An extraction process with high active ingredient retention

Abstract

The invention discloses an extraction process for retaining highly active ingredients, belonging to the field of natural product extraction. The extraction raw material is maqui fruit, and the extraction method is that the maqui fruit is first subjected to ester extraction, and then the residue is subjected to cellulose enzymatic hydrolysis, Pichia yeast primary fermentation, and Aspergillus oryzae secondary fermentation. The extraction method provided by the invention provides a new way for the reuse of maqui fruit.

Description

Technical Field

[0001] The invention belongs to the field of natural product extraction, and in particular relates to an extraction process for retaining highly active components. Background Art

[0002] Maqui Berry, a unique super fruit originating from South America, has attracted extensive attention in the fields of nutrition, medicine and cosmetics in recent years because of its rich content of various bioactive ingredients, such as high concentrations of flavonoids, vitamins and minerals. These bioactive ingredients not only have powerful antioxidant, anti-inflammatory and immune regulation functions, but also show significant benefits in cardiovascular health, skin care and anti-aging.

[0003] However, traditional extraction methods often have problems such as low extraction efficiency, large loss of effective ingredients, and high impurity content, which limit the application potential of maqui fruit extract in various fields. In order to overcome these challenges and improve the quality and utilization value of maqui fruit extract, researchers continue to explore and optimize the extraction process.

[0004] Compared with traditional extraction methods, the combined extraction method of ester extraction, enzymatic hydrolysis and fermentation can not only maximize the retention rate of active products, but also retain the natural activity of the extract as much as possible through the composite extraction mode of the three. Summary of the invention

[0005] The invention provides an extraction process for retaining highly active ingredients, and fully extracts the biologically active substances in the maqui fruit through enzymolysis and fermentation technology.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] Firstly, the present invention performs ester extraction on the maqui fruit.

[0008] S1: Wash the maqui fruit, remove impurities, freeze-dry, grind into powder, and store in a sealed and dark place at 5-8℃ before use.

[0009] S2: The maqui fruit powder was subjected to ester extraction using ethyl acetate, and then filtered to obtain a filtrate A1 and a residue B1.

[0010] S3: freeze-drying the ester extraction product A1 to obtain freeze-dried powder A2.

[0011] Preferably, the ethyl acetate in the ester extraction is water-saturated ethyl acetate, the extraction temperature is 40-65° C., and the number of extraction reflux times is 3-6 times.

[0012] Secondly, the present invention performs enzymatic hydrolysis on the B1 residue.

[0013] T1: washing, drying and crushing the residue of maqui ester extraction to obtain residue powder B2;

[0014] T2: Use cellulase, pectinase, protease and glucanase to enzymolyze B2, and filter to obtain the enzymolysis product A3, wherein the order of addition is first adding cellulase and pectinase for enzymolysis for 3±0.5; then adding glucanase for enzymolysis for 2±0.3h; then adding protease for enzymolysis for 4±0.2h, and then obtaining the enzymolysis product A3.

[0015] Preferably, the total added amount of the enzyme accounts for 8-10% of the raw material mass; the mass fraction ratio of the cellulase, pectinase, proteolytic enzyme and glucanase is 1:1-1.5:3:1.

[0016] Furthermore: The present invention performs a fermentation on the enzymatic hydrolysate A3

[0017] V1: The enzymatic hydrolysate A3 is fermented using yeast to obtain a fermentation product A4.

[0018] Preferably, the yeast strain used is Pichia pastoris, the V1 fermentation starting temperature is 28±2°C, the fermentation starting pH is 5.5±0.5; the fermentation starting oxygen content is 18±2%; the strain mass percentage accounts for 8-10% of the raw material mass; and the fermentation time is 16±4h.

[0019] Finally: The present invention performs secondary fermentation on the fermentation product A4

[0020] V2: After filtering and sterilizing the fermentation product A4, 2-4% of the mass percentage of the filtered fermentation product A4 was added with the maqui extract A2, and then fermented with Aspergillus, and filtered to obtain the fermentation product A5 and mycelium B3.

[0021] Preferably, the Aspergillus species used is Aspergillus oryzae, the fermentation starting temperature is 29±1°C, the fermentation starting pH is 5.5±0.5; the fermentation starting relative humidity is 92±2.5%; the mass percentage of the strain accounts for 6-10% of the raw material mass, the fermentation time is 48±12h, and the fermentation stirring speed is 45r / min.

[0022] V3: The mycelium B3 is enzymatically hydrolyzed using proteolytic enzymes and cellulase, and then freeze-dried to obtain product A6.

