Preparation method and application of ginseng fermentation liquid

Abstract

The present invention provides a preparation method and application of ginseng fermented liquid, comprising the following steps: obtaining fermentation raw materials, using ginseng as raw materials, and obtaining fermentation raw materials after crushing and puffing treatment; primary fermentation, enzymolysis with amylase and amyloglucosidase to obtain enzymolysis liquid A; secondary fermentation, then adding Rhizopus oryzae and lactobacillus mixed liquid to enzymolysis liquid A, first aerobic fermentation for 45 to 48 hours, and then anaerobic fermentation for 28 to 35 hours to obtain fermentation liquid B; fermentation liquid B is filtered through a filter bag, sterilized, allowed to stand, centrifuged, and filtered through a membrane, and the filtrate is collected to obtain ginseng fermentation filtrate. The ginseng fermentation composition prepared by the present invention has a significant effect on skin anti-wrinkle and firming.

Description

Technical Field

[0001] The invention belongs to the field of natural product extraction, and particularly relates to ginseng fermented liquid, a preparation method of the ginseng fermented liquid, and a composition containing the ginseng fermented liquid for anti-wrinkle and skin tightening. Background Art

[0002] It is well known that aging and environmental stresses cause changes in the appearance, elasticity, and thickness of the skin. Specifically, aging and environmental factors can lead to skin conditions, including but not limited to fine lines, wrinkles, dry skin, enlarged pores, skin discoloration, reduced elasticity, excess hair, thinning of the skin, purpura, actinic keratosis, pruritus, eczema, acne, rosacea, erythema, telangiectasia, actinic telangiectasia, skin cancer, and hypertrophic rosacea. As a result, consumers are increasingly turning to skin care products to reduce the appearance of wrinkles, even out skin tone, provide moisture, reduce the appearance of dark circles under the eyes, and the like.

[0003] Traditional cosmetics are mainly synthesized by the chemical industry, with functions such as cleaning, whitening, and skin care. However, many consumers are hesitant to use chemically synthesized products, as they believe that such products are not friendly or safe to the body. Therefore, it is urgent to provide an anti-wrinkle and firming product with good safety and natural ingredients to meet consumer needs. Summary of the invention

[0004] The present invention has been made in view of the above-mentioned problems.

[0005] One object of the present invention is to provide a ginseng fermented liquid filtrate for improving skin conditions, specifically for helping the skin to resist wrinkles and tighten, and reducing the loss of skin collagen and elastin.

[0006] Another object of the present invention is to provide a ginseng fermentation liquid composition containing the ginseng fermentation filtrate.

[0007] The inventors have found that after ginseng is puffed, Rhizopus oryzae and Lactobacillus are used in combination, and there is a synergistic effect between the two, and the ginseng filtrate produced can improve the physiological state of the skin.

[0008] In particular, the present invention is achieved by the following preparation method:

[0009] The method for preparing ginseng fermentation filtrate comprises the following steps:

[0010] (1) obtaining a fermentation raw material, using ginseng as the raw material, and crushing and puffing the raw material to obtain the fermentation raw material;

[0011] (2) a primary fermentation, enzymatic hydrolysis with α-amylase and amyloglucosidase to obtain an enzymatic hydrolyzate A;

[0012] (3) secondary fermentation, then adding Rhizopus oryzae and Lactobacillus to the enzymatic hydrolysate A, aerobically fermenting at 28-30° C. for 45-48 hours, and anaerobically fermenting at 26-29° C. for 28-35 hours, to obtain fermentation liquid B;

[0013] (4) The fermentation liquid B is filtered through a filter bag, sterilized, allowed to stand, centrifuged, and filtered through a membrane in sequence, and the filtrate is collected to obtain a ginseng fermentation filtrate.

[0014] In a preferred embodiment of the present invention, the aerobic fermentation temperature can be 28°C, 29°C, or 30°C. In some embodiments, the aerobic fermentation temperature is controlled at 28°C.

[0015] In a preferred embodiment of the present invention, the aerobic fermentation time is 45 hours, 46 hours, 47 hours, or 48 hours. In some embodiments, the aerobic fermentation time is 48 hours.

