A preparation method of a cavac extract based on enzyme cutting technology, and a product and application thereof

Abstract

This invention relates to the field of daily chemical technology, and in particular to a method for preparing kava extract based on enzymatic hydrolysis technology, its products, and applications. The preparation method comprises the following steps: S01 low-temperature treatment of kava rhizomes, followed by pulverization and sieving; S02 enzymatic hydrolysis of the kava rhizome powder; S03 fermentation treatment of the kava rhizome enzymatic hydrolysis extract obtained in S02; and S04 stabilization treatment of the fermented kava rhizome extract obtained in S03. This invention employs a combination of multi-enzyme synergistic pretreatment and probiotic co-fermentation, conducted entirely in an aqueous environment, eliminating the need for organic solvents such as ethanol and methanol. The operation is safe and environmentally friendly, aligning with the development trend of green raw materials in cosmetics.

Description

Technical Field

[0001] This invention relates to the field of daily chemical technology, and in particular to a method for preparing kava extract based on enzymatic hydrolysis technology, as well as its products and applications. Background Technology

[0002] Kava, scientifically known as *Piper methysticum*, is a plant belonging to the Piperaceae family, primarily distributed in the South Pacific islands. The roots of kava have been used for centuries in local tradition as a natural sedative and a ritualistic beverage, exhibiting significant calming, nerve-soothing, and muscle-relaxing effects. With in-depth research into its pharmacological activity, kava has gradually attracted attention from the modern medical, nutritional, and cosmetic fields.

[0003] Modern research indicates that the main functional components of kava are kavalactones, including kavain, dihydrokavain, and yangonin, as well as flavonoids, polyphenols, and sterols. These components exhibit anti-anxiety, anti-inflammatory, antioxidant, and neurosoothing physiological activities in vitro and in vivo through mechanisms such as affecting GABA receptors, regulating neurotransmitter release, and inhibiting neuroinflammation. Therefore, kava extract is considered to have potential benefits in skin soothing, sensitivity treatment, and relief of neuroinflammation.

[0004] However, traditional kava extracts are typically extracted using ethanol or organic solvents, which not only suffers from poor stability of active ingredients and a high risk of residual dissolution, but also has drawbacks such as low bioavailability and potential increased irritation when added to skincare products. Furthermore, some countries have restrictions on the use of oral kava products, further driving its development towards "topical, natural, and gentle" formulations.

[0005] In recent years, with the rise of concepts such as "plant microecological fermentation," "post-biotics," and "bioactive peptides" in the cosmetics industry, extracting kava pepper using green biotransformation methods such as enzymatic hydrolysis and microbial fermentation has become a feasible strategy to improve its bioactivity, stability, and skin compatibility. For example, by synergistically releasing bound effective substances through plant cell wall degrading enzymes, or by fermenting kava root powder with safe strains such as lactic acid bacteria and yeast, not only can kava lactones and polyphenolic components be enriched, but post-biotic components with skin-soothing functions can also be induced, such as γ-aminobutyric acid (GABA), short-chain fatty acids, and small molecule glycopeptides.

[0006] Currently, there are no systematic reports in the published literature on the preparation method of kava pepper root with "multi-enzyme synergistic pretreatment + compound probiotic fermentation" as the core, and there is also a lack of specific application research on such kava fermentation extracts in the raw materials of soothing and repairing cosmetics.

[0007] Therefore, there is an urgent need to develop a green, safe, and efficient method for preparing kava pepper extract, which can not only significantly improve the release rate and stability of its active ingredients, but also endow it with better skin-soothing, anti-allergic, and anti-inflammatory activities to meet the development needs of modern functional skin care products and microecological skin care. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a method for preparing kava extract based on enzymatic digestion technology. The steps of the preparation method are as follows:

[0009] S01 Low-temperature treatment of Kava rhizomes, followed by crushing and sieving;

[0010] S02 enzyme digestion treatment of Kava rhizome powder;

[0011] Kava rhizome enzymatically digested extract obtained by fermenting S02 with S03;

[0012] S04 stabilizes the fermented extract of kava rhizome obtained from S03.

[0013] As one embodiment of the present invention, the detailed steps of step S01 are as follows: the freshly obtained kava rhizomes are washed, sliced, dried at a low temperature of 30-45°C, pulverized and passed through a 600-mesh sieve, and then sealed and stored.

[0014] As one embodiment of the present invention, the detailed steps of step S02 are as follows: the kava rhizome powder obtained in S01 is mixed with deionized water at a solid-liquid ratio of 1:15, and the pH is adjusted to 5.2 with lactate buffer; then enzymatic digestion is carried out in a constant temperature water bath at 35-45℃. Cellulase and pectinase are added at 0h; protease is added at 1.5h; mannanase is added at 3h; the water bath is continued until 4h is completed, and then the temperature is heated to 85℃ and maintained for 10 minutes to completely inactivate the enzymes.

