CN117414319A

CN117414319A - Nanometer eye cream of Aronia melanocarpa extract fermented by lactobacillus plantarum and preparation method thereof

Info

Publication number: CN117414319A
Application number: CN202311389706.7A
Authority: CN
Inventors: 王家明; 赵春然; 郑加栋; 张红梅; 王颖卓
Original assignee: Xinyi Tianjin Biotechnology Co ltd; Tianjin University of Science and Technology
Current assignee: Xinyi Tianjin Biotechnology Co ltd; Tianjin University of Science and Technology
Priority date: 2023-10-25
Filing date: 2023-10-25
Publication date: 2024-01-19

Abstract

The invention belongs to the technical field of cosmetics, and discloses a nano eye cream of a Aronia melanocarpa extract after fermentation of lactobacillus plantarum, wherein the lactobacillus plantarum is lactobacillus plantarum OPB15, and the name of the lactobacillus plantarum is: OPB15, class name: lactobacillus plantarum (Lactobacillus), accession number: CGMCC No.27735, date of preservation: 2023, 6, 29, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No. 1, 3, the Korean region of Beijing, and the China general microbiological culture Collection center. The invention applies the active ingredients of the Aronia melanocarpa after fermentation to the design of skin care products such as eye cream and the like, mixes and encapsulates the active substances of the Aronia melanocarpa and the chamomile extract together by using a liposome technology, optimizes and screens the technological parameters of the liposome nanotechnology, and exerts the skin care effect to the greatest extent.

Description

Nanometer eye cream of Aronia melanocarpa extract fermented by lactobacillus plantarum and preparation method thereof

Technical Field

The invention belongs to the technical field of cosmetics, and particularly relates to a nano eye cream of a Aronia melanocarpa extract after lactobacillus plantarum fermentation and a preparation method thereof.

Background

The eye cream is a skin care cosmetic product for periocular skin, has the effects of improving skin moisturizing and barrier functions, and sometimes has the effects of tightening eye skin, relieving dark circles, improving wrinkles and fine lines according to the addition of functional components and active substances of different products.

The eye skin of a person is weak and is more vulnerable to external environment, and is one of the most susceptible parts to aging. The aging is characterized by a series of phenomena such as skin thickness reduction, moisture content reduction, skin relaxation, fine wrinkles increase, black eye circles, skin pigmentation around eyes, darkness and the like on the skin of eyes, and the reasons are that the antioxidant capacity of the skin is reduced, lipofuscin accumulation, collagen synthesis capacity is reduced, hyaluronic acid content is reduced, skin moisture content is reduced and the like.

Aronia melanocarpa (academic name: aronia melanocarpa (Michx.) Elliott) has antioxidant, antitumor, antiinflammatory, antibacterial, etc. physiological activities, and flavone, anthocyanin, and polyphenol in fruit are the highest in known plants, and exceed those of blueberry, etc., and have high polysaccharide content. The Aronia melanocarpa extract has special effect in treating cancer, diabetes, cardiovascular and cerebrovascular diseases, and can improve immunity, regulate blood sugar and blood lipid, improve liver function, and prevent and treat cardiovascular diseases.

The prior eye cream with functions of repairing eye skin, resisting wrinkles, resisting oxidization and the like has no obvious common effect, takes effect after long-time use on the functions of tightening, resisting wrinkles, repairing and the like, is sensitive and fragile to the eye skin, and is easy to cause skin allergy and skin discomfort due to a large amount of chemical substance raw materials contained in the eye cream.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a Aronia melanocarpa extract nanometer eye cream fermented by lactobacillus plantarum and a preparation method thereof.

The technical scheme adopted for solving the technical problems is as follows:

the lactobacillus plantarum is lactobacillus plantarum OPB15, and the name of the lactobacillus plantarum is that of the lactobacillus plantarum is Aronia melanocarpa extract nanometer eye cream after lactobacillus plantarum fermentation: OPB15, class name: lactobacillus plantarum (Lactobacillus plantarum), accession number: CGMCC No.27735, date of preservation: 2023, 6, 29, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No. 1, 3, the Korean region of Beijing, and the China general microbiological culture Collection center.

The preparation method of the Aronia melanocarpa extract nanometer eye cream comprises the following steps of:

(1) Mixing active polypeptide, ginsenoside, flos Matricariae Chamomillae extract, aronia melanocarpa polysaccharide, and fermented Aronia melanocarpa extract with camel milk liposome under stirring, incubating at 35+ -1deg.C for 10+ -1 min, and continuously stirring; spin-drying water to form a honeycomb membrane, adding puer tea extract, dissolving the honeycomb membrane, and performing ultrasonic treatment for 10 minutes under the pressure of 2500psi to obtain camel milk liposome encapsulating active ingredients; heating camel milk liposome encapsulated with active ingredients to 70+/-1 ℃;

wherein the active polypeptide: ginsenoside: chamomile extract: aronia melanocarpa polysaccharide: and (3) fermenting the Aronia melanocarpa extract: camel milk liposome: the mass ratio of the puer tea extract is 1:1:1:2:3:8:1, a step of;

(2) Adding acacia and sodium alginate into water, stirring to obtain gel matrix, adding active ingredient-entrapped camel milk liposome into the gel matrix at 70+ -1deg.C, stirring to uniformity, and adding vitamin C and vitamin E; continuously stirring until the mixture is uniform, and preserving the temperature for 10+/-1 minutes to obtain a mixture;

wherein, acacia: sodium alginate: camel milk liposome encapsulating active ingredients: vitamin C: the mass ratio of the vitamin E is 1:1:4:1:1, a step of;

(3) Cooling the mixture to 30-45 ℃, adding ultrapure water into hyaluronic acid, trehalose, tea tree essential oil and clove essential oil, mixing, stirring uniformly, adding into the mixture prepared in the step (2), shaking uniformly, standing until no precipitate exists, adding ultrapure water and acacia gum, and regulating the viscosity of eye cream, thus obtaining the fermented black chokeberry extract nanometer eye cream;

wherein, hyaluronic acid: trehalose: tea tree essential oil: butyl essential oil: ultrapure water added for the first time: the mass ratio of the mixture prepared in the step (2) is 0.8:1:1:1:5:10.

further, the preparation method of the puer tea extract in the step (1) comprises the following steps:

weighing puer tea, crushing, and drying until the quality is unchanged, wherein the liquid-to-material ratio is 1:30 adding ultrapure water, heating and refluxing at 80 ℃ for 1.5 hours, cooling, vacuum filtering to obtain filtrate, repeatedly extracting filter residues for three times, combining the filtrates, pre-freezing, freeze-drying in a freeze dryer, and freeze-drying into powder after 12 hours to obtain the puer tea extract.