[0023] V4 mixed with A5 and A6 to obtain the final product A7.

[0024] Preferably, the mass fraction ratio of the proteolytic enzyme to the β-glucosidase is 1:1.5-2; the mass percentage of the proteolytic enzyme and the β-glucosidase accounts for 3-5% of the mass of the raw material.

[0025] In the present invention: the present invention uses the maqui fruit as the extract, freeze-drying and crushing the maqui fruit, firstly ester extraction is performed on it, and then the ester extraction residue is enzymatically hydrolyzed, and then the enzymatic hydrolysis product is subjected to secondary fermentation to fully extract and preserve the effective substances in the maqui fruit. The present invention fully extracts and preserves the effective substances in the maqui fruit, firstly ethyl acetate is used as an extractant to extract the natural active substances in the maqui fruit that are easily soluble in ester or water, and preserves the natural active ingredients that are easily oxidized, and secondly for the residue after extraction, which also contains rich polysaccharides, heterocyclic hydrocarbons, proteins and polypeptides, cellulase, pectinase, proteolytic enzyme, and glucanase are used to enzymatically hydrolyze the residue, firstly cellulase and pectinase are used to decompose the cell walls in the plant cells, and the macromolecules such as cellulose and pectin in the residue are decomposed, and then glucanase is used to decompose the long-chain glucan obtained by decomposing cellulose, pectin, etc. into glycosides, and then proteolytic enzymes are used to decompose the protein in the residue into amino acids, in preparation for the next fermentation. Finally, the enzymatic hydrolysis product is fermented. First, a primary fermentation is performed, using Pichia pastoris as the fermentation strain to obtain related secondary metabolites through the metabolism of Pichia pastoris, and then a secondary fermentation is performed using Aspergillus oryzae. Before the secondary fermentation and after the primary fermentation, the previously prepared maqui fruit extract needs to be added to the fermentation product, and the phenols and ketones in the extract are used to stimulate Aspergillus oryzae, and the metabolic pathway of Aspergillus oryzae is regulated to achieve the enrichment of flavonoid products.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1: The extraction method provided by the present invention can effectively preserve the biologically active substances in the maqui fruit;

[0028] 2: The extraction method provided by the present invention can effectively enrich flavonoids in maqui fruit;

[0029] 3: The extraction method provided by the present invention can effectively improve the efficacy of traditional maqui fruit extract in whitening, moisturizing, anti-aging and antibacterial aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Example 1 Chromatogram of Ester Timaqi Fruit Extract A1.

[0031] Figure 2 Example 1 Chromatogram of the final product of Maqui fruit extract A7. DETAILED DESCRIPTION

[0032] In order to better understand the present invention, the present invention is further described below in conjunction with specific examples, wherein the terms used in the examples are for describing specific embodiments and do not constitute limitations on the scope of protection of the present invention.

[0033] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0034] Some raw materials and their sources are as follows:

[0035] Maqi fruit: purchased from Chongqing Hongjiu Fruit Co., Ltd.

[0036] Pichia pastoris: purchased from Ningbo Testo Biotechnology Co., Ltd.

[0037] Aspergillus oryzae: purchased from Ningbo Testo Biotechnology Co., Ltd.

[0038] Saccharomyces cerevisiae was purchased from Ningbo Testo Biotechnology Co., Ltd.

[0039] Black mold: purchased from Ningbo Testo Biotechnology Co., Ltd.

[0040] Preparation, comprising the following steps:

[0041] Embodiment 1:

[0042] S1: Wash the maqui fruit, remove impurities, freeze-dry, grind into powder, and store in a sealed and dark place at 5-8℃ before use.

[0043] S2: The maqui fruit powder was subjected to ester extraction using ethyl acetate, and then filtered to obtain a filtrate A1 and a residue B1.

[0044] S3: freeze-drying the ester extraction product A1 to obtain freeze-dried powder A2.

[0045] T1: washing, drying and crushing the residue of maqui ester extraction to obtain residue powder B2;

[0046] T2: Use cellulase, pectinase, protease and glucanase to enzymolyze B2, and filter to obtain the enzymolysis product A3, wherein the order of addition is first adding cellulase and pectinase for enzymolysis 3; then adding glucanase for enzymolysis 2; then adding protease for enzymolysis for 4h, and then obtaining the enzymolysis product A3.

