A method for preparing oat beta-glucan and its application and composition

Abstract

The application provides a preparation method and application and composition of oat beta-glucan, and relates to the technical field of cosmetics.The preparation method comprises the following steps: (1) oat is crushed, mixed with alkaline water, and subjected to subcritical water extraction at 150-180 DEG C to obtain a crude extract; (2) the pH of the crude extract is adjusted to be acidic, the crude extract is allowed to stand, and the supernatant is taken; (3) the pH of the supernatant is adjusted to be neutral, then amylase and neutral protease are added to perform enzymatic reaction, and the filtrate is obtained through filtration; and (4) the filtrate is purified through Sephacryl S-200 gel resin, concentrated, and oat beta-glucan is obtained.The method significantly improves the yield of oat beta-glucan, maximally retains the anti-aging activity of oat beta-glucan, and has better anti-aging, anti-wrinkle and skin elasticity improvement effects; oat beta-glucan and other anti-aging ingredients are mutually matched, and the effect of significantly removing DPPH free radicals is brought.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a method for preparing oat β-glucan and its application and composition. Background Technology

[0002] Oat β-glucan is an unbranched linear polysaccharide found in the cell walls of oat tissues. It is a water-soluble fiber, comprising approximately 2.5%-7.8% of oat content. It is an important active ingredient in oats, possessing functional activities such as lowering blood sugar and cholesterol, regulating immunity, and regulating gastrointestinal health. It is widely used in food, health products, and cosmetics. Extraction methods for oat β-glucan generally include alkaline extraction, water extraction, and enzymatic extraction. The extraction process involves steps to remove oat starch and protein to obtain oat β-glucan with high purity and high content.

[0003] Oat bran itself contains certain oils and other substances, which affect the dissolution and purity of β-glucan. Therefore, during the extraction process, it is often necessary to add a certain amount of organic solvent to dissolve these remaining substances before purification. Even after purification, some organic solvents and other substances may remain in the β-glucan, resulting in high extraction costs, limited purity, and poor extraction rates. Furthermore, the residual organic solvents are somewhat toxic, affecting its application in cosmetics. Researchers have conducted extensive studies on the preparation process of oat β-glucan.

[0004] Chinese invention patent CN111978430A discloses a method for preparing oat β-glucan, including the following steps: first, microwave drying and supercritical carbon dioxide extraction of oat bran; then, dispersing the oat bran in water and electrolyzing it using a carbon rod electrode; next, ultrasonicating the electrolyzed mixture, adding a mixed enzyme for repeated enzymatic hydrolysis to obtain an enzymatic hydrolysate; adjusting the pH of the enzymatic hydrolysate to 3-6, heating, allowing it to settle, and separating the supernatant; adding chitosan and sodium alginate to the supernatant, filtering and separating it using ultrafiltration membranes with different molecular weight cutoffs in a series membrane separation method, collecting the retentate in segments, adding ethanol to the retentate, collecting the precipitate after alcohol precipitation, and vacuum drying the precipitate to obtain high-purity oat β-glucan of different molecular weights. This method involves complex processing steps, requiring not only microwave and supercritical carbon dioxide extraction but also electrolysis and ultrasonication, increasing the processing cost.

[0005] Chinese invention patent CN1300323A discloses a method for preparing oat β-glucan, including the following steps: (1) oats are ground into oat bran; (2) oat bran is mixed with water at 45-65℃ and the pH is adjusted to 9-11; (3) after separation, the filtrate is collected, α-amylase is added, and the mixture is kept at 70-90℃ for 1-2 hours for enzymatic hydrolysis; (4) the mixture is cooled to 10-30℃, the pH is adjusted to 4.5-5.0, stirred, and allowed to stand to precipitate proteins; (5) the supernatant is obtained by centrifugation, ultrafiltration is performed to concentrate the supernatant, and isopropanol is added to the concentrate to obtain a β-glucan gel precipitate; (6) the β-glucan gel precipitate is centrifuged and vacuum dried to obtain the β-glucan product. In the above method, although no organic solvent is used in the process of removing oil, ethanol or isopropanol is used in the final alcohol precipitation step, which still has the drawback of using organic solvents.