Further, the preparation method of the chamomile extract in the step (1) comprises the following steps:

weighing stems and leaves of chamomile, drying until the mass is no longer changed, crushing into powder, and mixing according to a liquid-to-material ratio of 1:15 adding ultrapure water, heating and refluxing for 3 hours at 85 ℃, cooling, vacuum filtering to obtain a filtrate, repeatedly extracting filter residues for three times, combining the filtrates, pre-freezing, freeze-drying in a freeze dryer, and freeze-drying into powder after 12 hours to obtain the chamomile extract.

Further, the preparation method of the ginsenoside in the step (1) comprises the following steps:

weighing ginseng, crushing, and drying until the quality is unchanged, wherein the liquid-to-material ratio is 1:10 adding ultrapure water, heating and refluxing at 77 ℃ for 3 hours, cooling, vacuum filtering to obtain filtrate, repeatedly extracting filter residues for three times, combining the filtrates, pre-freezing, and then freeze-drying in a freeze dryer for 12 hours to obtain ginsenoside dry powder, thus obtaining the ginsenoside extract.

Further, the preparation method of the Aronia melanocarpa polysaccharide in the step (1) comprises the following steps:

1) Taking Aronia melanocarpa, placing in a filter paper bucket, folding the opening of the filter paper bucket, sealing, placing in a Soxhlet extractor, extracting with diethyl ether at 54 deg.C until the diethyl ether is clear, taking out the filter paper wrapped with black fruit, and placing in a fume hood to volatilize the residual diethyl ether;

2) Extracting black fruit with water at 77 deg.C for 90 min, precipitating with ethanol to obtain filtrate, extracting the residue once again, mixing the filtrates, and filtering to obtain clear liquid; concentrating, namely concentrating the Aronia melanocarpa to 10ml per 15g, and precipitating the Aronia melanocarpa with 92% ethanol overnight; centrifuging at 7000rpm for 10 min, removing supernatant, washing precipitate with diethyl ether and ethanol for 2 times, and dissolving with hot water after volatilizing solvent;

3) After the addition of sevage reagent (chloroform: the volume ratio of n-butanol is 4: 1) Deproteinizing, shaking for 30min, standing for separating protein, removing organic phase and protein between water phase and organic phase for 5-6 times; dialyzing with dialysis bag (MD 44-10000) for 12 hr, and lyophilizing to obtain Aronia melanocarpa polysaccharide.

Further, the preparation method of the Aronia melanocarpa extract after fermentation in the step (1) comprises the following steps:

taking Aronia melanocarpa fruits and soybeans, crushing, drying until the fruits are unchanged, sterilizing, adding sterile water to be sticky, adding lactobacillus plantarum OPB15 bacteria powder, shaking uniformly, culturing in a constant-temperature incubator for 48 hours, taking out, soaking with 5-10 times of ethanol, performing ultrasonic extraction, centrifuging, filtering to obtain filtrate, cooling to room temperature, repeatedly extracting filter residues with ethanol for three times, mixing the filtrates, pouring the filtrate into a round-bottom flask, adding 5-10 times of ethanol, shaking uniformly, filtering with a 0.45um filter membrane, and taking the filtrate to obtain the Aronia melanocarpa fruit-soybean beverage;

wherein, the Aronia melanocarpa fruit: and (3) soybean: the mass ratio of the lactobacillus plantarum OPB15 bacterial powder is 1:0.01:5.

further, the preparation method of the camel milk liposome in the step (1) comprises the following steps:

firstly, incubating camel milk at 35+/-1 ℃ for 10+/-1 min, continuously stirring, spin-drying water by a film dispersion method to form a honeycomb film, and performing ultrasonic treatment for 10+/-1 min under the pressure of 2500psi to prepare the camel milk liposome.

Further, the preparation method of the active polypeptide in the step (1) is as follows:

crushing hickory, wheat, peas, pollen pini and peach kernels, adding water, adjusting the pH value to be slightly alkaline pH=8.7+/-0.5, extracting protein by an alkali-dissolution acid precipitation method, freeze-drying, centrifuging at 8000rpm for 10 minutes after protease enzymolysis, and freeze-drying supernatant to obtain the active polypeptide extract.

Further, the tea tree essential oil and the clove essential oil have synergistic preservative effect, and are plant source preservatives.

The invention has the advantages and positive effects that:

1. the invention applies the active ingredients of the Aronia melanocarpa after fermentation to the design of skin care products such as eye cream and the like, mixes and encapsulates the active substances of the Aronia melanocarpa and the chamomile extract together by using a liposome technology, optimizes and screens the technological parameters of the liposome nanotechnology, and exerts the skin care effect to the greatest extent.

2. The invention applies the Aronia melanocarpa polysaccharide to the cosmetics for the first time to improve the antioxidation effect of the cosmetics, and the lactobacillus plantarum OPB15 is used for fermenting the products for the first time, and the fermenting products have stable and good effects, can enhance the antioxidation function and have excellent skin feel.

3. The Aronia melanocarpa polysaccharide used in the invention has the function of enhancing antioxidation. The Aronia melanocarpa polysaccharide is also applied to cosmetics for the first time.

4. The Aronia melanocarpa used by the invention has the effects of resisting oxidation, resisting aging, resisting radiation, relieving visual fatigue and the like. And organic acid generated by lactobacillus plantarum OPB15 used for fermentation in the fermentation process can prevent degradation of phenolic substances, so that the total phenolic content is increased. The acidic environment caused by the generated organic acid of the fermentation is favorable for stabilizing the phenolic substances. During fermentation, certain enzymes are produced, and polysaccharide and protein on the cell wall of the Sorbus nigra are hydrolyzed, so that phenolic substances combined with the polysaccharide and protein are released. Increasing its antioxidant capacity.

5. The eye cream has the effects of improving skin moisture, barrier function, tightening eye skin, relieving dark circles, improving wrinkles and fine lines, reducing edema and keeping skin fresh.

6. The eye cream of the invention uses liposomes as vehicles. Liposomes (lipomes) are miniature spherical carriers formed by encapsulating an active ingredient within a lipid bilayer, the soluble ingredient may be encapsulated in the inner aqueous phase of a phospholipid, and the lipid-soluble ingredient may be encapsulated between phospholipid bilayers. The liposome is a typical representative of a nano drug-carrying system, has passive targeting, protects the drug from being destroyed, increases the stability and the solubility of insoluble drugs, can control the release rate of the drug, prolongs the circulation time of the liposome in the body, and improves the curative effect of the drug. Is friendly to the sensitive people of the skin, and the finished product has no harmful chemical substances.

