Preparation method and application of general composite enzymolysis suitable for warm-nourishing raw materials

Abstract

The application discloses a preparation method and application of general composite enzymolysis suitable for warm and tonic raw materials, and belongs to the technical field of enzymolysis and daily body care product processing. The application mixes the warm and tonic raw materials suitable for the application with composite enzymes, uniformly mixes the warm and tonic raw materials suitable for the application with the composite enzymes, crushes and sieves the warm and tonic raw materials suitable for the application with the composite enzymes, then mixes the warm and tonic raw materials suitable for the application with the composite enzymes with water to obtain a slurry; the obtained slurry is subjected to twice enzymolysis, enzyme inactivation and reduced-pressure concentration to obtain a product. The method of the application first breaks the plant cell wall to release components, and then directionally cracks phenolic glycoside bonds / phenolic ester bonds to generate liposoluble small-molecule phenolic active ingredients; the application can convert difficult-to-penetrating macromolecules into 280-420 Da liposoluble small molecules, and simultaneously realizes high skin permeation and long-acting warm and soothing. The method of the application can be suitable for all warm and tonic natural plant external raw materials containing phenolic glycosides / phenolic esters, and is general and compatible. The product prepared by using the application can realize long-acting warm and skin soothing synergistic effect.

Description

Technical Field

[0001] This invention relates to the field of enzymatic hydrolysis and processing technology of daily body care products, specifically to a general compound enzymatic hydrolysis preparation method and application applicable to mild and nourishing raw materials. Background Technology

[0002] Gentle and nourishing natural ingredients are the core raw materials for topical care patches, ointments, gels, and creams. Transdermal absorption efficiency and skin feel are key product indicators. Current processing techniques primarily rely on physical pulverization and hot water extraction, resulting in the active ingredients remaining in a large molecular state, unable to generate fat-soluble active ingredients, leading to extremely low skin dissolution and penetration efficiency. Existing enzymatic hydrolysis technologies are mostly designed for oral food ingredients, lacking a universal enzymatic hydrolysis + warming and soothing dual-effect process specifically for gentle and nourishing ingredients. Current technologies all use a single enzyme, a one-step enzymatic hydrolysis, and are only suitable for a single raw material, lacking a design for the targeted cleavage of phenolic glycosides and phenolic ester bonds.

[0003] Currently, traditional warming and nourishing ingredients are extracted only through crushing and boiling, resulting in large molecular forms of active ingredients with low skin dissolution rates and poor transdermal penetration. Existing topical products often use chemical penetration enhancers, which can easily cause skin redness and irritation, lacking gentle biological penetration-enhancing solutions. Current technologies do not yet offer a universal enzymatic hydrolysis penetration-enhancing + warming and soothing dual-effect process for warming and nourishing ingredients; they are only suitable for single ingredients and fixed formulas, resulting in extremely poor versatility.

[0004] Typically, the raw materials and carriers of topical care products have poor compatibility, resulting in short skin retention time, low utilization efficiency, and no lasting warming sensation. Warming and nourishing topical products lack core technological barriers and are easily imitated based solely on their formulas, making it impossible to quantify their warming, soothing, and transdermal effects using objective physicochemical indicators. Summary of the Invention

[0005] In view of this, the present invention provides a universal compound enzymatic hydrolysis preparation method suitable for warming and nourishing raw materials, and a long-lasting warming and soothing external application. It can convert macromolecular phenolic glycosides and phenolic esters into lipid-soluble small-molecule phenolic active ingredients, achieving a triple effect of high transdermal penetration, long-lasting warmth, and skin soothing, making it suitable for large-scale production of external care products.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A general compound enzymatic hydrolysis preparation method applicable to warming and nourishing raw materials includes the following steps: (1) Take suitable warming and nourishing raw materials and compound enzymes, mix them evenly, and then crush and sieve them; The complex enzyme is composed of the following raw materials in parts by weight: 10-30 parts cellulase, 10-30 parts pectinase, 10-20 parts keratinase, 10-20 parts β-glucosidase, 5-20 parts phenol esterase, 3-10 parts laccase, 3-10 parts glucanase, 3-10 parts lipase, and 3-10 parts melibiase. (2) Mix the sieved material with water and stir evenly to obtain a slurry; (3) The obtained slurry was subjected to a first enzymatic hydrolysis at 45-50℃ and pH 5.0-5.5; (4) The slurry after the first enzymatic hydrolysis is subjected to a second enzymatic hydrolysis at 37-40℃ and pH 5.5-6.5; (3) The slurry after the second enzymatic hydrolysis is inactivated and concentrated under reduced pressure to obtain a general compound enzymatic hydrolysis product suitable for warm supplement raw materials.