[0015] In one embodiment of the present invention, the mass ratio of cellulase, pectinase, protease, mannanase and kava root powder is (1.0-1.5):(0.5-0.8):(0.3-0.5):(0.1-0.3):100.

[0016] As one embodiment of the present invention, the detailed steps of step S03 are as follows: the enzyme digestion product obtained in step S02 is rotary evaporated to 1 / 3 of the volume of the raw material, then heated to 35°C, and three activated bacterial solutions are added, with a total inoculum of 10%, and fermented at low speed for 72 hours; then heated to 70°C and held for 10 minutes, cooled to room temperature, centrifuged at 4000 rpm for 10 minutes, and the supernatant is collected.

[0017] As one embodiment of the present invention, the preparation steps of the three activated bacterial solutions are as follows:

[0018] Each bacterial strain was cultured separately in activation medium until a concentration of 10 was reached. 9 CFU / mL; then mix the three bacterial cultures in a 1:1:1 volume ratio; let stand for 30 minutes;

[0019] The activation medium was MRS medium, and the three bacterial strains were Lactobacillus plantarum, Bifidobacterium longum, and Saccharomyces cerevisiae.

[0020] As one embodiment of the present invention, the detailed steps of step S04 are as follows:

[0021] (3) In a rotary evaporation flask, phospholipids, cholesterol and vitamin E are dissolved in ethanol and the solvent is removed by rotary evaporation at 50°C to form a thin film;

[0022] (4) Add kava extract and mix slowly at a low temperature below 30°C for 30 min; then repeatedly freeze-thaw (from -20°C to room temperature, 3 times) to form uniform microbubbles; then sonicate for 10-15 min (100W) under ice bath conditions; then filter at 0.45 μm to remove impurities and freeze dry to make powder.

[0023] As one embodiment of the present invention, a kava extract is prepared by the preparation method described above.

[0024] As one embodiment of the present invention, the kava extract is used in the daily chemical industry.

[0025] As one embodiment of the present invention, a soothing water contains the aforementioned kava extract.

[0026] By adopting the above technical solution, the present invention has the following beneficial effects:

[0027] This invention employs a combination of multi-enzyme synergistic pretreatment and probiotic compound fermentation, conducted entirely in an aqueous environment. It eliminates the need for organic solvents such as ethanol and methanol, making the operation safe and environmentally friendly, and aligning with the development trend of green raw materials for cosmetics.

[0028] By adding cellulase, pectinase and protease for enzymatic hydrolysis in the early stage, the cell wall structure of kava root powder can be effectively destroyed, releasing more bound kava lactones, polyphenols and flavonoids, providing a sufficient substrate source for subsequent fermentation.

[0029] In the compound microbial fermentation system, Lactobacillus plantarum can efficiently produce γ-aminobutyric acid (GABA), Bifidobacterium longum can regulate the stability of the microbial community, and Saccharomyces cerevisiae can release a variety of vitamins and small molecule peptides, synergistically producing a variety of post-biotic metabolites, significantly enhancing the skin-soothing, calming, and barrier-repairing effects of the raw materials.

[0030] The organic acids produced during fermentation (such as lactic acid and short-chain fatty acids) can naturally regulate the pH of the system to a slightly acidic environment, which helps to inhibit harmful microorganisms, stabilize easily degradable components such as calvalactone and GABA, and at the same time have good preservative effects, improving the product's storage stability.

[0031] Compared to traditional kava alcohol extract, the fermented kava extract prepared by this invention has no solvent residue, a moderate pH, low irritation, and minimal toxicity risk. It is especially suitable for people with sensitive skin, sunburned skin, and skin prone to redness, and has higher skin tolerance. Attached Figure Description

[0032] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 The test diagram is for test 3, where ① to ⑥ are examples 1 to 6 respectively. Detailed Implementation

[0034] This invention provides a method for preparing kava extract based on enzymatic digestion technology. The steps of the preparation method are as follows:

[0035] S01 Wash the freshly obtained kava rhizomes, slice them, dry them at a low temperature of 30-45℃, pulverize them through a 600-mesh sieve, and then seal and store them.

[0036] S02: The kava rhizome powder obtained from S01 was mixed with deionized water at a solid-liquid ratio of 1:15, and the pH was adjusted to 5.2 with lactate buffer. Then, enzymatic digestion was carried out in a constant temperature water bath at 35-45℃. Cellulase and pectinase were added at 0h; protease was added at 1.5h; mannanase was added at 3h; and the water bath was continued for 4h. Then, the temperature was heated to 85℃ and held for 10 minutes to completely inactivate the enzymes.

[0037] The mass ratio of cellulase, pectinase, protease, mannanase, and kava root powder is 1.2:0.6:0.3:0.2:100.

