CN215873329U - Multi-layer grain structure of nutrition composition for promoting skin cell activation and resisting oxidation - Google Patents
Multi-layer grain structure of nutrition composition for promoting skin cell activation and resisting oxidation Download PDFInfo
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Abstract
The present disclosure provides a multi-layer grain structure of a nutritional composition for promoting skin cell activation and oxidation resistance, which comprises a nutritional composition core, an oxidation resistant layer, a functional coating layer and a structural protection layer in sequence from the center to the outer side, wherein the components and the flavor of the multi-layer grain structure are protected through layer-by-layer coating, and the multi-layer grain structure is made to be spherical; the nutritional composition comprises fermented grape yeast, aronia melanocarpa extract, comprehensive berry fruit enzyme, dunaliella salina, emblic leafflower fruit extract and perilla seed extract; the multilayer crystal grain structure can be made into health products such as drinks, powder bags, colloids, capsules or pastilles, and can improve the whiteness of skin, the elasticity of skin, the water content of skin and reduce skin spots and wrinkles.
Description
Technical Field
The present disclosure relates to a beverage containing a multi-layered grain structure, and more particularly to a multi-layered grain structure of a nutritional composition for promoting skin cell activation and oxidation resistance.
Background
Skin care and skin whitening care are gradually regarded by modern people, and in order to pursue good skin quality, a plurality of skin-related data including skin spots, skin elasticity, skin whiteness, skin wrinkles, skin moisture content and the like are focused, and the skin quality is reduced, wherein one important reason is that the skin is protected from being damaged by oxidative stress due to oxidative stress including aging, improper diet, sunlight irradiation and the like, and the important problem of skin care and care is that the skin is protected from being damaged by the oxidative stress.
Fibroblast cells of the skin are the most important cells in the dermis layer of the skin, and extracellular matrix (ECM) secreted by fibroblast cells is an important substance for maintaining the elasticity of the skin, resisting wrinkles and protecting the skin, so that the proliferation of fibroblast cells has important functions of repairing damaged skin and maintaining the health of the skin in addition to participating in the wound healing process.
However, with aging and the accumulation of external adverse factors, the number of skin fibroblasts is reduced, so that the protective ability of the skin is reduced, and when the loss rate of the skin fibroblasts is higher than the proliferation rate of the skin fibroblasts, the skin is mottled, wrinkled and flaccid.
In the existing skin health care products, the problems of difficult absorption, damage to active substances in gastric juice or poor efficacy and the like are often generated after eating, and whether a user has better flavor, aroma and taste is also an important subject to be solved; in addition, the concept of natural organic health products is gradually emerging, and the natural components of the skin health products, including fruits, plants, natural enzymes and the like, can be developed while achieving the effect of skin health, so that the natural organic health products have better benefits for human health.
SUMMERY OF THE UTILITY MODEL
Accordingly, the present disclosure provides a multi-layered grain structure of a nutritional composition for promoting skin cell activation and oxidation resistance, which comprises a nutritional composition core, an oxidation-resistant layer, a functional coating layer and a structural protection layer in order from the center to the outer side, wherein the multi-layered grain structure is coated layer by layer to protect the components and flavor of the multi-layered grain structure and is made to be spherical.
In one embodiment, the present disclosure provides a multi-layered grain structure of a nutritional composition for promoting skin cell activation and oxidation resistance, wherein the particle size of the nutritional composition core is 1 to 5mm, and the thickness of the oxidation resistant layer is 0.5 to 1 mm.
In one embodiment, the multi-layered grain structure of the nutritional composition for promoting skin cell activation and oxidation resistance provided by the present disclosure, wherein the core of the nutritional composition is a composition of fermented grape yeast and integrated berry and fruit ferment, and the antioxidant layer is a composition of aronia melanocarpa extract, dunaliella salina, emblic leafflower fruit extract and perilla seed extract.
In one embodiment, the present disclosure provides a multi-layered grain structure of a nutritional composition for promoting skin cell activation and oxidation resistance, wherein the oxidation resistant layer is characterized by a strong ability to neutralize free radicals, so as to protect important active ingredients of the nutritional composition core coated by the oxidation resistant layer in the multi-layered grain structure.
In one embodiment, the multi-layered grain structure of the nutritional composition for promoting skin cell activation and oxidation resistance provided by the present disclosure, wherein the functional coating layer is made of soybean lecithin, has a thickness of 0.08 to 0.1mm, is located outside the oxidation resistant layer, and has hydrophobicity, and the functional coating layer has a plurality of micropores filled with colloid, which can increase adhesion of the functional coating layer, make the structural composition of the functional coating layer more stable, and can strongly protect the nutritional composition core and the oxidation resistant layer coated therewith, so that the main effective components are not damaged by external environment, and maintain the configuration of the multi-layered grain structure.
The soybean lecithin is a nontoxic surfactant with good tolerance, and in a liquid environment, the phospholipid of the soybean lecithin can form a lipid bilayer structure to separate the components in the antioxidation layer and the structure protective layer, and can maintain the attachment capacity between layers in the multilayer crystal grain structure, so that the spherical configuration of the multilayer crystal grain structure is more stable and more favorable for human body absorption.