[0047] V1: The enzymatic hydrolysate A3 is fermented with Pichia pastoris to obtain a fermentation product A4; the V1 fermentation starting temperature is 28° C., the fermentation starting pH is 5.5; the fermentation starting oxygen content is 18; the mass percentage of the strain is 9% of the raw material mass; and the fermentation time is 16 hours.

[0048] V2: The fermentation product A4 is filtered and sterilized, and then 3% of the mass percentage of the filtered fermentation product A4 is added with the maqui fruit extract A2, and then fermented with Aspergillus oryzae, and filtered to obtain the fermentation product A5, with the fermentation product A5 as the final product; the fermentation starting temperature is 29°C, the fermentation starting pH is 5.5; the fermentation starting relative humidity is 92%; the mass percentage of the strain is 8% of the raw material mass, the fermentation time is 48h, and the fermentation stirring speed is 45r / min.

[0049] V2: After filtering and sterilizing the fermentation product A4, 2-4% of the mass percentage of the filtered fermentation product A4 was added with the maqui extract A2, and then fermented with Aspergillus, and filtered to obtain the fermentation product A5 and mycelium B3.

[0050] V3: The mycelium B3 is enzymatically hydrolyzed using proteolytic enzymes and cellulase, and then freeze-dried to obtain product A6.

[0051] V4: Mix A5 and A6 to obtain the final product A7.

[0052] Embodiment 2:

[0053] S1: Wash the maqui fruit, remove impurities, freeze-dry, grind into powder, and store in a sealed and dark place at 5-8℃ before use.

[0054] S2: The maqui fruit powder was subjected to ester extraction using ethyl acetate, and then filtered to obtain a filtrate A1 and a residue B1.

[0055] S3: freeze-drying the ester extraction product A1 to obtain freeze-dried powder A2.

[0056] T1: washing, drying and crushing the residue of maqui ester extraction to obtain residue powder B2;

[0057] T2: B2 was enzymatically hydrolyzed using cellulase, pectinase, protease and glucanase, and the enzymatic product A3 was obtained by filtration, wherein the order of addition was first adding cellulase and pectinase for hydrolysis for 3.5 h; then adding glucanase for hydrolysis for 2.3 h; then adding protease for hydrolysis for 4.2 h, and then obtaining the enzymatic product A3.

[0058] V1: The enzymatic hydrolysate A3 is fermented with Pichia pastoris to obtain a fermentation product A4; the V1 fermentation starting temperature is 30° C., the fermentation starting pH is 6; the fermentation starting oxygen content is 20%; the mass percentage of the strain is 10% of the raw material mass; and the fermentation time is 20 hours.

[0059] V2: The fermentation product A4 was filtered and sterilized, and then 4% of the mass percentage of the filtered fermentation product A4 was added with maqui extract A2, and then fermented with Aspergillus oryzae, and filtered to obtain fermentation product A5 and mycelium B3; the fermentation starting temperature was 30°C, the fermentation starting pH was 6; the fermentation starting relative humidity was 94.5%; the mass percentage of the strain was 10% of the raw material mass, the fermentation time was 60h, and the fermentation stirring speed was 45r / min.

[0060] V3: The mycelium B3 is enzymatically hydrolyzed using proteolytic enzymes and cellulase, and then freeze-dried to obtain product A6.

[0061] V4: Mix A5 and A6 to obtain the final product A7.

[0062] Embodiment 3:

[0063] S1: Wash the maqui fruit, remove impurities, freeze-dry, grind into powder, and store in a sealed and dark place at 5-8℃ before use.

[0064] S2: The maqui fruit powder was subjected to ester extraction using ethyl acetate, and then filtered to obtain a filtrate A1 and a residue B1.

[0065] S3: freeze-drying the ester extraction product A1 to obtain freeze-dried powder A2.

[0066] T1: washing, drying and crushing the residue of maqui ester extraction to obtain residue powder B2;

[0067] T2: B2 was enzymatically hydrolyzed using cellulase, pectinase, protease and glucanase, and the enzymatic product A3 was obtained by filtration, wherein the order of addition was first adding cellulase and pectinase for hydrolysis for 2.5 h; then adding glucanase for hydrolysis for 1.7 h; then adding protease for hydrolysis for 3.8 h, and then obtaining the enzymatic product A3.

[0068] V1: The enzymatic hydrolysate A3 is fermented with Pichia pastoris to obtain a fermentation product A4; the V1 fermentation starting temperature is 26° C., the fermentation starting pH is 5; the fermentation starting oxygen content is 16%; the mass percentage of the strain is 8% of the raw material mass; and the fermentation time is 12 h.

