CN110638681A

CN110638681A - Preparation method of mussel antiallergic peptide and application of mussel antiallergic peptide in cosmetics

Info

Publication number: CN110638681A
Application number: CN201910970096.7A
Authority: CN
Inventors: 盘学平
Original assignee: Guangzhou Shangzi Chemical Technology Co Ltd
Current assignee: Guangzhou Shangzi Chemical Technology Co Ltd
Priority date: 2019-10-12
Filing date: 2019-10-12
Publication date: 2020-01-03
Anticipated expiration: 2039-10-12
Also published as: CN110638681B

Abstract

The invention belongs to the field of marine biological component extraction, relates to a preparation method of a mussel antiallergic extract and an application of the mussel antiallergic extract in cosmetics, in particular to a preparation method of mussel antiallergic peptide and an application of the mussel antiallergic peptide in cosmetics, and particularly relates to an application of mussel antiallergic extract as an effective component in preparation of medicines, skin care products or/and cosmetics. The invention comprises the following steps: pretreatment, homogenate, ultrahigh pressure extraction, subcritical extraction, enzymolysis and ultrafiltration, and can also comprise vacuum concentration and drying steps. The invention applies the normal temperature and ultrahigh pressure technology and the subcritical extraction technology to the preparation of the mussel antiallergic extract, so that the produced extract has the characteristics of high extraction efficiency, high component activity, uniform chain scission and controllable molecular weight within 3KD, and the content of mussel small peptide can reach 89.79 percent at most. The mussel extract has excellent antiallergic ability and strong antioxidation.

Description

Preparation method of mussel antiallergic peptide and application of mussel antiallergic peptide in cosmetics

Technical Field

The invention belongs to the field of marine biological component extraction, relates to a preparation method of a mussel antiallergic extract and an application of the mussel antiallergic extract in cosmetics, in particular to a preparation method of mussel antiallergic peptide and an application of the mussel antiallergic peptide in cosmetics, and particularly relates to an application of mussel antiallergic extract as an effective component in preparation of medicines, skin care products or/and cosmetics.

Background

From the medical point of view, skin allergy mainly refers to the symptoms of pricking pain, burning, itching and the like when the skin is subjected to various external stimuli such as cosmetics, pollen, polluted air, chemical agents and the like with adverse reactions, and is accompanied with mild abnormal phenomena such as desquamation, erythema, allergic dermatitis and the like. Skin cells are damaged when skin is allergic, a sebum membrane is split to secrete incomplete skin cells, various sensitive skins are formed, and various sequelae such as long-term skin allergy, pigmentation, redness and swelling, large pores, acne, color spots, wrinkles and the like seriously influence the comfort and appearance of people. The problem to be solved urgently in the cosmetic industry is to find an antiallergic product which is safe to use, mild in action and good in efficacy.

Mussel resources are abundant, and the annual yield in the world is over 100 ten thousand tons. Mussels are rich in various trace elements such as manganese, zinc, selenium, iodine and the like, wherein the selenium content is particularly rich, and the mussels have the effects of cancer prevention, cancer resistance, aging resistance and the like. In addition, mussels are rich in unsaturated fatty acids, proteins and amino acids, and have various activities of promoting metabolism, blood circulation, resisting radiation, promoting wound healing, etc. And as a natural marine biological resource, the prepared mussel peptide has excellent biocompatibility. Therefore, the effective components in the mussel body have wide application prospect, and great commercial value is further generated.

The Chinese patent application 201711122919.8 discloses a bioactive polypeptide GLNYYQQKPVAL and a preparation method and application thereof, the invention relates to the field of protein, and in particular relates to a bioactive polypeptide GLNYYQQKPVAL and a preparation method and application thereof, wherein the amino acid sequence of the bioactive polypeptide GLNYYQQKPVAL is Gly-Leu-Asn-Tyr-Tyr-Gln-Gln-Lys-Pro-Val-Ala-Leu. In vitro antioxidant experiments and in vivo anti-aging experiments prove that the polypeptide GLNYYQQKPVAL has good antioxidant biological activity and anti-aging activity, and on one hand, the bioactive polypeptide GLNYYQQKPVAL has good antioxidant activity, can remove free radicals in organisms and improve the quality of life; on the other hand, the activity of an anti-peroxidase system in vivo can be improved, and the function of resisting exogenous stimulation of the organism is enhanced, so that the probability of aging, aging and illness of the organism is reduced, and the method has very important significance for developing foods, health-care products and medicines with antioxidant and anti-aging functions.

Chinese patent application 201910229397.4 discloses an antiallergic cosmetic composition containing plant active peptide and a preparation process thereof, wherein the cosmetic composition comprises, by weight, 8-12 parts of honeysuckle, 4-10 parts of chamomile, 3-9 parts of anthocyanin, 1-5 parts of carboxymethyl cellulose, 2-8 parts of benzoyl peroxide, 4-10 parts of herba lycopi extract, 5-15 parts of fructus alpiniae oxyphyllae extract, 6-12 parts of rose extract, 8-18 parts of portulaca oleracea extract, 2-6 parts of 1-methylhydantoin-2-imide, 3-9 parts of xanthan gum and 14-10 parts of oligopeptide.

The processing process of the prepared active peptide has the defects of low extraction efficiency, damaged functional components, residual organic solvent and the like, and has the defects of difficult acquisition of raw materials, high cost, complex preparation process and the like, and does not relate to actual application process technology and formula, and has no report on the application of the mussel peptide in the antiallergic cosmetics. Therefore, there is a need in the research and practice to develop a novel method for preparing mussel extract and use the extract as an antiallergic ingredient in skin care products or cosmetics.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides a preparation method of a mussel extract with antiallergic activity and application of the mussel extract as an effective component in preparing cosmetics.

The invention is realized by the following technical scheme:

a preparation method of shellfish antiallergic extract comprises the following steps:

a pretreatment step: taking the useful part of the fresh and live shellfish, and putting the useful part into the pretreatment liquid to obtain a fresh and live shellfish mixture; and (3) homogenizing: homogenizing the mixture of the fresh and live shellfish to obtain homogenate; ultrahigh pressure extraction: mixing the homogenate with an extraction solvent for soaking treatment, and performing ultrahigh pressure extraction treatment to obtain an initial extract; a subcritical extraction step: performing subcritical extraction treatment on the primary extract to obtain a protein extract; an enzymolysis step: carrying out enzymolysis treatment, enzyme deactivation treatment and centrifugal treatment on the protein extract to obtain shellfish protein hydrolysate; an ultrafiltration step: and carrying out ultrafiltration treatment on the shellfish protein enzymolysis liquid to obtain the shellfish antiallergic extract ultrafiltrate.

In some embodiments, the pretreatment solution is an aqueous vitamin C solution; preferably, in the pretreatment step, the concentration of the vitamin C aqueous solution is 0.1-0.5% by mass, and preferably 0.2%; preferably, the mass ratio of the useful part to the vitamin C aqueous solution is 1: (1-2), preferably 1: 1; preferably, the useful part is a mantle part; preferably, the shellfish is mussel.

In some embodiments, the homogenization step is performed at a stirring rate of 2000-10000rpm, preferably 8000rpm, for a period of 0.5-10min, preferably 5 min.

In some embodiments, in the ultra-high pressure extraction step, the extraction solvent is one or both of an aqueous solution, an alcoholic solution of a phosphate buffer system; preferably, the concentration of the aqueous solution of the phosphate buffer system is 0.02-0.02M, and the alcoholic solution is 70-80% by volume of ethanol aqueous solution.

In some embodiments, in the ultra-high pressure extraction step, the soaking treatment is performed for 1 to 3 hours.

In some embodiments, in the ultra-high pressure step, in the ultra-high pressure treatment: extracting at the pressure of 100-800MPa for 1-5 times at normal temperature, wherein each time lasts for 15-25 min, and the volume of the extraction solvent is 6-18 times, preferably 10 times that of the homogenate or the residue obtained in the last extraction.

In some embodiments, in the subcritical extraction step, the subcritical extraction treatment comprises: placing the primary extract in an extraction tank, vacuumizing, and adding a subcritical extraction solvent into the primary extract to extract for 1-5 times; the mass ratio of the extraction solvent to the initial extract or the semi-solid extracted last time is (3-15): 1, preferably 10: 1; preferably, the total time of the subcritical extraction treatment is 40-120 minutes, the temperature of each time is 10-50 ℃, and preferably 40-50 ℃; preferably, the subcritical extractant comprises: water, methanol and ethanol, wherein the volume ratio of the components is (5-10): (1-3): 1, preferably 7: 2: 1.

in some embodiments, in the step of enzymolysis, in the enzymolysis treatment, 5-15 times, preferably 10 times, the mass of water is added to the protein extract to form an enzymolysis reaction system; the mass ratio of the dry enzyme weight to the enzymolysis reaction system is 1-5%, and the total enzymolysis time is 1-7 h; preferably, the enzyme is an enzyme preparation of 10-30 ten thousand U/g, preferably 20 ten thousand U/g; preferably, the enzyme comprises one or more of pepsin, trypsin and subtilisin; more preferably, the enzymolysis condition of the pepsin is pH 2-4 and 25-40 ℃, the enzymolysis condition of the trypsin is pH 7.5-9.0 and 37-45 ℃, and the enzymolysis condition of the subtilisin is pH 7-8 and 37-50 ℃; the enzymolysis time of the pepsin is 10-90min, the enzymolysis time of the trypsin is 30-120min, and the enzymolysis time of the subtilisin is 30-180 min.