Drawings

FIG. 1 is a chromatogram of a mixed monosaccharide control of the present invention;

FIG. 2 is a chromatogram of the polysaccharide hydrolysis monosaccharide of Aronia melanocarpa according to the present invention;

FIG. 3 shows NaIO of different concentrations in the present invention ₄ The standard curve is prepared;

FIG. 4 is a gas chromatogram of the present invention after standard derivatization;

FIG. 5 is a gas chromatogram of the invention after sample degradation derivatization;

FIG. 6 shows the polysaccharide of Aronia melanocarpa in the present invention ¹ H NMR spectrum;

FIG. 7 shows the polysaccharide of Aronia melanocarpa in the present invention ¹³ C NMR spectrum;

FIG. 8 is a Fourier infrared plot of Aronia melanocarpa polysaccharide of the present invention;

FIG. 9 is a graph showing the molecular weight standards of the present invention for different molecular weight standards;

FIG. 10 is a differential chromatogram of Aronia melanocarpa polysaccharide of the present invention;

FIG. 11 is a solid state fermentation diagram of Aronia melanocarpa fruit fermented by Lactobacillus plantarum OPB15 according to the invention;

FIG. 12 shows ultraviolet and mass spectra (+) of unfermented Aronia melanocarpa;

FIG. 13 shows ultraviolet and mass spectra (-) of unfermented Aronia melanocarpa;

FIG. 14 shows the ultraviolet and mass spectrum (+) of Aronia melanocarpa fermented by Lactobacillus plantarum OPB15 according to the invention;

FIG. 15 is a graph showing the ultraviolet and mass spectra (-) of Aronia melanocarpa fermented by Lactobacillus plantarum OPB15 according to the invention;

FIG. 16 is a colony morphology of Lactobacillus plantarum OPB15 according to the invention;

FIG. 17 is a microscopic photograph of Lactobacillus plantarum OPB15 according to the invention;

FIG. 18 is a graph of BLAST results versus tree for the Lactobacillus plantarum OPB15 genomic sequence of the invention.

Detailed Description

The invention will now be further illustrated by reference to the following examples, which are intended to be illustrative, not limiting, and are not intended to limit the scope of the invention.

The various experimental operations involved in the specific embodiments are conventional in the art, and are not specifically noted herein, and may be implemented by those skilled in the art with reference to various general specifications, technical literature or related specifications, manuals, etc. before the filing date of the present invention.

preferably, the preparation method of the puer tea extract in the step (1) comprises the following steps:

Preferably, the preparation method of the chamomile extract in the step (1) comprises the following steps:

Preferably, the preparation method of the ginsenoside in the step (1) comprises the following steps:

Preferably, the preparation method of the Aronia melanocarpa polysaccharide in the step (1) comprises the following steps:

Preferably, the preparation method of the Aronia melanocarpa extract after fermentation in the step (1) comprises the following steps:

preferably, the preparation method of the camel milk liposome in the step (1) comprises the following steps:

Preferably, the method for preparing the active polypeptide in the step (1) comprises the following steps:

Preferably, the tea tree essential oil and the clove essential oil have synergistic preservative effect, and are plant source preservatives.

Specifically, the related preparation and detection are as follows:

comparative example 1

Vitamin C standard working solution: accurately weighing l0mg pure vitamin C, fully dissolving in 1ml ultrapure water to prepare 10mg/ml vitamin C solution, and carrying out gradient dilution to 20, 15, 10, 6 and 2ug/ml Vc standard working solution.

Example 1 unfermented Aronia melanocarpa extract was prepared as follows:

taking a certain amount of Aronia melanocarpa and a small amount of soybean, pulverizing, oven drying until no change occurs, sterilizing, adding a small amount of sterile water to a viscous state, adding a proper amount of lactobacillus plantarum OPB15 powder, shaking uniformly, immediately soaking with 5-10 times of ethanol, performing ultrasonic extraction and centrifugation, filtering to obtain filtrate, cooling to room temperature, repeatedly extracting filter residues with ethanol for three times, mixing the filtrates, pouring the filtrate into a round-bottom flask, adding ethanol to scale marks, shaking uniformly, filtering with a 0.45um filter membrane, and taking the filtrate to obtain the Aronia melanocarpa extract. Wherein, the Aronia melanocarpa fruit: and (3) soybean: the mass ratio of the lactobacillus plantarum OPB15 bacterial powder is 1:0.01:5

Example 2 the preparation method of the Aronia melanocarpa extract after fermentation is as follows:

taking the same amount of Aronia melanocarpa fruits and a small amount of soybeans as unfermented, crushing, drying until no change occurs, sterilizing, adding a small amount of sterile water to be sticky, adding a proper amount of lactobacillus plantarum OPB15 bacteria powder, shaking uniformly, culturing in a constant temperature incubator for 48 hours, taking out, immediately soaking with 5-10 times of ethanol, carrying out ultrasonic extraction, centrifuging, filtering to obtain filtrate, cooling to room temperature, repeatedly extracting filter residues with ethanol for three times, mixing the filtrates, pouring the filtrate into a round bottom flask, adding 5-10 times of ethanol, shaking uniformly, filtering with a 0.45um filter membrane, and taking the filtrate to obtain the Aronia melanocarpa fruit. Wherein, the Aronia melanocarpa fruit: and (3) soybean: the mass ratio of the lactobacillus plantarum OPB15 bacterial powder is 1:0.01:5.

example 3 preparation method of Aronia melanocarpa polysaccharide comprises the following steps:

1) 15g of Aronia melanocarpa is taken out in a filter paper bucket, the opening of the filter paper bucket is folded and sealed, and the filter paper bucket is put into a Soxhlet extractor and is treated with diethyl ether at 54 ℃. Extraction was started until the diethyl ether extracted was clear. The black fruit wrapped filter paper fume hood was removed and the remaining ether was evaporated to dryness.

2) And (5) extracting the black fruits with water and precipitating with ethanol after the black fruits are taken out from the fume hood. The temperature was 77℃for 90 minutes. And (3) extracting the filter residue once again, combining the two filtrates, filtering, and taking clear liquid. Concentrate to 10ml,92% ethanol was ethanol precipitated overnight. Centrifugation at 7000rpm for 10 min, the supernatant was discarded. The precipitate was washed 2 times with diethyl ether and ethanol, and dissolved in hot water after the solvent had evaporated.

3) After the addition of sevage reagent (chloroform: n-butanol=4: 1) Deproteinizing, then shaking for about 30min, standing for separating protein, removing organic phase and protein between aqueous phase and organic phase, and repeating for 5 to 6 times. Dialyzing with dialysis bag (MD 44-10000) for 12 hr, and lyophilizing to obtain black fruit polysaccharide.