In one aspect, the present disclosure provides a multi-layered grain structure of a nutritional composition for promoting skin cell activation and oxidation resistance, wherein the colloid is selected from the group consisting of acacia gum, corn glyco-colloid, or other edible colloids.
In one embodiment, the present disclosure provides a multi-layered grain structure of a nutritional composition for promoting skin cell activation and oxidation resistance, wherein a flavoring layer, a flavor layer and a fragrance layer are further disposed between the functional coating layer and the structural protection layer.
In one embodiment, the multi-layer grain structure of the nutritional composition for promoting skin cell activation and oxidation resistance provided by the present disclosure, wherein the thickness of the flavoring layer is 0.1 to 0.4mm, the thickness of the flavor layer is 0.8 to 1mm, and the thickness of the flavor layer is 0.06 to 0.1mm, so as to sequentially release flavor, sour and sweet flavor, and fruit flavor.
In one embodiment, the present disclosure provides a multi-layered grain structure of a nutritional composition for promoting skin cell activation and oxidation resistance, wherein the structural protection layer has a thickness of 0.08 to 0.12mm and is located outside the functional coating layer, so as to protect and maintain the configuration of the multi-layered grain structure and prevent the multi-layered grain structure from being damaged by gastric juice.
In one aspect, the present disclosure provides a multi-layered grain structure of a nutritional composition for promoting skin cell activation and oxidation resistance, wherein the structural protective layer is selected from the group consisting of maltodextrin, starch, cellulose, pectin, or other edible excipients.
In one embodiment, the present disclosure provides a multi-layered grain structure of a nutritional composition for promoting skin cell activation and oxidation resistance, which is soluble in water to form a solution.
In one embodiment, the multi-layered grain structure of the nutritional composition for promoting skin cell activation and oxidation resistance provided by the present disclosure can be made into health products such as drinks, powder packets, colloids, capsules or tablets.
In summary, the present disclosure provides a multi-layered grain structure of a nutritional composition for promoting skin cell activation and oxidation resistance, wherein the multi-layered grain structure is not easily damaged by gastric juice, so that a human body can effectively absorb effective ingredients, and practical experience of a user is improved through multi-layered flavor configuration, and the nutritional composition has the functions of promoting skin fibroblast activation and skin repair, and protecting skin fibroblast from oxidation pressure. The multi-layer grain structure of the nutritional composition can be used for preparing drinks so as to achieve the effects of protecting skin fibroblasts from oxidation pressure and promoting the activation of the skin fibroblasts, and the nutritional composition can effectively improve the quality of skin and achieve the effect of skin health care.
By means of the technical characteristics of the disclosure, the skin health can be effectively promoted, the defects that the conventional health-care products are easy to damage, difficult to absorb, bad in edible flavor and the like when passing through the stomach are overcome, and the nutritional composition is a natural organic component, is beneficial to human health and does not have negative effects.
The detailed features and advantages of the present disclosure are described in detail in the following detailed description, which is sufficient for any person skilled in the art to understand the technical content of the present disclosure and to implement the same, and the objects and advantages related to the present disclosure can be easily understood by any person skilled in the art from the disclosure, claims and drawings of the present disclosure.
Drawings
Fig. 1 is a perspective partial cross-sectional view of one embodiment of a multi-layer die structure according to the present disclosure.
Fig. 2 is a quantification of pre-apoptotic images of the multi-layered grain structure of the present disclosure to protect skin fibroblasts from oxidative stress.
Fig. 3 is a quantification of images of the late apoptotic stage of the protection of dermal fibroblasts from oxidative stress for the multi-layered grain structure of the present disclosure.
Fig. 4 is a graph of relative cell activation ratios for a multilayered grain structure of the present disclosure that promotes skin fibroblast activation.
FIG. 5 is a graph of relative ROS production ratios for the disclosed multilayered grain structure to promote skin fibroblast oxidation resistance.
Fig. 6 is a graph showing the results of average skin whiteness changes for 4 weeks and 8 weeks after the test subjects drink the grape yeast composite fruit and vegetable beverage of the present disclosure.
Fig. 7 is a graph showing the average skin spot change results of the subjects who drink the grape yeast composite fruit and vegetable beverage of the present disclosure for 4 weeks and 8 weeks.
Fig. 8 is a graph showing the results of the average skin moisture content change for the test subjects after drinking the grape yeast composite fruit and vegetable beverage of the present disclosure for 4 weeks and 8 weeks.
Fig. 9 is a graph showing the results of the average skin elasticity changes of the test subjects after drinking the grape yeast composite fruit and vegetable beverage of the present disclosure for 4 weeks and 8 weeks.
Fig. 10 is a graph showing the average skin wrinkle change results for the test subjects who consumed the grape yeast composite fruit and vegetable beverage of the present disclosure for 4 weeks and 8 weeks.
[ List of reference numerals ]
Multilayer grain structure of a nutritional composition for promoting skin cell activation and oxidation resistance
11 core of nutritional composition
12 antioxidation layer
13 functional coating layer
14 structural protective layer
21 seasoning layer
22 flavor layer
23 fragrance layer
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1, the present disclosure provides a multi-layered grain structure 1 of a nutritional composition for promoting skin cell activation and oxidation resistance, which comprises a nutritional composition core 11, an oxidation-resistant layer 12, a functional coating layer 13 and a structural protection layer 14 in sequence from the center to the outside, wherein the ingredients and flavors of the multi-layered grain structure are protected by layer-by-layer coating, and the multi-layered grain structure is made to be spherical. Wherein, the spherical structure can ensure that the absorption efficiency is better.