In some embodiments, in the step of enzymolysis, the inactivation treatment is performed at 90-100 ℃ for 5-10 min; in the enzymolysis step, the rotation speed of the centrifugal treatment is 8000-10000rpm, and the time is 5-15 min.

In some embodiments, in the ultrafiltration step, the pressure in the ultrafiltration treatment is 0.02 to 0.25MPa, preferably 0.12 MPa; preferably, the ultrafiltration treatment employs an ultrafiltration membrane having a molecular cut-off of 3000 Da.

In some embodiments, the method of making further comprises: vacuum concentration step: carrying out vacuum concentration treatment on the shellfish antiallergic extract ultrafiltrate to obtain a shellfish antiallergic extract concentrated solution; the weight percentage of the shellfish antiallergic extract concentrated solution to the shellfish antiallergic extract ultrafiltrate is 7-13 wt%.

In some embodiments, the method of making further comprises: and (3) drying: and (3) carrying out spray drying treatment and sterilization treatment on the shellfish antiallergic extract liquid to obtain shellfish extract powder with antiallergic activity.

The shellfish antiallergic extract obtained by the preparation method can be applied to preparation of medicines, skin care products or/and cosmetics with antiallergic, antioxidant or skin barrier function repairing promoting effects.

In some embodiments, the shellfish anti-allergic extract has a minimum effective concentration of 5-12 g/mL.

In some embodiments, the shellfish antiallergic extract accounts for 5-15 wt% of the skin care product or/and the cosmetic.

In some embodiments, the skin care or/and cosmetic formulation comprises: creams, lotions, lipsticks, powders, gels, sprays and masks.

A shellfish antiallergic extract is prepared by the above preparation method.

A composition comprising, as starting materials: the shellfish antiallergic extract and medicinal adjuvants.

In some embodiments, the pharmaceutical adjuvant comprises an oil phase material, an aqueous phase material, a pharmaceutical polysaccharide, and a preservative; preferably, the shellfish antiallergic extract, the oil phase material, the water phase material, the medicinal polysaccharide and the preservative are used in the following weight ratio: (6-14) (12-24) (9-15) (1-3) (0-1), more preferably 10:18:12:2: 1; preferably, the oil phase material is white vaseline, glycerol, stearic acid, stearyl alcohol and stearyl cetyl alcohol; more preferably, the white vaseline, the glycerol, the stearic acid, the stearyl alcohol and the stearyl cetyl alcohol are used in the following weight ratio: (5-10): (1-2): (1-2): (1-2): (1-2), more preferably 5:1:1:1: 1; preferably, the water phase material is glycerol and trehalose, and more preferably, the glycerol and the trehalose are used in the following weight ratio: (1-3): 1, more preferably, 2: 1; preferably, the medicinal polysaccharide is one or a mixture of xanthan gum, agar, arabic gum and chitin; preferably, the preservative is one or a mixture of salicylic acid and sorbic acid.

A method for preparing a composition by preparing the composition from the materials of the composition, comprising the steps of:

s1: preparing an oil phase and an aqueous phase simultaneously or sequentially; the steps for preparing the oil phase are as follows: mixing all the oil phase materials, and heating to obtain an oil phase; the steps for preparing the aqueous phase are: mixing all the water phase materials, and heating to obtain a water phase; s2: mixing the oil phase and the aqueous phase to obtain a homogeneous mixture; s3: mixing the homogeneous mixture, the shellfish antiallergic extract, the pharmaceutical polysaccharide, and the preservative to obtain the composition; preferably, in step S1: in the step of preparing the oil phase, heating to 75-85 ℃, preferably 80 ℃; more preferably, in step S1: in the step of preparing the aqueous phase, the aqueous phase is heated to 75-85 ℃, preferably 80 ℃; more preferably, in step S2: mixing the oil phase and the water phase at 75-85 ℃ for 10-20min, and cooling to 50-60 ℃ to obtain the homogeneous mixture; more preferably, in step S2: and mixing the oil phase and the water phase at 80 ℃ for 15min, and cooling to 55 ℃ to obtain the homogeneous mixture.

Compared with the prior art, the invention has the following advantages:

1. the method of the invention adopts the normal temperature and ultrahigh pressure technology combined with the subcritical extraction technology to prepare the mussel antiallergic extract, so that the produced extract has the characteristics of high extraction efficiency, high component activity, uniform chain scission and controllable molecular weight within 3KD, and the content of mussel small peptide can reach 89.79 percent at most. After passive skin anaphylactic reaction experiments of mice, the mussel extract is verified to have excellent anti-allergic capability. H₂O₂The experiment of inducing the oxidative damage of the epidermal cells proves that the mussel extract has stronger antioxidation.

2. The preparation method of the invention adopts reasonable steps and parameters, and the synergistic effect is achieved, thereby further improving the extraction effect.

3. The invention provides a perfect shellfish antiallergic preparation process, which comprises the following steps: the preparation method is simple and easy to realize industrial production.

4. The invention realizes the high-value utilization of marine biological resources and overcomes the industrial problems of simple processing and low added value of the current shellfish.

5. The mussel allergic peptide prepared by the method has the characteristics of naturalness, mildness, safety, high efficiency, easiness in storage and the like, can effectively repair sensitive skin and improve the allergic symptoms of the skin; and has the excellent effects of promoting the removal of free radicals, healing wounds, repairing skin barrier functions and the like, and has very important significance for the development of novel functional cosmetics.

Drawings

FIG. 1: transmission electron microscopy images of the normal group (without any drug added) of the antioxidant activity experiments of mussel antiallergic extract in test 5.

FIG. 2: transmission electron micrograph of experimental group (group to which mussel antiallergic extract was added) for test of antioxidant activity of mussel antiallergic extract in 5.

FIG. 3: test for antioxidant Activity of mussel antiallergic extract in 5H₂O₂Transmission electron micrographs of the group (skin epidermal cell injury group).

Detailed Description

In a first aspect, the present invention provides a method for preparing an antiallergic extract of shellfish, the method comprising the steps of:

step one, pretreatment: cutting the shell of the fresh and live shellfish with a cutter, separating the shell and the soft body part, taking the useful part of the fresh and live shellfish, preferably the mantle part, and putting the useful part into the vitamin C water solution to obtain the mixture of the fresh and live shellfish.

The concentration of the vitamin C aqueous solution is 0.1-0.5% by mass, preferably 0.2% by mass. The aqueous vitamin C solution is used to prevent oxidation of the useful fraction; the mass ratio of the useful part of the shellfish to the vitamin C aqueous solution is 1: (1-2) (for example: 1, 1:1.2, 1:1.3, 1:1.5, 1:1.8, 1:2 are acceptable), preferably 1:1.

the shellfish is preferably mussel, for example: mytilus coruscus, Mytilus edulis; the preferred place of production of shellfish is Sixian in Zhoushan city of Zhejiang province.

Step two, homogenizing: and (3) homogenizing the mixture of the fresh and live shellfish to obtain homogenate.

The stirring rate of the above homogenization treatment is 2000-10000rpm (for example, 2000rpm, 5000rpm, 7500rpm, 10000rpm is acceptable), preferably 8000rpm, and the time is 0.5-10min (for example, 0.5min, 1min, 2min, 4min, 6min, 8min, 10min is acceptable), preferably 5 min.

Step three, ultrahigh pressure extraction: mixing the homogenate with an extraction solvent for soaking treatment, and then putting the mixture into an extraction container of an ultrahigh pressure extraction device for ultrahigh pressure extraction treatment to obtain an initial extract.

The soaking time is 1-3 hr (for example, 1 hr, 1.5 hr, 2 hr, 2.5 hr, 3 hr), and the extraction solvent is water solution or alcohol solution of phosphate buffer system; the volume of the extraction solvent is 6 to 18 times (for example, 6, 8, 10, 12, 15, 16, 18 times) that of the homogenate, and preferably 10 times.

The aqueous solution of the above phosphate buffer system is preferably: the pH value is neutral, the concentration is 0.02-0.05M (for example: 0.02, 0.03, 0.04, 0.05M), and the preparation method comprises: mixing 0.02-0.05 mol/L each of disodium hydrogen phosphate and sodium dihydrogen phosphate according to a volume ratio of 1:1, adjusting the pH value to 6.8-7, and storing at-4 ℃; the alcohol solution is preferably an aqueous ethanol solution having a volume ratio of 70 to 80% (for example, 70%, 72%, 75%, 78%, 80%).