[0069] V2: The fermentation product A4 was filtered and sterilized, and then 2% of the mass percentage of the filtered fermentation product A4 was added with maqui extract A2, and then fermented with Aspergillus oryzae, and filtered to obtain fermentation product A5 and mycelium B3; the fermentation starting temperature was 28°C, the fermentation starting pH was 5.0; the fermentation starting relative humidity was 90%; the mass percentage of the strain accounted for 6% of the raw material mass, the fermentation time was 36h, and the fermentation stirring speed was 45r / min.

[0070] V3: The mycelium B3 is enzymatically hydrolyzed using proteolytic enzymes and cellulase, and then freeze-dried to obtain product A6.

[0071] V4: Mix A5 and A6 to obtain the final product A7.

[0072] Comparative Example 1: S1-S3 of Example 1 were followed to obtain freeze-dried powder A2 as the final product.

[0073] Comparative Example 2 follows S1-S3 and T1-T2 of Example 1, with enzymatic hydrolysis product A3 as the final product.

[0074] Comparative Example 3 follows S1-S3, T1-T2, and V1 of Example 1, with the primary fermentation product A4 as the final product.

[0075] Comparative Example 4 follows S1-S3, T1-T2, V1-V2 of Example 1, with the secondary fermentation product A5 as the final product.

[0076] Comparative Example 5 follows S1-S3, T1-T2, and V1-V3 of Example 1, with mycelium enzymatic hydrolysis product A6 as the final product.

[0077] Comparative Example 6 follows S1-S3, V1-V4 of Example 1, but differs from Example 1 in that the residue B2 is not subjected to enzymatic hydrolysis, but is directly fermented, and the remaining steps are the same as those of Example 1 to obtain the fermentation product C1.

[0078] Comparative Example 7 follows S1-S3, T1-T2, and V1-V4 of Example 1. The difference from Example 1 is that the yeast used in V1 is Saccharomyces cerevisiae, and the remaining steps are the same as those of Example 1 to obtain a fermentation product C2.

[0079] Comparative Example 8 follows S1-S3, T1-T2, and V1-V4 of Example 1. The difference from Example 1 is that the Aspergillus used in V2 is black mold, and the remaining steps are the same as in Example 1 to obtain fermentation product C3.

[0080] Comparative Example 9 follows S1-S3, T1-T2, and V1-V1 of Example 1. The difference from Example 1 is that V2 does not add the maqui extract A1. The remaining steps are the same as Example 1 to obtain the fermentation product C4.

[0081] Cytotoxicity assay

[0082] Take the human immortalized epidermal cell line (HaCaT) with stable growth state and adjust the cell concentration to 1×10 5 / mL, 100μL per well. Inoculate into 96-well cell culture plates (except the outermost wells and blank control wells). Add 200μL of PBS to the outermost wells of the cell culture plate to reduce the evaporation of the culture medium. Place in a carbon dioxide incubator and culture at 37℃±1℃, CO2 concentration 5.0%±1% until the cell fusion reaches more than 90%. Add samples, the sample concentration is 10mg / L, set up blank control and sample groups, 100μL per well. Start adding samples (sample group) and PBS (blank control) at the same time interval between each two columns to ensure that the intervention time of each test substance on the cells is 5min. After 5min, remove the test substances in turn, add PBS to each well to wash the test substances, and wash twice. When using a vacuum suction pump or a discharge gun to remove the liquid in the well, be careful to avoid sucking away the surviving cells and affecting the experimental results. Add 100μL of complete culture medium and 10μL of CCK-8 solution to each well, and place in an incubator to culture for about 2h. At a wavelength of 450 nm, the absorbance (OD) value was measured and the cell viability was calculated; the results are shown in Table 1.

[0083]

[0084] Table 1 Cytotoxicity test

[0085] Experimental Group HaCaT survival rate Example 1 98.34±0.22％ Example 2 97.27±0.47％ Example 3 98.15±0.17％ blank 100％

[0086] According to the experimental results, the cell survival rate is maintained above 90%. The extract prepared by the preparation process provided in this experiment has little effect on cell viability.

[0087] Hyaluronidase inhibition assay

[0088] Hyaluronic acid, also known as hyaluronic acid, is a powerful moisturizer. It can attract and lock in moisture, maintain skin moisture, and improve dry, rough and dehydrated skin conditions. Hyaluronic acid can also enhance the biological activity of cells and has a strong synergistic effect on repairing and protecting the skin. Therefore, by inhibiting hyaluronidase, the decomposition rate of hyaluronic acid in the skin is reduced, thereby achieving long-term moisture retention and repair of the skin, which is of great significance.