[0038] S03 The enzyme digestion product obtained in step S02 is rotary evaporated to 1 / 3 of the volume of the raw material, then heated to 35°C, and three activated bacterial cultures are added. The total inoculum is 10 vol%, and fermentation is carried out at low speed for 72 hours. Then, it is heated to 70°C and held for 10 minutes, cooled to room temperature, centrifuged at 4000 rpm for 10 minutes, and the supernatant is collected.

[0039] The preparation steps for the three activated bacterial solutions are as follows:

[0040] Each bacterial strain was cultured separately in activation medium until a concentration of 10 was reached. 9 CFU / mL; then mix the three bacterial solutions in a 1:1:1 volume ratio; let stand for 30 minutes; the activation medium is MRS medium.

[0041] The three bacterial strains are Lactobacillus plantarum, Bifidobacterium longum, and Saccharomyces cerevisiae; respectively:

[0042] Lactobacillus plantarum 25125;

[0043] Bifidobacterium longum 6259;

[0044] Saccharomyces cerevisiae 1406.

[0045] S04 evaporates the supernatant under low pressure to 1 / 3 of its original volume. In a rotary evaporation flask, phospholipids, cholesterol, and vitamin E are dissolved in ethanol and the solvent is removed by rotary evaporation at 50°C to form a thin film. The ratio of phospholipids, cholesterol, vitamin E, and ethanol is 70mg:25mg:5mg:2mL.

[0046] Add kava extract (23 mg) and 115 mL of asiaticoside buffer (0.03 wt%, pH 5.5), mix slowly at a low temperature below 30 °C for 30 min; then freeze (-20 °C) and thaw (room temperature) 3 times to form uniform microbubbles; then sonicate for 10-15 min (100 W) under ice bath conditions; then filter through 0.45 μm to remove impurities, and freeze dry to obtain powder.

[0047] An instant soothing water containing kava extract, the raw materials for its preparation include:

[0048] Phase A: 84.78 parts water, 10.00 parts butanediol, 0.02 parts disodium EDTA, 0.50 parts p-hydroxyacetophenone, and 0.20 parts carbomer;

[0049] Phase B: 0.20 parts arginine, 1.80 parts water;

[0050] Phase C: 0.50 parts hexanediol, 2.0 parts kava extract.

[0051] The preparation method of the instant soothing water containing kava extract described in this invention is as follows: After mixing phase B and phase C evenly, phase A is added.

[0052] This invention utilizes an enzymatic digestion and fermentation method to prepare kava extract, increasing the release rate of kava lactones and generating more small molecules and phytic acids, thus promoting skin absorption and enhancing soothing effects. Simultaneously, it combines a polyphenol buffer solution to enhance the extract's stability through hydrogen bonding. While the extract obtained using this two-step process is highly effective, its stability suffers a sharp decline due to the increased susceptibility of fermented products to hydrolysis, photolysis, oxidants, and surfactants. The final stabilization treatment in this invention not only improves efficacy but also enhances stability.

[0053] The present invention will be further explained below with reference to specific embodiments.

[0054] Example 1

[0055] This embodiment provides an instant soothing water containing kava extract, the raw materials for which are prepared include:

[0056] Phase A: 84.78 parts water, 10.00 parts butanediol, 0.02 parts disodium EDTA, 0.50 parts p-hydroxyacetophenone, and 0.20 parts carbomer;

[0057] Phase B: 0.20 parts arginine, 1.80 parts water;

[0058] Phase C: 0.50 parts hexanediol, 2.0 parts kava extract.

[0059] The preparation method of the kava extract includes the following steps:

[0060] S01 Wash the freshly obtained kava rhizomes, slice them, dry them at a low temperature of 30-45℃, pulverize them through a 600-mesh sieve, and then seal and store them.

[0061] S02: The kava rhizome powder obtained from S01 was mixed with deionized water at a solid-liquid ratio of 1:15, and the pH was adjusted to 5.2 with lactate buffer. Then, enzymatic digestion was carried out in a constant temperature water bath at 35-45℃. Cellulase and pectinase were added at 0h; protease was added at 1.5h; mannanase was added at 3h; and the water bath was continued for 4h. Then, the temperature was heated to 85℃ and held for 10 minutes to completely inactivate the enzymes.

[0062] The mass ratio of cellulase, pectinase, protease, mannanase, and kava root powder is 1.2:0.6:0.3:0.2:100.

[0063] S03 The enzyme digestion product obtained in step S02 is rotary evaporated to 1 / 3 of the volume of the raw material, then heated to 35°C, and three activated bacterial cultures are added. The total inoculum is 10 vol%, and fermentation is carried out at low speed for 72 hours. Then, it is heated to 70°C and held for 10 minutes, cooled to room temperature, centrifuged at 4000 rpm for 10 minutes, and the supernatant is collected.

[0064] The preparation steps for the three activated bacterial solutions are as follows:

[0065] Each bacterial strain was cultured separately in activation medium until a concentration of 10 was reached. 9 CFU / mL; then mix the three bacterial solutions in a 1:1:1 volume ratio; let stand for 30 minutes; the activation medium is MRS medium.