[0006] To address the shortcomings of existing methods for preparing oat β-glucan, which are complex and often involve the use of organic solvents, a method is needed that does not use organic solvents, is simple in procedure, efficiently removes proteins, and ultimately yields oat β-glucan with high purity and high activity of active factors. Summary of the Invention

[0007] This invention addresses the problems existing in the prior art by providing a method for preparing oat β-glucan, its application, and a composition thereof. Oats are pulverized and then subjected to subcritical water extraction to obtain a crude extract rich in oat β-glucan. The supernatant of the crude extract is then enzymatically hydrolyzed using amylase and neutral protease to efficiently remove impurities. The hydrolysate is purified using Sephacryl S-200 gel resin to obtain oat β-glucan with high yield, high purity, and high activity of active factors. The oat β-glucan obtained by the above method has superior anti-aging, anti-wrinkle, and skin elasticity-improving effects.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] First, this invention provides a method for preparing oat β-glucan, comprising the following steps:

[0010] (1) Oat powder is mixed with alkaline water for subcritical water extraction at an extraction temperature of 150-180℃ to obtain crude extract;

[0011] (2) Adjust the pH of the crude extract to acidic, let it stand, and take the supernatant;

[0012] (3) Adjust the pH of the supernatant to neutral, then add amylase and neutral protease to carry out enzymatic hydrolysis, and filter to obtain filtrate;

[0013] (4) The filtrate was purified by passing it through Sephacryl S-200 gel resin and concentrated to obtain oat β-glucan.

[0014] Preferably, in step (1), the oats are ground to 100-200 mesh.

[0015] Preferably, in step (1), the pH of the alkaline water is 9-10.

[0016] More preferably, in step (1), the pH of the alkaline water is 9.5.

[0017] Preferably, in step (1), the alkaline water used is not limited to sodium bicarbonate, sodium carbonate, or sodium hydroxide.

[0018] Preferably, in step (1), the subcritical water extraction is performed at an extraction temperature of 160°C.

[0019] Preferably, in step (1), the subcritical water extraction takes 20-50 minutes.

[0020] More preferably, in step (1), the subcritical water extraction takes 30 minutes.

[0021] Preferably, in step (1), the subcritical water extraction uses a solid-liquid ratio of oats to water of 1:15-25 g / mL.

[0022] More preferably, in step (1), the subcritical water extraction uses an oat-to-water solid-liquid ratio of 1:20 g / mL.

[0023] Preferably, in step (2), the pH is 3.8-4.3.

[0024] More preferably, in step (2), the pH is 4.0.

[0025] Preferably, in step (2), the acidic substance used to adjust the pH is selected from at least one of citric acid, malic acid, and acetic acid.

[0026] More preferably, in step (2), the acidic substance used to adjust the pH is citric acid.

[0027] Preferably, in step (2), the settling time is 8-16 hours.

[0028] More preferably, in step (2), the settling time is 12 hours.

[0029] Preferably, in step (3), the alkaline substance used to adjust the pH is not limited to sodium bicarbonate, sodium carbonate, or sodium hydroxide.

[0030] Preferably, in step (3), the mass ratio of amylase to neutral protease is 3-5:1.

[0031] More preferably, in step (3), the mass ratio of the amylase to the neutral protease is 4:1.

[0032] Preferably, in step (3), the amount of amylase added is 5-15% of the weight of oats.

[0033] More preferably, in step (3), the amount of amylase added is 9-11% of the weight of the oats.

[0034] More preferably, in step (3), the amount of amylase added is 10% of the weight of the oats.

[0035] Preferably, in step (3), the enzymatic hydrolysis reaction is specifically: an enzymatic hydrolysis reaction with stirring at 45-55℃ for 2-6 hours.

