Soybean composite functional factor, preparation method thereof and application of soybean composite functional factor in cosmetics

Abstract

The invention discloses a soybean composite functional factor, a preparation method thereof and application of the soybean composite functional factor in cosmetics, and relates to the technical field of extraction and development of functional active ingredients. The natural coexistence characteristic of soybean protein and oligosaccharide in low-temperature defatted soybean meal is utilized, and composite separation strategies and process integration means such as organic solvent residue-free defatting treatment, a modern double-enzymolysis segmentation technology, gradient acid regulation and protein precipitation, ion exchange under a neutral condition, target peak detection by HPLC (High Performance Liquid Chromatography), membrane separation, freeze drying and the like are adopted, so that the low-temperature defatted soybean meal is obtained. And high-purity precise separation is realized on the premise of keeping a composite form. According to the preparation method disclosed by the invention, macromolecular soybean protein can be degraded into soybean polypeptide and soybean oligosaccharide of which the relative molecular mass is less than 5000, and the water-soluble soybean compound functional factor of high-purity soybean polypeptide, oligosaccharide and trace soybean saponin is obtained and is applied to cosmetics; and a personal skin care product with multiple effects of resisting wrinkles, tightening, blocking sun, moisturizing and repairing can be provided.

Description

Technical Field

[0001] The present invention belongs to the technical field of extraction and development of functional active ingredients, and in particular relates to a soybean composite functional factor, a preparation method thereof, and application in cosmetics. Background Art

[0002] Soybeans are a high-protein crop, containing 36% to 40% protein. Soy protein contains a wide range of amino acids, earning it the nickname "high-quality protein source." Numerous studies have confirmed the physiological functions and potential applications of soybean peptides (relative molecular weight <5000), soybean oligosaccharides (stachyose and raffinose), and soybean saponins.

[0003] 1. Current status of research on the development of soybean composite functional factors

[0004] Leading companies like Shuangta Food and Huaxi Biology are accelerating the development of complex products, and the preference of the post-90s and Generation Z generations for "natural" and "functional" labels is driving market demand to approximately 3.5 billion RMB in 2023, representing 20% ​​of the total domestic soybean peptide market. This figure is expected to exceed 5 billion RMB in 2025. Therefore, water-soluble complex products of soybean peptides, oligosaccharides, and saponins hold enormous market potential in China. However, due to limitations in the hydrolysis method, the finished products of complex soybean peptides, oligosaccharides, and saponins have a strong odor and low functional activity. Furthermore, the soy oligosaccharides and saponins cannot be effectively retained, resulting in a wide range of molecular weight distributions in the finished products. Furthermore, the separation and purification of the complex components is challenging, and precise control of enzymatic hydrolysis conditions and membrane separation parameters is impossible, resulting in extremely low purity of the soybean peptides and oligosaccharides. Finally, high-purity soy saponins are irritating to the skin and cannot be used in cosmetics.

[0005] For example, patent document CN110564795B discloses soybean polypeptides, their preparation method, and their application in cosmetics. This method uses water-soluble, puffed soybean meal as a fermentation substrate, which is then inoculated with Bacillus subtilis for fermentation. The soybean polypeptides are then sterilized, centrifuged, decolorized, ultrafiltered, concentrated, and freeze-dried to obtain the soybean polypeptides. Although this method achieves the extraction of white, odorless soybean polypeptides, the relative molecular mass of the soybean polypeptides is wide, with a purity of <30%, and the content of soybean oligosaccharides is close to zero due to ultrafiltration removal. Therefore, products with a wide relative molecular mass and a single active ingredient cannot achieve a synergistic effect when used in cosmetics. For another example, patent document CN102617702A discloses a method for producing peptidoglycan. This method uses defatted, low-temperature soybean meal as raw material. After acid precipitation of protein, separation, water washing, alkaline protease hydrolysis, and enzyme inactivation, the product undergoes plate and frame filtration, electrodialysis, resin column purification, and concentration to produce a peptide liquid. After acid precipitation, the product undergoes plate and frame filtration, electrodialysis, resin column purification, and concentration at a pH of 3.0 to produce oligosaccharides. The peptides and oligosaccharides are then mixed in the desired ratio, and finally, the product is treated with activated carbon and spray-dried to obtain the peptidoglycan product. Although this method produces a water-soluble complex of soybean peptides and oligosaccharides, the acidic conditions (pH 3.0) may cause partial hydrolysis of the polysaccharide, reducing the oligosaccharide yield. Furthermore, due to the high viscosity and strong hygroscopicity of soybean oligosaccharides, a drag reducer (such as maltodextrin) must be added during the spray-drying process to dry the liquid soybean oligosaccharides. However, the addition of a drag reducer significantly reduces the purity of the soybean oligosaccharides in the finished product.

[0006] 2. Current status of the cosmetics market

[0007] With the continuous changes in the lifestyles and values ​​of Chinese consumers, the cosmetics market is developing towards high-end, functional and natural directions. Consumers' demand for skin care products that have multiple functions (such as anti-wrinkle and firming, sun protection, moisturizing, repair, and anti-oxidation) and are safe, natural and gentle has increased significantly.

[0008] (1) Single function limitation: Traditional facial creams mostly focus on a single function (such as only moisturizing or only anti-wrinkle), which is difficult to meet the demand of modern consumers for "multiple functions in one bottle";

[0009] (2) Insufficient synergy of active ingredients: Commercially available products often simply stack multiple ingredients and lack a scientific compatibility system, making it difficult to achieve synergistic effects between ingredients;

[0010] (3) Bottlenecks in the application of natural ingredients: Plant active ingredients have technical difficulties such as poor stability, low transdermal absorption, and insufficient bioavailability.

[0011] 3. Physiological functions of soybean peptides on the skin

[0012] Due to their relatively small molecular weight, soy peptides can penetrate the skin barrier (stratum corneum) and act directly on the dermis. Some short-chain peptides (such as the tripeptide-glycine-glutamic acid) can bind to receptors on the surface of keratinocytes, enhancing cell permeability. Soy peptides act on the skin through multiple targets and pathways, combining moisturizing, anti-wrinkle firming, and skin barrier repair functions, making them a preferred natural functional ingredient.

[0013] 4. Physiological functions of soybean oligosaccharides on the skin

[0014] (1) Physiological functions of cottonseed sugar on the skin

[0015] Raffinose forms a protective film on the skin, locking in moisture and keeping it supple and moisturizing. Furthermore, raffinose possesses anti-inflammatory properties, alleviating inflammation and improving skin condition. This is particularly true for allergy-prone skin, alleviating redness, swelling, itching, and pain. Long-term use of skincare products containing raffinose can delay aging, maintain elasticity and radiance, and enhance skin's delicate appearance. It also moisturizes and locks in moisture without irritation, while also providing anti-allergic and acne-fighting properties. Therefore, cosmetics containing raffinose are highly safe.

[0016] (2) Physiological functions of stachyose on the skin

[0017] Stachyose also has moisturizing properties, forming a protective layer on the skin's surface to prevent water loss and keep it moisturized. Its high solubility can improve rough skin, creating a smoother appearance, and it's non-irritating. Furthermore, stachyose has antioxidant properties, scavenging free radicals in the body and protecting cells from oxidative damage, thereby maintaining skin elasticity and preventing aging.

[0018] (3) Soybean saponins

[0019] The hydroxyl (-OH) and carboxyl (-COOH) groups in saponin molecules combine with water molecules to increase the moisture content of the skin. They enhance the epidermal barrier function by regulating the differentiation of keratinocytes. They enhance the skin's endogenous antioxidant capacity. They inhibit inflammatory factors and relieve redness and itching caused by oxidative stress. They activate the MAPK / ERK pathway: they promote the synthesis of collagen and elastic fibers and reduce wrinkles. The conjugated double bonds in saponins can absorb UVA / UVB, reduce DNA damage, and inhibit the UV-induced inflammatory cascade. The hydrophobic micelle structure of saponins destroys bacterial cell membranes and inhibits Propionibacterium acnes. By inhibiting the activity of 5α-reductase, they reduce androgen-induced sebum secretion and the formation of blackheads and whiteheads.