[0016] In a preferred embodiment of the present invention, the anaerobic fermentation temperature can be 26°C, 27°C, 28°C, or 29°C. In some embodiments, the anaerobic fermentation temperature is 28°C.

[0017] In preferred embodiments of the present invention, the anaerobic fermentation time is 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, and 35 hours. In some embodiments, the anaerobic fermentation time is 34 hours.

[0018] Furthermore, the ginseng includes one or more compositions of ginseng root, ginseng leaf, ginseng stem or ginseng root.

[0019] Furthermore, the added amount of the amylase is 1-10 KNU / g, and the added amount of the amyloglucosidase is 10-40 U / g.

[0020] Further, Rhizopus oryzae and Lactobacillus are mixed in a ratio of 1:5.

[0021] Furthermore, the amount of the mixed solution of Rhizopus oryzae and Lactobacillus is 0.1-2% of the total mass of the fermentation solution.

[0022] Furthermore, the amount of fermentation raw materials added is 0.5-2% of the total mass of the fermentation liquid.

[0023] Furthermore, the ginseng fermentation filtrate is prepared by the method according to any one of claims 1-6.

[0024] A ginseng fermented liquid composition, characterized in that it comprises:

[0025] Ginseng ferment filtrate;

[0026] 1,2-Hexanediol; and

[0027] 1,2-Pentanediol.

[0028] Furthermore, the ginseng fermentation filtrate accounts for 1-5% of the total mass of the ginseng fermentation composition.

[0029] The invention discloses an application of a ginseng fermented liquid composition in preparing beauty and skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a schematic diagram of the anti-wrinkle effect of a preferred embodiment of the present invention;

[0031] Figure 2 It is a schematic diagram of the firming effect of a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0032] The embodiments of the present invention are described in detail below. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0033] The present invention relates to a ginseng fermentation filtrate for improving skin condition, specifically helping the skin to resist wrinkles and tighten, and reducing the loss of skin collagen and elastin, a preparation method of the ginseng fermentation filtrate, and a ginseng fermentation filtrate composition containing the ginseng fermentation filtrate for helping the skin to resist wrinkles and tighten, and reducing the loss of skin collagen and elastin.

[0034] The present invention will be described in detail below.

[0035] The method for preparing ginseng fermentation filtrate comprises the following steps:

[0036] (1) obtaining a fermentation raw material, using ginseng as the raw material, and crushing and puffing the raw material to obtain the fermentation raw material;

[0037] (2) a primary fermentation, enzymatic hydrolysis with amylase and amyloglucosidase to obtain an enzymatic hydrolyzate A;

[0038] (3) secondary fermentation, then adding Rhizopus oryzae and Lactobacillus to the enzymatic hydrolysate A, aerobically fermenting at 28-30° C. for 45-48 hours, and anaerobically fermenting at 26-29° C. for 28-35 hours, to obtain fermentation liquid B;

[0039] (4) The fermentation liquid B is filtered through a filter bag, sterilized, allowed to stand, centrifuged, and filtered through a membrane in sequence, and the filtrate is collected to obtain a ginseng fermentation liquid composition.

[0040] Ginseng not only refers to ginseng roots, but also refers to ginseng leaves, ginseng stems or ginseng roots, which are rarely used today.

[0041] The pulverization can be done by dry pulverization, where the medicine is properly dried and then pulverized, and then pulverized again by mixing and grinding. Wet pulverization can also be done by using liquid as the grinding medium, soaking the ginseng in a solvent for a period of time, and then pulverizing it. Airflow pulverization can also be used, using an airflow pulverizer to directly pulverize the cleaned ginseng raw materials. High-frequency vibration screen pulverization can also be used, where the ginseng raw materials are repeatedly impacted by high-frequency oscillation to achieve pulverization. It can also be dried and heated in an electric blast drying oven and then pulverized with a universal pulverizer: first, the dried ginseng is beaten into a pulp with water, and then dried in the sun to become cooked ginseng powder.