In the ultrahigh pressure extraction treatment: extracting at 100-800MPa (such as 100MPa, 200MPa, 400MPa, 500MPa, 600MPa, 800MPa) at room temperature for 1-5 times (such as 1, 2, 3, 4, 5 times), each time for 15-25 min (such as 15min, 17.5min, 20min, 22.5min, 25 min); preferably, the extraction time is the same for each extraction and the extraction temperature is the same. Obtaining effluent liquid and residues in each extraction; in the extraction after the 1 st time, adding 6-18 times, preferably 10 times of volume of the extraction solvent into the residues extracted for the previous time each time, and combining the effluent obtained in each extraction to obtain the initial extract.

Step four, subcritical extraction: and (3) placing the initial extract in an extraction tank for subcritical extraction treatment to obtain a protein extract.

The subcritical extraction treatment comprises the following steps: placing the primary extract in an extraction tank, vacuumizing, and adding a subcritical extraction solvent into the extraction tank, wherein the mass ratio of the extraction solvent to the primary extract is (3-15): 1 (for example, 3:1, 5:1, 6:1, 10:1, 12:1, 15:1), extracting for 1-5 times (for example, 1, 2, 3, 4, 5 times), and separating to obtain protein extract.

The subcritical extracting agent comprises water, methanol and ethanol, and the volume ratio of the water to the methanol to the ethanol is (5-10): (1-3): 1, may be 5:3:1, 10:1:1, 5:1:1, 8:3:1, 9:2:1, preferably 7: 2: 1.

the total time of the subcritical extraction treatment is 40-120 min (for example, 40min, 50min, 60min, 80min, 100min, 120min), and the temperature is 10-50 ℃ (for example, 10min, 20min, 25min, 30min, 40min, 50 min); preferably, the extraction time is the same for each extraction and the extraction temperature is the same. In the extraction after the 1 st time, an extraction solvent is added into the semi-solid matter extracted last time, and the mass ratio of the extraction solvent to the semi-solid matter extracted last time is (3-15): 1 (e.g., 3:1, 5:1, 7.5:1, 8:1, 10:1, 12.5:1, 15: 1); and after extraction is finished, combining the extract liquor obtained each time, and performing reduced pressure evaporation separation on the combined extract liquor to obtain the semi-solid protein extract.

Step five, enzymolysis step: performing enzymolysis treatment on the protein extract, performing enzyme deactivation treatment by using boiling water, and performing centrifugation treatment to obtain shellfish protease hydrolysate.

In the above-mentioned enzymatic hydrolysis treatment, 5 to 15 times (for example, 5, 7.5, 8, 10, 12, 15 times), preferably 10 times, by mass of water is added to the protein extract enzyme to form an enzymatic hydrolysis reaction system, the mass ratio of the enzyme to the system is 1 to 5% (for example, 1%, 2%, 3%, 4%, 5%), and the total enzymatic hydrolysis time is 1 to 7 hours.

The enzyme is 10-30 ten thousand U/g (for example, 10 ten thousand U/g, 12.5 ten thousand U/g, 15 ten thousand U/g, 17.5 ten thousand U/g, 23 ten thousand U/g, 25 ten thousand U/g, 30 ten thousand U/g), preferably 20 ten thousand U/g, one or more of pepsin, trypsin, and subtilisin can be used, preferably the combination of the three enzymes is used. Wherein the enzymolysis condition of the pepsin is pH 2-4, the temperature is 25-40 ℃, and the pH value of an enzymolysis reaction system can be adjusted by adopting 0.1mol/L HCl solution; the enzymolysis condition of the trypsin is pH 7.5-9.0, 37-45 ℃, and the pH value of an enzymolysis reaction system can be adjusted by adopting 0.2mol/L NaOH solution; the enzymolysis condition of the subtilisin is pH 7-8, 37-50 ℃. The above three enzymes were used for the enzymatic treatment because: most of trace elements such as iron, manganese, zinc, selenium and the like in the shellfish exist in the insoluble protein, and the shellfish allergy peptide is hydrolyzed by adopting a method of combining a plurality of enzymes, so that the complementation and the synergism of various enzymes can be realized, and the insoluble macromolecular protein is converted into soluble protein, amino acid and the like, so that the shellfish allergy peptide which is rich in a plurality of active components and has high performance can be obtained.

The enzymolysis time of pepsin is 10-90min (for example, 10min, 20min, 30min, 40min, 50min, 60min, 70min, 80min, 90min), the enzymolysis time of trypsin is 30-120min (for example, 30min, 40min, 50min, 60min, 70min, 80min, 90min, 100min, 110min, 120min), and the enzymolysis time of subtilisin is 30-180min (for example, 30min, 40min, 50min, 60min, 70min, 80min, 90min, 100min, 110min, 120min, 130min, 140min, 150min, 160min, 170min, 180 min).

The operations of enzymolysis, inactivation and centrifugation are as follows: firstly, adjusting the pH value and temperature of a reaction system, adding a first enzyme until enzymolysis is finished, then inactivating the first enzyme by boiling water, and then cooling the reaction system and adjusting the pH value to be used as an enzymolysis reaction system of a second enzyme; adding a second enzyme into the mixture until enzymolysis is finished, inactivating the mixture by boiling water, and then cooling the reaction system and adjusting the pH value to be used as an enzymolysis reaction system of a third enzyme; adding a third enzyme, inactivating with boiling water after enzymolysis, cooling the reaction system to 40-50 deg.C, centrifuging, and collecting supernatant as shellfish protein enzymolysis liquid.

In the inactivation treatment, the temperature is 90-100 deg.C (such as 90 deg.C, 92 deg.C, 96 deg.C, 98 deg.C, 100 deg.C), and the time is 5-10min (such as 5min, 6min, 7min, 8min, 9min, 10 min); the centrifugation is carried out at 8000-10000rpm (for example, 8000rpm, 8500rpm, 9000rpm, 9500rpm, 10000rpm) for 5-15 min.

Step six, pressurized ultrafiltration: and (3) performing pressurized ultrafiltration treatment on the shellfish protein enzymolysis liquid to obtain the shellfish antiallergic extract ultrafiltrate.

The pressure of the pressure ultrafiltration is 0.02-0.25MPa (for example, 0.02MPa, 0.05MPa, 0.08MPa, 0.1MPa, 0.12MPa, 0.15MPa, 0.17MPa, 0.2MPa, 0.22MPa, 0.25 MPa). The ultrafiltration treatment was carried out using an ultrafiltration membrane having a molecular cut-off of 3000 Da.

The above preparation method may further comprise the steps of:

step seven, vacuum concentration: performing vacuum concentration treatment on the shellfish antiallergic extract ultrafiltrate by adopting a scraper concentrator to obtain a shellfish antiallergic extract concentrated solution; the weight percentage of the shellfish anti-allergic extract concentrated solution and the shellfish anti-allergic extract ultrafiltrate is 7-13 wt% (for example, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%).

The concentrated solution of shellfish antiallergic extract can be used as effective component for cosmetic.

Step eight, drying: spray drying the shellfish antiallergic extract, and sterilizing to obtain shellfish extract powder with antiallergic activity.

The sterilization treatment is carried out by ultraviolet sterilization at 85-95 deg.C (e.g., 85 deg.C, 88 deg.C, 90 deg.C, 92 deg.C, 95 deg.C) for 2-5 min (e.g., 2min, 2.5min, 3min, 4min, 5 min).

The parameters are as follows: the homogenizing stirring speed, the pressure intensity of ultrahigh pressure treatment, the extraction times, the extraction time, the subcritical extraction times, the extraction treatment time, the temperature, the enzymolysis time, the temperature and the PH value are within the numerical range, and the active small peptide with high extraction rate and uniform chain scission can be obtained.

In a second aspect, the invention provides an application of the shellfish antiallergic extract obtained by the preparation method in the first aspect, wherein the application comprises the following steps: can be used for preparing skin care products and/or cosmetics with antiallergic, antioxidant, and skin barrier function repairing effects.

The minimum effective concentration of the shellfish antiallergic extract is 5-12g/mL (e.g. 5g/mL, 6g/mL, 7g/mL, 8g/mL, 9g/mL, 10g/mL, 11g/mL, 12 g/mL).

The shellfish antiallergic extract accounts for 5-15 wt% (for example, 5 wt%, 7 wt%, 8 wt%, 10 wt%, 12 wt%, 13 wt%, 15 wt%) of the skin care product or/and the cosmetic.

The skin care product or/and cosmetic comprises: cream, aqua, lotion, lipstick, powder, gel, spray or mask and the like.

In a fourth aspect, the present invention provides a composition comprising the mussel antiallergic extract of the first aspect.

A composition comprising, as raw materials: the shellfish antiallergic extract of the first aspect and a pharmaceutical excipient.

The medicinal adjuvants comprise oil phase material, water phase material, medicinal polysaccharide and antiseptic;

the dosage weight ratio of the shellfish antiallergic extract, the oil phase material, the water phase material, the medicinal polysaccharide and the preservative is as follows: (6-14) (12-24) (9-15) (1-3) (0-1), more preferably 10:18:12:2: 1;

preferably, the oil phase material is white vaseline, glycerol, stearic acid, stearyl alcohol, and stearyl cetyl alcohol; more preferably, the white petrolatum, the glycerin, the stearic acid, the stearyl alcohol and the cetostearyl alcohol are used in the following weight ratios: (5-10): (1-2): (1-2): (1-2): (1-2), more preferably 5:1:1:1: 1;

preferably, the water phase material is glycerol and trehalose, and more preferably, the glycerol and the trehalose are used in a weight ratio of: (1-3): 1, more preferably, 2: 1;

preferably, the medicinal polysaccharide is one or more of xanthan gum, agar, arabic gum and chitin;

preferably, the preservative is one or a mixture of salicylic acid and sorbic acid.