[0089] (1) Take four groups of clean test tubes, label them A, B, C, and D, add 50 μL of the sample (10 mg / L) to test tubes A and B, add 50 μL of distilled water to test tubes C and D, add 50 μL of 500 U / mL hyaluronidase solution to test tubes A and C, add 50 μL of pH 5.6 acetate buffer to test tubes B and D, and place the test tubes in a 37°C incubator for 20 min.

[0090] (2) Add 10 μL of 2.5 mol / L CaCl2 solution to each of the four test tubes and place them in an incubator at 37°C for 20 min.

[0091] (3) Add 50 μL of 0.5 mg / mL sodium hyaluronate solution to test tubes A and C, and add 50 μL of pH 5.6 acetate buffer to test tubes B and D. Place the test tubes in a 37°C incubator for 40 min and then take them out and place them at room temperature for 10 min.

[0092] (4) Add 50 μL of distilled water, 10 μL of 5 mol / L NaOH solution, and 50 μL of acetylacetone solution to the four test tubes respectively. Place the test tubes in a boiling water bath for 30 min, then in an ice bath for 10 min, and finally at room temperature for 10 min.

[0093] (5) Add 100 μL of P-DAB reagent to each test tube, and then measure the absorbance at 530 nm using an ELISA reader.

[0094]

[0095] Wherein: A is the OD value of the sample solution; B is the OD value of the sample blank; C is the OD value of the control sample solution; D is the OD value of the control sample blank.

[0096] Table 2 Hyaluronidase inhibition rate

[0097] Experimental Group Hyaluronidase inhibition rate Example 1 94.5±0.79％ Example 2 92.4±0.17％ Example 3 91.4±0.41％ Comparative Example 1 37.28±1.14％ Comparative Example 2 26.52±0.92％ Comparative Example 3 38.17±0.47％ Comparative Example 4 42.15±1.44％ Comparative Example 5 37.26±0.93％ Comparative Example 6 23.27±0.42％ Comparative Example 7 19.27±2.54％ Comparative Example 8 14.24±0.75％ Comparative Example 9 35.52±0.47％

[0098] According to the analysis of the results: From the results of Examples 1-3, it can be seen that the extracts prepared by the preparation process provided in the embodiments of the present invention have good hyaluronidase inhibitory efficacy, especially the effect of Example 1 is better, indicating that the process defined by the present invention can effectively improve the inhibitory effect of the extract on hyaluronidase.

[0099] From the results of Example 1 and Comparative Examples 1-5, it can be seen that the products of any stage of this process cannot achieve the effects specified by the process of the present invention.

[0100] Comparing the results of Example 1 with Comparative Examples 6-9, it can be seen that changing the strains used in this process or changing the steps used in this process cannot achieve the effect achieved by this process.

[0101] Zebrafish tail moisturizing test

[0102] The zebrafish epidermis has a certain osmotic pressure tolerance range, and water loss will occur if the tolerance range is exceeded. Using a high osmotic pressure solution to treat zebrafish will cause its tail to shrink and become smaller due to water loss. Therefore, an induced model of zebrafish hydration and moisturizing can be established to evaluate the effect of moisturizing products.

[0103] (1) Breeding: Zebrafish were paired at a male-female ratio of 2:1, and the eggs were collected and incubated overnight (28°C incubator).

[0104] (2) Experimental groups: The experiment set up a blank control group and an experimental group. The experimental groups included a model group, a positive group, and a sample group, with 8 2 dpf fry in each group.

[0105] (3) Solution preparation: Accurately weigh 1 mg of the samples of the examples and comparative examples and prepare the sample solution to be tested, and use urea (0.05%) as the positive control for the experiment, adding 1 mg to each well. After the administration is completed, place the 6-well plate in a 28° C. incubator in the dark for 24 hours.

[0106]

[0107] Test results: as shown in Table 3 below.