[0020] Soy saponins, with their multi-target mechanism of action, have the potential to deliver five benefits in skin care: anti-inflammatory, antioxidant, moisturizing, anti-wrinkle, firming, and oil control. With advancements in formulation technology, they are expected to become a core ingredient in high-end functional skincare products. However, quality control of soy saponins in practical applications requires careful attention; concentrations greater than 0.5% can cause itching or stinging.

[0021] In summary, it is necessary to seek a method for preparing a water-soluble composite functional factor of soybean peptides, soybean oligosaccharides and soybean saponins, which can not only improve the separation efficiency and reduce solvent consumption, but also significantly improve the purity of soybean peptides and oligosaccharides, effectively play the synergistic effect between the ingredients, and enable the soybean composite functional factor product to be well used in cosmetics, bringing consumers safer, more efficient and personalized personal care products. Summary of the Invention

[0022] In order to overcome the problems existing in the prior art (such as the low purity of soybean polypeptides and soybean oligosaccharide complexes, which make it difficult to exert their efficacy, insufficient synergy of active ingredients, and irritation of high-purity polypeptides and soybean saponins to the skin, which cannot be used in cosmetics), the present invention provides a method for preparing a soybean composite functional factor, which uses a supercritical CO2 extraction method without organic solvent residue for degreasing, double-enzyme segmented enzymatic hydrolysis, gradient acidification protein precipitation, ion exchange under neutral conditions, HPLC detection of target peaks, membrane separation and freeze drying and other key processes to extract soybean polypeptides and oligosaccharides, which can significantly improve the purity of soybean polypeptides and oligosaccharides, while controlling the soybean saponins below a reasonable mass fraction, effectively exerting the synergistic effect of polypeptide + oligosaccharide + saponin composite functional factors, which is better than single functional ingredient products. By using liposome encapsulation technology, soybean saponins will not irritate the skin, while improving the transdermal absorption rate of high-purity soybean polypeptides, exerting a multi-functional synergistic effect, and can be used as a mild, safe, and non-toxic cosmetic raw material to obtain a soybean composite functional factor product that can be used in cosmetics and can bring anti-wrinkle, firming, sun protection, moisturizing, and repair multi-functionality.

[0023] In order to achieve the above object, the present invention adopts the following technical solutions:

[0024] One of the purposes of the present invention is to provide a method for preparing a soybean composite functional factor, comprising the following steps:

[0025] (1) Pretreatment of soybean raw materials

[0026] The soybeans are freeze-dried and then crushed to 60-80 mesh size, and then defatted using supercritical CO2 extraction to obtain soybean meal. The freeze-drying conditions are: pre-freezing at -80°C for 30-60 minutes, main drying at -40--30°C, and gradually heating to 0°C after drying. The extraction conditions are: temperature 40-50°C, pressure 40-50 MPa, and time 3-5 hours.

[0027] (2) Water dissolution and enzymatic hydrolysis

[0028] The soybean meal is added to purified water at a material-liquid ratio of 1:12 to 1:15, and 0.1 to 0.5% of a β-galactosidase inhibitor is added. After extraction for 60 to 90 minutes, the pH is dynamically adjusted and a dual-enzyme segmented enzymatic hydrolysis is performed. The dual enzymes include a neutral protease and a flavor protease, and the addition amount is 2% to 6%. The neutral protease is added first, and the flavor protease is added later. The dynamic pH adjustment method is: 6.5 → 7.2 → 6.8, and the addition ratio of the neutral protease to the flavor protease is 5:1.

[0029] (3) Enzyme inactivation and separation

[0030] The enzymatically hydrolyzed liquid is subjected to enzyme inactivation treatment and then rapidly cooled. After centrifugation, the supernatant is taken for ultrafiltration to retain polypeptides with a relative molecular mass of less than 5000.

[0031] (4) Weakly acidic protein precipitation

[0032] The supernatant obtained after separation was subjected to gradient acid adjustment with magnetic stirring; the acid adjustment conditions were: 6.0→4.5, pH drop of 0.5 / 10min, 0→0.8M, rate of 0.02M / min;

[0033] (5) Ion exchange chromatography and target peak collection

[0034] A gradient elution procedure was used to elute oligosaccharides first and then polypeptides. The overlapping region of the oligosaccharide peak and the polypeptide peak was collected, and finally a light yellow liquid was obtained by nanofiltration. The gradient elution procedure was as follows: in the first stage, the NaCl concentration was adjusted to 0-0.4M to elute oligosaccharides, in the second stage, the NaCl concentration was adjusted to 0.6-0.8M to elute polypeptides, and in the third stage, the NaCl concentration was adjusted to 0.9-1.0M to elute impurities.

[0035] (6) Activated carbon decolorization and deodorization

[0036] Adjust the pH value of the liquid and add activated carbon twice in a gradient manner while stirring: first add 2% to 3% of the liquid and treat for 25 to 35 minutes, then add 2% to 3% and treat for 5 to 15 minutes, the temperature is 40 to 50 ° C, and the stirrer speed is 400 to 600 r / min;

[0037] (7) Filtration, sterilization, and concentration

[0038] The liquid is centrifuged to obtain the supernatant, which is then filtered, sterilized by ultra-high temperature and concentrated by reverse osmosis (RO).

[0039] (8) Freeze-drying

[0040] After pre-freezing at -80°C for 30 minutes, main drying is performed: temperature is increased stepwise from -40 to -30°C and 0.005 to 0.015 MPa, and dehydration is performed at a controlled rate to obtain the soybean composite functional factor.

[0041] Furthermore, the method for adding the double enzymes in step (2) is: first add neutral protease at 55-58°C for enzymatic hydrolysis for 60-70 minutes, then add flavor protease at 50-60°C for enzymatic hydrolysis for 60-80 minutes, stirring while adding flavor protease, and rotating speed 60-80 r / min.

[0042] The water-soluble soybean meal of the present invention is simultaneously added with a beta-galactosidase inhibitor to prevent oligosaccharides from being hydrolyzed into monosaccharides. After extraction, double-enzyme segmented enzymolysis is performed, wherein a neutral protease is first added to preferentially cut off the hydrophobic groups, and then a flavor protease is added to supplement the hydrolysis of the hydrophobic peptide segments and improve the flavor.

[0043] Furthermore, the enzyme inactivation conditions in step (3) are: temperature 90-100° C., time 8-12 min; and centrifugation conditions are: speed 4000-5000 r / min, time 13-15 min.

[0044] In this method, the enzymatically hydrolyzed liquid is inactivated and rapidly cooled to reduce peptide degradation caused by excessive protein denaturation and to reduce high-temperature degradation of soybean nucleic acids. After centrifugation, the supernatant is ultrafiltered to retain peptides with a relative molecular mass of less than 5000, ensuring efficient peptide retention and removing unhydrolyzed protein, cellulose, soybean nucleic acids, and the like.

[0045] Furthermore, the stirring conditions in step (4) are: temperature 60-64° C., rotation speed 550-650 r / min, stirring for 30-35 min, and then centrifuging at a speed of 10,000 r / min for 20-25 min.

[0046] In the present invention, the supernatant obtained by separation is subjected to gradient acidification, stirred with a magnetic stirrer at a speed of 600 r / min, and then centrifuged at a speed of 10,000 r / min to remove denatured protein precipitates and part of the saponin+protein complex.

[0047] Furthermore, the collection conditions of the overlapping region of the oligosaccharide peak and the polypeptide peak in step (5) are as follows: an initial flow rate of 0.4 to 0.6 mL / min, increased to 1.8 to 2.2 mL / min at a NaCl concentration of 0.4 to 0.6 M, a treatment time of 50 to 60 min, and detection by HPLC, conditions C18 column, mobile phase 0.1% phosphoric acid water-methanol = 85:15; the relative molecular mass of the substance retained by nanofiltration is ≤550.

[0048] The present invention adopts a packed resin gradient elution procedure: oligosaccharides are eluted first, then polypeptides are eluted, and the collected liquid is subjected to nanofiltration with a relative molecular mass of ≤550 to remove monosaccharides and free amino acids, thereby further improving the purity of oligosaccharides.