[0042] Puffing means putting the crushed ginseng powder into the puffing machine, setting the puffing temperature to 120-170℃, the puffing pressure to 3.92-4.90MPa, and controlling the feed motor voltage to about 50V, and the puffing machine starts working. As the heating and pressurization proceed, the water molecules inside the ginseng powder are in an overheated state. When a certain high pressure is reached, it instantly turns into normal pressure. The overheated water molecules inside the ginseng powder vaporize and explode at the same time. The huge puffing pressure changes the external form and internal structure of the ginseng powder, making it swell and loose, forming a sponge-like hollow mesh structure to obtain fermentation raw materials, which is convenient for subsequent reactions.

[0043] Amylase is an enzyme that generally acts on α-1,4-glucans such as soluble starch, amylose, and glycogen to hydrolyze α-1,4-glycosidic bonds. Amylase has the function of decomposing amylose and amylopectin, and its decomposition products include maltose, glucose, maltotriose, dextrin, etc.

[0044] Amyloglucosidase is a nearly white to light brown amorphous powder, or a light brown to dark brown liquid, which can be dispersed in food-grade diluents or carriers. It can catalyze the hydrolysis of α-1,4- and α-1,6-glycosidic bonds of polysaccharides (starch, glycogen, etc.) to form glucose.

[0045] The addition amount of amylase is 1-10 KNU / g, and the addition amount of amyloglucosidase is 10-40 U / g. The addition amount of the two enzymes is calculated based on the amount of ginseng fermentation raw materials. The use of the two enzymes for enzymatic hydrolysis can fully saccharify.

[0046] Rhizopus oryzae belongs to the Zygomycota, Zygomycetes, Mucorales, Mucoraceae, Rhizopus. The colony is loose or dense, initially white, then gray-brown or dark brown. The hyphae creep and are colorless. The rhizoids are well developed, branched in finger-like or root-like shapes, and brown. The sporangiophores are erect or slightly curved, 2-4 plants in a bundle, opposite to the rhizoids, sometimes swollen or branched, brown, 210-2500μm long, 5-18μm in diameter, the spindle is spherical or nearly spherical or oval, light brown, 30-200μm in diameter. The spindle is wedge-shaped. The sporangium is spherical or nearly spherical, black when old, and has a diameter of 60-250μm. The sporangium spores are oval and spherical, with different shapes and sizes, and no zygospores are found. The fungus can grow at 37-40℃

[0047] Lactobacillus is a Gram-positive bacillus, long, short, sometimes rod-shaped, slender, but without branches. No spores. The colony is round, white (sometimes yellow), translucent, smooth and convex with neat edges, rough and matte surface, and uneven edges.

[0048] Take the ampoule tube of plant lactobacillus, disinfect the outer surface of the ampoule tube with alcohol absorbent cotton in a clean bench, heat the top with a flame, and drop a small amount of sterile water to the top of the heated ampoule to break it. Knock off the top of the broken ampoule, take an appropriate amount of liquid culture medium and drop it into the tube, shake it gently to dissolve the freeze-dried bacteria into a suspended state. Absorb all the bacterial suspension, transplant it into the slope, culture it at 37℃ for 24h, and subculture it twice in a row to obtain the activated strain of lactobacillus.

[0049] A ring of activated plant lactobacillus slant is picked and inoculated into a liquid culture medium, and cultured at 35-37°C for 20-24 hours. Then 3-5% of the bacteria are taken from the cultured bacterial liquid, transferred to a liquid culture medium, and cultured at 35-37°C for 20-24 hours to prepare a lactobacillus breeding seed liquid.

[0050] The Rhizopus oryzae strain is stored in a tube, streaked with PDA solid culture medium for recovery, cultured at 26-28° C. for 3-5 days, a single colony is picked from the bottom of the plate and streaked to be inoculated into an eggplant bottle filled with PDA solid culture medium, and cultured in an incubator at 26-28° C. for 5-7 days. When the fungus moss fills the eggplant bottle and produces a large number of spores, the spores can be eluted with sterile physiological saline and the spore concentration is adjusted to 100 million CFU / mL, which is the Rhizopus oryzae seed solution.