In a fifth aspect, the present invention provides a method for preparing the composition of the fourth aspect, comprising the steps of:

the method comprises the following steps: the oil phase and the aqueous phase are prepared simultaneously or sequentially.

Preparing an oil phase: mixing all the above oil phase materials, and heating to 75-85 deg.C (preferably 80 deg.C) to obtain oil phase;

preparing an aqueous phase: mixing all the above water phase materials, and heating to 75-85 deg.C (preferably 80 deg.C) to obtain water phase;

step two, mixing the oil phase and the water phase at 75-85 deg.C (preferably 80 deg.C) for 10-20min (preferably 15min), and cooling to 50-60 deg.C (preferably 55 deg.C) to obtain homogeneous mixture.

And step three, mixing the homogeneous mixture, the shellfish antiallergic extract, the medicinal polysaccharide and the preservative to obtain the composition.

The following detailed description is provided for the purpose of illustrating the embodiments and the advantageous effects thereof, and is not intended to limit the scope of the present disclosure.

Mussels for use in the various embodiments of the invention are produced in Sixian, Zhoushan, Zhejiang.

Example 1

The preparation method of the mussel antiallergic extract comprises the following steps:

(1) mussel pretreatment: cutting fresh mussels by a cutter, and separating shells and a soft body part to obtain a mantle part; and adding the mixture into a vitamin C aqueous solution with the same mass and the mass fraction of 0.2% to obtain a fresh live mussel mixture.

(2) And (3) homogenizing treatment: the mixture was stirred (homogenized) (8000rpm/5min) to obtain a homogenate.

(3) Ultrahigh pressure treatment: adding 10 times volume of extraction solvent (0.02-0.05M phosphate buffer system water solution) into the mixture, soaking for 3 hr, placing into an extraction container of ultrahigh pressure extraction device (L1-600/10, available from Tianjin Huataisen vast bioengineering technology GmbH, under 600MPa, extracting at room temperature for 5 times, each time for 25 min; wherein, the 2 nd to 5 th extraction are all that the extraction solvent with 10 times volume of the homogenate liquid obtained in the step (2) is added into the extraction residue of the last extraction, and the effluent liquid of 5 times of extraction is combined to obtain the primary extract.

(4) And (3) subcritical extraction process: placing the mussel primary extract in an extraction tank (manufactured by Henan subcritical extraction technical research institute, Inc., model: CBE-Z-10L), vacuumizing (vacuum degree of 0.5MPa), adding subcritical extraction solvent (volume ratio of water, methanol and ethanol is 7: 2: 1) and extracting for 4 times, wherein the total extraction time is 120 minutes, each time is the same, and each time temperature is 45 ℃; wherein the 2 nd-4 th extraction is to add an extraction solvent into the semisolid matter (semisolid matter) obtained by the last extraction, and the mass ratio of the extraction solvent to the initial extract and/or the semisolid matter obtained after the last extraction is 10: and 1, combining the extraction solutions for 4 times after the extraction is finished, and evaporating the combined extraction solutions under reduced pressure to obtain the semi-solid protein extract.

(5) And (3) enzymolysis treatment: adding 10 times of water into the extract to form an enzymolysis reaction system; selecting 0.1mol/L HCl or 0.2mol/L NaOH solution to adjust the PH of the reaction, adding 20 ten thousand U/g pepsin, 20 ten thousand U/g trypsin and 20 ten thousand U/g subtilisin with certain mass ratio (solid powder enzyme: the reaction system is 5 percent), and respectively carrying out enzymolysis for 30-90min, 60-120min and 60-180min under the conditions of pH2, pH7.5, pH7, 37 ℃, 45 ℃ and 40 ℃ in sequence.

Inactivating after each enzymolysis at 95 deg.C for 5 min; after the third enzymolysis and inactivation, the enzymolysis system is cooled to 50 ℃, and then centrifuged for 5min at 8000rpm, and the supernatant is collected as the protein enzymolysis liquid.

(6) Pressure ultrafiltration: and (3) carrying out pressurized ultrafiltration treatment on the protein enzymolysis liquid, wherein the pressure is 0.12MPa, and the molecular interception is 3000Da, so as to obtain the mussel antiallergic extract ultrafiltrate.

(7) And (3) vacuum concentration: vacuum concentrating the mussel antiallergic extract ultrafiltrate to 7-13 wt% concentrated solution by adopting a scraper concentrator, and adding the concentrated solution into cosmetics as an effective component.

(8) And (3) drying treatment: spray drying the obtained concentrated solution, and ultraviolet sterilizing at 90 deg.C for 5min to obtain mussel extract powder S1 with antiallergic activity.

Example 2

The preparation method of the mussel antiallergic extract, which adopts the same equipment as the example 1, comprises the following steps:

(2) And (3) homogenizing treatment: the mixture was stirred (homogenized) (5000rpm/5min) to obtain a homogenate.

(3) Ultrahigh pressure treatment: adding 10 times volume of extraction solvent (0.02-0.05M phosphate buffer system water solution), soaking for 3 hr, placing into extraction container of ultrahigh pressure extraction device, extracting at normal temperature for 3 times (each time for 25min) under 300 MPa; wherein, the 2 nd to 3 rd extractions are all that the extraction solvent with the volume 10 times of the homogenate liquid obtained in the step (2) is added into the residue of the last extraction, and the effluent of the 3 times of extraction is combined to obtain the initial extract.

(4) And (3) subcritical extraction process: placing the mussel primary extract in an extraction tank, vacuumizing (vacuum degree of 0.5MPa), adding a subcritical extraction solvent (volume ratio of water to methanol to ethanol is 7: 2: 1) and extracting for 4 times, wherein the total extraction time is 60 minutes, each time is the same, and each time temperature is 45 ℃; wherein the 2 nd-4 th extraction is to add an extraction solvent into the semi-solid (semi-solid) obtained in the last extraction, and the weight ratio of the extraction solvent to the initial extract and/or the semi-solid obtained after the last extraction is 10: and 1, combining the extraction solutions for 4 times after the extraction is finished, and evaporating the combined extraction solutions under reduced pressure to obtain the semi-solid protein extract.

(5) And (3) enzymolysis treatment: adding 10 times of water by mass into the protein extract to form an enzymolysis reaction system; adjusting the pH of the reaction by selecting 0.1mol/L HCl or 0.2mol/L NaOH solution, adding 20 ten thousand U/g pepsin, 20 ten thousand U/g trypsin and 20 ten thousand U/g subtilisin in a certain mass ratio (powdery enzyme: homogenate is 1%), and performing enzymolysis for 30-90min, 60-120min and 60-180min at the conditions of pH2, pH7.5, pH7, 37 ℃, 45 ℃ and 40 ℃ respectively.

(7) And (3) vacuum concentration: vacuum concentrating the mussel antiallergic extract ultrafiltrate to 7-13 wt% of an extracting solution by adopting a scraper concentrator, and adding the extracting solution into cosmetics as an effective component.

(8) And (3) drying treatment: spray drying the obtained extractive solution, and ultraviolet sterilizing at 90 deg.C for 5min to obtain mussel extract powder S2 with antiallergic activity.

Example 3

(2) And (3) homogenizing treatment: the mixture was stirred (8000rpm/5min) to obtain a homogenate.

(3) Ultrahigh pressure treatment: adding 10 times volume of extraction solvent (0.02-0.05M phosphate buffer system water solution), soaking for 3 hr, placing into extraction container of ultrahigh pressure extraction device, extracting at normal temperature under 600MPa for 5 times, each for 25 min; wherein, the 2 nd to 5 th extraction are all that adding the extraction solvent with 10 times volume of the homogenate liquid obtained in the step (2) into the residue of the last extraction, and combining the 5 times extraction effluent to obtain the initial extract.

(5) And (3) enzymolysis treatment: adding 10 times of water by mass into the protein extract to form an enzymolysis reaction system; adjusting the pH of the reaction by selecting 0.1mol/L HCl or 0.2mol/L NaOH solution, adding 20 ten thousand U/g pepsin, trypsin and subtilisin with a certain mass ratio (solid powdered enzyme: homogenate is 1%), and performing enzymolysis for 30-90min, 60-120min and 60-180min at pH2, pH7.5, pH7, 37 ℃, 45 ℃ and 40 ℃ respectively.

(8) And (3) drying treatment: spray drying the obtained extractive solution, and ultraviolet sterilizing at 90 deg.C for 5min to obtain mussel extract powder S3 with antiallergic activity.

Example 4

the method comprises the following steps:

(2) And (3) homogenizing treatment: the mixture was stirred (5000rpm/5min) to obtain a homogenate.