EXAMPLE 4 preparation method of unfermented Aronia melanocarpa nanometer eye cream

(1) The active polypeptide, the ginsenoside, the chamomile extract, the Aronia melanocarpa polysaccharide prepared in example 3, the unfermented Aronia melanocarpa extract prepared in example 1 and the camel milk liposome are stirred and mixed uniformly, incubated for about 10 minutes at about 35 ℃, and stirred continuously. Spin-drying water to obtain honeycomb membrane, adding puer tea extract, dissolving the honeycomb membrane, and ultrasonic treating for 10 min under 2500psi to obtain camel milk liposome containing active ingredients. Heating camel milk liposome coated with active ingredients to about 70deg.C. Wherein the active polypeptide: ginsenoside: chamomile extract: the Aronia melanocarpa polysaccharide prepared in example 3: the unfermented Aronia melanocarpa extract prepared in example 1: camel milk liposome: the mass ratio of the puer tea extract is 1:1:1:2:3:8:1, a step of;

(2) Adding acacia and sodium alginate into water according to a certain proportion, stirring to obtain a gel matrix, adding active ingredient-entrapped camel milk liposome into the gel matrix at about 70deg.C, stirring to uniformity, and adding vitamin C and vitamin E. Continuously stirring until the mixture is uniform, and preserving the temperature for about 10 minutes to obtain a mixture; wherein, the mass ratio of the acacia, the sodium alginate, the camel milk liposome encapsulating the active ingredients, the vitamin C and the vitamin E is 1:1:4:1:1, a step of;

(3) And (3) reducing the temperature of the mixture to 30-45 ℃, adding ultrapure water into hyaluronic acid, trehalose, tea tree essential oil and clove essential oil, mixing, stirring uniformly, adding into the mixture prepared in the step (2), shaking uniformly, standing until no precipitate exists, adding a proper amount of ultrapure water and acacia to regulate the viscosity of the eye cream, and thus obtaining the unfermented Aronia melanocarpa nano eye cream. Wherein, hyaluronic acid: trehalose: tea tree essential oil: butyl essential oil: ultrapure water added for the first time: the mass ratio of the mixture prepared in the step (2) is 0.8:1:1:1:5:10.

wherein, the preparation method of the puer tea extract in the step (1) comprises the following steps:

The preparation method of the chamomile extract in the step (1) comprises the following steps:

weighing stems and leaves of chamomile, drying until the mass is no longer changed, crushing into powder, and mixing the liquid with the material in a ratio of 1:15 adding ultrapure water, heating and refluxing for 3 hours at 85 ℃, cooling, vacuum filtering to obtain a filtrate, repeatedly extracting filter residues for three times, combining the filtrates, pre-freezing, freeze-drying in a freeze dryer, and freeze-drying into powder after 12 hours to obtain the chamomile extract.

The preparation method of the ginsenoside in the step (1) comprises the following steps:

weighing a proper amount of ginseng, crushing, and drying until the quality is unchanged, wherein the liquid-to-material ratio is 1:10 adding ultrapure water, heating and refluxing at 77 ℃ for 3 hours, cooling, vacuum filtering to obtain filtrate, repeatedly extracting filter residues for three times, combining the filtrates, pre-freezing, and then freeze-drying in a freeze dryer for 12 hours to obtain ginsenoside dry powder, thus obtaining the ginsenoside extract.

The preparation method of the camel milk liposome in the step (1) comprises the following steps:

firstly, the camel milk is incubated at about 35 ℃ for about 10 minutes, and is continuously stirred to spin dry the water into a honeycomb membrane by a film dispersion method, and the camel milk liposome is prepared by ultrasonic treatment for about 10 minutes under the pressure of 2500 psi.

The camel milk liposome is diluted by 10-100 times with distilled water before being mixed with other active ingredients.

The preparation method of the active polypeptide in the step (1) comprises the following steps:

taking a certain amount of hickory, wheat, peas, pollen pini and peach kernels, crushing, adding water, adjusting the pH value to be slightly alkaline (pH=8.7), extracting protein by an alkali-dissolution acid precipitation method, freeze-drying, centrifuging at 8000rpm for 10 minutes after enzymolysis by protease, and freeze-drying supernatant to obtain the active polypeptide extract.

The tea tree essential oil and the clove essential oil have the functions of corrosion prevention and are plant source preservatives.

Example 5 preparation method of fermented Aronia melanocarpa nanometer eye cream comprises the following steps:

(1) The active polypeptide, the ginsenoside, the chamomile extract, the Aronia melanocarpa polysaccharide prepared in example 3 and the fermented Aronia melanocarpa extract prepared in example 2 are stirred and mixed uniformly, incubated for about 10 minutes at about 35 ℃, and stirred continuously. Spin-drying water to obtain honeycomb membrane, adding puer tea extract, dissolving the honeycomb membrane, and ultrasonic treating for 10 min under 2500psi to obtain camel milk liposome containing active ingredients. Heating camel milk liposome coated with active ingredients to about 70deg.C. Wherein the active polypeptide: ginsenoside: chamomile extract: the Aronia melanocarpa polysaccharide prepared in example 3: the fermented Aronia melanocarpa extract prepared in example 2: camel milk liposome: the mass ratio of the puer tea extract is 1:1:1:2:3:8:1, a step of;

(3) Cooling to 30-45deg.C, adding ultrapure water into hyaluronic acid, trehalose, tea tree essential oil and flos Caryophylli essential oil, mixing, stirring to uniformity, adding into the mixture prepared in step (2), shaking, standing until no precipitate exists, adding appropriate amount of ultrapure water and acacia to adjust viscosity of eye cream, and obtaining fermented Aronia melanocarpa nanometer eye cream. Wherein, hyaluronic acid: trehalose: tea tree essential oil: butyl essential oil: ultrapure water added for the first time: the mass ratio of the mixture prepared in the step (2) is 0.8:1:1:1:5:10.

the preparation method of the puer tea extract in the step (1) comprises the following steps:

taking a certain amount of hickory, wheat, peas, pollen pini and peach kernels, crushing, adding water, adjusting the pH value to be about alkalescent pH=8.7, extracting protein by an alkali-dissolution acid precipitation method, freeze-drying, centrifuging at 8000rpm for 10 minutes after enzymolysis by protease, and freeze-drying supernatant to obtain the active polypeptide extract.

EXAMPLE 6 Aronia melanocarpa polysaccharide Structure

(1) Monosaccharide composition determination

1) Derivatization of a control: absorbing 400ul of mixed monosaccharide (mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose) solution, placing the mixed monosaccharide (mannose, rhamnose, glucuronic acid, glucose, galactose, xylose and arabinose) solution in a nut test tube according to the mass ratio of 1:1:1:1:1:1:1:1), sequentially adding 400ul of 0.3mol/L sodium hydroxide and 400ul of 0.5mol/L PMP solution, sealing, uniformly mixing, placing the mixture in a 70-DEG C water bath for 70min, taking out the mixture, cooling the mixture to room temperature, adding 400ul of 0.3mol/L hydrochloric acid to regulate the solution to be neutral, uniformly mixing the mixture, adding 1mL of chloroform to extract three times to remove PMP, taking out a water layer, and filtering the mixture by a 0.45um filter membrane.