[0036] More preferably, in step (3), the enzymatic hydrolysis reaction is specifically: an enzymatic hydrolysis reaction with stirring at 50°C for 3 hours.

[0037] Preferably, in step (3), the solid-liquid ratio of the enzymatic hydrolysis reaction is 1:20-30 g / mL, and the solid mass refers to the mass of oats.

[0038] More preferably, in step (3), the solid-liquid ratio of the enzymatic hydrolysis reaction is 1:25 g / mL.

[0039] Preferably, in step (4), the purification process is carried out at a flow rate of 1-3 BV / h.

[0040] More preferably, in step (4), the purification process is carried out at a flow rate of 2 BV / h.

[0041] Then, the present invention provides oat β-glucan prepared by the above preparation method.

[0042] Furthermore, the present invention provides the application of the above-mentioned oat β-glucan in the preparation of cosmetic raw materials or cosmetics.

[0043] Preferably, the cosmetic used in the application is a cosmetic with anti-wrinkle, wrinkle-reducing, skin elasticity-enhancing, and skin texture-improving properties.

[0044] Furthermore, the present invention provides a cosmetic ingredient comprising the following components: the above-mentioned oat β-glucan, a moisturizer, a skin conditioning agent, a preservative, and water.

[0045] Finally, the present invention provides an anti-aging composition comprising the following components: the above-mentioned oat β-glucan, resveratrol, ferulic acid and vitamin E.

[0046] Preferably, in the anti-aging composition, the mass ratio of oat β-glucan, resveratrol, ferulic acid and vitamin E is 10:5-8:3-6:1-3.

[0047] More preferably, in the anti-aging composition, the mass ratio of oat β-glucan, resveratrol, ferulic acid and vitamin E is 10:7:5:2.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] 1. This invention employs subcritical water extraction and enzymatic hydrolysis with amylase and neutral protease to improve the yield of oat β-glucan and reduce polysaccharide degradation. Furthermore, this invention purifies the extracted oat β-glucan using gel resin, efficiently removing impurities and significantly improving the purity of oat β-glucan.

[0050] 2. The oat β-glucan prepared by this invention has high activity, excellent effect on the proliferation of human skin fibroblasts, and better repair and anti-aging effects.

[0051] 3. The oat β-glucan prepared in this invention is used in the preparation of cosmetics. It interacts with resveratrol, ferulic acid and vitamin E to synergistically enhance the effects and bring about significant anti-aging, moisturizing and anti-wrinkle effects. Attached Figure Description

[0052] Figure 1 This is a graph showing the comparison results of DPPH free radical scavenging rates of anti-aging compositions. Detailed Implementation

[0053] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0054] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0055] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention are obtained through conventional commercial means.

[0056] In the following examples, the neutral protease was purchased from Shandong Longket Enzyme Preparation Co., Ltd., and its enzyme activity was 100,000 U / g;

[0057] The papain was purchased from Nanning Shanwan Biotechnology Co., Ltd., and its enzyme activity was 100,000 U / g.

[0058] The above components do not have a significant impact on the effect depending on the manufacturer.

[0059] Example 1

[0060] A method for preparing oat β-glucan, comprising the following steps:

[0061] (1) Oat powder is sieved to collect oat flour with a particle size of 100-200 mesh; oat flour is mixed with sodium carbonate solution with pH=9.5 for subcritical water extraction with a solid-liquid ratio of 1:20 g / mL, extracted at 160℃ for 30 min, and filtered to obtain crude extract;

[0062] (2) The pH of the crude extract was adjusted to 4.0 with citric acid, allowed to stand for 12 hours, filtered, and the supernatant was collected.

[0063] (3) The pH of the supernatant was adjusted to neutral using 2wt% sodium hydroxide solution. Then, amylase and neutral protease were added in a mass ratio of 4:1 for enzymatic hydrolysis. The amount of amylase added was 10% of the oat mass. The enzymatic hydrolysis was carried out at 50℃ for 3 hours with stirring. The solid-liquid ratio (oat mass g / liquid volume mL) was 1:25 g / mL. After the enzymatic hydrolysis was completed, the mixture was filtered to obtain the filtrate.