(4) And (3) subcritical extraction process: placing the mussel primary extract in an extraction tank, vacuumizing (vacuum degree of 0.5MPa), adding a subcritical extraction solvent (volume ratio of water to methanol to ethanol is 7: 2: 1) and extracting for 4 times, wherein the total extraction time is 120 minutes, each time is the same, and each time temperature is 45 ℃; wherein the 2 nd-4 th extraction is to add an extraction solvent into the semi-solid (semi-solid) obtained in the last extraction, and the weight ratio of the extraction solvent to the initial extract and/or the semi-solid obtained after the last extraction is 10: and 1, combining the extraction solutions for 4 times after the extraction is finished, and evaporating the combined extraction solutions under reduced pressure to obtain the semi-solid protein extract.

(8) And (3) drying treatment: spray drying the obtained concentrated solution, and ultraviolet sterilizing at 90 deg.C for 5min to obtain mussel extract powder S4 with antiallergic activity.

Example 5

(5) And (3) enzymolysis treatment: adding 10 times of water by mass into the protein extract to form an enzymolysis reaction system; adjusting the pH of the reaction by selecting 0.1mol/L HCl or 0.2mol/L NaOH solution, adding 20 ten thousand U/g pepsin, trypsin and subtilisin with a certain mass ratio (solid powdered enzyme: homogenate is 1%) at pH2, pH7.5, pH7, 37 ℃, 45 ℃ and 40 ℃ for 30-90min, 60-120min and 60-180min respectively.

(6) Pressure ultrafiltration: and (3) carrying out pressurized ultrafiltration treatment on the protein enzymolysis liquid, wherein the molecular interception is 3000Da under the pressure of 0.12MPa, so as to obtain the mussel antiallergic extract ultrafiltrate.

(8) And (3) drying treatment: spray drying the obtained concentrated solution, and ultraviolet sterilizing at 90 deg.C for 5min to obtain mussel extract powder S5 with antiallergic activity.

Example 6

(2) And (3) homogenizing treatment: the mixture was stirred (10000rpm/3min) to obtain a homogenate.

(3) Ultrahigh pressure treatment: adding 10 times volume of extractive solution extraction solvent (0.02-0.05M phosphate buffer system water solution), soaking for 3 hr, placing into extraction container of ultrahigh pressure extraction device, extracting at normal temperature under 200MPa for 4 times, each for 20 min; wherein, the 2 nd to 4 th extraction are all that the extraction solvent with the volume 10 times of the homogenate liquid obtained in the step (2) is added into the residue of the last extraction, and the effluent of the 4 times of extraction is combined to obtain the initial extract.

(4) And (3) subcritical extraction process: placing the mussel primary extract in an extraction tank, vacuumizing (vacuum degree of 0.5MPa), adding a subcritical extraction solvent (volume ratio of water to methanol to ethanol is 7: 2: 1) and extracting for 2 times, wherein the total extraction time is 120 minutes, each time is the same, and each time temperature is 45 ℃; wherein the 2 nd extraction is to add an extraction solvent into the semi-solid (semi-solid) obtained in the last extraction, and the weight ratio of the extraction solvent to the initial extract and/or the semi-solid obtained after the last extraction is 10: and 1, combining the extraction liquid for 2 times after the extraction is finished, and evaporating the combined extraction liquid under reduced pressure to obtain the semi-solid protein extract.

(6) Pressure ultrafiltration: and (3) carrying out pressurized ultrafiltration treatment on the protein enzymolysis liquid, wherein the molecular interception is 3000Da under the pressure of 0.14MPa, so as to obtain the mussel antiallergic extract ultrafiltrate.

(8) And (3) drying treatment: spray drying the obtained concentrated solution, and ultraviolet sterilizing at 90 deg.C for 5min to obtain mussel extract powder S6 with antiallergic activity.

Example 7

(2) And (3) homogenizing treatment: the mixture was stirred (5000rpm/8min) to obtain a homogenate.

(3) Ultrahigh pressure treatment: adding 10 times volume of extraction solvent (0.02-0.05M phosphate buffer system water solution), soaking for 3 hr, extracting at normal temperature for 25min under 800MPa in an extraction container of ultrahigh pressure extraction device for 1 time, and collecting eluate to obtain primary extract.

(4) And (3) subcritical extraction process: placing the mussel primary extract in an extraction tank, vacuumizing (vacuum degree of 0.5MPa), adding a subcritical extraction solvent (volume ratio of water to methanol to ethanol is 7: 2: 1) and extracting for 5 times, wherein the total extraction time is 40 minutes, each time is the same, and each time temperature is 45 ℃; wherein the 2 nd-5 th extraction is to add an extraction solvent into the semi-solid (semi-solid) obtained in the last extraction, and the weight ratio of the extraction solvent to the initial extract and/or the semi-solid obtained after the last extraction is 10: and 1, combining the extraction solutions for 5 times after the extraction is finished, and evaporating the combined extraction solutions under reduced pressure to obtain a semi-solid extract.

(5) And (3) enzymolysis treatment: adding 10 times of water by mass into the protein extract to form an enzymolysis reaction system; adjusting the pH of the reaction with 0.1mol/L HCl or 0.2mol/L NaOH solution, adding pepsin, trypsin and subtilisin with a certain mass ratio (solid powdered enzyme: homogenate: 1%) of 20 ten thousand U/g, and performing enzymolysis at pH2, pH7.5, pH7, 37 deg.C, 45 deg.C and 40 deg.C for 30-90min, 60-120min and 60-180min, respectively.

(6) Pressure ultrafiltration: and (3) carrying out ultrafiltration treatment on the protein enzymolysis liquid under the pressure of 0.05MPa and the molecular interception amount of 3000Da to obtain the mussel antiallergic extract ultrafiltrate.

(8) And (3) drying treatment: spray drying the obtained concentrated solution, and ultraviolet sterilizing at 90 deg.C for 5min to obtain mussel extract powder S7 with antiallergic activity.

Example 8

This example is an antiallergic composition containing the anti-allergic active mussel extract powder S1 of example 1.

(1) Preparing an oil phase: weighing 10 parts by weight of white vaseline, 2 parts by weight of glycerol, 2 parts by weight of stearic acid, 2 parts by weight of octadecanol and 2 parts by weight of stearyl cetyl alcohol, adding the weighed materials into an oil phase pot, heating to 80 ℃, and uniformly stirring;

(2) preparing an aqueous phase: weighing 8 parts by weight of glycerol and 4 parts by weight of trehalose, adding the glycerol and the trehalose into a water phase pot, heating to 80 ℃, and uniformly stirring;

(3) mixing: mixing the oil phase and the water phase, reacting in an emulsifying pot for 15min under heat preservation, stirring until the mixture is uniformly dispersed, cooling to 55 ℃, continuing stirring under heat preservation for 5min, adding 10 parts by weight of mussel antiallergic extract (S1 prepared in example 1), 2 parts by weight of xanthan gum and 0.4 part by weight of salicylic acid (as antiseptic), stirring uniformly, standing and cooling to room temperature to obtain the composition containing the mussel extract with antiallergic activity.

Example 9 the operations and parameters of steps (1), (3) to (8) were the same as those of example 1.

With the difference that step (2): stirring (homogenizing) treatment (2000rpm/0.5min)

Finally obtaining mussel extract powder S9 with antiallergic activity.

Example 10 the operations and parameters of steps (1), (2), (4) to (8) were the same as those of example 1.

With the difference that step (3): the pressure was 100MPa, and the other operations and parameters were the same as those of example 1.

Finally obtaining mussel extract powder S10 with antiallergic activity.

Example 11 the operations and parameters of steps (1) - (3), (5) - (8) were the same as in example 1.

With the difference that step (4): the number of extractions was 1, and the other operations and parameters were the same as in example 1.

Finally obtaining mussel extract powder S11 with antiallergic activity.

Example 12 the operations and parameters of steps (1) - (4), (6) - (8) are the same as in example 1.

With the difference that step (5): in the enzymolysis treatment, the enzymolysis time of pepsin, trypsin and subtilisin is respectively as follows: 10min, 30min, 30min, and other operations and parameters were the same as in example 1.

Finally obtaining mussel extract powder S12 with antiallergic activity.

Example 13 the operations and parameters of steps (1) - (4), (6) - (8) are the same as those of example 1.

With the difference that step (5): in the enzymatic treatment, only pepsin was used as an enzyme, and the other operations and parameters were the same as in example 1.

Finally obtaining mussel extract powder S13 with antiallergic activity.

Example 14 the operations and parameters of steps (1) - (4), (6) - (8) are the same as in example 1.

With the difference that step (6): in the pressure ultrafiltration treatment, the pressure was 0.25MPa, and the operation and parameters were the same as those in example 1.

Finally obtaining mussel extract powder S14 with antiallergic activity.

Comparative example 1

Another method for preparing an anti-allergic extract of mussels, not involving the ultra-high pressure step, is to take a mixture of fresh mussels of the same batch as in examples 1-5.

Mussel pretreatment and homogenization treatment in the step (1) and the step (2): the parameters and operation were the same as in example 1.

And (3) subcritical extraction process: the homogenate was placed in an extraction tank for extraction, with the parameters and operation identical to those of example 1.