[0066] The three bacterial strains are Lactobacillus plantarum, Bifidobacterium longum, and Saccharomyces cerevisiae; respectively:

[0067] Lactobacillus plantarum 25125;

[0068] Bifidobacterium longum 6259;

[0069] Saccharomyces cerevisiae 1406.

[0070] S04 evaporates the supernatant under low pressure to 1 / 3 of its original volume. In a rotary evaporation flask, phospholipids, cholesterol, and vitamin E are dissolved in ethanol and the solvent is removed by rotary evaporation at 50°C to form a thin film. The ratio of phospholipids, cholesterol, vitamin E, and ethanol is 70mg:25mg:5mg:2mL.

[0071] Add kava extract (23 mg) and 115 mL of asiaticoside buffer (0.03 wt%, pH 5.5), mix slowly at a low temperature below 30 °C for 30 min; then freeze (-20 °C) and thaw (room temperature) 3 times to form uniform microbubbles; then sonicate for 10-15 min (100 W) under ice bath conditions; then filter through 0.45 μm to remove impurities, and freeze dry to obtain powder.

[0072] The preparation method of the instant soothing water containing kava extract described in this embodiment is as follows:

[0073] The preparation method of the instant soothing water containing kava extract described in this invention is as follows: After mixing phase B and phase C evenly, phase A is added.

[0074] Example 2

[0075] This embodiment provides an instant soothing water containing kava extract, the raw materials for which are prepared include:

[0076] Phase A: 84.78 parts water, 10.00 parts butanediol, 0.02 parts disodium EDTA, 0.50 parts p-hydroxyacetophenone, and 0.20 parts carbomer;

[0077] Phase B: 0.20 parts arginine, 1.80 parts water;

[0078] Phase C: 0.50 parts hexanediol, 2.0 parts kava extract.

[0079] The preparation method of the kava extract includes the following steps:

[0080] S01 Wash the freshly obtained kava rhizomes, slice them, dry them at a low temperature of 30-45℃, pulverize them through a 600-mesh sieve, and then seal and store them.

[0081] S02: The kava rhizome powder obtained from S01 was mixed with deionized water at a solid-liquid ratio of 1:15, and the pH was adjusted to 5.2 with lactate buffer. Then, enzymatic digestion was carried out in a constant temperature water bath at 35-45℃. Cellulase and pectinase were added at 0h; protease was added at 1.5h; mannanase was added at 3h; and the water bath was continued for 4h. Then, the temperature was heated to 85℃ and held for 10 minutes to completely inactivate the enzymes.

[0082] The mass ratio of cellulase, pectinase, protease, mannanase, and kava root powder is 1.2:0.6:0.3:0.2:100.

[0083] S03 The enzymatic digestion product obtained in step S02 is rotary evaporated to a volume of 1 / 3 of the raw material, and then freeze-dried to produce powder.

[0084] The preparation method of the instant soothing water containing kava extract described in this embodiment is as follows:

[0085] The preparation method of the instant soothing water containing kava extract described in this invention is as follows: After mixing phase B and phase C evenly, phase A is added.

[0086] Example 3

[0087] This embodiment provides an instant soothing water containing kava extract, the raw materials for which are prepared include:

[0088] Phase A: 84.78 parts water, 10.00 parts butanediol, 0.02 parts disodium EDTA, 0.50 parts p-hydroxyacetophenone, and 0.20 parts carbomer;

[0089] Phase B: 0.20 parts arginine, 1.80 parts water;

[0090] Phase C: 0.50 parts hexanediol, 2.0 parts kava extract.

[0091] The preparation method of the kava extract includes the following steps:

[0092] S01 Wash the freshly obtained kava rhizomes, slice them, dry them at a low temperature of 30-45℃, pulverize them through a 600-mesh sieve, and then seal and store them.

[0093] S02: The kava rhizome powder obtained in S01 was mixed with water to the same volume, heated to 35°C, and three activated bacterial solutions were added. The total inoculum was 10 vol%, and fermented at low speed for 72 hours. Then, it was heated to 70°C and held for 10 minutes, cooled to room temperature, centrifuged at 4000 rpm for 10 minutes, and the supernatant was collected.

[0094] The preparation steps for the three activated bacterial solutions are as follows:

[0095] Each bacterial strain was cultured separately in activation medium until a concentration of 10 was reached. 9 CFU / mL; then mix the three bacterial solutions in a 1:1:1 volume ratio; let stand for 30 minutes; the activation medium is MRS medium.

[0096] The three bacterial strains are Lactobacillus plantarum, Bifidobacterium longum, and Saccharomyces cerevisiae; respectively:

[0097] Lactobacillus plantarum 25125;

[0098] Bifidobacterium longum 6259;

[0099] Saccharomyces cerevisiae 1406.