[0064] (4) The filtrate was purified by passing it through Sephacryl S-200 gel resin at a flow rate of 2 BV / h. After elution, the solution was concentrated under reduced pressure and dried to obtain oat β-glucan.

[0065] Example 2

[0066] A method for preparing oat β-glucan, comprising the following steps:

[0067] (1) Oat powder is sieved to collect oat flour with a particle size of 100-200 mesh; oat flour is mixed with sodium carbonate solution with pH=9 for subcritical water extraction, the solid-liquid ratio is 1:25 g / mL, extraction is carried out at 150℃ for 50 min, and the crude extract is obtained by filtration.

[0068] (2) The pH of the crude extract was adjusted to 3.8 with citric acid, allowed to stand for 8 hours, filtered, and the supernatant was collected.

[0069] (3) The pH of the supernatant was adjusted to neutral using 2wt% sodium hydroxide solution. Then, amylase and neutral protease were added in a mass ratio of 3:1 for enzymatic hydrolysis. The amount of amylase added was 9% of the oat mass. The enzymatic hydrolysis reaction was carried out at 45℃ for 6 hours with stirring. The solid-liquid ratio (oat mass g / liquid volume mL) was 1:30 g / mL. After the enzymatic hydrolysis was completed, the mixture was filtered to obtain the filtrate.

[0070] (4) The filtrate was purified by passing it through Sephacryl S-200 gel resin at a flow rate of 1 BV / h. After elution, the solution was concentrated under reduced pressure and dried to obtain oat β-glucan.

[0071] Example 3

[0072] A method for preparing oat β-glucan, comprising the following steps:

[0073] (1) Oat powder is sieved and oat flour with a particle size of 100-200 mesh is collected; oat flour is mixed with sodium hydroxide solution with pH=10 for subcritical water extraction, the solid-liquid ratio is 1:15 g / mL, extraction is carried out at 180℃ for 20 min, and the crude extract is obtained by filtration.

[0074] (2) The pH of the crude extract was adjusted to 4.3 with citric acid, allowed to stand for 16 hours, filtered, and the supernatant was collected.

[0075] (3) The pH of the supernatant was adjusted to neutral using 2wt% sodium hydroxide solution. Then, amylase and neutral protease were added in a mass ratio of 5:1 for enzymatic hydrolysis. The amount of amylase added was 11% of the oat mass. The enzymatic hydrolysis reaction was carried out at 55℃ for 2 hours with stirring. The solid-liquid ratio (oat mass g / liquid volume mL) was 1:20 g / mL. After the enzymatic hydrolysis was completed, the mixture was filtered to obtain the filtrate.

[0076] (4) The filtrate was purified by passing it through Sephacryl S-200 gel resin at a flow rate of 3 BV / h. After elution, the solution was concentrated under reduced pressure and dried to obtain oat β-glucan.

[0077] Comparative Example 1

[0078] Unlike Example 1, step (1) is different, specifically as follows:

[0079] (1) Oat powder is sieved to collect oat flour with a particle size of 100-200 mesh; oat flour is mixed with citric acid, pH is adjusted to 4.0, and subcritical water extraction is performed with a solid-liquid ratio of 1:20 g / mL, extraction at 200℃ for 10 min, and then filtered to obtain crude extract;

[0080] (2) Let the crude extract stand for 12 hours, filter, and take the supernatant;

[0081] Steps (3)-(4) are the same as in Example 1.

[0082] Comparative Example 2

[0083] Unlike Example 1, the subcritical water extraction time in step (1) is 90 min. Everything else is the same as in Example 1.

[0084] Comparative Example 3

[0085] Unlike Example 1, the mass ratio of amylase to neutral protease in step (3) is 1:1. Everything else is the same as in Example 1.

[0086] Comparative Example 4

[0087] The difference from Example 1 is that the neutral protease in step (3) is replaced with papain. Everything else is the same as in Example 1.