[0007] In this invention, warming and nourishing raw materials refer to natural plant and animal raw materials that are rich in phenolic glycosides and phenolic ester precursors and can produce a long-lasting warming and soothing sensation after enzymatic hydrolysis, such as mugwort, epimedium, cinnamon, cistanche, clove, etc., which is a classification description of raw materials.

[0008] Lipid-soluble small molecule phenolic active ingredients: Natural active ingredients with a molecular weight of 280-420 Da and a lipid-water partition coefficient of 2.0-3.5, which can easily penetrate the stratum corneum of the skin, generated by enzymatic hydrolysis in this invention.

[0009] The fat-soluble small molecules are small molecule phenolic active ingredients (phenolic aglycones, small molecule polyphenols) of mild and nourishing natural raw materials. Its molecular formula conforms to: Molecular weight 120~420 Da; Its core general structural formula conforms to: ; Wherein, Ar is the benzene ring core, OH is the phenolic hydroxyl group, R is the C1~C3 short chain substituent, m=1~3, n=0~2; The complete structural formula conforms to: ; Wherein, -Ph: benzene ring; -x=1~3: number of phenolic hydroxyl groups; -R: H, CH3, OCH3; -no glycosidic syl group → lipid-soluble small molecule phenolic aglycone).

[0010] The lipid-soluble small molecule is characterized by the absence of glycosidic saccharides, a benzene ring core, phenolic hydroxyl groups, and short lipid-soluble side chains, and possesses high lipid solubility, high transdermal permeability, and warming and soothing biological activity.

[0011] The long-lasting warming effect of this invention is manifested in the following ways: after external application, the local skin temperature is stably increased by 0.5-1.5℃, and the warming effect lasts for 4-6 hours, which can be quantitatively detected by an infrared thermal imaging instrument as a physical skin sensation index; the skin soothing effect of this invention is manifested in the following ways: relieving discomfort such as skin tightness and coldness, and improving the daily care effect of skin comfort; the transdermal penetration promotion effect of this invention is manifested in the following ways: using compound enzymes to biodegrade warming and nourishing raw materials, improving the dissolution of ingredients, transdermal efficiency and care activity.

[0012] Furthermore, the applicable warming and tonifying ingredients are any one or more of the following: mugwort, angelica, evodia, clove, ginger, borneol, and seahorse.

[0013] Preferably, its composition includes the following raw materials in parts by weight: 10-50 parts of Artemisia argyi, 5-30 parts of Angelica sinensis, 5-30 parts of Evodia rutaecarpa, 5-15 parts of cloves, 5-30 parts of ginger, 1-10 parts of borneol, and 1-10 parts of seahorse.

[0014] Furthermore, the mass ratio of the suitable warming and nourishing raw materials to the compound enzyme is 1:0.002-0.012.

[0015] Furthermore, the sieving in step (1) is sieving through an 80-mesh sieve.

[0016] Furthermore, in step (2), the ratio of the screened material to the water is 1:6-10.

[0017] Furthermore, the first enzymatic hydrolysis time in step (3) is 90-180 min; the second enzymatic hydrolysis time in step (4) is 120-240 min.

[0018] The present invention involves two enzymatic hydrolysis processes: the first process breaks the cell wall, and the second process directionally cleaves the phenolic glycoside / phenolic ester bonds.

[0019] Furthermore, the enzyme inactivation described in step (5) is performed at 75-85℃ for 15 minutes to retain the lipid-soluble small molecule active ingredients; The vacuum concentration is performed at 65-75°C. The present invention also provides the application of the general-purpose composite enzymatic hydrolysate suitable for warming and nourishing raw materials prepared by the above method in long-lasting warm and soothing external application. The method is as follows: the obtained general-purpose composite enzymatic hydrolysate suitable for warming and nourishing raw materials is mixed with the dressing matrix to make an external care patch, ointment, gel or lotion.