[0049] In step (6) of the present invention, polypeptides (charged) and oligosaccharides (polar) compete with saponins and isoflavones for adsorption sites. Therefore, high-purity, impurity-free, powdered food-grade wood activated carbon with a specific surface area of ​​≥1000m 2 / g. Selectivity is enhanced by controlling the activated carbon dosage (≥3%), maintaining a neutral pH environment, and maintaining a high specific surface area to optimize the hydrophobic interactions between saponins, isoflavones, and activated carbon, thereby preferentially adsorbing saponins, isoflavones, and carotenoids in the aqueous phase. Activated carbon is added twice in a gradient to increase surface activity, accelerate the adsorption of saponins and isoflavones, and avoid high-temperature damage to peptides. A stirrer ensures full contact between the activated carbon and the liquid, reduces local concentration differences, and prevents activated carbon agglomeration. The treated liquid is milky white.

[0050] Furthermore, the sterilization conditions in step (7) are: temperature of 125-135° C., time of 4-6 seconds, and pressure of 15-20 MPa; and the reverse osmosis (RO) concentration conditions are: The retentate concentration is ≥10%.

[0051] In the present invention, the milky white liquid is centrifuged to remove activated carbon particles, and the supernatant is filtered, sterilized instantaneously at ultrahigh temperature and concentrated by reverse osmosis (RO) to degrade nucleic acid.

[0052] In step (8) of the present invention, the concentrated liquid is freeze-dried and then heated in steps, and then dehydrated at a controlled rate to obtain a water-soluble soybean composite functional factor with soybean polypeptides and soybean oligosaccharides as main components and a mixture of trace soybean saponins. The powder has no obvious odor and is a white loose powder. The polypeptide content is ≥55-65% (dry basis), stachyose+raffinose ≥25-35% (dry basis), and soybean saponins ≤1.3% (dry basis). Other components include trace amounts of amino acids, sucrose, monosaccharides, fats, carbohydrates, vitamins and minerals.

[0053] The second purpose of the present invention is to provide a soybean composite functional factor, which is prepared according to the preparation method.

[0054] The third object of the present invention is to provide a novel step-by-step packaging method for soybean composite functional factors, wherein the soybean composite functional factor package is the soybean composite functional factor described above, and the method comprises the following steps:

[0055] Step 1: New liposome encapsulation

[0056] (1) 4-6% phosphatidylethanolamine + 1-2% sodium cholesterol sulfate are dissolved in supercritical CO2 to obtain a lipid solution;

[0057] (2) The soybean composite functional factor was dissolved in phosphate buffer and injected into the microfluidic chip at a volume ratio of (0.1-1.5):(2.5-3.5) with the lipid solution;

[0058] (3) low-temperature vacuum drying to form liposome precursors;

[0059] Step 2: Water phase gelation wrapping

[0060] (1) Gellan gum and Tween 80 are dissolved in deionized water to a concentration of 4.5-5.5% and 1.5-2.5%, respectively, and heated to form a thixotropic gel;

[0061] (2) mixing the liposome precursor with the gel solution and forming liposome-in-water composite particles by ultrasound-assisted emulsification;

[0062] Step 3: Purification and concentration

[0063] (1) Use a composite membrane with a molecular weight cut-off of 900 to 1100 and a pressure of 1.5 to 2.0 bar;

[0064] (2) Freeze-drying and adding trehalose as a protective agent. After freeze-drying, the liposome encapsulation efficiency is ≥90%, and the particle size is controlled at 150-200 nm.

[0065] Furthermore, the vacuum drying conditions in the first step are: -45 to -55°C, 0.05 to 0.15 MPa; the ultrasound-assisted emulsification conditions in the second step are: 18 to 22 kHz, 4 to 6 min; and the amount of trehalose added in the third step is 4 to 6%.

[0066] The above method achieves efficient preparation, stable encapsulation and long-term storage of liposomes through the multi-step synergistic effect of new liposome encapsulation, aqueous phase gelation encapsulation, purification and concentration, from material composition, process parameters to post-processing technology. Compared with traditional methods, the new step-by-step encapsulation technology has greatly improved stability.

[0067] From a material perspective, the above method combines PE and sodium cholesterol sulfate to improve the membrane's antioxidant and mechanical strength, while supercritical CO2 dissolution maximizes the retention of active ingredients. From a process perspective, microfluidics precisely controls particle size distribution, gellan gum thixotropic gel provides a shear barrier, and TFF efficient purification reduces impurity-induced degradation. From a protective perspective, the vitrification protection of trehalose significantly outperforms traditional freeze-drying processes, ensuring long-term storage stability. Therefore, this novel method surpasses traditional methods in physical stability (particle size control, mechanical strength), chemical stability (antioxidant, anti-enzymatic hydrolysis), and long-term storage performance, making it more suitable for delivering soy-based active functional ingredients that are sensitive to heat, oxidation, and mechanical stress.

[0068] The fourth object of the present invention is to provide a soybean composite functional factor liposome encapsulated material, wherein the soybean composite functional factor liposome encapsulated material is prepared according to the method described above.

[0069] The fifth object of the present invention is to provide a method for preparing the soybean composite functional factor, the soybean composite functional factor, the novel soybean composite functional factor step-by-step encapsulation method, or the use of the soybean composite functional factor liposome encapsulation in the preparation of cosmetics.

[0070] Compared with the prior art, the present invention has the following technical effects:

[0071] 1. The present invention avoids oligosaccharide degradation or protein denaturation caused by high temperature of enzymatic hydrolysis; achieves high-purity separation by combining ultrafiltration and chromatography; integrates enzymatic hydrolysis, ultrafiltration, and chromatography steps into a continuous process to reduce intermediate product loss and energy consumption; and combines HPLC with membrane separation technology to ensure that the relative molecular mass distribution of the product meets the standards for cosmetic physiological efficacy.

[0072] 2. The product of the present invention has multiple functions and is safe, suitable for use on all skin types: The color of the soybean composite functional factor of the present invention is white, high in purity, has no obvious odor, and does not contain synthetic pigments, plastic powders, mineral oils and other skin-damaging ingredients. The present invention has high DPPH free radical scavenging rate and elastase inhibition rate, strong antioxidant activity, and can significantly inhibit the activity of elastase, indicating that it has anti-wrinkle and firming effects; the SPF of the present invention measured by UV-visible spectrophotometry reaches 30, indicating that it has sun protection effect; by measuring the change rate and repair rate of transepidermal water loss (TEWL) of the soybean composite functional factor, the test results of the soybean composite functional factor group far exceeded those of the positive control group (1% ceramide), indicating that it has moisturizing and repairing effects. The present invention adopts a novel liposome encapsulation technology to better play the multi-functional synergistic effect of high-purity soybean peptides, oligosaccharides and saponins. It is mild in nature, non-irritating, and has no toxic side effects. It can be directly added to cosmetics, providing consumers with excellent anti-wrinkle, firming, sun protection, moisturizing, and repair multi-dimensional skin care effects, and has good market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 The content of each component in the soybean composite functional factor prepared in Comparative Example 1. DETAILED DESCRIPTION

[0074] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps, or conditions of the present invention are intended to fall within the scope of the present invention. The reagents and instruments used in the following examples are all commercially available, and the methods used in the examples are consistent with conventional methods unless otherwise specified.

[0075] The technical solution of the present invention is further elaborated in detail below in conjunction with embodiments.

[0076] Example 1 The soybean composite functional factor of the present invention and its preparation method

[0077] 1. Pretreatment of soybean raw materials

[0078] The washed soybeans (soybean variety Hefeng 50, purchased from Shunxin Seed Store in Nong'an Town, Nong'an County) were freeze-dried and then crushed to 60 mesh. The freeze-drying conditions were: pre-freezing at -80°C for 30 min, main drying (-40 to -30°C), and then heating to 0°C. The soybean powder was then defatted using supercritical CO2 extraction. The extraction conditions were: temperature 40°C, pressure 40 MPa, and time 3 h.