[0108] Table 3 Zebrafish moisturizing effect test

[0109] Experimental Group Zebrafish tail area reduction inhibition rate / % Positive group 1 blank 0 Example 1 85.42 Example 2 86.51 Example 3 85.37 Comparative Example 1 16.42 Comparative Example 2 47.35 Comparative Example 3 45.73 Comparative Example 4 41.52 Comparative Example 5 53.52 Comparative Example 6 22.73 Comparative Example 7 54.17 Comparative Example 8 51.04 Comparative Example 9 47.34

[0110] According to the results analysis in Table 3: From the results of Examples 1-3, it can be seen that the process provided by the embodiments of the present invention has a good extraction effect, especially the effect of Example 1 is better, indicating that the process and formula combination defined by the present invention can effectively improve the inhibitory effect of the extract on the reduction of the zebrafish tail area.

[0111] From the results of Example 1 and Comparative Examples 1-5, it can be seen that the products of any stage of this process cannot achieve the effects specified by the process of the present invention.

[0112] Comparing the results of Example 1 with Comparative Examples 6-9, it can be seen that changing the strains used in this process or changing the steps used in this process cannot achieve the effect achieved by this process.

[0113] Antibacterial effect on Propionibacterium acnes

[0114] Referring to WS / T 650-2019, the inhibition ring test was used to wash the 24-hour fresh slant culture of P. acnes with PBS and dilute it to 5.0×10 5 CFU / mL-5.0×10 6 CFU / mL is reserved.

[0115] Preparation of antibacterial sheets: Cut filter paper sheets with a diameter of 5 mm and a thickness not exceeding 4 mm, and immerse each group of 4 sheets in 10 mL of sample solution.

[0116] Preparation of negative control samples: Take samples of the same material, make discs (blocks) of the same size as the test group, and soak them in sterile water.

[0117] Use a sterile cotton swab to apply 5.0×10 5 CFU / mL-5.0×10 6 CFU / mL acne bacteria suspension is evenly applied on the surface of a suitable culture medium plate for 3 times. Each time it is applied, the plate should be rotated 60°, and finally the cotton swab should be applied around the edge of the plate. Cover the plate and dry it at room temperature for 5 minutes. For each test, place 1 infected plate, 4 test samples and 1 negative control sample on each plate, for a total of 5 samples. Use sterile tweezers to take the sample and paste it on the surface of the plate. The negative control sample is pasted in the center of the plate, and the test sample is pasted around. After pasting, use sterile tweezers to gently press the sample to make it close to the surface of the plate. The distance between the centers of each sample is more than 25mm, and the distance from the periphery of the plate is more than 15mm. Cover the plate, incubate at 36℃±1℃ for 16h~18h to observe the results, and repeat the test 3 times. Use a vernier caliper to measure the diameter of the antibacterial ring (including the patch) and record it. When measuring the antibacterial ring, a uniform and completely sterile antibacterial ring should be selected, and the diameter should be measured based on the outer edge of the antibacterial ring.

[0118] The negative control sample should not produce an antibacterial ring. If the diameter of the antibacterial ring of the test sample is greater than 7mm, it is judged to have an antibacterial effect; if the diameter of the antibacterial ring is ≤7mm, it is judged to have no antibacterial effect. If the antibacterial effect is found in the three repeated tests, it is judged to be qualified. See Table 4 for specific results.

[0119] Table 4 Propionibacterium acnes inhibition test

[0120]

[0121]

[0122] The results show that: the comparison between the embodiment and the comparative example shows that the embodiment is superior to the comparative example in terms of antibacterial performance. Therefore, the effect achieved by the present invention cannot be achieved by preparing the product according to the process ratio specified in the present invention, and any default will fail to achieve the effect achieved by the present invention.

[0123] Inflammatory factor inhibition test

[0124] TNF-α is the most common inflammatory factor in inflammatory reactions. It can induce the phenotype of vascular endothelial cells to change to an inflammatory type, leading to changes in the body's metabolism and hemodynamics, and promoting the production of inflammatory mediators. Inflammatory mediators can increase vascular permeability, causing local congestion and edema, and may stimulate nerve endings to cause itching and pain. Therefore, the inhibition of TNF-α helps soothe and repair the skin.

[0125] RAW264.7 cells grown to the logarithmic phase were cultured at 1.5×10 5Cells were inoculated at a density of 100 μg / mL in a 24-well plate, with 800 μL of cell suspension per well, and cultured in a 37°C, 5% CO2 incubator for 24 hours. When the cells grew to near fusion, 50 μL of the sample concentration of 10 mg / L was administered, with 3 replicates per group. The positive control was 50 μL of 10 μg / mL dexamethasone, and 1 μg / mL lipopolysaccharide (LPS) was used for stimulation 1 hour later. After incubation in a cell culture incubator for 24 hours, the cell culture fluid was transferred to a centrifuge tube, centrifuged at 5000 r / min for 10 minutes, and the cell supernatant was collected and tested according to the instructions of the mouse TNF-α enzyme-linked immunosorbent assay kit.