2) Hydrolysis of Aronia melanocarpa polysaccharide: the Aronia melanocarpa polysaccharide prepared in example 3 was dissolved, placed in a screw flask, 1mL of trifluoroacetic acid was added, and the mixture was uniformly mixed and hydrolyzed at 120℃for 4.5 hours. Taking out and cooling after hydrolysis. Evaporated to dryness in a water bath at 80℃and 1mL of methanol was added thereto to remove trifluoroacetic acid. This procedure was repeated three times. The residue was dissolved in water under heating.

3) Polysaccharide PMP derivatization and reference derivatization: absorbing 400ul of the hydrolysis liquid of the Aronia melanocarpa polysaccharide prepared in the example 3, placing the hydrolysis liquid into a nut test tube, sequentially adding 400ul of 0.3mol/L sodium hydroxide and 400ul of 0.5mol/L PMP solution, sealing, uniformly mixing, placing the mixture into a water bath kettle at 70 ℃ for 70min, taking out, cooling to room temperature, adding 400ul of 0.3mol/L hydrochloric acid to adjust the solution to be neutral, uniformly mixing, adding 1mL of chloroform to extract for three times to remove PMP, taking out a water layer, and filtering with a 0.45um filter membrane.

4) Chromatographic conditions: inertSustatin C18 column (4.6X250 mm,5 um). Acetonitrile-0.05 mol/L phosphate buffer (pH=6.8) as mobile phase, flow rate of 1mL/min, detection wavelength of 254nm, column temperature of 35 and sample injection amount of 10ul.

Conclusion: the fructus Piperis has eight monosaccharide chromatographic peaks, which are respectively mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, and arabinose (shown in figures 1 and 2). The external standard method calculates the molar ratio of each monosaccharide by peak area, and the molar ratio of each saccharide is: 1.93:0.87:0.86:1.99:27.27:3.36:1.75:1.48.

(2) Periodate oxidation and Smith degradation

1) Standard curve establishment: preparing 30mmol/LNaIO4, packaging with current tinfoil, taking 6 dry test tubes, sequentially adding 30mmol/LNaIO4 solution 0, 0.5, 1, 1.5, 2 and 4mL, mixing and shaking the test tubes uniformly, taking 0.1mL to 25mL volumetric flasks for each tube, fixing the volume, and measuring the OD value at 223 nm. The abscissa represents concentration, the ordinate represents absorbance, and a standard curve (see fig. 3) is drawn.

2) Oxidation of periodic acid: 12.5mg of example 3 were weighed, dissolved in 12.5ml of ultrapure water, put into a 25ml volumetric flask, and NaIO at a concentration of 15mmol/L was added ₄ . The OD value was measured at 223nm by shaking reaction in a flask of 100ul to 25ml in 0, 7, 12, 24, 48, 72, 96h with a pipette, respectively, to a constant volume with ultrapure water, at room temperature until the OD value was no longer changed.

3) Terminating the reaction: when the OD value was not changed, 2ml of ethylene glycol was added to the reaction system to terminate the reaction. And allowed to stand for 20min. 2ml of the reaction solution was removed by a pipette, and the amount of formic acid released was determined by dropping with 0.01mol/L NaOH standard solution using phenolphthalein as an indicator. The remainder was transferred to dialysis bag (3000 Da) for dialysis.

4) Smith degradation: and (3) dialyzing for 60 hours, changing water once for 4 hours, concentrating to about 10mL under reduced pressure after dialyzing, adding 70mg of sodium borohydride, uniformly mixing and reacting for 24 hours in a dark place. After the reaction is completed, 50% acetic acid solution (v/v) is dripped to adjust the PH to 5-7, the ultrapure water is dialyzed for 60 hours, water is replaced for 4 hours as much as possible, and the dialysate is evaporated to dryness under reduced pressure after dialysis. 10mg hydroxylamine hydrochloride and 1mL pyridine were added and the reaction was continued with shaking in a 90℃water bath for 30min until the precipitate disappeared. After cooling to room temperature, adding 1mL of acetic anhydride, carrying out oscillation reaction for 30min in a 90-DEG water bath, taking out, cooling to room temperature, finally generating the volatile sugar nitrile acetate derivative, adding 1mL of chloroform, and then carrying out GC analysis by 0.22um coating (shown in figures 4 and 5).

Conclusion: 1mol of hexose consumed 1.42mol of periodic acid to produce 0.6493 formic acid indicated that it contained 64.93% of 1-or 1-6 bonds (ortho-trihydroxy), 12.14% of 1-2, 1-2.6,1-4 or 1-2.4 glycosidic bonds (ortho-dihydroxyl) and 22.93% of 1-3, 1-3.4,1-3.6,1-2.4,1-2.3,1-2.3.4 glycosidic bonds (and no ortho-trihydroxy nor ortho-dihydroxyl).

The products of the Aronia melanocarpa after Smith degradation include glycerol, erythritol and ethylene glycol. Indicating the presence of a large number of 1.fwdarw.6, 1.fwdarw.1.fwdarw.2, 1.fwdarw.2, 6, 1.fwdarw.4 or 1.fwdarw.4, 6 glycosidic bonds;

the product contains a small amount of mannose and glucose, which indicates that the Sorbus nigra contains non-oxidized 1-3, 1-2, 3, 1-3, 6, 1-2, 4, 1-2, 3,4 glycosidic bonds; the existence of mannose and glucose indicates that the mannose and the glucose contain non-oxidized glycosidic bonds, rhamnose is detected in degradation products, the partial rhamnose is not oxidized by periodic acid, and the conclusion that a part of connecting bonds in the Sorbus nigra are 1-2 or 1-2, 6 is made; galactose was detected in the Smith degradation products, indicating that the black fruit contained 1.fwdarw.3, 1.fwdarw.2, 3, 1.fwdarw.2, 4, 1.fwdarw.3, 6 or 1.fwdarw.2, 3,4 glycosidic linkages, i.e., galactose residues.

(3) Nuclear magnetic resonance measurement

15mg of the Aronia melanocarpa polysaccharide sample prepared in example 3 was weighed and dissolved in 1mLD ₂ O, go through ¹ H and ¹³ determination of C spectra

Conclusion: in comparison with other natural components, the saccharide is ¹ The signals in HNMR spectra are within a very narrow range, the polysaccharide signals are generally concentrated in the delta 3.0-6.0 range, and more than 5.0ppm of alpha-configuration pyranose is generally smallAt 5.0ppm is beta-configuration pyranose. The 1H NMR spectrum of Aronia melanocarpa polysaccharide shows that the signal of the hydrogen at different heads appears at delta 5.0-6.0, so that Aronia melanocarpa polysaccharide has alpha-configuration glycosidic bond (shown in figure 6).