[0088] The oat β-glucan prepared in Example 1 was used in the preparation of the following anti-aging composition.

[0089] Application Example 1

[0090] An anti-aging composition comprising, by weight: 10 parts oat beta-glucan, 7 parts resveratrol, 5 parts ferulic acid and 2 parts vitamin E.

[0091] The components are mixed to obtain an anti-aging composition.

[0092] Application Example 2

[0093] An anti-aging composition comprising, by weight: 10 parts oat beta-glucan, 5 parts resveratrol, 6 parts ferulic acid and 3 parts vitamin E.

[0094] The components are mixed to obtain an anti-aging composition.

[0095] Application Example 3

[0096] An anti-aging composition comprising, by weight: 10 parts oat beta-glucan, 8 parts resveratrol, 3 parts ferulic acid and 1 part vitamin E.

[0097] The components are mixed to obtain an anti-aging composition.

[0098] Application Comparative Example 1

[0099] Unlike Application Example 1, the anti-aging composition consists of the following components by weight: 20 parts oat β-glucan, 2 parts resveratrol, 1 part ferulic acid, and 1 part vitamin E.

[0100] Application Comparative Example 2

[0101] Unlike Application Example 1, in the anti-aging composition, oat β-glucan is replaced with wolfberry polysaccharide.

[0102] An anti-aging composition comprising, by weight: 10 parts of wolfberry polysaccharide, 7 parts of resveratrol, 5 parts of ferulic acid and 2 parts of vitamin E.

[0103] Experiment 1: Oat β-glucan yield

[0104] The yield of the extracted oat β-glucan was calculated.

[0105] Yield (%) = Mass of extracted oat β-glucan (g) / Theoretical mass of oat β-glucan (g) × 100%.

[0106] The yields of oat β-glucan prepared in Examples 1-3 and Comparative Examples 1-4 were calculated, and the experiments were repeated in triplicate. The results are shown in Table 1.

[0107] Table 1

[0108]

[0109] In Table 1, # P < 0.05 indicates that the comparative example is significantly different from Example 1.

[0110] As shown in Table 1, the preparation method of this invention, through specific subcritical water extraction conditions and enzymatic hydrolysis with a combined complex enzyme, significantly improves the yield of oat β-glucan, achieving a yield of over 90%. This invention offers a significantly improved yield compared to the traditional acid / alkaline extraction method, which has a yield of approximately 50%. Comparative Example 1 used a higher temperature acidic solution for oat β-glucan extraction; Comparative Example 2 extended the extraction time, leading to the degradation of oat β-glucan and a reduced product yield; Comparative Examples 3 and 4 altered the type or ratio of enzymes, disrupting the synergistic effect between enzymes and thus reducing the extraction efficiency of oat β-glucan.

[0111] Experiment 2: Anti-aging effects of oat β-glucan

[0112] MTT assay for fibroblast proliferation

[0113] Human skin fibroblasts (HDF) (purchased from Thermo Fisher Scientific (China) Co., Ltd.) were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2 in an aseptic incubator under relative humidity conditions. Cell growth was observed daily, and cells were passaged when approximately 80% of the cells adhered to the culture medium. For passage, the supernatant was discarded, and the cells were washed 2-3 times with PBS. The PBS was discarded, and 2 mL of 0.25% trypsin was added for digestion for 3 min. Digestion was stopped by adding serum-containing culture medium. The adherent cells were gently aspirated to form a cell suspension, centrifuged at 1400 rpm for 4 min, the supernatant was discarded, and fresh culture medium was added to resuspend the centrifuged cells into a single-cell suspension. The passage ratio was 1:3, and the cells were transferred to culture flasks for further culture. Cells from passages 6-12 were used for subsequent experiments.