[0079] 2. Water-soluble and enzymatic hydrolysis

[0080] Soybean meal was added to purified water at a material-to-liquid ratio of 1:12, and 0.1% by mass of 1-deoxygalactonojirimycin (Sigma-Aldrich: Product No. D1415) was added. After extraction for 60 minutes, food-grade hydrochloric acid was added to dynamically adjust the pH (6.5→7.2→6.8). The enzyme addition amount was 2% by mass (neutral protease: flavor protease = 5:1). Bacillus subtilis enzyme (Novozymes: Product No. P2610) was first added for enzymatic hydrolysis at 55°C for 70 minutes, followed by papain (Worthington Biochemical: Product No. LS003119) at 50°C for further enzymatic hydrolysis for 80 minutes. Stirring was performed while adding the protease at a speed of 60 r / min.

[0081] 3. Enzyme inactivation, separation, and acid precipitation

[0082] The enzyme-inactivated liquid was quickly placed in an ice bath and centrifuged at 4000 r / min for 13 min. The supernatant was filtered through a polyvinylidene fluoride (PVDF) membrane and then subjected to gradient acid adjustment (6.0→4.5, pH decrease of 0.5 / 10 min, 0→0.8 M, rate of 0.02 M / min). Acid was added while stirring, and the mixture was stirred at 60°C and 650 r / min for 30 min, followed by centrifugation at 10000 r / min for 20 min.

[0083] 4. Ion exchange chromatography and target peak collection

[0084] Filled with Q-Sepharose HP highly cross-linked agarose matrix resin, oligosaccharides are eluted first, then peptides. The oligosaccharide peak appears in the low salt range (0-0.4M NaCl), and the peptide peak appears in the high salt range (0.6-0.8M NaCl). Impurities are eluted at a NaCl concentration of 0.9-1.0M, and the overlapping area of ​​the oligosaccharide peak and the peptide peak is collected. After a treatment time of 50 minutes, a light yellow liquid is obtained by nanofiltration.

[0085] Specifically, the initial flow rate was 0.5 mL / min, which was increased to 2 mL / min at a NaCl concentration of 0.5 M, and the treatment time was 50-60 min. HPLC was performed using a C18 column and a mobile phase of 0.1% phosphoric acid water-methanol = 85:15. The collected soybean polypeptides and soybean oligosaccharide solutions were filtered through nanofiltration with a molecular weight cutoff of ≤550.

[0086] 5. Activated carbon adsorption and filtration

[0087] The collected liquid was adjusted to pH value (6.0→7.0), and activated carbon (Calgon Carbon Company: Model 400) first add 2% volume fraction of feed liquid, process for 30 minutes, add 2% volume fraction, and process for another 10 minutes, temperature 40 ° C, stirrer speed 400 r / min, centrifuge the milky white feed liquid at a speed of 10000 r / min for 20 minutes, and take the supernatant and filter it through a microporous filter (0.22 μm filter membrane);

[0088] 6. Sterilization, concentration, freeze drying

[0089] The transparent liquid obtained by filtration was sterilized at 125°C, 4s, 15MPa, and then concentrated (reverse osmosis (RO) concentration conditions: The retentate concentration is ≥10%) and freeze-dried (pre-freezing at 80°C for 30 minutes followed by main drying: stepwise heating at -40°C and 0.01 MPa, with controlled dehydration) to obtain a white, odorless, water-soluble soybean composite functional factor.

[0090] Example 2 The soybean composite functional factor of the present invention and its preparation method (for the unspecified part, see Example 1)

[0091] 1. Pretreatment of soybean raw materials

[0092] The washed soybeans were freeze-dried and then crushed to 80 mesh. The freeze-drying conditions were: pre-freeze at -80°C for 60 minutes, perform main drying (-40 to -30°C), and then heat to 0°C. The soybean powder was then defatted. The extraction conditions were: temperature 50°C, pressure 50 MPa, and time 5 hours.

[0093] 2. Water-soluble and enzymatic hydrolysis

[0094] Soybean meal was added to purified water at a material-liquid ratio of 1:15, and 1-deoxygalactonojirimycin (1-deoxygalactonojirimycin) was added at a mass fraction of 0.5%. After extraction for 90 minutes, food-grade hydrochloric acid was added to dynamically adjust the pH. The enzyme addition amount was 6% by mass. Bacillus subtilis enzyme was first added to hydrolyze at 58°C for 60 minutes, and then papain was added to hydrolyze at 60°C for another 60 minutes. Stirring was carried out while adding the protease at a speed of 80 r / min.

[0095] 3. Enzyme inactivation, separation, and acid precipitation

[0096] The enzyme-inactivated liquid was quickly placed in an ice bath and centrifuged at 5000 r / min for 15 min. The supernatant was filtered through a polyvinylidene fluoride (PVDF) membrane and then subjected to gradient acid adjustment (6.0 → 4.5). Acid was added while stirring, and the mixture was stirred at 64°C and 550 r / min for 35 min, followed by centrifugation for 25 min.

[0097] 4. Ion exchange chromatography and target peak collection

[0098] Filled with Q-Sepharose HP highly cross-linked agarose matrix resin, oligosaccharides are eluted first, then peptides. The oligosaccharide peak appears in the low salt range (0-0.4M NaCl), and the peptide peak appears in the high salt range (0.6-0.8M NaCl). Impurities are eluted at a NaCl concentration of 0.9-1.0M, and the overlapping area of ​​the oligosaccharide peak and the peptide peak is collected. After a treatment time of 60 minutes, a light yellow liquid is obtained by nanofiltration.

[0099] 5. Activated carbon adsorption and filtration

[0100] The pH value of the collected liquid was adjusted, and the volume fraction of the liquid was added at 3% for 30 minutes, and then the volume fraction was added at 3% for another 10 minutes. The temperature was 50°C and the stirrer speed was 600 r / min. The milky white liquid was centrifuged for 25 minutes, and the supernatant was filtered through a microporous filter.

[0101] 6. Sterilization, concentration, freeze drying

[0102] The transparent liquid obtained by filtration is sterilized under the conditions of 135° C., 6 s, and 20 MPa, and then concentrated and freeze-dried to obtain a white, odorless, water-soluble soybean composite functional factor.

[0103] Example 3 The soybean composite functional factor of the present invention and its preparation method (for the unexplained part, see Example 1)

[0104] 1. Pretreatment of soybean raw materials

[0105] The washed soybeans were freeze-dried and then crushed to 70 mesh. The freeze-drying conditions were: pre-freeze at -80°C for 45 minutes, perform main drying (-40 to -30°C), then heat to 0°C. The soybean powder was then defatted. The extraction conditions were: temperature 45°C, pressure 45 MPa, and time 4 hours.

[0106] 2. Water-soluble and enzymatic hydrolysis

[0107] Soybean meal was added to purified water at a material-liquid ratio of 1:14, and 1-deoxygalactonojirimycin (1-deoxygalactonojirimycin) was added at a mass fraction of 0.3%. After extraction for 75 minutes, food-grade hydrochloric acid was added to dynamically adjust the pH. The enzyme addition amount was 3% by mass. Bacillus subtilis enzyme was first added for enzymatic hydrolysis at 56°C for 65 minutes, followed by papain at 55°C for a further 70 minutes. Stirring was performed while the enzyme was added at a speed of 70 rpm.

[0108] 3. Enzyme inactivation, separation, and acid precipitation

[0109] The enzyme-inactivated liquid was quickly placed in an ice bath and centrifuged at 4500 r / min for 14 min. The supernatant was filtered through a polyvinylidene fluoride (PVDF) membrane and then subjected to gradient acid adjustment (6.0 → 4.5). Acid was added while stirring, and the mixture was stirred at 62°C and 600 r / min for 33 min, followed by centrifugation for 23 min.

[0110] 4. Ion exchange chromatography and target peak collection

[0111] Filled with Q-Sepharose HP highly cross-linked agarose matrix resin, oligosaccharides are eluted first, then peptides. The oligosaccharide peak appears in the low salt range (0-0.4M NaCl), and the peptide peak appears in the high salt range (0.6-0.8M NaCl). Impurities are eluted at a NaCl concentration of 0.9-1.0M, and the overlapping area of ​​the oligosaccharide peak and the peptide peak is collected. After a treatment time of 55 minutes, a light yellow liquid is obtained by nanofiltration.