In a preferred embodiment of the present disclosure, the particle size of the nutritional composition core 11 is 1 to 5mm, and the thickness of the oxidation resistant layer 12 is 0.5 to 1mm, wherein the nutritional composition core 11 is a composition of fermented grape yeast and integrated berry and fruit ferment, and the oxidation resistant layer 12 is a composition of aronia melanocarpa extract, dunaliella salina, emblic leafflower fruit extract and perilla seed extract.
The aronia melanocarpa is also called as aronia melanocarpa, has high-concentration high-nutrition-value components such as polyphenols, anthocyanin, flavonoid, vitamin C, beta-carotene and the like, and has strong antioxidant free radical capacity. Dunaliella contains abundant beta-carotene, alpha-carotene, lycopene, lutein and zeaxanthin, which are called natural comprehensive carotenoids, and can effectively resist free radicals. The emblic leafflower fruit is sour and slightly astringent in taste, has the effects of clearing heat and cooling blood, helping digestion and strengthening spleen, and promoting the production of body fluid to quench thirst, is mainly used for treating blood heat and blood stasis, dyspepsia, abdominal distension, cough, laryngalgia, dry mouth, vitamin C deficiency and the like, has a strong antioxidation effect, and can effectively help whitening and reduce harmful substances generated by aging. The fructus Perillae extract contains multiple polyphenols, has antioxidant and anti-inflammatory (lipoxygenase inhibiting) effects, and can resist free radicals, relieve inflammation, inhibit histamine release, and eliminate red swelling.
A preferred embodiment of the present disclosure is characterized in that the fermented grape yeast comprises bordeaux farina and swiss yeast extract.
The French Bordeaux red wine powder is brewed from Mello grape (Merlot, the production place is located on the right bank of Bordeaux in France), contains various polyphenols such as gallic acid, catechin, quercetin, procyanidine, resveratrol and the like, has strong antioxidation effect, and is helpful for resisting skin oxidation pressure. The swiss yeast extract (from EVOLVA company) contains rich antioxidant substances, is natural and non-genetically modified in the extraction process, has the public security food certification (GRAS) of the American Food and Drug Administration (FDA), can be used as a dietary supplement in European Union, and can achieve the effects of removing harmful substances in vivo, improving the activity of longevity genes, improving the circulation of blood vessel endings and the like.
In a preferred embodiment of the present disclosure, the comprehensive berry and fruit ferment is extracted from a combination of blueberry, white grape, cranberry, apple, carrot, red grape, waxberry, mulberry, sugarcane, passion fruit, pineapple, lemon, broccoli, celery, asparagus, red pomegranate, blackcurrant, and purple carrot.
The comprehensive raspberry and fruit enzyme (from TCI company) utilizes fermentation technology to extract effective trace elements, polysaccharides and the like in the comprehensive raspberry and fruit, and simultaneously retains high-content superoxide dismutase (SOD) in the process, and has multiple effects of delaying aging, keeping skin activity, resisting oxidation pressure and the like.
In a preferred embodiment of the present disclosure, the functional coating layer 13 is composed of soybean lecithin, has a thickness of 0.08 to 0.1mm, is located outside the antioxidation layer 12, has hydrophobicity, and is used to coat the nutrition composition core 11 and the antioxidation layer 12, and the thickness design can protect the main effective components of the multi-layer grain structure without hindering absorption by human body; the soybean lecithin is composed of a choline head group, a glycerophosphate and two fatty acids, and has hydrophobicity, so that the functional coating layer 13 can maintain the configuration of the antioxidation layer 12, and meanwhile, the structural protection layer 14 is firmly connected, and the phospholipid component can further maintain the stability of the multi-layer grain structure of the present disclosure; in addition, the functional coating layer 13 of the present disclosure has a plurality of micropores filled with a colloid, which can greatly improve the adhesion of the functional coating layer 13, make the structural composition thereof more stable, and enhance the protection of the effective components from the external environment, and maintain the configuration of the multilayer crystal grain structure.
A preferred embodiment of the present disclosure is characterized in that, when the multi-layer grain structure of the present disclosure is made into a beverage, the soybean lecithin of the functional coating layer 13 forms lipid bilayers in a liquid environment, the structure of the lipid bilayers is tightly arranged, which can further stabilize the configuration of the multi-layer grain structure of the present disclosure, so that the multi-layer grain structure of the present disclosure maintains a spherical shape, and the spherical multi-layer grain structure can effectively coat and protect the effective components in the multi-layer grain structure, and improve the absorption rate of the effective components by the human body; furthermore, the lipid bilayer structure of the functional coating layer can further improve the attachment capability between layers in the multilayer grain structure.
A preferred embodiment of the present disclosure is characterized in that the colloid is selected from the group consisting of acacia, corn syrup or other edible colloid, which improves the adhesion of the functional coating layer 13 of the present disclosure, making the coating layer more compact.
In a preferred embodiment of the present disclosure, the multi-layer grain structure may further include a seasoning layer 21, a flavor layer 22 and a fragrance layer 23 between the functional coating layer and the structural protection layer.