Sugar is in ¹³ The signal of the terminal carbon in the C NMR spectrum is within the range of delta 95-110 ppm. The delta values of the alpha-D and beta-L type glycosidic linkages of monosaccharides are typically less than 100ppm, and the delta values of the alpha-L and beta-D type glycosidic linkages are typically greater than 100ppm, which can be reduced to delta 98ppm in the case of tertiary alcohol and ester glycosides, and individual phenolic glycosides. Thus, according to polysaccharide ¹³ The number of saccharides and the configuration of glycosidic linkages in the polysaccharide are deduced from the number of carbon signals and the delta value in the range of delta 95-110ppm in the C NMR spectrum. The signal of the exocarpium of Aronia melanocarpa polysaccharide has 2 absorption peaks in the delta 95-110ppm range, and it is presumed that 2 sugar residues exist in the repeating unit of the polysaccharide sugar chain in Aronia melanocarpa polysaccharide. The peak of the signal of the anomeric carbon in the range of delta 70-80ppm is a characteristic peak of the pyran ring (see FIG. 7).

(4) Fourier infrared measurement

Taking the Aronia melanocarpa polysaccharide prepared in example 3 and dry KBr according to the mass of 1:100, fully grinding, tabletting and infrared 4000-400 cm ^-1 Scanning is carried out in the wave band.

Conclusion: at 3415cm ^-1 The absorption peak shown there may be a telescopic vibration absorption peak of O-H; 2925cm ^-1 The weak absorption peak at the point may be a C-H bending vibration absorption peak; 1650cm ^-1 Bending vibration absorption peaks which may be O-H; 1375cm ^-1 A variable angle vibration peak which may be C-H; 1032cm ^-1 A bending vibration absorption peak which may be a C-O-C connection; 1200-1000cm ^-1 Peaks indicate that it is likely to be a pyran polysaccharide; and at 882cm ^-1 The absorption peaks of (a) indicate that they may be linked by β -type glycosidic linkages (see fig. 8).

(5) Determination of polysaccharide molecular weight of Aronia melanocarpa

Chromatographic conditions: the experiment adopts high-efficiency GEL permeation chromatography to determine the molecular weight distribution of Aronia melanocarpa polysaccharide, and the chromatographic column is TSK-GEL G4000 PWXL GEL column (7.8 mm×30.0 cm). The mobile phase is ultrapure water; the flow rate is 0.8mL/min; the column temperature is 35 ℃; the detector used was a differential refractive detector.

Establishing a molecular weight correction standard curve: dextran standard substances with different molecular weights are matched into standard liquid with the concentration of 1.0mg/mL by using a mobile phase, and the sample injection amount is 1g/L. Molecular weight standard curves were prepared (FIG. 9).

The retention time was calculated by sampling 1g/L of the Aronia melanocarpa polysaccharide solution (FIG. 10), and the retention time was calculated to give a molecular weight of Mr= 10876Da by the formula Qu Gong.

Example 7: screening of Lactobacillus plantarum OPB15

Isolation and screening of Lactobacillus

(1) 0.1ml of a yogurt sample (from the home self-brewed yogurt of the farmers in the Awa-tikou province of Kaisha) was inoculated into a skim milk medium and placed in a 37℃incubator for 48h. A small amount of bacterial liquid is picked by an inoculating loop, streaked and inoculated in an MRS agar culture medium plate.

(2) After 48 hours, different colonies are picked up by an inoculating loop according to the shape, size, color and the like of the colonies, and streaked separation and purification are carried out.

(3) Gram-positive bacteria and catalase-negative bacteria were retained by gram staining and catalase analysis.

(4) Obtaining thallus and colony characteristics of the final strain: gram-positive rod-shaped bacteria, non-sporulating, non-motile bacteria (fig. 16); the diameter is about 1.0-1.2 mm, the front face is round, the center is convex, the edge is neat, the surface is moist and smooth, milky white, opaque and slightly sour (figure 17).

Molecular biological identification of Lactobacillus

(1) Single genome extraction

(A) Culturing the screened lactobacillus overnight;

(B) Using a Soxhaust bacteria genome DNA extraction kit, the product number is 1600, taking 1ml of the bacterial suspension cultured overnight, centrifuging in a 1.5ml centrifuge tube at 12000r/min for 1min, and sucking out the supernatant as much as possible;

(C) Adding 200ul of lysozyme with a final concentration of 20mg/ml into thalli, and treating for 40min at 37 ℃;

(D) Adding solution A (10 mmol/L, pH =8.0 Tris-HCl and 1mmol/L EDTA mixed solution, SDS with concentration of 0.08g/mL and NaCl with concentration of 1.5 mol/L) into the mixed solution obtained in the step (C), blowing by a pipette to fully suspend the thalli, adding 20ul rNaseA (10 mg/mL) into the suspension, fully reversing and uniformly mixing, and standing at room temperature for 30min;

(E) Adding 20ul proteinase K into the tube, mixing well, and digesting at 55deg.C for 60min; inverting the centrifuge tube for several times during digestion until the sample is clear and viscous, and proving that the sample is completely digested;

(F) Adding 200ul of solution B (mixed solution of phenol and isoamyl alcohol=25:24:1 by volume ratio) into the inquiry tube, fully inverting and uniformly mixing, if white precipitation appears, standing at 75 ℃ for 15-30min, wherein the precipitation can disappear;

(G) Adding 200ul of absolute ethyl alcohol into a tube, fully and uniformly mixing, wherein flocculent precipitate can be generated at the moment without influencing DNA extraction, adding the solution and the flocculent precipitate into an adsorption column, and standing for 2min;

(H) Centrifuging at 12000rpm for 2min, discarding the waste liquid, and placing the adsorption column into a collecting pipe;

(I) Adding 600ul of rinsing liquid (75% ethanol) into the adsorption column, centrifuging at 12000rpm for 1min, discarding the waste liquid, placing the adsorption column into a collecting pipe, and repeating for one time;

(J) Centrifuging at 12000rpm for 2min, and placing the adsorption column in a 50 deg.C incubator for several minutes;

(K) Placing the adsorption column into a sterilized 1.5ml centrifuge tube, suspending and dripping 100ul of the washing liquid (general purpose) preheated by 65 ℃ water bath into the center of the adsorption film, standing for 5min at room temperature, and centrifuging for 1min at 12000 rpm;

and (L) adding the eluent obtained by centrifugation into an adsorption column, standing for 2min at room temperature, and centrifuging at 12000rpm for 2min to obtain the genome DNA of the lactobacillus.