Performing enzymolysis treatment in the step (4), performing pressurized ultrafiltration in the step (5), performing vacuum concentration in the step (6), and performing drying treatment in the step (7) with the same parameters and operation as those in the step (1) to obtain mussel extract powder D1 with antiallergic activity.

Comparative example 2

Another method for preparing an anti-allergic extract of mussels, not including a subcritical extraction step, is to take a mixture of fresh mussels of the same batch as in examples 1-5.

Mussel pretreatment, homogenate treatment in the step (2) and ultrahigh pressure treatment in the step (3): the parameters and operation were the same as in example 1.

Step (4), enzymolysis treatment: and (3) adding 10 times of water by mass into the primary extract to form an enzymolysis reaction system, wherein the parameters and operation are the same as those in the embodiment 1.

The steps of (5) pressure ultrafiltration, (6) vacuum concentration and (7) drying treatment parameters and operation are the same as those in the embodiment 1, and the mussel extract powder D2 with antiallergic activity is obtained.

Comparative example 3

Another method for preparing an anti-allergic mussel extract, which does not involve ultra-high pressure and sub-critical extraction steps, is to take a mixture of fresh mussels of the same batch as in examples 1-5.

Step (3) enzymolysis treatment: water in an amount of 10 times the mass of the homogenate was added to form an enzymatic reaction system, and the parameters and operation were the same as those in example 1.

The steps of (4) pressure ultrafiltration, (5) vacuum concentration and (6) drying treatment parameters and operation are the same as those of the embodiment 1, and the mussel extract powder D3 with antiallergic activity is obtained.

Comparative example 4

The operation and parameters of steps (2) - (8) are the same as those of example 1, and the comparative example is to take fresh live mussels belonging to the same batch as examples 1-5.

With the difference that step (1): the adductor muscle of mussel was taken for manipulation.

Finally obtaining mussel extract powder D4 with antiallergic activity.

Detection example:

detection 1: and (3) content detection: the test method is that trichloroacetic acid is used as protein precipitant, protein after enzymolysis and peptide with longer peptide chain are precipitated, short chain small peptide is dissolved out by acid, and the protein quality is measured after centrifugation, filtration, digestion and distillation. The method is obtained by revising the light marine fish oligopeptide powder (QB/T2879-2007) of the people's republic of China.

The protein hydrolysate (including peptides and free amino acids) with lower molecular weight is soluble in trichloroacetic acid solution, and the high molecular weight protein is easy to precipitate in trichloroacetic acid solution. And (3) dissolving the sample by trichloroacetic acid, centrifuging to separate out a precipitate, and subtracting the content of free amino acid from the content of acid-soluble protein in clear liquid to obtain the content of oligopeptide.

The specific operation comprises the following steps: a sample (mussel extract prepared in each example and comparative example) 2.00g was taken, 10ml of 15 wt% TCA (trichloroacetic acid) was added, mixed well and allowed to stand for 5 min. The solution was quantitatively transferred, centrifuged at 4000rpm for 10min and the whole supernatant was taken. Soluble protein is measured according to the method GB/T5009.5, the protein conversion coefficient is 6.25, and free amino acid is measured according to the method GB/T5009.5. Each sample was run in triplicate and the average recorded. Oligopeptide content (%) — soluble protein content-free amino acid content.

The mussel antiallergic extract obtained in example 1 has an active small peptide content of 89.79 wt%;

the mussel antiallergic extract obtained in example 2 has an active small peptide content of 69.25 wt%;

the mussel antiallergic extract obtained in example 3 has an active small peptide content of 73.69 wt%;

the mussel antiallergic extract obtained in example 4 has an active small peptide content of 76.24 wt%;

the mussel antiallergic extract obtained in example 5 has an active small peptide content of 74.26 wt%;

the content of active small peptide in the mussel antiallergic extract obtained in examples 6 and 7 is 79.75 wt% and 69.75 wt%, respectively.

The content of the active extract of the mussel antiallergic peptide prepared in example 9 is 88.95 wt%;

the content of the active extract of the mussel antiallergic peptide prepared in example 10 is 69.03 wt%;

the content of the active extract of the mussel antiallergic peptide prepared in example 11 is 68.29 wt%;

the content of the active extract of the mussel antiallergic peptide prepared in example 12 is 67.92 wt%;

the content of the active extract of the mussel antiallergic peptide prepared in example 13 is 65.32 wt%;

the content of the active extract of the mussel antiallergic peptide prepared in example 14 is 75.12 wt%;

the content of the active extract of the mussel antiallergic peptide prepared in comparative example 1 is 55.12 wt%;

the content of the active extract of the mussel antiallergic peptide prepared in comparative example 2 is 60.23 wt%;

the content of the active extract of the mussel antiallergic peptide prepared in comparative example 3 is 41.25 wt%;

the content of the active extract of the mussel antiallergic peptide prepared in comparative example 4 was 9.12 wt%.

The results show that the preparation method of the mussel antiallergic extract can ensure that the extraction rate of the active small peptide is up to 89.79 wt%, which is obviously higher than that of the mussel extract prepared by the existing method, the broken chains of the small peptide are uniform, the molecular weight range is within 3KD, the quick absorption of skin epidermal cells is facilitated, the allergic symptoms are effectively relieved, and the application prospect is good.

And (3) detection 2: toxicology test of mussel antiallergic extract prepared by the above method

First, experiment method

1. Principle of experiment

The CAM method is commonly used in the detection of skin irritation. The stimulatory capacity of a test substance can be assessed using specific information presented by the CAM membrane or blood vessels, such as bleeding, angiolysis, coagulation, minor changes in small vessel diameter, and the like.

2. Experimental Material

Specific pathogen free grade "white henry" fertilized chicken eggs were purchased from Zhejiang poultry breeding, Inc.; fluorescein sodium (analytically pure) was purchased from chinese reagent company; silicone rubber ring (diameter 10m m, Zhejiang province disease prevention and control center).

3. Statistical analysis

Data results were statistically analyzed.

4. The experimental method comprises the following steps:

the incubation period of the fertilized hen eggs is carried out under the conventional temperature and humidity conditions, and the number of the experimental hen embryos of each group is respectively 15. On the chorioallantoic membrane of each chick embryo, 1 ring of silicone rubber was placed, to which 35mg of the sample (powder) was added. The skylight was sealed with clear adhesive and placed in a (37.5 + -0.5) deg.C incubator. After loading 40min, the chick embryos were removed from the incubator and the transparent gel and egg shells around the skylights were removed for viewing. Observing the conditions of silicone rubber ring and chick embryo chorioallantoic membrane with magnifying lens lamp, removing chick embryo with abnormal condition outside ring, and calculating RC₅₀The values, experimental results are shown in table 1.

Table 1 skin irritation test results for the products prepared

Wherein, the mussel mixed liquid (namely the 'fresh mussel mixture' in the step (1)) belonging to the same batch as the mussel mixed liquid in the examples 1-5 is taken, and the other experimental conditions are kept consistent with the examples (see the comparative example 1) without ultrahigh pressure treatment, so as to obtain the mussel active peptide powder D1.

Note: in CAM VA experimental fractionation, RC₅₀Less than 1.0%, judging that the chemical is light stimulation or above, and setting as 'I'; RC (resistor-capacitor) capacitor₅₀1.0-20.0%, judging the chemical substance to be micro-stimulation, and setting as 'II'; RC (resistor-capacitor) capacitor₅₀More than 20.0 percent, and judging that the chemical is non-irritantAnd is set to "III".

The experimental results show that the mussel antiallergic extract prepared by the various methods has no irritation to the skin, and is a natural, safe and efficient product.

And (3) detection: antiallergic effect experiment of mussel peptide

Firstly, experimental reagents and materials: kunming mouse, provided by Beijing Wittiulihua laboratory animal technology Co.

The license number is SCXK (Jing) 2002-; guinea pig, license number SCXK (Jing), 2002-: sodium cromoglycate, dexamethasone acetate and the like.

Second, Experimental methods

(1) Mice were tested for allogenic passive cutaneous hypersensitivity (type i allergy): mice were pretreated to be in a sensitized state. The preparation method of the mouse allergy model comprises the following steps: adding 1mL of fresh egg white into 9mL of physiological saline to prepare 10% egg white liquid; taking about 10 healthy mice of about 30g, injecting 0.1 mL/foot of 10% egg white liquid at two back soles of the mice, and simultaneously injecting 0.1 mL/mouse of aluminum hydroxide gel in the abdominal cavity; 12d after sensitization, blood is taken from the orbit and centrifuged at low speed (2000 rpm); 0.5mL of mouse antiserum is taken and added with 2.5mL of physiological saline to prepare antiserum diluent.

(2) 30 healthy mice were divided into 3 groups by weight, namely a blank control group (C), a mussel antiallergic extract group (S1) and a cromolyn sodium group positive control group (D), and 10 mice in each group were selected. Each group was administered by continuous gavage for 7d 1 time daily, at doses given in table 2. After the first administration, antiserum diluent is required to be injected into abdominal wall skin of each mouse, the dose is 0.06mL, and after the last administration, evans blue egg white staining solution (0.5%) is injected into abdominal wall skin of each group intravenously, the mice are killed, the blue staining skin soaking solution is taken, and the optical density value is measured at 610nm by using a spectrophotometer. The results are shown in Table 2.