[0100] S03 evaporates the supernatant under low pressure to 1 / 3 of its original volume. In a rotary evaporation flask, phospholipids, cholesterol, and vitamin E are dissolved in ethanol and the solvent is removed by rotary evaporation at 50°C to form a thin film. The ratio of phospholipids, cholesterol, vitamin E, and ethanol is 70mg:25mg:5mg:2mL.

[0101] Add kava extract (23 mg) and 115 mL of asiaticoside buffer (0.03 wt%, pH 5.5), mix slowly at a low temperature below 30 °C for 30 min; then freeze (-20 °C) and thaw (room temperature) 3 times to form uniform microbubbles; then sonicate for 10-15 min (100 W) under ice bath conditions; then filter through 0.45 μm to remove impurities, and freeze dry to obtain powder.

[0102] The preparation method of the instant soothing water containing kava extract described in this embodiment is as follows:

[0103] The preparation method of the instant soothing water containing kava extract described in this invention is as follows: After mixing phase B and phase C evenly, phase A is added.

[0104] Example 4

[0105] This embodiment provides an instant soothing water containing kava extract, the raw materials for which are prepared include:

[0106] Phase A: 84.78 parts water, 10.00 parts butanediol, 0.02 parts disodium EDTA, 0.50 parts p-hydroxyacetophenone, and 0.20 parts carbomer;

[0107] Phase B: 0.20 parts arginine, 1.80 parts water;

[0108] Phase C: 0.50 parts hexanediol, 2.0 parts kava extract.

[0109] The preparation method of the kava extract includes the following steps:

[0110] S01 Wash the freshly obtained kava rhizomes, slice them, dry them at a low temperature of 30-45℃, pulverize them through a 600-mesh sieve, and then seal and store them.

[0111] S02: The kava rhizome powder obtained from S01 was mixed with deionized water at a solid-liquid ratio of 1:15, and the pH was adjusted to 5.2 with lactate buffer. Then, enzymatic digestion was carried out in a constant temperature water bath at 35-45℃. Cellulase and pectinase were added at 0h; protease was added at 1.5h; mannanase was added at 3h; and the water bath was continued for 4h. Then, the temperature was heated to 85℃ and held for 10 minutes to completely inactivate the enzymes.

[0112] The mass ratio of cellulase, pectinase, protease, mannanase, and kava root powder is 1.2:0.6:0.3:0.2:100.

[0113] S03 The enzyme digestion product obtained in step S02 is rotary evaporated to 1 / 3 of the volume of the raw material, then heated to 35°C, and three activated bacterial cultures are added. The total inoculum is 10 vol%, and fermentation is carried out at low speed for 72 hours. Then, it is heated to 70°C and held for 10 minutes, cooled to room temperature, centrifuged at 4000 rpm for 10 minutes, and the supernatant is collected.

[0114] The preparation steps for the three activated bacterial solutions are as follows:

[0115] Each bacterial strain was cultured separately in activation medium until a concentration of 10 was reached. 9 CFU / mL; then mix the three bacterial solutions in a 1:1:1 volume ratio; let stand for 30 minutes; the activation medium is MRS medium.

[0116] The three bacterial strains are Lactobacillus plantarum, Bifidobacterium longum, and Saccharomyces cerevisiae; respectively:

[0117] Lactobacillus plantarum 25125;

[0118] Bifidobacterium longum 6259;

[0119] Saccharomyces cerevisiae 1406.

[0120] S04 involves evaporating the supernatant under low pressure to one-third of its original volume, followed by freeze-drying to produce powder.

[0121] The preparation method of the instant soothing water containing kava extract described in this embodiment is as follows:

[0122] The preparation method of the instant soothing water containing kava extract described in this invention is as follows: After mixing phase B and phase C evenly, phase A is added.

[0123] Example 5

[0124] This embodiment provides an instant soothing water containing kava extract, the raw materials for which are prepared include:

[0125] Phase A: 84.78 parts water, 10.00 parts butanediol, 0.02 parts disodium EDTA, 0.50 parts p-hydroxyacetophenone, and 0.20 parts carbomer;

[0126] Phase B: 0.20 parts arginine, 1.80 parts water;

[0127] Phase C: 0.50 parts hexanediol, 2.0 parts kava extract.

[0128] The preparation method of the kava extract includes the following steps:

[0129] S01 Wash the freshly obtained kava rhizomes, slice them, dry them at a low temperature of 30-45℃, pulverize them through a 600-mesh sieve, and then seal and store them.

[0130] S02: The kava rhizome powder obtained from S01 was mixed with deionized water at a solid-liquid ratio of 1:15, and the pH was adjusted to 5.2 with lactate buffer. Then, enzymatic digestion was carried out in a constant temperature water bath at 35-45℃. Cellulase and pectinase were added at 0h; protease was added at 1.5h; mannanase was added at 3h; and the water bath was continued for 4h. Then, the temperature was heated to 85℃ and held for 10 minutes to completely inactivate the enzymes.