[0126]

[0127] Table 5 Results of RAW secretion of TNF-α

[0128]

[0129]

[0130] From the analysis of the results in Table 5: It can be seen from the results of Examples 1-3 that the extracts prepared by the process provided in the embodiments of the present invention have good TNF-α inhibitory efficacy, especially the effect of Example 1 is better, indicating that using Example 1 as the fermentation process parameter can effectively improve the inhibitory effect of the maqui fruit extract on TNF-α.

[0131] From the results of Example 1 and Comparative Examples 1-5, it can be seen that the products of any stage of this process cannot achieve the effects specified by the process of the present invention.

[0132] Comparing the results of Example 1 with Comparative Examples 6-9, it can be seen that changing the strains used in this process or changing the steps used in this process cannot achieve the effect achieved by this process.

[0133] Cell migration rate

[0134] The degree of cell migration has an important impact on wound healing and skin repair. In the process of wound healing, cell migration is an important step for epidermal cells to cover the wound, so the higher the cell migration rate, the faster the wound heals. At the same time, cell migration is also involved in the repair process after skin damage. If cell migration is impaired, it will lead to skin repair disorders, which in turn cause various skin diseases.

[0135] Inoculate cells into 12-well plates at the corresponding inoculation density and incubate overnight at 37°C in a CO2 incubator. When the cell plating rate in the 12-well plate reaches about 80%, scratch the sample group and the solvent control group. After the scratching, wash the cells with PBS to remove dead cells, add fresh EGF-free keratinocyte culture medium, add 50μL of the sample at 10mg / L, and take photos and record using an inverted microscope. Incubate in a 37°C, 5% CO2 incubator for 24h, take photos and record again, and use ImageJ software for statistical analysis.

[0136] Table 6 Hacat cell migration rate

[0137]

[0138]

[0139] From the analysis of the results in Table 6: It can be seen from the results of Examples 1-3 that the extracts prepared by the process provided in the embodiments of the present invention have the effect of significantly improving the cell migration rate, especially the effect of Example 1 is better, indicating that using Example 1 as the process parameter can effectively improve the cell migration rate.

[0140] From the results of Example 1 and Comparative Examples 1-5, it can be seen that the products of any stage of this process cannot achieve the effects specified by the process of the present invention.

[0141] Comparing the results of Example 1 with Comparative Examples 6-9, it can be seen that changing the strains used in this process or changing the steps used in this process cannot achieve the effect achieved by this process.

[0142] Tyrosinase activity inhibition assay

[0143] Tyrosinase is a key enzyme in the synthesis of melanin. Inhibiting it can effectively reduce the synthesis of melanin and play a whitening role.

[0144] Solution preparation:

[0145] Prepare an enzyme solution with a concentration of 5U / L, dispense it into 1.5mL EP tubes, and store it in liquid nitrogen. Before use, dilute it with PBS to prepare a 0.5U / L tyrosinase solution, and prepare it before use. Accurately weigh L-tyrosine and L-dopa, add an appropriate amount of PBS solution, and dissolve them by ultrasonic at room temperature for 30 minutes to prepare a substrate solution with a concentration of 3.0mmol / L; Kojic acid (positive control) solution Accurately weigh 1g of kojic acid, dissolve it in DMSO, add an appropriate amount of PBS solution to dilute it, and prepare it to the concentration required for the experiment, with a final DMSO content of 0.5%; weigh 1mg of the extract, dissolve it in 10mLDMSO, and dilute it with an appropriate amount of PBS solution, with a final DMSO content of 0.5%.

[0146] Determination of tyrosinase activity inhibition rate:

[0147] 40 μL of sample, 10 μL of PBS, and 10 μL of enzyme solution were mixed in sequence, incubated at 37 degrees Celsius for 10 minutes, 50 μL of substrate solution was added, and incubated for 25 minutes. The absorbance value at 475 nm was detected on a multifunctional microplate reader, and the inhibition rate was calculated based on the absorbance value.

[0148]

[0149] Table 7 Tyrosinase inhibition rate

[0150] Experimental Group Inhibition rate / % Example 1 89.33％ Example 2 93.27％ Example 3 91.24％ Comparative Example 1 47.38％ Comparative Example 2 33.17％ Comparative Example 3 31.14％ Comparative Example 4 22.55％ Comparative Example 5 14.27％ Comparative Example 6 24.22％ Comparative Example 7 20.92％ Comparative Example 8 20.47％ Comparative Example 9 37.24％ Kojic Acid 100％

[0151] According to the results of Examples 1-3 in Table 7, the extracts prepared by the process provided by the embodiments of the present invention have a good inhibitory effect on tyrosinase, especially Example 1 has a better effect.