TABLE 2 Effect of mussel antiallergic extract on passive cutaneous anaphylaxis of the same mouse species

Note that compared with the control group,^*P<0.05。

the experimental result shows that compared with a control group, the mussel antiallergic extract has obvious inhibition effect on the same passive skin allergic reaction of mice.

(3) Experiment on cutaneous vasculitic response to guinea pig Forssman (type ii allergy): a certain amount of Sheep Red Blood Cell (SRBC) resisting serum is prepared, 30 healthy mice are taken and randomly divided into 3 groups according to weight, wherein the 3 groups are respectively a blank control group (C), a mussel antiallergic extract group (S1) and a cromolyn sodium group positive control group (D), 10 mice are in each group, and the back of the mice is shaved 24 hours before the experiment. Each group was gavaged 1 time at the doses shown in table 3, and each guinea pig was post-dosed with an antiserum dilution (previously described anti-sheep red blood cell serum) intradermally injected into the back of the mouse at a dose of 0.2mL, followed by intravenous injection of 0.5% evans blue staining solution, after which the mice were sacrificed and the blue-stained skin soak solution was removed and the optical density value was measured at 610nm using a spectrophotometer. The results are shown in Table 3.

TABLE 3 Effect of mussel antiallergic extracts on cutaneous vasculitic reaction in guinea pigs Forssman

Note that compared with the control group,^*P<0.05,^**P<0.01。

the experimental result shows that compared with a control group, the mussel antiallergic extract group has obvious inhibition effect on the skin vasculitis reaction of the Forssman of the guinea pig.

(3) (type IV allergy) test on delayed type allergy to 2, 4-dinitrochlorobenzene in mice

40 healthy mice were divided into 4 groups by weight, namely a blank control group (C), a model control group, a mussel anti-allergic extract group (S1) and a dexamethasone acetate positive control group (D), and 10 mice in each group. Each group was administered 1 time per day with continuous gavage for 5 days, with distilled water for the blank control group and the model control group at doses given in table 4, and dexamethasone acetate administered 1 time the day of the experiment. Except for the model control group, the abdomen of each group of mice was shaved and a certain amount (thin coating uniformly) of 1% 2, 4-dinitrochlorobenzene solution was applied from the 1 st day of gavage, and the next day was again applied for strengthening, for a total of 2 applications. On the 5 th day after sensitization (30 min after the last administration of all groups), 0.01mL of 2, 4-dinitrochlorobenzene solution was applied to the right ear of the mouse, the mouse was sacrificed by dislocation, the two ears were cut along the base line of the auricle, and the same part of each ear was cut off to obtain the mass. The difference in the quality of the left and right ear was used as the degree of swelling, and the results are shown in Table 4.

TABLE 4 Effect of mussel antiallergic extract on delayed allergy to 2, 4-dinitrochlorobenzene (ear swelling in mice)

The experimental result shows that compared with a control group, the mussel antiallergic extract group has obvious inhibition effect on the delayed allergic otitis media of mice caused by 2, 4-dinitrochlorobenzene.

(4) Mouse ear swelling experiment 30 healthy mice purchased from Beijing vitamin Tonglihua laboratory animal technology, Inc. were randomly divided into 3 groups by weight, which were a blank control group (C), a mussel antiallergic extract group (S1), and a compound dexamethasone acetate cream (dermatipide) positive control group (D), 10 mice per group. The application program of the mussel antiallergic extract group (S1) is as follows: smearing mussel antiallergic extract 0.2g/kg, and the positive control group medication program is as follows: the liniment comprises dermatipidine (999: dermatipidine, compound dexamethasone acetate cream, and Sanjiu medicine) 10.0mg/kg, and the administration volume is 20 mL/kg. After 3 days of administration, each group of mice was coated with xylene-induced inflammation (0.02 mL/mouse) on the front and back sides of the right ear, and the test drugs were applied to the right ear at 0h, 0.5h, 1h, 1.5h, and 2h, respectively, with the same dose of the drug. And (4) after 4h, dislocating the cervical vertebra of the mouse, cutting off two ears, punching round ear pieces at the same parts of the left ear and the right ear by using an 8mm puncher, weighing on an analytical balance, taking the weight difference of the two ear pieces as the swelling degree of inflammation, and calculating the swelling inhibition rate.

The experimental results are as follows:

TABLE 5 Effect of mussel antiallergic extract on xylene-induced swelling of mouse pinna

As can be seen from Table 5, the degree of ear swelling in mice in both experimental groups was compared with that in the blank control group: the difference between the two experimental groups has no significant significance, and both the two experimental groups have significant anti-inflammatory efficacy.

In conclusion, the mussel antiallergic extract has certain treatment effect on I, II and type III anaphylaxis. And the ointment has certain anti-inflammatory and detumescence effects, and really has excellent effects on relieving skin pruritus and skin allergy symptoms.

And (4) testing: antiallergic composition comprising mussel antiallergic extract as effective component and used in cosmetic

Experimental groups: the composition containing mussel antiallergic extract prepared in example 8. Control group:

the mussel antiallergic extract is absent, and other raw materials and procedures are consistent.

Experimental subject: selecting 100 patients with skin allergy history such as urticaria and the like, wherein the age of the 100 patients is 15-25 years, and 50 patients are male students; the test group was divided into 50 individuals and a control group of 50 individuals at random according to sex, and each group had 25 boys and 25 girls.

③ the experimental effect: after the test group and the control group were applied with the cream prepared above every day for 10 days (2 times a day, the application dose was about 0.1ml per square centimeter of skin), the test groups were exposed to the allergens, respectively, and the test results were as follows:

in the experimental group, 12 people had allergic symptoms such as pruritus and rash, 7 people had allergic reactions such as slight rash, and 31 people had no allergic reactions. The effective rate of antianaphylaxis is 62%.

While 27 persons in the control group developed severe allergic symptoms and 12 persons had mild allergic reactions. The effective rate of antianaphylaxis is 0%.

The results show that the mussel antiallergic extract is feasible to be applied to the preparation of cosmetics as an antiallergic active ingredient.

And (5) testing: antioxidant activity test of mussel antiallergic extract

By H₂O₂Inducing oxidative damage of epidermal cells, observing the protective effect of the mussel antiallergic extract on the skin cell ultrastructure by adopting a transmission electron microscope, and discussing the mechanism and influence of the mussel antiallergic extract on the antioxidant capacity of the skin epidermal cells.

The experimental method comprises the following steps:

(1) isolation of epidermal cells of experimental animals: taking a Wistar rat suckling mouse to obtain breast skin epidermal cells; adding trypsin solution, digesting in a constant-temperature water bath kettle at 37 ℃ for 10min by shaking, standing for precipitation, and removing supernatant; the trypsin solution was added again, digestion was repeated until the debris was completely digested, and finally D-Hank's solution was added to stop digestion. 1000rpm min^-1Repeatedly centrifuging and cleaning, removing supernatant, and making final precipitate into cell suspension.

(2) Cell inoculation: subpackaging the prepared cell suspension into culture bottles, adding 10mL of cell culture solution containing 15% calf serum and antibiotics, mixing, covering with bottle cap, and adding CO at 37 deg.C₂Culturing in an incubator. After 1.5 hours, the flask was removed and the cell culture broth was placed in another flask and continued to be CO at 37 ℃₂Culturing in an incubator. After 24 hours, the culture medium was changed to remove nonadherent cells. After 72 hours, those with good growth were selected for the experiment and the beating of single cells or monolayers was visualized by inverted microscopy.

(3) Grouping experiments:

taking epidermal cells with good growth state, and culturing at a ratio of 1 × 10⁶The concentration of cells/mL was randomly divided into 3 groups of 6 flasks of cells each, 3 groups were:

normal group: no medicine is added;

H₂O₂group (2): adding H into the culture solution₂O₂To a final concentration of 100. mu. mol. L^-1Continuously culturing for 3 h;

experimental groups: adding into culture solutionMussel antiallergic extract to give final concentration of 10 mg.L^-1Pre-cultured for 12H, and then treated with the same H₂O₂And (4) grouping.

(4) Imaging by an electron microscope:

culturing the above 3 groups at 1000 r.min^-1Centrifuging for 5min, collecting cells by discarding supernatant, fixing with 2.5% glutaraldehyde aqueous solution at 4 deg.C for 10min, 2000 r.min^-1Centrifuging for 10min, discarding the supernatant, collecting precipitate, dehydrating with ethanol (analytically pure, greater than or equal to 95%) and acetone (analytically pure, greater than or equal to 99.5%), embedding with epoxy resin, making ultrathin section, staining with uranium acetate-lead citrate, observing with transmission electron microscope, and taking photograph.

As shown in FIGS. 1-3, from the above transmission electron microscopy, it can be seen visually that the mussel antiallergic extract has been shown to be responsible for H₂O₂Recovery of epidermal cells of the skin during injury. The normal group of skin epidermal cells has clear ultrastructure, and the arrangement of the mitochondrial ridges is regular without obvious damage. H₂O₂The group was seen to have mitochondrial swelling, broken cristae, and massive vacuoles in the cytoplasm. The experimental group shows that the skin epidermal cell morphology is already normal under the protection of the mussel antiallergic extract.