[0020] The beneficial effects of this invention are as follows: The universal compound enzyme formulation in this invention is customized for common components such as phenolic glycosides, phenolic esters, cellulose, and pectin in warming and nourishing raw materials, without relying on any fixed formula; moreover, the method of this invention first breaks down plant cell walls to release components, and then directionally cleaves phenolic glycoside / phenolic ester bonds to generate lipid-soluble small-molecule phenolic active ingredients; this invention can convert large molecules that are difficult to penetrate the skin into 280-420 Da lipid-soluble small molecules, simultaneously achieving high transdermal penetration and long-lasting warming and soothing effects. The method of this invention is applicable to all warming and nourishing natural plant external raw materials containing phenolic glycosides / phenolic esters, and is universally compatible. The product prepared using this invention can achieve a synergistic effect of long-lasting warming and skin soothing. Detailed Implementation

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1 A general compound enzymatic hydrolysis preparation method applicable to warming and nourishing raw materials (1) Take suitable warming and nourishing raw materials and compound enzymes, mix them evenly and then crush them through an 80-mesh sieve; The ingredients suitable for warming and tonifying are: 25 parts mugwort, 15 parts angelica, 20 parts evodia, 10 parts clove, 25 parts ginger, 6 parts borneol, and 6 parts seahorse. The complex enzyme composition is as follows: 20 parts cellulase, 20 parts pectinase, 15 parts keratinase, 15 parts β-glucosidase, 17 parts phenol esterase, 8 parts laccase, 6 parts glucanase, 6 parts lipase, and 6 parts melibiase. The appropriate mass ratio of warming and nourishing raw materials to the compound enzyme is 1:0.008; (2) Mix the sieved material with water and stir evenly to obtain a slurry; the ratio of sieved material to water is 1:8. (3) The obtained slurry was enzymatically hydrolyzed at 47℃ and pH 5.2 for 150 min; (4) The slurry after the first enzymatic hydrolysis was enzymatically hydrolyzed for 180 min at 38℃ and pH 6.0; (3) The slurry after the second enzymatic hydrolysis was inactivated at 75°C for 15 min and concentrated under reduced pressure at 65°C to obtain a general compound enzymatic hydrolysis product suitable for warm supplement raw materials.

[0023] Example 2 A general compound enzymatic hydrolysis preparation method applicable to warming and nourishing raw materials (1) Take suitable warming and nourishing raw materials and compound enzymes, mix them evenly and then crush them through an 80-mesh sieve; The ingredients suitable for warming and tonifying are: 25 parts mugwort, 15 parts angelica, 20 parts evodia, 10 parts clove, 25 parts ginger, 6 parts borneol, and 6 parts seahorse. The complex enzyme composition is as follows: 10 parts cellulase, 30 parts pectinase, 10 parts keratinase, 10 parts β-glucosidase, 20 parts phenol esterase, 10 parts laccase, 3 parts glucanase, 3 parts lipase, and 10 parts melibiase. The appropriate mass ratio of warming and nourishing raw materials to the compound enzyme is 1:0.012; (2) Mix the sieved material with water and stir evenly to obtain a slurry; the ratio of sieved material to water is 1:10. (3) The obtained slurry was enzymatically hydrolyzed at 50℃ and pH 5.5 for 180 min; (4) The slurry after the first enzymatic hydrolysis was enzymatically hydrolyzed at 40℃ and pH 6.5 for 240 min; (3) The slurry after the second enzymatic hydrolysis was inactivated at 85°C for 15 min and concentrated under reduced pressure at 65°C to obtain a general compound enzymatic hydrolysis product suitable for warm supplement raw materials.