[0152] From the results of Example 1 and Comparative Examples 1-5, it can be seen that the products of any stage of this process cannot achieve the effects specified by the process of the present invention.

[0153] Comparing the results of Example 1 with Comparative Examples 6-9, it can be seen that changing the strains used in this process or changing the steps used in this process cannot achieve the effect achieved by this process.

[0154] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present application.

Claims

1. An extraction process for retaining highly active ingredients, characterized in that: The extract A2 and the residue B1 are obtained by ester extraction of the maqui fruit, and then the residue B1 is hydrolyzed with cellulase to obtain the hydrolyzate A3, the hydrolyzate A3 is subjected to secondary fermentation to obtain the fermentation product A5 and mycelium B3, the mycelium B3 is hydrolyzed to obtain the hydrolyzate A6, and A5 and A6 are mixed to obtain the maqui fruit extract A7; The specific steps of ester extraction are as follows: S1: Wash the maqui fruit and crush it to get A1: S2: reflux extraction of A1 using ethyl acetate to obtain extract A2 and residue B1; The cellulase enzymolysis specifically uses cellulase, pectinase, proteolytic enzyme, and glucanase; The specific steps of the secondary fermentation are as follows: V1: ferment the enzymatic hydrolysate A3 with yeast, and filter to obtain the fermentation product A4; V2: fermentation product A4 was treated with Aspergillus, and the filtrate was filtered and freeze-dried to obtain fermentation product A5 and mycelium B3; V3: enzymatically hydrolyzing mycelium B3, filtering, and freeze-drying the filtrate to obtain enzymatic hydrolyzate A6; V4: Mix A5 and A6 to obtain maqui fruit extract A7; The yeast is Pichia pastoris; the Aspergillus is Aspergillus oryzae; The V2 fermentation process also includes 2-4% of the mass percentage of the raw material mass of maqui ester extract A2; The enzymes used in the V3 enzymolysis are protease and cellulase.

2. The extraction process according to claim 1, characterized in that: The reflux extraction times are 3-6 times, and the extraction temperature is 40-65°C.

3. The extraction process according to claim 1, characterized in that: T1: washing, drying and crushing the residue B1 to obtain the residue B2; T2: B2 is enzymatically hydrolyzed using cellulase, pectinase, protease and glucanase, and then filtered to obtain the enzymatic hydrolysis product A3.

4. The extraction process according to claim 1, characterized in that: The initial temperature of enzymatic hydrolysis is 35±3°C; the mass percentage of the enzyme is 8-10% of the mass of the raw material; the mass fraction ratio of the cellulase, pectinase, proteinase and glucanase is 1:1-1.5:3:1; the order of adding the cellulase, pectinase, proteinase and glucanase is to first add the cellulase and pectinase for enzymatic hydrolysis for 3±0.5h, then add the glucanase for enzymatic hydrolysis for 2±0.3h, and then add the proteinase for enzymatic hydrolysis for 4±0.2h.

5. The extraction process according to claim 1, characterized in that: The starting temperature of the V1 fermentation is 28±2°C, the starting pH of the fermentation is 5.5±0.5; the starting oxygen content of the fermentation is 18±2%; the mass percentage of the strain is 8-10% of the mass of the raw materials; the fermentation time is 16±4h; the starting temperature of the V2 fermentation is 29±1°C, the starting pH of the fermentation is 5.5±0.5; the starting relative humidity of the fermentation is 92±2.5%; the mass percentage of the strain is 6-10% of the mass of the raw materials, the fermentation time is 48±12h, and the fermentation stirring speed is 45r / min.

6. The extraction process according to claim 1, characterized in that: The mycelium B3 is enzymatically hydrolyzed with proteolytic enzyme and cellulase, wherein the mass fraction ratio of the proteolytic enzyme to the cellulase is 1:1.5-2; and the mass percentage of the proteolytic enzyme and the cellulase accounts for 3-5% of the mass of the raw material.

7. An application of a maqui fruit extract, characterized in that: The invention relates to the use of the maqui fruit extract prepared by the extraction process described in claims 1 to 6 in the preparation of cosmetics or medicines.

Images