TTAGGCGGCTGGTTCCTAAAAGGTTACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGCATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAAGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTAGCCGTGGCTTTCTGGTAATACCGTCATACCTGAACAGTTACTCTCAGATATGTTCTTCTTTAACAACNGAGTTTTACGAGCCGAAACCCTTCTCACTGCTATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTCGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGTTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAACTCAAGGAATTGACGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGACCTTACCNNTCTGACATACTATGCAAATCTAGAGAT

(2)16S r DNA PCR

Bacterial 16S r DNA 50. Mu. LPCR reaction System: 24 mu L of Taq enzyme; dd H ₂ O: 18. Mu.L; 27F, 2. Mu.L; 1492R, 2. Mu.L; and (3) a template: 4. Mu.L.

PCR conditions: 94℃for 5min (i.e.step 1); 94℃for 30s (i.e.step 2); 55℃for 30s (i.e.step 3); 30s at 72 ℃ (i.e. step 4); step2 to 4 30×; and at 72℃for 5min.

Preparing 1% agarose gel, mixing the PCR product with 10000×loading buffer, loading 2 μl, running at 120V for 30min, and performing gel imaging;

the obtained PCR product was sent to a professional sequencing company, and the obtained sequencing result was subjected to searching and similarity alignment in Gen Bank using BLAST (see FIG. 18), and identified as a strain of Lactobacillus plantarum, and stored at-80 ℃.

Example 8: lactobacillus plantarum OPB15 has good tolerance to simulated gastrointestinal fluids

In vitro simulated gastric fluid: the pH value of the 0.1mol/L potassium phosphate buffer solution is regulated to 2.5, pepsin (10 g/L) is added, and the mixture is uniformly mixed and then passes through a 0.22 mu m sterile film, so that simulated gastric juice can be obtained.

In vitro simulated intestinal juice: the pH value of the 0.1mol/L potassium phosphate buffer solution is regulated to 6.8, pancreatin (10 g/L) and pig bile salt (3 g/L) are added, and the mixture is uniformly mixed and then passes through a 0.22 mu m sterile film, thus obtaining the simulated intestinal juice.

Gastric digestion was simulated:

3.0mL of lactobacillus plantarum OPB15 culture solution is taken and centrifuged for 3min, mycelium is collected and placed in 3.0mL of gastric juice simulating environment with pH value of 2.5, and gastric juice digestion is simulated under anaerobic condition at 37 ℃ and 90 r/min. Samples were taken at 0, 1.0, 2.0, 3.0 hours, viable bacteria were counted using a dilution-coated plate count method, and survival rates were calculated.

Intestinal digestion was simulated:

centrifuging 3.0mL of lactobacillus plantarum OPB15 culture solution for 3min, collecting mycelium, and placing in 3.0mL of simulated intestinal juice environment with pH value of 6.8 at 37 ℃ under anaerobic condition of 120r/min to simulate intestinal juice digestion. Samples were taken at 0, 2.0, 4.0, 6.0, 8.0 hours, viable bacteria were counted using a dilution-coated plate count method, and survival rates were calculated.

The survival rate (%) was calculated as the ratio of the number of viable bacteria at the time of sampling to the number of viable bacteria at the time of 0h in the culture solution, expressed as%. The experimental results are shown in table 1, and the results show that lactobacillus plantarum OPB15 has better tolerance to artificial simulated gastrointestinal fluids.

TABLE 1 tolerance of Lactobacillus plantarum OPB15 in artificial simulated gastric fluid

TABLE 2 tolerance of Lactobacillus plantarum OPB15 in artificially simulated intestinal juice

Example 9: lactobacillus plantarum OPB15 has no toxic or side effect on SFP-class Balb/c mice

Suspending Lactobacillus plantarum OPB15 in 100g/L skim milk solution to give 4.0X10 g concentration ⁹ Bacterial suspension of CFU/m L. SPF-class Balb/c mice are bred in an environment with 40 males, the body mass of 18-22 g, the temperature of 20-25 ℃ and the relative humidity (50+/-5)%, and the illumination time of 12h/12 h. The lactobacillus plantarum OPB15 group and the control group are divided. The lactobacillus plantarum OPB15 group was given 0.3mL of this concentrated bacterial suspension once daily for gavage, and the control group was gavaged with the same volume of 100g/L skim milk solution without lactobacillus plantarum OPB15, and the death and weight were recorded for one week.

The results of these tests are shown in Table 3. These results indicate that the feeding concentration is 1.2X10 ⁹ The CFU/lactobacillus plantarum OPB15 has no obvious influence on mice, no obvious change of weight and no death phenomenon. The appearance of the mice has no obvious pathological symptoms.

TABLE 3 weight changes and death in mice

The correlation detection is as follows:

1. antioxidant ability evaluation- -DPPH radical scavenging experiment

1. The unfermented Aronia melanocarpa extract prepared in example 1 was diluted to a certain concentration, and mixed with a nitrogen radical extract at a ratio of 1:9, and centrifuging at 1000rpm for 10min, collecting supernatant, and storing at 4deg.C.

2. The fermented Aronia melanocarpa extract prepared in example 2 was diluted to the same concentration as the unfermented Aronia melanocarpa extract prepared in example 1, and the concentration was adjusted to 1:9, and centrifuging at 1000rpm for 10min, collecting supernatant, and storing at 4deg.C.

3. 10mg/ml vitamin C solution is diluted to 20, 15, 10, 8, 6, 4 and 2ug/ml Vc standard working solution by nitrogen free radical extracting solution.

4. A blank tube, a sample measuring tube, a positive control tube and a solution were set in the above order according to Table 4, and the mixture was stirred uniformly and allowed to stand at room temperature for 30 minutes.

TABLE 4 description of the addition of substances to the tubes

5. The experimental group comprises fermented Aronia melanocarpa ethanol solution and unfermented Aronia melanocarpa ethanol solution; control group comparative example 1 (vitamin C ethanol solution)

6. Taking a 96-well plate, sequentially sucking 300ul of each tube of solution into the 96-well plate, and detecting absorbance values (517 nm) of each tube by using an enzyme-labeled instrument, and sequentially marking the absorbance values as A0, A1, A2 and A3.

7. Positive control DPPH clearance (%) = (A0-A3)/A0 x 100%

8. DPPH clearance (%) = [ A0- (A1-A2) ]/A0 of the sample to be tested is 100%

9. Wherein: a0 Absorbance values for the blank tube. A1 Absorbance values of the tube were measured for the samples. A2 Absorbance values for the control tube of the samples. A3 Absorbance of a positive control tube.