[0114] The oat β-glucan prepared in Examples 1-3, and the oat β-glucan prepared in Comparative Examples 1, 2, and 4, were dissolved in DMEM medium without bovine serum to prepare 5% solutions. These solutions were then diluted with PBS to a concentration of 0.01%, and finally filtered through a 0.22 μm sterile membrane for later use. Fibroblasts were cultured at a concentration of 3 × 10⁻⁶. 4 Cells were seeded into 96-well plates, with each sample replicated in 6 wells. Oat β-glucan solutions from different examples and comparatives were added, and samples were taken after 1, 3, 5, and 7 days of culture to detect the number of fibroblasts.

[0115] The results of the fibroblast count detection are shown in Table 2.

[0116] Table 2

[0117]

[0118] As can be seen from Table 2, the oat β-glucan prepared by the method of the present invention has a better effect on the proliferation of human skin fibroblasts compared with the comparative example, and thus exhibits higher anti-aging and modification activities.

[0119] Experiment 3: Anti-aging efficacy of the anti-aging composition

[0120] Test samples: Anti-aging compositions of Examples 1-3 and Comparative Examples 1-2.

[0121] Sample solution preparation: The anti-aging composition was mixed with distilled water to prepare a 1 mg / mL sample solution.

[0122] Dissolve DPPH in a small amount of toluene and then prepare a 0.1 g / L DPPH solution with 50% ethanol. Take 0.1 mL of each sample solution with a concentration of 1.0 mg / mL and 0.1 mL of distilled water, and add them to 1.9 mL of DPPH solution respectively. Mix quickly and let stand at room temperature for 10 min. Then, scan the absorption spectrum in a scanning UV-Vis spectrophotometer (using 50% ethanol solution as a reference solution for zeroing).

[0123] The scavenging rate of the sample against DPPH free radicals = 1 - [(AB) / A0] × 100%;

[0124] A0 represents the absorbance of DPPH without DPPH (1.9 mL DPPH + 0.1 mL 50% ethanol), A represents the absorbance of the sample after reaction with DPPH, and B represents the absorbance of the blank sample (0.1 mL sample + 1.9 mL 50% ethanol). Each sample was measured in triplicate, and the average value was taken. The results of DPPH free radical scavenging rate are shown in […]. Figure 1 .

[0125] from Figure 1 As can be seen, the anti-aging composition of the present invention achieves a DPPH free radical scavenging rate of over 95%, which is significantly improved compared to Comparative Example 1 and Comparative Example 2. The anti-aging composition of the present invention with specific components and proportions exhibits excellent anti-aging and antioxidant effects.

[0126] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing oat β-glucan, characterized in that, Including the following steps: (1) Oat powder is mixed with alkaline water for subcritical water extraction at an extraction temperature of 150-180℃ and an extraction time of 20-50 min to obtain crude extract; the pH of the alkaline water is 9-10; the solid-liquid ratio of oat to water in the subcritical water extraction is 1:15-25 g / mL. (2) Adjust the pH of the crude extract to acidic, let it stand, and take the supernatant; (3) Adjust the pH of the supernatant to neutral, then add amylase and neutral protease to carry out enzymatic hydrolysis, and filter to obtain filtrate; the mass ratio of amylase to neutral protease is 3-5:1; the amount of amylase added is 5-15% of the mass of oats; (4) The filtrate was purified by passing it through Sephacryl S-200 gel resin and concentrated to obtain oat β-glucan.

2. The preparation method according to claim 1, characterized in that, In step (1), the subcritical water extraction is performed at an extraction temperature of 160°C.

3. The preparation method according to claim 1, characterized in that, In step (3), the enzymatic hydrolysis reaction is carried out by stirring at 45-55℃ for 2-6 hours; the solid-liquid ratio of the enzymatic hydrolysis reaction is 1:20-30 g / mL. In step (4), the purification process is carried out at a flow rate of 1-3 BV / h.

4. An anti-aging composition, characterized in that, The product comprises: oat β-glucan, resveratrol, ferulic acid and vitamin E prepared by the preparation method according to any one of claims 1-3; wherein the mass ratio of oat β-glucan, resveratrol, ferulic acid and vitamin E is 10:5-8:3-6:1-3.

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