A preferred embodiment of the present disclosure is characterized in that the thickness of the flavoring layer 21 is 0.1 to 0.4mm, the thickness of the flavor layer 22 is 0.8 to 1mm, the thickness of the flavor layer 23 is 0.06 to 0.1mm, the thickness and arrangement are designed to sequentially release flavor, sour and sweet flavor and fruity taste, when the multilayer grain structure of the present disclosure is taken, the flavor layer 23 will be released from the mouth first, then release the blended sour and sweet taste in the flavor layer 22, and finally release the fruity taste in the flavoring layer 21, so that the consumers can feel multi-level flavor and taste through the sequential release, thereby improving the taste experience of the users.
In a preferred embodiment of the present disclosure, the seasoning layer 21 is selected from the group consisting of red grape juice, concentrated pomegranate juice, concentrated prunus wilsonii fruit juice, blackcurrant fruit juice, concentrated purple radish juice, concentrated blueberry juice, or other edible fruit juice; said flavor layer 22 is selected from the group consisting of lactitol, xylitol, potassium sorbate, citric acid, sodium citrate, sucralose, or other edible excipients; the flavour layer 23 is selected from the group consisting of natural red grape liquid flavour, cranberry flavour or other edible flavour.
In a preferred embodiment of the present disclosure, the structural protection layer 14 has a thickness of 0.08 to 0.12mm and is located outside the functional coating layer 13, and the thickness is designed to protect and maintain the configuration of the multi-layered grain structure and prevent the multi-layered grain structure from being damaged by gastric juice, and at the same time, ensure that the main effective components of the multi-layered grain structure can be absorbed by the human body normally.
A preferred embodiment of the present disclosure is characterized in that the structural protective layer 14 is selected from the group consisting of maltodextrin, starch, cellulose, pectin or other edible excipients.
In a preferred embodiment of the present disclosure, the multi-layered grain structure 1 of the skin cell activation and oxidation resistance promoting nutritional composition is soluble in water to form a solution; and it can be made into health products in the form of beverage, powder pack, colloid, capsule or lozenge.
Example one, fluorescent staining detection of skin cell damage
In the early stage of apoptosis, Phospholipid Serine (PS) inside a cell membrane turns to the outer side of the cell membrane, phospholipid binding protein (Annexin V) is combined with PS which turns to the outer side of the membrane in the early stage of apoptosis with high affinity, when the Annexin V with an Alexa Fluor TM 488 label (green fluorescence signal) is used, the apoptosis phenomenon and degree can be observed through a fluorescence microscope, meanwhile, cells with completely damaged cell membranes are dyed by matching with Propidium Iodide (PI), the PI can penetrate the damaged and incomplete cell membranes and enter the cells to be combined with cell nuclei to present red fluorescence signals, and the combination of the Annexin V and the PI can be used for observing the apoptosis and damaged conditions in the early stage and the late stage.
In this embodiment, hydrogen peroxide (simulated oxidation pressure) is used to trigger cells to move to an apoptosis pathway, and whether the multi-layer grain structure of the nutritional composition disclosed in the present disclosure has the effects of protecting cells and reducing apoptosis indexes is detected. In order to confirm the protective power of the multi-layer grain structure of the nutritional composition disclosed by the present disclosure against skin aging, the skin fibroblasts (CCD-966SK) were treated through the multi-layer grain structure of the nutritional composition disclosed by the present disclosure, and the damage of cells was examined to determine the protective effect on skin.
Materials and instruments
1. Cell lines: skin fibroblasts (CCD-966SK), obtained from American Type Culture Collection (ATCC), product number: cat. CRL-1881.
2. Culture medium: minimal Essential Medium (MEM, Minimum Essential Medium, available from Gibco, 61100-.
3. Phosphate buffered saline (PBS solution): purchased from Gibco, product No. 10437-.
4. Propidium iodide staining solution: purchased from BD Pharmingen, product No. 51-66211E.
5. Hoechst stain (Hoechst 33342): purchased from Thermo, product number cat.62249.
6. Annexin V stain (Alexa Fluor 488): purchased from Invitrogen, product number cat. a 13201.
7. Annexin V binding buffer: purchased from eBioscience, product No. 00-0055-43.
Experimental procedure
The experiment will be performed in three groups, an experimental group (multi-layered grain structure with the nutritional composition of the present disclosure added), a control group, and a control group, wherein the experimental group is subdivided into an experimental group a (the concentration of the sample in the culture medium is 0.0625%) and an experimental group B (the concentration of the sample in the culture medium is 0.125%), and each group is subjected to three replicates:
1. the skin fibroblasts were added at 2X 10 per well4In individual format, the culture was inoculated into 24-well plates containing 1ml of medium per well.
2. Adding a sample to be tested to the experimental group, wherein the sample is a multi-layer grain structure of the nutritional composition disclosed by the disclosure and is in 5% CO2And cultured at 37 ℃ for 24 hours.
3. After 24 hours, hydrogen peroxide (H) was added to the mixture at a final concentration of 500. mu.M2O2) In the experimental group and the control group, the treatment was carried out for 6 hours.
4. Cells were stained with propidium iodide staining solution (1: 250) and annexin V stain (1: 250) in annexin V binding buffer for 15-30 min.