[0112] 5. Activated carbon adsorption and filtration

[0113] The pH value of the collected liquid was adjusted, and 2% of the volume fraction of the liquid was added for 30 minutes, and then 3% of the volume fraction was added for another 10 minutes. The temperature was 45°C and the stirrer speed was 500 r / min. The milky white liquid was centrifuged for 23 minutes, and the supernatant was filtered through a microporous filter.

[0114] 6. Sterilization, concentration, freeze drying

[0115] The transparent liquid obtained by filtration is sterilized under the conditions of 130° C., 5 s, and 17 MPa, and then concentrated and freeze-dried to obtain a white, odorless, water-soluble soybean composite functional factor.

[0116] Example 4 The soybean composite functional factor of the present invention and its preparation method (for the unexplained part, see Example 1)

[0117] 1. Pretreatment of soybean raw materials

[0118] The washed soybeans were freeze-dried and then crushed to 60 mesh. The freeze-drying conditions were: pre-freeze at -80°C for 50 minutes, then conduct main drying (-40 to -30°C) and then heat to 0°C. The soybean powder was then defatted. The extraction conditions were: temperature 46°C, pressure 48 MPa, and time 4.5 hours.

[0119] 2. Water-soluble and enzymatic hydrolysis

[0120] Soybean meal was added to purified water at a material-liquid ratio of 1:13, and 1-deoxygalactonojirimycin (1-deoxygalactonojirimycin) was added at a mass fraction of 0.4%. After extraction for 80 minutes, food-grade hydrochloric acid was added to dynamically adjust the pH. The enzyme addition amount was 4% by mass. Bacillus subtilis enzyme was first added for enzymatic hydrolysis at 57°C for 62 minutes, followed by papain at 52°C for a further 75 minutes. Stirring was performed while the protease was added at a speed of 60 r / min.

[0121] 3. Enzyme inactivation, separation, and acid precipitation

[0122] The enzyme-inactivated liquid was quickly placed in an ice bath and centrifuged at 4800 r / min for 13 min. The supernatant was filtered through a polyvinylidene fluoride (PVDF) membrane and then subjected to gradient acid adjustment (6.0 → 4.5). Acid was added while stirring, and the mixture was stirred at 60°C and 600 r / min for 30 min, followed by centrifugation for 22 min.

[0123] 4. Ion exchange chromatography and target peak collection

[0124] Filled with Q-Sepharose HP highly cross-linked agarose matrix resin, oligosaccharides are eluted first, then peptides. The oligosaccharide peak appears in the low salt range (0-0.4M NaCl), and the peptide peak appears in the high salt range (0.6-0.8M NaCl). Impurities are eluted at a NaCl concentration of 0.9-1.0M, and the overlapping area of ​​the oligosaccharide peak and the peptide peak is collected. After a treatment time of 58 minutes, a light yellow liquid is obtained by nanofiltration.

[0125] 5. Activated carbon adsorption and filtration

[0126] The pH value of the collected liquid was adjusted, and 3% of the volume fraction of the liquid was added for 30 minutes, and then 2% of the volume fraction was added for another 10 minutes. The temperature was 48°C and the stirrer speed was 500 r / min. The milky white liquid was centrifuged for 21 minutes, and the supernatant was filtered through a microporous filter.

[0127] 6. Sterilization, concentration, freeze drying

[0128] The transparent liquid obtained by filtration is sterilized under the conditions of 132° C., 6 s, and 16 MPa, and then concentrated and freeze-dried to obtain a white, odorless, water-soluble soybean composite functional factor.

[0129] Comparative Example 1: Method for processing soybean composite functional factors using by-products produced by a soybean protein concentrate production plant

[0130] (1) Low-temperature soybean meal (LT-DDSF, food grade, protein ≥50%, purchased from Tongyu Yifahe Soybean Products Co., Ltd.) as raw material was ground into 50 mesh soybean meal coarse powder, and then mixed with 60% to 70% food-grade ethanol solvent at a material-liquid ratio of 1:7 to 1:10, at a temperature of 50°C to 60°C, and stirred for no less than 4 hours;

[0131] (2) The slurry mixture was centrifuged at a speed of 4000 r / min, and then vacuum distilled, plate and frame filtered, double-effect concentrated, sterilized, and spray dried to obtain a light brown, powdery, water-soluble soybean composite functional factor. Vacuum concentration conditions: vacuum degree of 66.7kpa~73.3kpa, temperature of 80℃. Plate and frame filtration conditions: working pressure of 0.2Mpa~0.5Mpa. Double-effect concentration conditions: first-effect evaporation temperature of 62℃, second-effect evaporation temperature of 46℃, and flow time of about 3 minutes. Sterilization conditions: steam pressure ≈200kpa, temperature ≈130℃, and time of about 10 seconds. Spray drying conditions: inlet air temperature of 170℃~180℃, exhaust air temperature of 70℃~80℃.

[0132] The difference between Comparative Example 1 and the present invention is that: because the soy isoflavones and carotenoids are not removed, the color of the soy composite functional factor is darker and cannot be used in cosmetics; in addition, the ingredient content table does not contain soy peptides, and when used in cosmetics, the various ingredients cannot synergize. The content of each ingredient is as follows Figure 1 shown.

[0133] Comparative Example 2: Control Test of Changing the Process Parameters in Examples 1-4

[0134] The cleaned soybeans are crushed into 50 meshes and defatted by supercritical CO2 extraction at a temperature of 30-40°C and a pressure of 30-40 MPa for 2 hours. The soybean meal is added to purified water at a material-liquid ratio of 1:10, and flavor protease is added after extraction for 40-50 minutes. The insoluble matter in the liquid after enzyme inactivation, separation, and acid precipitation is then filtered to obtain a light brown solution, and the filtrate with a relative molecular mass of less than 5000 is collected through an ultramicrofiltration membrane. The obtained liquid is added with 2% activated carbon for adsorption decolorization and deodorization, and a water-soluble, light brown, and odorous soybean composite functional factor dry powder is obtained through filtration, sterilization, concentration, and freeze-drying, wherein the soybean polypeptide content is 25-35%, the soybean oligosaccharide content is 3-9%, and the soybean saponin content is ≤3%.

[0135] The differences between Comparative Example 2 and the present invention are as follows: the CO2 extraction method used insufficient pressure, temperature, and time, resulting in incomplete defatting and affecting the subsequent enzymatic hydrolysis step; the absence of a β-galactosidase inhibitor during the solubilization of the soybean meal resulted in partial hydrolysis of oligosaccharides, reducing purity; the short extraction time resulted in a low protein yield; the absence of a neutral protease during the enzymatic hydrolysis step resulted in incomplete enzymatic hydrolysis and reduced soybean polypeptide purity; and the addition of activated carbon with insufficient adsorption capacity resulted in only 2%; the process parameters for the remaining steps remained the same. These deficiencies in the process parameters resulted in a dark-colored, strongly odorous, and low-purity soybean composite functional factor.

[0136] Example 5 Multifunctional Test of the Soybean Composite Functional Factor of the Present Invention - Determination of the Anti-wrinkle and Firming Efficacy of the Soybean Composite Functional Factor

[0137] The anti-wrinkle and firming effects of the soybean composite functional factors were tested by measuring the DPPH free radical scavenging activity and elastase inhibition rate.

[0138] (1) Determination of DPPH free radical scavenging activity

[0139] The DPPH free radical reacts with the soybean complex functional factor, causing the purple color to fade, and the antioxidant capacity is evaluated by the change in absorbance at 517nm. The specific steps are as follows:

[0140] The soybean composite functional factor in Example 3 was dissolved in 10 mL of 70% ethanol aqueous solution and prepared into 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL and 1.0 mg / mL sample solutions respectively. Ultrasound assisted dissolution was performed and the supernatant was centrifuged for standby use. 200 μL of sample solution and 2000 μL of 0.1 m mol / L DPPH ethanol solution were fully mixed and allowed to stand at room temperature in the dark for 30 min. The blank control was 200 μL of 70% ethanol aqueous solution. The free radical scavenging activity of the sample was quantified by the absorbance (A value) of DPPH at a wavelength of 517 nm on a spectrophotometer, and the half maximum scavenging concentration (SC 50 As the concentration of soybean composite functional factors increases, the antioxidant activity becomes stronger. The linear relationship is fitted to obtain the regression equation: y = 66.82x + 17.76 (R 2 =0.95), EC 50 Value 0.483mg / mL.