[0131] The mass ratio of cellulase, pectinase, protease, mannanase, and kava root powder is 1.2:0.6:0.3:0.2:100.

[0132] S03 The enzyme digestion product obtained in step S02 is rotary evaporated to 1 / 3 of the volume of the raw material, then heated to 35°C, and three activated bacterial cultures are added. The total inoculum is 10 vol%, and fermentation is carried out at low speed for 72 hours. Then, it is heated to 70°C and held for 10 minutes, cooled to room temperature, centrifuged at 4000 rpm for 10 minutes, and the supernatant is collected.

[0133] The preparation steps for the three activated bacterial solutions are as follows:

[0134] Each bacterial strain was cultured separately in activation medium until a concentration of 10 was reached. 9 CFU / mL; then mix the three bacterial solutions in a 1:1:1 volume ratio; let stand for 30 minutes; the activation medium is MRS medium.

[0135] The three bacterial strains are Lactobacillus plantarum, Bifidobacterium longum, and Saccharomyces cerevisiae; respectively:

[0136] Lactobacillus plantarum 25125;

[0137] Bifidobacterium longum 6259;

[0138] Saccharomyces cerevisiae 1406.

[0139] S04 After evaporating the supernatant under low pressure to 1 / 3 of its original volume, the cyclodextrin and water are mixed and heated to 40°C to dissolve completely, yielding an 8wt% aqueous solution of hydroxypropyl-β-cyclodextrin. The concentrated supernatant of the kava extract is added to the hydroxypropyl-β-cyclodextrin aqueous solution, stirred evenly, allowed to stand, and filtered. The solution is then freeze-dried to obtain powder. The ratio of kava extract to hydroxypropyl-β-cyclodextrin is 1:5.

[0140] The preparation method of the instant soothing water containing kava extract described in this embodiment is as follows:

[0141] The preparation method of the instant soothing water containing kava extract described in this invention is as follows: After mixing phase B and phase C evenly, phase A is added.

[0142] Example 6

[0143] This embodiment provides an instant soothing water containing kava extract, the raw materials for which are prepared include:

[0144] Phase A: 84.78 parts water, 10.00 parts butanediol, 0.02 parts disodium EDTA, 0.50 parts p-hydroxyacetophenone, and 0.20 parts carbomer;

[0145] Phase B: 0.20 parts arginine, 1.80 parts water;

[0146] Phase C: 0.50 parts hexanediol, 2.0 parts kava extract.

[0147] The preparation method of the kava extract includes the following steps:

[0148] S01 Wash the freshly obtained kava rhizomes, slice them, dry them at a low temperature of 30-45℃, pulverize them through a 600-mesh sieve, and then seal and store them.

[0149] S02: The kava rhizome powder obtained from S01 was mixed with deionized water at a solid-liquid ratio of 1:15, and the pH was adjusted to 5.2 with lactate buffer. Then, enzymatic digestion was carried out in a constant temperature water bath at 35-45℃. Cellulase and pectinase were added at 0h; protease was added at 1.5h; mannanase was added at 3h; and the water bath was continued for 4h. Then, the temperature was heated to 85℃ and held for 10 minutes to completely inactivate the enzymes.

[0150] The mass ratio of cellulase, pectinase, protease, mannanase, and kava root powder is 1.2:0.6:0.3:0.2:100.

[0151] S03 The enzyme digestion product obtained in step S02 is rotary evaporated to 1 / 3 of the volume of the raw material, then heated to 35°C, and the activated bacterial solution is added. The total inoculum is 10 vol%, and fermentation is carried out at low speed for 72 hours. Then, it is heated to 70°C and held for 10 minutes, cooled to room temperature, centrifuged at 4000 rpm for 10 minutes, and the supernatant is collected.

[0152] The preparation steps of the activated bacterial solution are as follows:

[0153] Each bacterial strain was cultured separately in activation medium until a concentration of 10 was reached. 9 CFU / mL; the activation medium was MRS medium.

[0154] The bacterial strain is *Lactobacillus plantarum*. 25125;

[0155] S04 evaporates the supernatant under low pressure to 1 / 3 of its original volume. In a rotary evaporation flask, phospholipids, cholesterol, and vitamin E are dissolved in ethanol and the solvent is removed by rotary evaporation at 50°C to form a thin film. The ratio of phospholipids, cholesterol, vitamin E, and ethanol is 70mg:25mg:5mg:2mL.

[0156] Add kava extract (23 mg) and 115 mL of asiaticoside buffer (0.03 wt%, pH 5.5), mix slowly at a low temperature below 30 °C for 30 min; then freeze (-20 °C) and thaw (room temperature) 3 times to form uniform microbubbles; then sonicate for 10-15 min (100 W) under ice bath conditions; then filter through 0.45 μm to remove impurities, and freeze dry to obtain powder.