5. Cells were stained with a hurst stain (1: 20000) for 3 minutes.
6. Rinse 2 times with 1 XPBS.
7. The results of skin cell damage were observed under a fluorescence microscope and analyzed for statistical significance between two values using the Student-t assay (. # indicates a P value <0.05 compared to the control group, # indicates a P value <0.01 compared to the control group, # indicates a P value <0.001 compared to the control group, # indicates a P value <0.05 compared to the control group, # indicates a P value <0.01 compared to the control group, and # indicates a P value <0.001 compared to the control group).
Results of the experiment
Please refer to fig. 2, which is a graph of a quantification of pre-apoptotic images processed by the multi-layered grain structure of the nutritional compositions of the present disclosure. As can be seen from the control group, after the apoptosis is induced by hydrogen peroxide, the expression level of Annexin V is obviously increased. The expression level of Annexin V of the control group is determined to be 100%, the expression level of Annexin V of the skin fibroblasts of the experimental group A is 87.17%, the skin cell damage rate of the experimental group A is lower than that of the control group, and the fact that the multilayer grain structure of the nutritional composition has the capacity of inhibiting early apoptosis caused by hydrogen peroxide is proved.
In another aspect, please refer to fig. 3, which is a graph illustrating quantification of post-apoptotic images of a nutritional composition according to the present disclosure. As can be seen from the control group, the cell apoptosis induced by hydrogen peroxide obviously induces the increase of the expression level of PI. The PI expression amount of the experimental group A is 77.46% and the PI expression amount of the experimental group B is 68.25% when the PI expression amount of the control group is determined to be 100%, the skin cell damage rate of the experimental group is lower than that of the control group, and the skin fibroblast damage rate of the experimental group B with the concentration of 0.125% is lower than that of the experimental group A with the concentration of 0.0625%, so that the multi-layer grain structure of the nutritional composition has the capability of inhibiting the later-stage apoptosis caused by hydrogen peroxide.
In conclusion, this example demonstrates that the multi-layered grain structure 1 of the nutritional composition for promoting skin cell activation and oxidation resistance of the present disclosure does have the efficacy of protecting skin fibroblasts from oxidative stress.
EXAMPLE two skin fibroblast activation test
In this embodiment, the degree of cell activation is detected by DNA synthesis, wherein an analog of thymine (Thymidine) is added during the cell culture period, when the cell cycle reaches the S phase of DNA synthesis, the analog can be inserted into newly synthesized DNA instead of thymine, and a fluorescent dye capable of combining the analog reacts with the analog, and the increase of fluorescence is detected by a flow cytometer to deduce the degree of cell activation; higher fluorescence intensity indicates higher degree of cell activation.
In this embodiment, in order to determine whether the multi-layered crystal grain structure of the nutritional composition of the present disclosure has the ability to promote cell activation, the skin fibroblast CCD-966SK is treated by penetrating through the multi-layered crystal grain structure of the nutritional composition, and DNA synthesis is detected by fluorescence to determine its effect on skin cell activation.
Materials and instruments
1. Cell lines: skin fibroblast cells CCD-966SK, from American Type Culture Collection (ATCC), product number Cat. CRL-1881.
2. Culture medium: minimal Essential Medium (MEM, Minimum Essential Medium, available from Gibco, 61100-.
3. Phosphate buffered saline (PBS solution): purchased from Gibco, product No. 10437-.
4. Cell proliferation ELISA (BrdU): available from Roche, product number 11647229001.
5. Enzyme immunoassay analyzer (ELISA reader): purchased from BioTek, product number FLx 800.
Experimental procedure
The experiments will be performed in experimental groups (groups of multi-layered grain structures to which the nutritional compositions of the present disclosure are added) and in control groups, each group being subjected to three replicates:
1. the skin fibroblasts were added at 3X 10 per well3In individual format, the cells were plated in 96-well plates containing 100. mu.l of medium per well in 5% CO2And cultured at 37 ℃ for 2 hours.
2. Adding a sample to be tested, which is a multi-layer grain structure of the nutritional composition disclosed by the disclosure, into the experimental group, so that the concentration of the sample in the culture medium is 0.0625%, and adding BrdU (5-bromo-2 '-deoxyuridine) 10 μ l with the concentration of 100uM into each group, and adding BrdU (5-bromo-2' -deoxyuridine) in 5% CO2And cultured at 37 ℃ for 24 hours.
3. The supernatants of each group were removed and 200 μ l of FixDenat reagent (in the cell activation assay kit) was added to each well and reacted for 30 minutes at room temperature.
4. The FixDenat reagent was removed and rinsed 1 time with 1 XPBS.
5. Mu.l of anti-BrdU-POD reagent (in the cell activation assay kit) was added to each well and reacted at room temperature for 30 minutes.
6. The anti-BrdU-POD reaction reagent was removed and rinsed 2 times with 200-300. mu.l of wash reagent (washing solution, said reagent in said cell activation detection kit).
7. Mu.l of substrate reagent (100 ml of tetramethyl-benzidine in the cell activation assay kit) was added to each well and reacted at room temperature for 5-30 minutes.
8. Add 25. mu.l of 1M H to each well2SO4And incubated with shaking at 300rpm for 1 minute.
9. The absorbance at OD450nm was measured and statistically analyzed, and the Student-t assay was used to analyze statistical significance between the two values (P value <0.05,. P value <0.01,. P value < 0.001).