[0141] DPPH free radical scavenging rate (%) = (1-A sample / A blank) × 100%.

[0142] (2) Elastase inhibition assay

[0143] Elastase hydrolyzes the substrate AAPF to release the yellow product p-nitroaniline, and the absorbance change is monitored spectrophotometrically (405 nm). The soybean functional complex reduces the absorbance increase by binding to the enzyme active site, and the inhibition rate reflects the inhibitory ability. The following are the specific steps:

[0144] The soybean composite functional factor in Example 3 was dissolved in 50 mmol / L Tris-HCl buffer (pH 8.0) containing 0.1% Tween 80 to prepare sample solutions of 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, and 5.0 mg / mL, respectively. Ultrasound-assisted dissolution was performed, and the supernatant was centrifuged and set aside. Elastase (porcine pancreas) ≥1000 U / mg was dissolved in Tris-HCl buffer to a final concentration of 0.5 U / mL. AAPF substrate was dissolved in DMSO to a final concentration of 2 mmol / L. Chromogenic substrate stock solution: 10 mmol / L APF was dissolved in DMSO. 20 μL of elastase solution was mixed with 160 μL Tris-HCl buffer and 20 μL of sample solution. After incubation in a 37°C constant temperature water bath for 5 minutes, 20 μL of AAPF substrate was added to initiate the hydrolysis reaction. A blank control was a solution of 20 μL elastase solution, 160 μL Tris-HCl buffer, and 20 μL AAPF substrate. The elastase inhibition assay of the samples was performed by monitoring the absorbance change (ΔA value / min) at a wavelength of 405 nm for 5 min using a spectrophotometer, and the sample concentration required to inhibit 50% of the enzyme activity (IC 50 ). As the concentration of soybean composite functional factors increases, the inhibition rate of elastase increases. The concentration and inhibition rate show a scatter plot. IC 50 The value was 2.3±0.3mg / mL.

[0145] Elastase inhibition rate (%) = (1 - ΔA sample / ΔA blank) × 100%. Where ΔA sample is the average absorbance of the sample group - initial absorbance; ΔA blank is the average absorbance of the blank group - initial absorbance.

[0146] The test results of the anti-wrinkle and firming effects of the soybean composite functional factors are shown in Table 1.

[0147] Table 1 Test results of the anti-wrinkle and firming efficacy of the soybean composite functional factor in Example 3

[0148]

[0149]

[0150] As shown in Table 1, the DPPH free radical scavenging rate reached 81.1%, indicating that the soybean composite functional factor has strong antioxidant activity, effectively neutralizing free radicals, reducing oxidative damage to collagen and elastin, and maintaining skin elasticity and firmness. Elastase activity is closely related to wrinkle formation, and the elastase inhibition rate reached 78.9%, indicating that the soybean composite functional factor significantly inhibited elastase activity and played a role in inhibiting wrinkle formation. Therefore, the soybean composite functional factor of the present invention has anti-wrinkle and firming effects.

[0151] Example 6 Multifunctional Test of the Soybean Composite Functional Factor of the Present Invention - Determination of the Sunscreen Efficacy of the Soybean Composite Functional Factor

[0152] The anti-ultraviolet activity of the soybean composite functional factor was determined by UV-visible spectrophotometry to measure the absorbance of the soybean composite functional factor in the ultraviolet band (200-400nm) to evaluate its sun protection effect. The following are the specific steps:

[0153] Dissolve 10.0 mg of the soybean composite functional factor from Example 3 in 10 mL of ultrapure water, vortex mix thoroughly, ultrasonicate at 40 kHz for 15 minutes, and centrifuge at 12,000 rpm for 10 minutes. The supernatant is filtered through a 0.22 μm filter membrane to obtain a 1 mg / mL stock solution. The stock solution is then prepared into solutions at 0.5 mg / mL, 1 mg / mL, and 2 mg / mL. Ultrapure water is used as a blank control. The absorbance (A) at 280-400 nm is recorded using a spectrophotometer. The maximum absorption peak is denoted by λmax, the absorption coefficient by ε, and the sun protection factor by SPF.

[0154] ε(L / mol)=△A / c. Where △A is the absorbance of the sample minus the absorbance of the blank, and c is the sample concentration (mol / L).

[0155] Where λ is the absorption peak wavelength, I(λ) is the standard solar spectrum, and e -τ(λ) is the transmittance (approximately 1-10 -A(λ) ).

[0156] The results of the anti-ultraviolet activity test of the soybean composite functional factors are shown in Table 2.

[0157] Table 2 Anti-ultraviolet activity test results of soybean composite functional factors in Example 3

[0158]

[0159] As shown in Table 2, the absorbance of the soybean composite functional factor in the ultraviolet band (280-315 nm) reaches 1.3, the SPF reaches 30, and it blocks 97% of UVB (280-315 nm) ultraviolet rays, which can meet the needs of daily activities and outdoor sports, indicating that the soybean composite functional factor of the present invention has sun protection effect.

[0160] Example 7 Multifunctional test of the soybean composite functional factor of the present invention - Determination of the moisturizing and repairing efficacy of the soybean composite functional factor

[0161] TEWL reflects the rate of water evaporation from the stratum corneum and is directly related to the barrier function of the skin. By measuring the transepidermal water loss rate of the soybean composite functional factor, its moisturizing and repairing effects can be evaluated. The following are the specific steps:

[0162] EpiDerm TM The three-dimensional skin model was placed in an incubator for 24 hours at a temperature of 25±1°C and a humidity of 50±5%. The soybean composite functional factor of Example 3 was dissolved in phosphate buffered saline (PBS) to prepare solutions with mass fractions of 1%, 5%, and 10%. The negative control was PBS, and the positive control was an emulsion containing 1% ceramide. The application amount was (2.0±0.1) mg / cm 2 After measuring the initial value (TEWL initial) of each skin model using a TEWL meter, the sample was evenly applied to the skin surface and allowed to absorb for 30 minutes. TEWL (g / h·m 2 The moisturizing effect of the soybean composite functional factor is expressed by the TEWL change rate, and the repairing effect of the soybean composite functional factor is expressed by the TEWL repair rate.

[0163] TEWL change rate (%) = ΔTEWL / initial TEWL × 100%, where ΔTEWL is the measured TEWL value of each group after treatment - initial TEWL value.

[0164] TEWL repair rate (%) = [(TEWL negative control - TEWL treatment group) / TEWL negative control] × 100%. In the formula, the TEWL negative control and the treatment group were collected at the same time point.

[0165] The test results of soybean composite functional factor TEWL are shown in Table 3.

[0166] Table 3 TEWL test results of soybean composite functional factor in Example 3

[0167]

[0168]

[0169] As can be seen from Table 3, although the 1% soybean composite functional factor group is close to the positive control (1% ceramide), the effect is weak, which is related to the low penetration efficiency and insufficient synergy of the formula. The 5% soybean composite functional factor group significantly repaired the skin, and the long-term effect was stable, but it was weaker than the 10% group. The best effect was the 10% soybean composite functional factor group, with a repair rate of 46.4%, far exceeding the positive control (26.8%), indicating that the high-quality fraction soybean composite factor has a significant advantage in long-term repair. Soybean peptides quickly penetrate into the stratum corneum, stimulate keratinocyte proliferation, fill lipid gaps, promote ceramide and cholesterol synthesis, and reduce TEWL; oligosaccharides maintain the hydration state of the stratum corneum, continuously moisturize, and increase the water content of the skin surface; saponins are anti-inflammatory, regulate the skin microecology, and assist in barrier repair. Continuous use of the complex of soybean peptides, soybean oligosaccharides and soybean saponins produces a synergistic effect, enhances short-term and long-term effects, promotes stratum corneum lipid synthesis, and the repair rate is significantly higher than the short-term. Therefore, the soybean composite functional factor of the present invention has moisturizing and repairing effects.