10. The above experimental results were repeated 3 times in parallel, i.e., n=3.

TABLE 5 evaluation results of the ability of vitamin C to clear DPPH

The Vc standard working solution has good linear relation at the speed of 2-8 ug/ml.

TABLE 6 evaluation results of DPPH-removing ability of fermented Aronia melanocarpa ethanol solution and unfermented Aronia melanocarpa ethanol solution

From Table 6, it can be seen that the fermented Aronia melanocarpa ethanol solution has a higher oxidation resistance than the unfermented Aronia melanocarpa ethanol solution.

2. Mass spectrum test of Aronia melanocarpa after fermentation and unfermented

1. The unfermented Aronia melanocarpa extract prepared in example 1 was diluted to a certain concentration, centrifuged at 1000rpm for 10min, and the supernatant was kept for use and 4 ℃ for further use.

2. The fermented Aronia melanocarpa extract prepared in example 2 was diluted to a certain concentration, centrifuged at 1000rpm for 10min, and the supernatant was kept for use at 4 ℃.

Liquid phase conditions:

instrument: waters HClass

Mobile phase: 0.1% formic acid B0.1% formic acid acetonitrile

Chromatographic column: waters BEH C181.7um 2.1 x 50um

Column temperature: 40 DEG C

TABLE 7 gradient elution

Time (min)	Flow rate (ml/min)	A(％)	B(％)
				0	0.4	95	5
1	0.4	95	5
				35	0.4	2	98
37	0.4	2	98
				37.1	0.4	95	5
40	0.4	95	5

Mass spectrometry conditions:

instrument: waters G2-XS Qtof

Positive mode: the voltage is 2kv, the ion source temperature is 110 ℃, the desolvation temperature is 400 ℃, the nitrogen flow rate is 800L/h, the acquisition mass number range is 50-1200, and the collision voltage is 20-40v.

Negative mode: the voltage is 1.5kv, the ion source temperature is 110 ℃, the desolvation temperature is 400 ℃, the nitrogen flow rate is 800L/h, the acquisition mass number range is 50-1200, and the collision voltage is 20-40v.

Table 8 mass spectral results

As can be seen from fig. 11 to 15 and table 8:

1. fig. 11-15 are total ion flow diagrams in positive and negative ion modes, and the total ion peak patterns in the positive and negative ion modes are symmetrical and have no obvious overlapping phenomenon, which shows that under the test conditions, the chemical composition of the dried sakura product can be well measured.

2. The chemical components of Aronia melanocarpa fruit mainly comprise flavonoids, anthocyanin, triterpenes, phenolic acids, sterols, sugar and glycosides.

3. The fructus Sorbi Pohuashanensis has high anthocyanin content, about 25% of total phenol content, and the main substances are 3-oxo-arabinoside, 3-oxo-glucoside, 3-oxo-xyloside, and 3-oxo-galactoside.

4. The accumulated polyphenol in the Aronia melanocarpa fruits is mainly synthesized by anthocyanin and glycoside compounds thereof.

5. The Aronia melanocarpa fruit contains abundant anthocyanin, and the proportion of the anthocyanin can reach about 25% of phenolic compounds. Anthocyanin is a common chemical component in natural plants. The anthocyanin can be hydrolyzed to the anthocyanidin under acidic conditions.

6. The flavonoid compound is mainly composed of quercetin and derivatives thereof, and the quercetin derivatives are mainly composed of isoquercetin and the like.

7. Organic acids produced during fermentation can prevent degradation of phenolic substances and thus lead to an increase in total phenol content. The acidic environment is favorable for stabilizing phenolic substances

8. The polysaccharide and protein on the cell wall of the Sorbus nigra are hydrolyzed by certain enzymes generated in the fermentation, so that the phenolic substances combined with the polysaccharide and protein are released, which may cause the improvement of the phenols.

Test example 3 sensory evaluation test of product:

two common eye creams were commercially available as control groups in examples 4 and 5 as experimental groups. Volunteers (25-55 years old) were selected 15 men and women each, and were randomized to average 3 groups, and after daily skin cleansing, the experimental and control groups were applied around the eyes and the effect was observed after 15 days. The different items were scored (0-100 points) according to skin condition changes, and the results are shown in table 9.

Table 9 evaluation results

	Example 5	Example 4	Control group 1	Control group 2
					Moisture retention	96.32±2.43	91.32±1.42	84.89±3.01	85.64±2.52
Refreshing property	98.56±2.12	95.65±1.67	85.56±1.24	91.83±1,53
					Permeability of	95.12±1.23	91.22±1.82	87.37±2.11	87.47±1.22
Wrinkle change	90.88±1.81	87.73±1.94	81.47±1.01	83.28±0.81
					Edema changes	91.56±0.21	89.31±0.22	79.84±0.31	86.96±0.11
Skin color change	81±0.23	79±1.22	77±0.12	77±0.21
					Comfort level	94±0.09	93±0.02	91±0.41	91±1.00

By combining the results, the Aronia melanocarpa liposome prepared by the invention is better than eye cream on the market, and each effect of the Aronia melanocarpa liposome after fermentation is optimal.

Although embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments.

Claims

1. A black chokeberry extract nanometer eye cream fermented by lactobacillus plantarum is characterized in that: the lactobacillus plantarum is lactobacillus plantarum OPB15, and the name of the lactobacillus plantarum is: OPB15, class name: lactobacillus plantarum (Lactobacillus plantarum), accession number: CGMCC No.27735, date of preservation: 2023, 6, 29, deposit unit: china general microbiological culture Collection center, north Chen Xi Lu No. 1, 3, the Korean region of Beijing, and the China general microbiological culture Collection center.

2. The method for preparing the black chokeberry extract nanometer eye cream according to claim 1, which is characterized in that: the method comprises the following steps:

3. the preparation method according to claim 2, characterized in that: the preparation method of the puer tea extract in the step (1) comprises the following steps:

4. The preparation method according to claim 2, characterized in that: the preparation method of the chamomile extract in the step (1) comprises the following steps:

5. The preparation method according to claim 2, characterized in that: the preparation method of the ginsenoside in the step (1) comprises the following steps:

6. The preparation method according to claim 2, characterized in that: the preparation method of the Aronia melanocarpa polysaccharide in the step (1) comprises the following steps:

7. The preparation method according to claim 2, characterized in that: the preparation method of the Aronia melanocarpa extract after fermentation in the step (1) comprises the following steps:

8. the preparation method according to claim 2, characterized in that: the preparation method of the camel milk liposome in the step (1) comprises the following steps:

9. The preparation method according to claim 2, characterized in that: the preparation method of the active polypeptide in the step (1) comprises the following steps:

10. The preparation method according to any one of claims 2 to 9, characterized in that: the tea tree essential oil and the clove essential oil have synergistic preservative effect and are plant source preservatives.