Results of the experiment
Please refer to fig. 4, which is a diagram illustrating a ratio of relative cell activation rates of the experimental group and the control group treated by the multi-layered grain structure of the nutritional composition of the present disclosure, wherein the cell activation rate of the control group is defined as 100%, and the cell activation rate of the experimental group is increased to 209.5%, indicating that the skin fibroblasts of the experimental group have better activation capability, which confirms that the multi-layered grain structure of the nutritional composition of the present disclosure has an effect of promoting the activation of the skin fibroblasts.
EXAMPLE three inhibition of ROS production (Hydrogen peroxide treatment)
In this case, the change of the content of active oxygen species in human dermal fibroblasts CCD-966SK treated with the multi-layer grain structure of the nutritional composition was measured by a fluorescent probe DCFH-DA in combination with a flow cytometer.
Materials and instruments
1. Cell lines: human dermal fibroblast CCD-966SK (center for biological resource preservation and research (BCRC), No.60153), hereinafter referred to as CCD-966SK cell.
2. Culture medium: basal medium containing 10 vol% FBS (total bone serum, from Gibco). Wherein the basal medium is prepared from Eagle's minimal essential medium (MEM, available from Gibco under product number 15188-.
3. Phosphate buffered saline (PBS solution): purchased from Gibco, product No. 10437-.
DCFH-DA solution: dichlorodihydrofluorescein diacetate (2, 7-dichoro-dihydro-fluorescein diacetate, DCFH-DA; product number SI-D6883, available from Sigma) was dissolved in dimethyl sulfoxide (DMSO, available from Sigma, product number SI-D6883-50MG) to prepare a 5MG/ml solution of DCFH-DA.
5. Flow cytometry (Flow cytometry) was purchased from Beckman, product model Catalog No. 660519.
6. Hydrogen peroxide (H2O 2): purchased from Sigma-Aldrich, product number 95299-1L.
7. Trypsin (Trypsin-EDTA): 10 XTrypsin-EDTA (from Gibco) was diluted 10-fold with 1XPBS solution.
Experimental procedure
The experiment will be divided into three groups of an experiment group, a control group (a group without adding the multilayer crystal grain structure of the nutrient composition of the utility model and without being treated by hydrogen peroxide), and a control group (a group without adding the multilayer crystal grain structure of the nutrient composition of the utility model but treated by hydrogen peroxide), and each group is respectively subjected to two repeated experiments:
1. CCD-966SK cells were plated at 1X 10 per well5In this manner, the culture medium was inoculated into 6-well plates each containing 2ml of the medium.
2. Place the culture dish in 5% CO2And cultured at 37 ℃ for 24 hours.
3. The medium was removed.
4.2 mL of the experimental medium was added to each well of the plate and incubated at 37 ℃ for 1 hour.
The experimental medium of the experimental group was 2mL of cell culture medium supplemented with 5 μ L of the multi-layered grain structure of the nutritional composition of the present invention (i.e., the multi-layered grain structure of the nutritional composition of the present invention was 0.25% by volume of the cell culture medium).
The experimental medium for the control group was a simple 2mL cell culture medium (i.e. without the multi-layered grain structure of the nutritional composition of the present invention).
The experimental medium of the control group was a simple 2mL cell culture medium (i.e. without the multi-layered grain structure of the nutritional composition of the present invention).
5. Add 5 u g/mL DCFH-DA solution 2 u L each hole in the cell culture medium, DCFH-DA treatment of cells for 15 minutes.
6. After DCFH-DA treatment, H was added to the experimental medium of the experimental group and the experimental medium of the control group, respectively2O2And reacted at 37 ℃ for 1 hour. Specifically, 35% wt of hydrogen peroxide was diluted to 100mM (10. mu.L of hydrogen peroxide was added to 990. mu.L of redistilled water), and then 20. mu.L of 100mM hydrogen peroxide was added to 2mL of cell culture plates.
7. After reaction, each well was rinsed 2 times with 1mL of 1XPBS solution.
8. 200 μ L of trypsin was added to each well and reacted in the dark for 5 minutes. After the reaction, 6mL of cell culture medium was added to terminate the reaction.
9. The cells and cell culture medium in each well were collected into a respective 1.5mL centrifuge tube, and the centrifuge tube containing the cells and medium was centrifuged at 400Xg for 10 minutes.
10. After centrifugation, the supernatant was removed and the cell pellet was back-lysed with 1X PBS solution.
11. Centrifuge at 400Xg for 10 min.
12. After centrifugation, the supernatant was removed and the cells were resuspended in 1mL of 1XPBS solution in the dark to obtain the test cell fluid.
13. And detecting the fluorescence signal of DCFH-DA in the cell fluid to be detected of each hole by using a flow cytometer. The excitation wavelength for fluorescence detection is 450-490nm, and the emission wavelength is 510-550 nm. Because DCFH-DA enters cells and is hydrolyzed into DCFH (dichlorodihydrofluorescein) and then oxidized into DCF (dichlorofluorescein) capable of emitting green fluorescence by active oxygen substances, the fluorescence intensity of the cells treated by DCFH-DA can reflect the content of the active oxygen substances in the cells, and the proportion of the number of the cells highly expressed by the active oxygen substances in the cells to the number of the original cells can be known. Since the experiment was conducted in duplicate, the measurement results of duplicate experiments of each group were averaged to obtain an average value, and then the average values of the control group and the experimental group were converted into relative ROS production amounts by taking the average value of the control group as 100%, as shown in fig. 5.