[0170] Example 8: Liposome encapsulation of the soybean composite functional factor in Experimental Example 3 of the present invention

[0171] This example achieves efficient preparation, stable encapsulation, and long-term storage of liposomes through the synergistic effects of novel liposome encapsulation, aqueous gel encapsulation, purification, and concentration, from material composition, process parameters, to post-processing technology. Compared with traditional methods, the novel step-by-step encapsulation technology significantly improves stability. The method is as follows:

[0172] Step 1: New liposome encapsulation

[0173] (1) Material dissolution

[0174] Phosphatidylethanolamine (PE, 5% by mass) and sodium cholesterol sulfate (1.5% by mass) were dissolved in supercritical CO2. The smaller polar head of PE promotes tighter molecular packing, while the sulfate groups of sodium cholesterol sulfate enhance hydration stability. Supercritical CO2 dissolution avoids residual organic solvents, significantly reducing degradation of the active ingredient.

[0175] (2) Microfluidic mixing

[0176] A soybean complex functional factor solution (pH 6.5 phosphate buffer) and a lipid solution were injected into a microfluidic chip at a volume ratio of 1:3 to form uniform liposomes. The mixing dynamics were precisely controlled by laminar flow to produce monodisperse liposomes (PDI < 0.1).

[0177] (3) Frozen intervention treatment

[0178] Low-temperature vacuum drying (-50°C, 0.1 MPa) forms liposome precursors to avoid high-temperature destruction of active ingredients and maintain membrane integrity.

[0179] Step 2: Water phase gelation wrapping

[0180] (1) Gel matrix preparation

[0181] Gellan gum (5% by mass, Sigma-Aldrich: Product No. G1910) and Tween 80 (2% by volume) were dissolved in deionized water and heated to 60° C. to form a thixotropic gel. The gellan gum was bound to the liposome precursor via hydrogen bonds.

[0182] (2) Composite wrapping

[0183] The liposome precursor was mixed with the gel solution and ultrasonically emulsified (20 kHz, 5 minutes) to form liposome-in-water composite particles. Ultrasonic emulsification induced the formation of a three-dimensional network in the gel matrix, which increased the mechanical strength (storage modulus G') by 3-5 times compared to the gum arabic system.

[0184] Step 3: Purification and concentration

[0185] (1) Tangential flow filtration (TFF)

[0186] Unencapsulated components are removed using a 1000 molecular weight cut-off composite membrane at a pressure of 1.5-2.0 bar. Continuous recirculation removes 99% of unencapsulated components. Dynamic membrane pressure control prevents membrane clogging and maintains liposome structural integrity.

[0187] (2) Freeze-drying

[0188] Adding trehalose (5%) as a protective agent results in a liposome encapsulation efficiency of ≥90% after lyophilization, with a particle size controlled between 150 and 200 nm. Trehalose forms hydrogen bonds with the polar head groups of phospholipids through a water displacement mechanism, raising the glass transition temperature (Tg) by 40°C. After lyophilization, the encapsulation efficiency is ≥90%, with a particle size increase of <10%.

[0189] From a material perspective, the above method combines PE and sodium cholesterol sulfate to improve the membrane's antioxidant and mechanical strength, while supercritical CO2 dissolution maximizes the retention of active ingredients. From a process perspective, microfluidics precisely controls particle size distribution, gellan gum thixotropic gel provides a shear barrier, and TFF efficient purification reduces impurity-induced degradation. From a protective perspective, the vitrification protection of trehalose significantly outperforms traditional freeze-drying processes, ensuring long-term storage stability. Therefore, this novel method surpasses traditional methods in physical stability (particle size control, mechanical strength), chemical stability (antioxidant, anti-enzymatic hydrolysis), and long-term storage performance, making it more suitable for delivering soy-based active functional ingredients that are sensitive to heat, oxidation, and mechanical stress.

[0190] Based on the test results of the soy composite functional factor's antioxidant activity, elastase activity, anti-UV activity, and TEWL change and repair rates, the soy composite functional factor, encapsulated in liposomes at a mass fraction of 8.00%, is used in cosmetics. This invention, with soy active ingredients as its core and combined with modern bioextraction technology, creates a skincare product with multiple benefits: anti-wrinkle, firming, sun protection, moisturizing, and repair. By optimizing the compatibility ratio of the soy composite functional factor, its skin permeability and bioavailability are enhanced.

[0191] Example 9 Application of the soybean composite functional factor of the present invention in cosmetics - multi-functional cream

[0192] Carbomer 1.00%, triethanolamine 0.20%, xanthan gum 0.1%, bisabolol 1.50%, glycerin 5.00%, sucrose fatty acid ester 3.00%, stearic acid 2.00%, hyaluronic acid 1.50%, octadecyl alcohol 0.20%, rosehip oil 1.00%, cyclopentasiloxane 1.00%, shea butter resin 4.00%, cetyl alcohol 3.00%, 0.10% phenoxyethanol + 0.35% octanediol + 0.05% disodium EDTA composite preservative, 8.00% of the liposome-encapsulated soybean composite functional factor in Example 8, and the balance is deionized water.

[0193] Example 10 Application of the soybean composite functional factor of the present invention in cosmetics - multi-functional essence

[0194] Butylene glycol 3.00%, glycerin 3.00%, pentylene glycol 2.50%, carbomer 0.3%, triethanolamine 0.05%, citric acid 0.1%, citric acid-sodium citrate buffer system 0.15%, bisabolol 1.00%, shea butter resin 4.00%, liposome-encapsulated soybean composite functional factor 8 8.00%, 0.10% phenoxyethanol + 0.35% octanediol + 0.05% disodium EDTA composite preservative, and the balance is deionized water.

[0195] Example 11 In vitro effect experiment of the multifunctional cream in Example 9 and the multifunctional essence in Example 10

[0196] 1. The anti-wrinkle and firming efficacy experiments of the multi-functional cream and the multi-functional essence were carried out using the operating steps of Example 5. The test results are shown in Table 4.

[0197] Table 4 Test results of the anti-wrinkle and firming effects of multi-functional cream and multi-functional essence

[0198]

[0199]

[0200] Results showed that both the cream and essence exhibited strong antioxidant activity, primarily attributable to the synergistic effects of liposome-encapsulated soybean peptides and soy saponins. The cream's clearance rate (67.1%) was higher than the essence's (62.4%), due to the stronger synergistic antioxidant effects of bisabolol (1.50%) and shea butter resin (4.00%) in the cream. However, the presence of butylene glycol and pentylene glycol in the essence may slightly interfere with the capture efficiency of DPPH free radicals. The cream's inhibition rate (73.3%) was higher than the essence's (66.7%), due to the competitive inhibition of the elastase active center by the soybean peptides in the cream by blocking the enzyme's active site. The essence's citric acid buffer (which reduces enzyme activity at pH 8.0) may indirectly affect the enzyme reaction kinetics. However, the presence of Tween 80 in the essence enhances ingredient permeability, resulting in a better skin absorption effect than the in vitro data.

[0201] 2. The anti-ultraviolet activity test of the multi-functional cream and the multi-functional essence was carried out using the operating steps of Example 6. The test results are shown in Table 5.

[0202] Table 5 Anti-ultraviolet activity test results of multi-functional cream and multi-functional essence

[0203]

[0204] The results showed that the SPF of multifunctional creams and essences (8.3-12.4) was lower than that of the soy complex functional factor SPF (30). This was because the concentration of the soy complex functional factor used in cosmetics was diluted, resulting in a decrease in SPF. In addition, multifunctional creams and essences containing liposomes were photodegraded after UV exposure, resulting in a decrease in protective ability, and their stability directly affected the SPF value. In addition, Tween 80 in the formula reduced the transdermal efficiency or absorbance of the active ingredients. Soy saponins and bisabolol can reduce UV-induced ROS generation. Liposome encapsulation technology can improve the transdermal efficiency of soy peptides. Soy saponins can reduce photodegradation. The SPF of 12.4 meets the requirements of daily commuting protection and repairs UV-induced barrier damage.