[0024] Example 3 A general compound enzymatic hydrolysis preparation method applicable to warming and nourishing raw materials (1) Take suitable warming and nourishing raw materials and compound enzymes, mix them evenly and then crush them through an 80-mesh sieve; The ingredients suitable for warming and tonifying are: 25 parts mugwort, 15 parts angelica, 20 parts evodia, 10 parts clove, 25 parts ginger, 6 parts borneol, and 6 parts seahorse. The complex enzyme composition is as follows: 30 parts cellulase, 10 parts pectinase, 20 parts keratinase, 20 parts β-glucosidase, 5 parts phenol esterase, 3 parts laccase, 10 parts glucanase, 10 parts lipase, and 3 parts melibiase. The appropriate mass ratio of mildly nourishing raw materials to the compound enzyme is 1:0.002-0.012. (2) Mix the sieved material with water and stir evenly to obtain a slurry; the ratio of sieved material to water is 1:6. (3) The obtained slurry was enzymatically hydrolyzed at 45℃ and pH 5.0 for 90 min; (4) The slurry after the first enzymatic hydrolysis was enzymatically hydrolyzed for 120 min at 37℃ and pH 5.5; (3) The slurry after the second enzymatic hydrolysis was inactivated at 75°C for 15 min and concentrated under reduced pressure at 65°C to obtain a general compound enzymatic hydrolysis product suitable for warm supplement raw materials.

[0025] Test case Efficacy Verification: Through in vitro physicochemical testing and skin compatibility verification, this invention confirms that the general-purpose composite enzymatic hydrolysis process, compared with the traditional pulverization process, has the following significant technical effects, meeting the requirements of efficient transdermal absorption and long-lasting retention for acupoint application: ① The yield of highly transdermal functional ingredients is significantly improved. The total polysaccharide content was determined by the phenol-sulfuric acid method, and the content of small molecule active ingredients was determined by the Folin-Ciocalteu colorimetric method. The proportion of small molecule components was determined by the alcohol precipitation fractionation method and the high performance gel filtration chromatography method.

[0026] In vitro testing showed that the extraction rate of small molecule phenolic active ingredients after enzymatic hydrolysis of the product in Example 1 increased by 3.5 times, and the proportion of small molecule ingredients increased from 22% to 89%.

[0027] ② Targeted optimization of component molecular weight Detection techniques and experimental methods: High-performance gel filtration chromatography was used with a TSK-G4000PWxl column, ultrapure water as the mobile phase, a flow rate of 0.8 mL / min, a column temperature of 35℃, and a differential refractive index detector. A molecular weight standard curve was plotted using dextran standard, and the molecular weight distribution of the samples was calculated.

[0028] Testing revealed that the molecular weight of functional components in traditional raw materials is mainly distributed between 10kDa and 120kDa. The solution of this invention can directionally degrade them into small molecule, highly transdermal components with a molecular weight of ≤5kDa, significantly improving skin penetration efficiency.

[0029] ③ Enhanced transdermal absorption rate The Franz diffusion cell method was used, with isolated mouse abdominal skin selected. The receiving solution was physiological saline-ethanol (9:1), the transdermal temperature was 37℃, the stirring speed was 300 r / min, and samples were taken at regular intervals to detect the transdermal amount. The skin retention time was evaluated using the skin residue assay.

[0030] In vitro transdermal testing showed that the transdermal permeability of the enzymatic hydrolysate of Example 1 of this invention was increased by 3.5 times and the skin residence time was extended by 2-3 times, which can form a stable permeable layer on the skin surface.

[0031] ④ Excellent skin compatibility Non-clinical skin safety verification was conducted in accordance with the human skin closed patch test method of the "Cosmetic Safety Technical Specifications (2015 Edition)". Thirty healthy adult volunteers (without skin diseases, no history of raw material allergies, and including those with sensitive skin) were selected. The enzymatic hydrolysis product extract of Example 1 was placed in the patch tester and fixed on the inner side of the subject's upper arm. The patch was continuously closed for 24 hours. After removing the patch tester, the skin reaction was observed at 30 minutes, 24 hours and 48 hours. The level of irritation / allergic reaction such as erythema, edema and itching was determined according to the standard.

[0032] The results showed that only 1 out of 30 subjects experienced transient mild redness, with no obvious edema, itching, allergies or other adverse reactions. The total adverse reaction rate was about 3.3%, with no moderate to severe irritation or allergic reactions. The product had very low overall skin irritation and good tolerance, and the skin felt gentle after penetration.