[0157] The preparation method of the instant soothing water containing kava extract described in this embodiment is as follows:

[0158] The preparation method of the instant soothing water containing kava extract described in this invention is as follows: After mixing phase B and phase C evenly, phase A is added.

[0159] Performance testing

[0160] Test 1:

[0161] Thermal stability test: The instant soothing waters obtained in Examples 1 to 6 were subjected to thermal stability tests to observe the product shape. They were placed at 0℃, 25℃, and 40℃ for 90 days respectively, and the product properties and color were observed. The test results are shown in Table 1.

[0162] Thermal cycling experiment: The instant soothing water prepared in Examples 1-6 was placed in a sealed container, ensuring complete sealing to prevent interference from external humidity or gases. The sample was placed in a constant temperature chamber set at 45°C and maintained for 12 hours. After removing the sample, it was quickly transferred to a low temperature chamber at -5°C and maintained for 12 hours. This process was repeated 6 times after completing one high-low temperature cycle; the physical properties of the sample were checked after each cycle.

[0163] Table 1. Experimental Results

[0164] name 0℃ 25℃ 40℃ Hot and cold circulation Example 1 No change No change No change No change Example 2 Slightly cloudy turbid turbid turbid Example 3 Slightly cloudy turbid turbid turbid Example 4 Slightly cloudy Slightly cloudy turbid turbid Example 5 No change No change Slightly cloudy turbid Example 6 No change No change No change No change

[0165] Test 2: Hyaluronidase Inhibition Rate

[0166] Hyaluronidase is a specific enzyme that cleaves hyaluronic acid and is involved in allergic reactions, showing a strong correlation with histamine release from mast cells. The soothing effect of a test sample can be determined using the hyaluronidase inhibition rate; a higher inhibition rate indicates a stronger soothing effect, and vice versa.

[0167] This test follows the laboratory method (HMC-WI-029 hyaluronidase inhibition rate). The results of the hyaluronidase inhibition rate test on the test sample and the negative control are compared. If the inhibition rate of the test sample is significantly higher than that of the negative control (P<0.05), the test sample can be considered to have a certain soothing effect. This test method is an in vitro method and is suitable for evaluating cosmetics that claim to achieve soothing effects by inhibiting hyaluronidase.

[0168] Instruments and equipment

[0169] BSA224S analytical balance, L6s ultraviolet spectrophotometer

[0170] reagents

[0171] Hyaluronidase, BR, Sodium Hyaluronate, BR

[0172] Test methods

[0173] (1) Treatment of control materials and test samples

[0174] Sample group: diluted with pure water to a sample concentration of 10%; Positive control (dipotassium glycyrrhizate, purity ≥98%): diluted with pure water to a positive control concentration of 3%; Negative control: pure water.

[0175] (2) Experimental Operation Procedures

[0176] Set up a sample group, a sample background group, a solvent group, and a solvent background group. Each group should have 3 replicates. Add different reagent solutions to each of the four groups, shake well, and let stand at room temperature for 30 minutes to develop color. Measure the absorbance value at a wavelength of 528 nm using a UV spectrophotometer.

[0177] Calculation formula

[0178] Hyaluronidase inhibition rate (%) = (1 - (CD) / (AB)) * 100%

[0179] In the formula: A—absorbance of the reaction solution without sample; B—absorbance of the reaction solution without sample and enzyme; C—absorbance of the reaction solution containing sample and enzyme; D—absorbance of the reaction solution containing sample and without enzyme.

[0180] Table 2. Experimental Results

[0181] name Experimental results p-value Example 1 56.631±0.152 ＜0.05 Example 2 39.517±1.237 ＜0.05 Example 3 42.936±0.426 ＜0.05 Example 4 36.254±1.125 ＜0.05 Example 5 44.714±2.415 ＜0.05 Example 6 50.382±4.315 ＜0.05 negative control -1.523±0.233 / Positive control 70.231±1.927 ＜0.05

[0182] Hyaluronidase inhibition rate is retained to three decimal places. P < 0.05 indicates that there is a significant difference between the sample and the positive control and the negative control.

[0183] Test 3: Evaluation of Mild (Non-irritating) Efficacy

[0184] This experiment tests the ability of a test substance to induce toxic changes in the chorioallantoic membrane of chicken embryos, evaluating the potential irritant factors and processes of the assessed substance. This method is applicable to the screening and detection of toxicity in cosmetics and cosmetic raw materials. The chorioallantoic membrane is a respiratory membrane surrounding the chicken embryo. Taking advantage of the intact, clear, and transparent vascular system of the chorioallantoic membrane in mid-stage hatching chicken embryos, a certain amount of the test substance is directly contacted with the chorioallantoic membrane. After a period of time, changes in chorioallantoic membrane toxicity indicators (such as hemorrhage, coagulation, and vascularization) are observed, and then a score is obtained by combining these results.