[0205] 3. The operating steps of Example 7 were used to test the moisturizing and repairing effects of the multi-functional cream and the multi-functional essence. The test results are shown in Table 6.

[0206] Table 6 TEWL test results of multi-functional cream and multi-functional essence

[0207]

[0208] Results showed that in the short-term moisturizing effect (1-8 hours), both the multi-functional cream and multi-functional essence achieved a >30% reduction in TEWL within 1 hour, indicating that the formulas contain strong occlusive ingredients such as shea butter resin (4%) and stearic acid (2%), which rapidly form a physical barrier, achieving both moisturizing and water absorption. The moisturizing and repair effects continued to increase over 2-4 hours, associated with the synergistic sustained-release effect of liposome-encapsulated soy peptides and soy oligosaccharides. The 28-day repair rate for both the multi-functional cream and essence reached >40%, significantly superior to the positive control, attributed to the synergistic effects of soy peptides promoting stratum corneum lipid synthesis and soy saponins' anti-inflammatory properties. The moisturizing and repair effects of the multi-functional essence were slightly weaker than those of the multi-functional cream due to the rapid evaporation of the butylene glycol / pentylene glycol moisturizers and their lower occlusive properties. This formula achieves comprehensive functional enhancement through a combination of physical barrier and biological repair and anti-inflammatory properties.

[0209] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a soybean composite functional factor, characterized in that: The following steps are involved: (1) Pretreatment of soybean raw materials The soybeans are freeze-dried and then crushed to 60-80 mesh size, and then defatted using supercritical CO2 extraction to obtain soybean meal. The freeze-drying conditions are: pre-freezing at -80°C for 30-60 minutes, main drying at -40--30°C, and gradually heating to 0°C after drying. The extraction conditions are: temperature 40-50°C, pressure 40-50 MPa, and time 3-5 hours. (2) Water dissolution and enzymatic hydrolysis Soybean meal is added to purified water at a material-liquid ratio of 1:12 to 1:15, and 0.1 to 0.5% of a β-galactosidase inhibitor is added. After extraction for 60 to 90 minutes, the pH is dynamically adjusted and a dual-enzyme segmented enzymolysis is performed. The dual enzymes include a neutral protease and a flavor protease, and the addition amount is 2% to 6%. The neutral protease is added first, and then the flavor protease is added. The dynamic pH adjustment method is: 6.5→7.2→6.8, the addition ratio of neutral protease and flavor protease is 5:1; (3) Enzyme inactivation and separation The enzymatically hydrolyzed liquid is subjected to enzyme inactivation treatment and then rapidly cooled. After centrifugation, the supernatant is taken for ultrafiltration to retain polypeptides with a relative molecular mass of less than 5000. (4) Weakly acidic protein precipitation The supernatant obtained after separation was subjected to gradient acid adjustment with magnetic stirring; the acid adjustment conditions were: 6.0→4.5, pH drop of 0.5 / 10min, 0→0.8M, rate of 0.02M / min; (5) Ion exchange chromatography and target peak collection A gradient elution procedure was used to elute oligosaccharides first and then polypeptides. The overlapping region of the oligosaccharide peak and the polypeptide peak was collected, and finally a light yellow liquid was obtained by nanofiltration. The gradient elution procedure was as follows: in the first stage, the NaCl concentration was adjusted to 0-0.4M to elute oligosaccharides, in the second stage, the NaCl concentration was adjusted to 0.6-0.8M to elute polypeptides, and in the third stage, the NaCl concentration was adjusted to 0.9-1.0M to elute impurities. (6) Activated carbon decolorization and deodorization Adjust the pH value of the liquid and add activated carbon twice in a gradient manner while stirring: first add 2% to 3% of the liquid and treat for 25 to 35 minutes, then add 2% to 3% and treat for 5 to 15 minutes, the temperature is 40 to 50 ° C, and the stirrer speed is 400 to 600 r / min; (7) Filtration, sterilization, and concentration The liquid is centrifuged to obtain the supernatant, which is then filtered through micropores, sterilized by ultra-high temperature and concentrated by reverse osmosis. (8) Freeze-drying After pre-freezing at -80°C for 30 minutes, main drying is performed: temperature is increased stepwise from -40 to -30°C and 0.005 to 0.015 MPa, and dehydration is performed at a controlled rate to obtain the soybean composite functional factor.

2. The preparation method according to claim 1, characterized in that The method for adding the double enzymes in step (2) is as follows: first add neutral protease at 55-58°C for enzymatic hydrolysis for 60-70 minutes, then add flavor protease at 50-60°C for enzymatic hydrolysis for 60-80 minutes, stirring while adding flavor protease, at a rotation speed of 60-80 r / min.

3. The preparation method according to claim 2, characterized in that The enzyme inactivation conditions in step (3) are: temperature 90-100° C., time 8-12 min; and centrifugation conditions are: speed 4000-5000 r / min, time 13-15 min.

4. The preparation method according to claim 3, characterized in that The collection conditions of the overlapping region of the oligosaccharide peak and the polypeptide peak in step (5) are as follows: an initial flow rate of 0.4-0.6 mL / min, increased to 1.8-2.2 mL / min at a NaCl concentration of 0.4-0.6 M, a processing time of 50-60 min, and detection by HPLC using a C18 column and a mobile phase of 0.1% phosphoric acid water-methanol = 85:15; the relative molecular mass of the substance retained by nanofiltration is ≤550.

5. The preparation method according to claim 4, characterized in that The sterilization conditions in step (7) are: temperature 125-135°C, time 4-6s, pressure 15-20MPa; the reverse osmosis concentration conditions are: The retentate concentration is ≥10%.

6. A soybean composite functional factor, characterized in that: The invention is prepared according to the preparation method according to any one of claims 1 to 5.

7. A novel step-by-step encapsulation method for soybean composite functional factors, characterized in that: The soybean composite functional factor comprises the soybean composite functional factor according to claim 6, and the method comprises the following steps: Step 1: New liposome encapsulation (1) 4-6% phosphatidylethanolamine + 1-2% sodium cholesterol sulfate are dissolved in supercritical CO2 to obtain a lipid solution; (2) The soybean composite functional factor was dissolved in phosphate buffer and injected into the microfluidic chip at a volume ratio of (0.1-1.5):(2.5-3.5) with the lipid solution; (3) low-temperature vacuum drying to form liposome precursors; Step 2: Water phase gelation wrapping (1) Gellan gum and Tween 80 are dissolved in deionized water to a concentration of 4.5-5.5% and 1.5-2.5%, respectively, and heated to form a thixotropic gel; (2) mixing the liposome precursor with the gel solution and forming liposome-in-water composite particles by ultrasound-assisted emulsification; Step 3: Purification and concentration (1) Use a composite membrane with a molecular weight cut-off of 900 to 1100 and a pressure of 1.5 to 2.0 bar; (2) Freeze-drying and adding trehalose as a protective agent. After freeze-drying, the liposome encapsulation efficiency is ≥90%, and the particle size is controlled at 150-200 nm.

8. The novel soybean composite functional factor step-by-step encapsulation method according to claim 7, characterized in that: The vacuum drying conditions in the first step are: -45 to -55°C, 0.05 to 0.15 MPa; the ultrasound-assisted emulsification conditions in the second step are: 18 to 22 kHz, 4 to 6 minutes; and the amount of trehalose added in the third step is 4 to 6%.

9. A soybean composite functional factor liposome encapsulated material, characterized in that: It is prepared according to the method according to any one of claims 7 to 8.

10. Use of the preparation method of the soybean composite functional factor according to any one of claims 1 to 5, the soybean composite functional factor according to claim 6, the novel step-by-step encapsulation method of the soybean composite functional factor according to any one of claims 7 to 8, or the soybean composite functional factor liposome encapsulation material according to claim 9 in the preparation of cosmetics.

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