In conclusion, the mussel antiallergic extract prepared by the research has good biocompatibility, has remarkable sterilizing and anti-inflammatory effects, and can effectively improve allergic symptoms of skin. In addition, various active ingredients contained in the mussel antiallergic extract can effectively promote the skin barrier repair function and accelerate skin metabolism, and can better play roles in resisting oxidation, promoting wound healing and the like. Therefore, the mussel antiallergic extract has wide application prospect and great commercial value when being used as an effective component in the preparation of cosmetics.

Claims

1. A preparation method of a shellfish antiallergic extract is characterized by comprising the following steps: the method comprises the following steps:

a pretreatment step: taking the useful part of the fresh and live shellfish, and putting the useful part into the pretreatment liquid to obtain a fresh and live shellfish mixture;

and (3) homogenizing: homogenizing the mixture of the fresh and live shellfish to obtain homogenate;

ultrahigh pressure extraction: mixing the homogenate with an extraction solvent for soaking treatment, and performing ultrahigh pressure extraction treatment to obtain an initial extract;

a subcritical extraction step: performing subcritical extraction treatment on the primary extract to obtain a protein extract;

an enzymolysis step: carrying out enzymolysis treatment, enzyme deactivation treatment and centrifugal treatment on the protein extract to obtain shellfish protein hydrolysate;

an ultrafiltration step: and carrying out ultrafiltration treatment on the shellfish protein enzymolysis liquid to obtain the shellfish antiallergic extract ultrafiltrate.

2. The method of claim 1, wherein:

the pretreatment solution is a vitamin C aqueous solution;

preferably, in the pretreatment step, the concentration of the vitamin C aqueous solution is 0.1-0.5% by mass, and preferably 0.2%;

preferably, the mass ratio of the useful part to the vitamin C aqueous solution is 1: (1-2), preferably 1: 1;

preferably, the useful part is a mantle part;

preferably, the shellfish is mussel.

3. The method of claim 1, wherein:

in the homogenizing step, the stirring rate of the homogenizing treatment is 2000-10000rpm, preferably 8000rpm, and the time is 0.5-10min, preferably 5 min.

4. The method of claim 1, wherein:

in the ultrahigh pressure extraction step, the extraction solvent is one or two of an aqueous solution and an alcoholic solution of a phosphate buffer system;

preferably, the concentration of the aqueous solution of the phosphate buffer system is 0.02-0.02M, and the alcoholic solution is 70-80% by volume of ethanol aqueous solution.

5. The production method according to claim 1 or 4, characterized in that:

in the ultrahigh pressure extraction step, the soaking treatment time is 1 to 3 hours.

6. The production method according to claim 1 or 4, characterized in that:

in the ultrahigh pressure step, in the ultrahigh pressure treatment: extracting at 100-800MPa for 1-5 times (15-25 min each time),

the volume of the extraction solvent is 6-18 times, preferably 10 times of the volume of the homogenate or the residue of the last extraction.

7. The method of claim 1, wherein:

in the subcritical extraction step, the subcritical extraction treatment includes: placing the primary extract in an extraction tank, vacuumizing, and adding a subcritical extraction solvent into the primary extract to extract for 1-5 times; the mass ratio of the extraction solvent to the initial extract or the semi-solid extracted last time is (3-15): 1, preferably 10: 1;

preferably, the total time of the subcritical extraction treatment is 40-120 minutes, the temperature of each time is 10-50 ℃, and preferably 40-50 ℃;

preferably, the subcritical extractant comprises: water, methanol and ethanol, wherein the volume ratio of the components is (5-10): (1-3): 1, preferably 7: 2: 1.

8. the method of claim 1, wherein:

in the enzymolysis step, in the enzymolysis treatment, 5-15 times, preferably 10 times of water by mass is added into the protein extract to form an enzymolysis reaction system; the mass ratio of the dry enzyme weight to the enzymolysis reaction system is 1-5%, and the total enzymolysis time is 1-7 h;

preferably, the enzyme is an enzyme preparation of 10-30 ten thousand U/g, preferably 20 ten thousand U/g;

preferably, the enzyme comprises one or more of pepsin, trypsin and subtilisin;

more preferably, the enzymolysis condition of the pepsin is pH 2-4 and 25-40 ℃, the enzymolysis condition of the trypsin is pH 7.5-9.0 and 37-45 ℃, and the enzymolysis condition of the subtilisin is pH 7-8 and 37-50 ℃; the enzymolysis time of the pepsin is 10-90min, the enzymolysis time of the trypsin is 30-120min, and the enzymolysis time of the subtilisin is 30-180 min.

9. The production method according to claim 1 or 8, characterized in that:

in the enzymolysis step, the inactivation treatment is carried out at 90-100 ℃ for 5-10 min.

10. The production method according to claim 1 or 8, characterized in that:

in the enzymolysis step, the rotation speed of the centrifugal treatment is 8000-10000rpm, and the time is 5-15 min.

11. The method of claim 1, wherein:

in the ultrafiltration step, the pressure is 0.02-0.25MPa, preferably 0.12MPa in the ultrafiltration treatment;

preferably, the ultrafiltration treatment employs an ultrafiltration membrane having a molecular cut-off of 3000 Da.

12. The method of claim 1, wherein: the preparation method also comprises the following steps:

vacuum concentration step: carrying out vacuum concentration treatment on the shellfish antiallergic extract ultrafiltrate to obtain a shellfish antiallergic extract concentrated solution; the weight percentage of the shellfish antiallergic extract concentrated solution to the shellfish antiallergic extract ultrafiltrate is 7-13 wt%.

13. The method of claim 12, wherein: the preparation method also comprises the following steps:

and (3) drying: and (3) carrying out spray drying treatment and sterilization treatment on the shellfish antiallergic extract liquid to obtain shellfish extract powder with antiallergic activity.

14. Use of shellfish antiallergic extract obtained by the preparation method according to any of claims 1-13 in the preparation of drugs, skin care products or/and cosmetics with antiallergic, antioxidant or skin barrier function repair promoting effects.

15. Use according to claim 14, characterized in that: the minimum effective concentration of the shellfish antiallergic extract is 5-12 g/mL.

16. Use according to claim 14, characterized in that: the shellfish antiallergic extract accounts for 5-15 wt% of the skin care product or/and the cosmetic.

17. Use according to any one of claims 14-16, wherein: the skin care product or/and the cosmetic preparation comprises the following components in parts by weight: creams, lotions, lipsticks, powders, gels, sprays and masks.

18. A shellfish antiallergic extract prepared by the preparation method according to any one of claims 1 to 13.

19. A composition comprising, as starting materials: the shellfish antiallergic extract of claim 18, together with a pharmaceutically acceptable excipient.

20. The composition of claim 19, wherein: the medicinal auxiliary materials comprise an oil phase material, a water phase material, medicinal polysaccharide and a preservative;

preferably, the shellfish antiallergic extract, the oil phase material, the water phase material, the medicinal polysaccharide and the preservative are used in the following weight ratio: (6-14) (12-24) (9-15) (1-3) (0-1), more preferably 10:18:12:2: 1;

preferably, the oil phase material is white vaseline, glycerol, stearic acid, stearyl alcohol and stearyl cetyl alcohol; more preferably, the white vaseline, the glycerol, the stearic acid, the stearyl alcohol and the stearyl cetyl alcohol are used in the following weight ratio: (5-10): (1-2): (1-2): (1-2): (1-2), more preferably 5:1:1:1: 1;

preferably, the water phase material is glycerol and trehalose, and more preferably, the glycerol and the trehalose are used in the following weight ratio: (1-3): 1, more preferably, 2: 1;

preferably, the medicinal polysaccharide is one or a mixture of xanthan gum, agar, arabic gum and chitin;

21. A method of making a composition by preparing the composition from the materials of the composition of claim 20, comprising the steps of:

s1: preparing an oil phase and an aqueous phase simultaneously or sequentially;

the steps for preparing the oil phase are as follows: mixing all the oil phase materials, and heating to obtain an oil phase;

the steps for preparing the aqueous phase are: mixing all the water phase materials, and heating to obtain a water phase;

s2: mixing the oil phase and the aqueous phase to obtain a homogeneous mixture;

s3: mixing the homogeneous mixture, the shellfish antiallergic extract, the pharmaceutical polysaccharide, and the preservative to obtain the composition;

preferably, in step S1: in the step of preparing the oil phase, heating to 75-85 ℃, preferably 80 ℃;

more preferably, in step S1: in the step of preparing the aqueous phase, the aqueous phase is heated to 75-85 ℃, preferably 80 ℃;

more preferably, in step S2: mixing the oil phase and the water phase at 75-85 ℃ for 10-20min, and cooling to 50-60 ℃ to obtain the homogeneous mixture; more preferably, in step S2: and mixing the oil phase and the water phase at 80 ℃ for 15min, and cooling to 55 ℃ to obtain the homogeneous mixture.