Results of the experiment
As shown in fig. 5, it is clear from the results of the control group and the control group that the relative amount of ROS produced (high fluorescence) was greatly increased (about 590.5%) after the hydrogen peroxide treatment; it shows that hydrogen peroxide treatment can indeed lead to the production of reactive oxygen species in the cells, which in turn can cause subsequent damage to skin fibroblasts. On the other hand, according to the results of the control group and the experimental group, when the cells are treated by the multi-layer grain structure of the nutritional composition, the relative ROS generation amount is obviously reduced by about 369.9%; it was shown that the multilayered grain structure of the present nutritional compositions can effectively reduce the production or accumulation of reactive oxygen species within cells. In other words, the multi-layered grain structure of the nutritional composition may act as an active oxygen species scavenger. That is, the multi-layered grain structure of the nutritional composition may reduce oxidative damage to cells caused by reactive oxygen species and the like by reducing the amount of reactive oxygen species within the cells.
Example four skin efficacy test of grape yeast composite fruit and vegetable beverage
In this embodiment, the actual skin efficacy of the grape yeast composite fruit and vegetable beverage prepared from the multi-layer grain structure 1 of the nutritional composition for promoting skin cell activation and oxidation resistance disclosed in the present disclosure is tested through human body experiments.
The experiment is divided into a placebo group and an experimental group, wherein 30 subjects of 40-70 years old respectively drink one bottle of drink every day, the subjects of the placebo group drink one bottle of placebo (without the multilayer crystal grain structure of the nutritional composition disclosed by the disclosure) every day according to instructions, and the experimental group drinks one bottle of grape yeast composite fruit and vegetable drink every day for 8 weeks continuously. Wherein changes in skin spots, skin elasticity, skin wrinkles and skin whiteness were measured with a VISIA skin tester (VISIA Complexion Analysis System, available from Canfield, usa) before (i.e. week 0), after 4 (i.e. week 4) and after 8 (i.e. week 8) drinking, and changes in skin moisture content were measured with a skin surface moisture tester (Corneometer CM825, available from C + K, germany), and statistical significance between the two values was analyzed with Student-t assay (P value <0.05 at week 0, < P value <0.01 at week 0, < P value <0.001 at week 0, # <0.05 at placebo, # <0.01 at placebo, # at P value <0.001 at placebo, # at.
Please refer to fig. 6. Which is the average percentage change of the skin whiteness of a subject after drinking the grape yeast composite fruit and vegetable beverage disclosed by the disclosure. When the average skin whiteness before drinking (week 0) of the test subjects in the experimental group was determined to be 100%, the average skin whiteness after 4 weeks of drinking was increased to 102.8%, and the average skin whiteness after 8 weeks of drinking was increased to 103.6%. In other words, after the test subjects drink the grape yeast composite fruit and vegetable beverage of the present disclosure for 4 weeks and 8 weeks, the average skin whiteness is respectively improved by 2.8% and 3.6%, and is statistically significant. Therefore, the grape yeast composite fruit and vegetable beverage disclosed by the utility model has the efficacy of improving the skin whiteness, and can achieve the whitening effect.
Please refer to fig. 7. Which is the average percent change in skin spots after a subject drinks the grape yeast composite fruit and vegetable beverage of the present disclosure. Wherein, when the average skin spots of the subjects in the experimental group before drinking (week 0) was defined as 100%, the average skin spots after drinking for 4 weeks was reduced to 95.8%, and the average skin spots after drinking for 8 weeks was reduced to 93%. In other words, after the subjects drink the grape yeast composite fruit and vegetable beverage of the present disclosure for 4 weeks and 8 weeks, the average skin spots are reduced by 4.2% and 7%, respectively, and the average skin spots are statistically significant. Therefore, the grape yeast composite fruit and vegetable beverage disclosed by the disclosure has the effect of reducing skin spots.
Please refer to fig. 8. Which is the average change percentage of the skin moisture content of a subject after drinking the grape yeast composite fruit and vegetable beverage disclosed by the disclosure. Wherein, when the average skin moisture content of the subjects of the experimental group before drinking (week 0) was determined to be 100%, the average skin moisture content after drinking for 4 weeks was increased to 102.8%, and the average skin moisture content after drinking for 8 weeks was increased to 104.9%. In other words, after the subjects drink the grape yeast composite fruit and vegetable beverage of the present disclosure for 4 weeks and 8 weeks, the average skin moisture content is increased by 2.8% and 4.9%, respectively, and is statistically significant. Therefore, the grape yeast composite fruit and vegetable beverage disclosed by the disclosure has the effect of increasing the water content of skin.
