined treatment group 1.76±0.15# 0.54±0.03* Positive control group 2.12±0.30# 0.68±0.05*

[0049] As can be seen from Table 2, the skin scores and total skin thickness of the treatment groups were significantly different from those of the model group (*#P<0.05). The polypeptide or polypeptide combined with lyophilized powder of the present invention can significantly reduce the skin score and total skin thickness, and has a good anti-wrinkle effect.

[0050] The mouse histamine ELISA kit was used to detect the level of histamine in mouse skin. 0.5g of skin from the experimental site was taken and dried with filter paper, then cut into pieces and ground into powder in a mortar filled with liquid nitrogen. Then 4.5mL of PBS buffer (0.01mol / L, PH7.4) was added and centrifuged at 5000r / min for 15min to prepare the supernatant. The histamine content in the supernatant was detected by ELISA. The specific results are as follows: Figure 2 shown.

[0051] from Figure 2It can be seen that the freeze-dried powder and polypeptide combined freeze-dried powder groups can significantly reduce the histamine content in the skin. The decrease is higher than that of the model group and is basically close to the level of the blank group, indicating that the freeze-dried powder of the present invention has a good anti-allergic effect.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antioxidant polypeptide named GSO-2, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. An anti-allergic drug, comprising the antioxidant polypeptide according to claim 1 and active probiotic freeze-dried powder; wherein: The active probiotic freeze-dried powder is obtained by mixing Lactobacillus rhamnosus ATCC9595 and Lactobacillus paracasei CGMCCNo.8903 in a ratio of 2:

1.

3. Use of the antioxidant polypeptide and active probiotic freeze-dried powder according to claim 1 in the preparation of anti-allergic drugs, wherein: The active probiotic freeze-dried powder is obtained by mixing Lactobacillus rhamnosus ATCC9595 and Lactobacillus paracasei CGMCCNo.8903 in a ratio of 2:1.

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