[0033] ⑤ Long-lasting warming effect verification: A randomized, single-blind, self-controlled human trial was conducted using a medical infrared thermal imager. The ambient temperature was 25±1℃ and the relative humidity was 50±5%. The local skin temperature was measured before application and at 0.5h, 1h, 2h, 4h, 6h, and 8h after application.

[0034] The results showed that the local skin temperature of the test group (the enzymatic hydrolysis product patch made from Example 1) steadily increased by 0.5~2.0℃, and the warming effect lasted for 4~6 hours, while the temperature rise of the control group (the patch made using the traditional pulverization process) was <0.5℃ and the warming effect lasted for <30 minutes.

[0035] ⑥ Verification of skin soothing effect: In accordance with the "Evaluation Specifications for Cosmetic Efficacy Claims", a randomized, double-blind, self-controlled human trial was conducted. The visual analog scale (VAS) was used to quantify the degree of skin coldness and tightness discomfort. After the subjects used the product of Example 1 for 3 consecutive days, the average relief rate of discomfort symptoms was ≥80%, and the skin comfort state was significantly improved.

[0036] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A general compound enzymatic hydrolysis preparation method applicable to warming and nourishing raw materials, characterized in that, Includes the following steps: (1) Take suitable warming and nourishing raw materials and compound enzymes, mix them evenly, and then crush and sieve them; The complex enzyme is composed of the following raw materials in parts by weight: 10-30 parts cellulase, 10-30 parts pectinase, 10-20 parts keratinase, 10-20 parts β-glucosidase, 5-20 parts phenol esterase, 3-10 parts laccase, 3-10 parts glucanase, 3-10 parts lipase, and 3-10 parts melibiase. (2) Mix the sieved material with water and stir evenly to obtain a slurry; (3) The obtained slurry was subjected to a first enzymatic hydrolysis at 45-50℃ and pH 5.0-5.5; (4) The slurry after the first enzymatic hydrolysis is subjected to a second enzymatic hydrolysis at 37-40℃ and pH 5.5-6.5; (5) The slurry after the second enzymatic hydrolysis is inactivated and concentrated under reduced pressure to obtain a general compound enzymatic hydrolysis product suitable for warm supplement raw materials.

2. The preparation method of a universal compound enzymatic hydrolysis applicable to warming and nourishing raw materials according to claim 1, characterized in that, The applicable warming and tonifying ingredients are any one or more of the following: mugwort, angelica, evodia, clove, ginger, borneol, and seahorse.

3. The preparation method of a universal compound enzymatic hydrolysis applicable to warming and nourishing raw materials according to claim 1 or 2, characterized in that, The mass ratio of the suitable warming and nourishing raw materials to the compound enzyme is 1:0.002-0.

012.

4. The preparation method of a universal compound enzymatic hydrolysis applicable to warming and nourishing raw materials according to claim 1, characterized in that, The sieving mentioned in step (1) is sieving through an 80-mesh sieve.

5. The preparation method of a universal compound enzymatic hydrolysis applicable to warming and nourishing raw materials according to claim 1, characterized in that, In step (2), the ratio of the screened material to water is 1:6-10.

6. The preparation method of a universal compound enzymatic hydrolysis applicable to warming and nourishing raw materials according to claim 1, characterized in that, The first enzymatic hydrolysis time in step (3) is 90-180 min; the second enzymatic hydrolysis time in step (4) is 120-240 min.

7. The preparation method of a universal compound enzymatic hydrolysis applicable to warming and nourishing raw materials according to claim 1, characterized in that, The enzyme inactivation described in step (5) is performed at 75-85℃ for 15 minutes; The vacuum concentration is carried out at 65-75°C.

8. The application of a universal compound enzymatic hydrolysate prepared by the method of any one of claims 1-7, suitable for warming and tonifying raw materials, in a long-lasting warming and soothing external application.

9. The application of a universal compound enzymatic hydrolysate of warming and tonifying raw materials according to claim 8 in a long-lasting warming and soothing external application, characterized in that, The resulting general-purpose enzymatic hydrolysis product matrix, suitable for warming and nourishing raw materials, is mixed to prepare external care patches, ointments, gels, or varnishes.