Please refer to fig. 9. Which is the average percent change in skin elasticity of a subject after drinking the grape yeast composite fruit and vegetable beverage of the present disclosure. Wherein, when the average skin elasticity of the subjects in the experimental group before drinking (week 0) was defined as 100%, the average skin elasticity improvement was 103.1% after drinking for 4 weeks and 105.4% after drinking for 8 weeks. In other words, after the subjects drink the grape yeast composite fruit and vegetable beverage of the present disclosure for 4 weeks and 8 weeks, the average skin elasticity is increased by 3.1% and 5.4%, respectively, and is statistically significant. Therefore, the grape yeast composite fruit and vegetable beverage disclosed by the disclosure has the effect of improving the skin elasticity.
Please refer to fig. 10. Which is the average percent change in skin wrinkles after a subject drinks the grape yeast composite fruit and vegetable beverage of the present disclosure. Wherein, when the average skin wrinkle of the subjects of the experimental group before drinking (week 0) was defined as 100%, the average skin wrinkle reduction after drinking for 4 weeks was 98.6%, and the average skin wrinkle reduction after drinking for 8 weeks was 81%. In other words, after the subjects drink the grape yeast composite fruit and vegetable drink disclosed by the disclosure for 4 weeks and 8 weeks, the average skin wrinkles are reduced by 1.4% and 19%, respectively, and the average skin wrinkles are statistically significant. Therefore, the grape yeast composite fruit and vegetable beverage disclosed by the disclosure has the effect of reducing skin wrinkles.
Referring to FIGS. 6 to 10, the average skin whiteness was significantly improved by 2.8%, skin spots were significantly reduced by about 4.2%, skin moisture content was improved by about 2.8%, skin elasticity was significantly improved by 3.1%, and skin wrinkles were reduced by 1.4% when the grape yeast composite fruit and vegetable beverage of the present disclosure was drunk for 4 weeks in comparison to the experimental group before drinking (week 0). And the average skin whiteness can be obviously improved by 3.6 percent, the skin spots can be obviously reduced by about 7 percent, the water content of the skin can be obviously improved by about 4.9 percent, the skin elasticity can be obviously improved by 5.4 percent, and the skin wrinkles can be obviously reduced by 19 percent by continuously drinking the grape yeast composite fruit and vegetable beverage disclosed by the utility model for 8 weeks. Therefore, the grape yeast composite fruit and vegetable beverage can improve the skin condition after long-term use, namely the grape yeast composite fruit and vegetable beverage prepared by the multi-layer crystal grain structure 1 of the nutritional composition for promoting skin cell activation and antioxidation really has the effect of improving the skin condition.
In conclusion, the multilayer grain structure 1 of the nutritional composition for promoting skin cell activation and oxidation resistance is proved to have the efficacy of protecting skin cells from oxidative stress and promoting skin cell activation through experiments; it can be further made into health products in the form of beverage, powder bag, colloid, capsule or lozenge; the multilayer crystal grain structure is not easy to be damaged by gastric juice, meanwhile, the active ingredients of the multilayer crystal grain structure can be absorbed by a human body, the skin whiteness, the skin elasticity and the skin water content can be improved, skin spots and wrinkles are reduced, the multilayer coating structure can sequentially release fragrance, sweet and sour flavor and fruity taste, and the dietary experience of a user is improved.
Although the present disclosure has been described with reference to the foregoing embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the disclosure, and therefore, the scope of the disclosure should be limited only by the appended claims.
Claims (7)
1. A multi-layer grain structure of a nutrition composition for promoting skin cell activation and oxidation resistance is characterized by sequentially comprising a nutrition composition core, an oxidation resistant layer, a functional coating layer and a structural protection layer from the center to the outer side, wherein the components and the flavor of the multi-layer grain structure are protected through layer by layer and are made to be spherical.
2. The multi-layered grain structure of the nutritional composition for promoting skin cell activation and antioxidation according to claim 1, wherein the particle size of the nutritional composition core is 1 to 5mm, and the thickness of the antioxidation layer is 0.5 to 1 mm.
3. The multi-layered grain structure of a nutritional composition for promoting skin cell activation and antioxidation according to claim 1, wherein the functional coating layer is composed of soybean lecithin, has a thickness of 0.08 to 0.1mm, is located outside the antioxidation layer, has hydrophobicity, and has a plurality of micropores filled with colloid, which can increase adhesion of the functional coating layer, make the structural composition thereof more stable, protect effective ingredients from external environment, and maintain the configuration of the multi-layered grain structure.
4. The multi-layered grain structure of claim 1, wherein the multi-layered grain structure further comprises a flavor layer, a flavor layer and a fragrance layer disposed between the functional coating layer and the structural protection layer.
5. The multi-layered grain structure of the skin cell activation and oxidation resistance promoting nutritional composition according to claim 4, wherein the thickness of the flavor layer is 0.1 to 0.4mm, the thickness of the flavor layer is 0.8 to 1mm, and the thickness of the aroma layer is 0.06 to 0.1mm, so as to sequentially release aroma, sweet and sour flavor and fruity taste.
6. The multi-layered grain structure of skin cell activation and oxidation resistance promoting nutritional composition according to claim 1, wherein the structural protective layer has a thickness of 0.08 to 0.12mm and is located outside the functional coating layer to protect and maintain the configuration of the multi-layered grain structure and prevent the multi-layered grain structure from being damaged by gastric juice.
7. The multi-layered crystalline structure of a skin cell activation and oxidation resistance promoting nutritional composition according to claim 1, which is in the form of a drink, a powder pack, a gel, a capsule or a lozenge health care product.
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