[0185] The indicators are shown in Table 3.

[0186] Table 3 Test Indicators

[0187]

[0188] Experimental preparation

[0189] The test samples were from Examples 1 to 6.

[0190] Chicken embryos: Fertilized chicken embryos of White Laihang chickens are selected. The quality of the chicken embryos meets the relevant standards and requirements. The supplier should be a designated production enterprise of SPF eggs for veterinary drug production and inspection recognized by the agricultural department.

[0191] Equipment: Incubator, serial number MCJY-171; Clean bench, serial number MCJY-159.

[0192] Test methods

[0193] Incubation conditions: Incubator temperature 37.5℃±0.5℃, relative humidity 55%~70%.

[0194] Experimental controls: The negative control was a 0.9% sodium chloride solution, and the positive control was a 0.1 mol / L sodium hydroxide solution. Each group contained at least 6 embryos, with an additional 1 chicken embryo each for the positive substance (reference material) and the solvent / carrier control.

[0195] Ten-day-old chicken embryos were removed, and the air cell was examined using an egg candler to determine its location. The upper layer of the eggshell above the air cell was peeled off with tweezers to expose the white shell membrane. An appropriate amount of 0.9% sodium chloride solution was added to moisten the shell membrane, which was then carefully removed with tweezers. The condition of the chorioallantoic membrane was recorded using a photographic device. The test sample was spread into a thin layer on the surface of a plastic film and placed over the chorioallantoic membrane for several minutes. After several minutes of treatment, the degree of chorioallantoic vascular damage was recorded using a photographic device, and a score was given based on the degree of vascular damage. The test results are shown below. Figure 1 And Table 4.

[0196] Table 4 shows the test results for Test 3.

[0197]

[0198]

[0199] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing kava extract based on enzymatic digestion technology, characterized in that, The preparation method comprises the following steps: S01 Low-temperature treatment of Kava rhizomes, followed by crushing and sieving; S02 enzyme digestion treatment of kava rhizome powder; Enzymatically digested extract of kava rhizome obtained by fermentation of S02 with S03; S04 Stabilization treatment of S03 yields kava rhizome fermentation extract; The detailed steps of step S02 are as follows: The kava rhizome powder obtained in S01 is mixed with deionized water at a solid-liquid ratio of 1:15, and the pH is adjusted to 5.2 with lactate buffer. Then, enzymatic digestion is carried out in a constant temperature water bath at 35~45℃. Cellulase and pectinase are added at 0 h; protease is added at 1.5 h; mannanase is added at 3 h; the water bath is continued for 4 hours, and then the temperature is heated to 85℃ and maintained for 10 minutes to completely inactivate the enzymes. The mass ratio of cellulase, pectinase, protease, mannanase, and kava root powder is (1.0-1.5):(0.5-0.8):(0.3-0.5):(0.1-0.3):100; The detailed steps of step S03 are as follows: The enzyme digestion product obtained in step S02 is rotary evaporated to 1 / 3 of the volume of the raw material, then heated to 35°C, and the three activated bacterial cultures are added, with a total inoculum of 10%. Fermentation is carried out at low speed with stirring for 72 hours; subsequently, the temperature is raised to 70°C and maintained for 10 minutes, then cooled to room temperature, centrifuged at 4000 rpm for 10 minutes, and the supernatant is collected. The preparation steps of the three activated bacterial cultures are as follows: Each bacterial strain was cultured separately in activation medium until a concentration of 10 was reached. 9 CFU / mL; then mix the three bacterial cultures in a 1:1:1 volume ratio; let stand for 30 minutes. The activation medium was MRS medium, and the three bacterial strains were Lactobacillus plantarum, Bifidobacterium longum and Saccharomyces cerevisiae. The detailed steps of step S04 are as follows: (1) In a rotary evaporation flask, phospholipids, cholesterol, and vitamin E are dissolved in ethanol, and the solvent is removed by rotary evaporation at 50°C to form a thin film; (2) Add kava extract and mix slowly at a low temperature below 30°C for 30 min; then repeatedly freeze-thaw to form uniform microbubbles; then sonicate at 100W for 10~15 min under ice bath conditions; then filter at 0.45μm to remove impurities, and freeze dry to make powder.

2. The preparation method of kava extract based on enzymatic digestion technology according to claim 1, characterized in that, The detailed steps of step S01 are as follows: Wash the freshly obtained kava rhizomes, slice them, dry them at a low temperature of 30-45℃, pulverize them through a 600-mesh sieve, and then seal and store them.

3. A kava extract, characterized in that, It is prepared by the preparation method described in claim 1 or 2.

4. The kava extract according to claim 3, characterized in that, Used in the daily chemical industry.

5. A soothing water, characterized in that, It contains the kava extract as described in claim 3.

Images