[0051] Mix the activated Rhizopus oryzae seed liquid and Lactobacillus seed liquid in proportion, and after mixing, take an appropriate amount of the mixed seed liquid and add it to the fermentation liquid A. Stir evenly, put it into a fermentation tank, and ferment it aerobically at 28-30℃ for 48 hours with stirring at 200r / h, the ventilation volume for the first 8 hours is 120L / h, the ventilation volume after 8-24 hours is 240Lh, the ventilation volume after 24 hours is 360L / h, and after 48 hours, it is converted to an anaerobic environment and fermented at 26-29℃ for 28-35 hours to obtain fermentation liquid B.

[0052] The fermentation liquid B is filtered through a membrane, and the pore size of the filter screen of the membrane filtration is 0.22-0.45 μm. In some specific embodiments, the pore size of the filter screen of the membrane filtration can be specifically 0.22 μm.

[0053] In some embodiments, the fermentation liquid B needs to be filtered through a filter bag first, and the mesh number of the filter bag for the filter bag filtration is 200-400 mesh; in some embodiments, the mesh number of the filter bag for the filter bag filtration can specifically be 200 mesh.

[0054] In some embodiments, the fermentation liquid B is further subjected to a standing treatment at 4° C. for 30 to 50 days; in some embodiments, the standing treatment at 4° C. may specifically be 30 days.

[0055] In some embodiments, the fermentation liquid B is further centrifuged at 6000-9000 rpm for 10-15 min. In some specific embodiments, the centrifugal speed may be 6000 rpm and the centrifugal time may be 15 min.

[0056] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment.It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0057] Example 1

[0058] The method for preparing ginseng fermentation filtrate comprises the following steps:

[0059] 1. Obtain fermentation raw materials, using ginseng as raw material, and obtain fermentation raw materials after crushing and puffing.

[0060] In this embodiment, ginseng roots are crushed by a high-speed powder grinder, and medium-powdered ginseng (30-80 mesh) is selected and placed in an extruder. The extrusion temperature is set to 120° C., the extrusion pressure is set to 4.90 MPa, and the feed motor voltage is controlled at about 50 V. After extrusion, the fermentation raw material is obtained.

[0061] 2. In the first fermentation, enzymatic hydrolysis is performed with amylase and amyloglucosidase to obtain enzymatic hydrolyzate A.

[0062] Weigh 100 g of ginseng fermentation raw material, place it in a container, add 1000 g of distilled water, stir to dissolve, then add amylase in an amount of 4 KNU / g, enzymolyze at 20°C for 1 hour, then add amyloglucosidase in an amount of 30 U / g, enzymolyze at 60°C for 3 hours to obtain an enzymolyzate.

[0063] 3. Secondary fermentation: adding Rhizopus oryzae and Lactobacillus to the enzymatic hydrolysate A, aerobically fermenting for 45 hours at 28-30°C and anaerobic fermenting for 28-35 hours at 30°C to obtain fermentation liquid B.

[0064] Take the ampoule tube of plant lactobacillus, disinfect the outer surface of the ampoule tube with alcohol absorbent cotton in a clean bench, heat the top with a flame, and drop a small amount of sterile water to the top of the heated ampoule to break it. Knock off the top of the broken ampoule, take 0.3mL of liquid culture medium and drop it into the tube, shake it gently to dissolve the freeze-dried bacteria into a suspended state. Absorb all the bacterial suspension, transplant it into the slant, culture it at 37℃ for 24h, and subculture it twice in succession to obtain the activated strain of lactobacillus.

[0065] A ring of activated plant lactobacillus slant was picked and inoculated into liquid culture medium, and cultured at 37°C for 24 hours. Then 5% of the bacteria were taken from the cultured bacterial liquid, transferred to liquid culture medium, and cultured at 37°C for 24 hours to prepare lactobacillus breeding seed liquid.

[0066] The Rhizopus oryzae strain preservation tube was streaked with PDA solid culture medium for recovery, and cultured at 28°C for 5 days. A single colony was picked from the bottom of the plate and streaked to be inoculated into an eggplant bottle containing 150 ml of PDA solid culture medium, and cultured in an incubator at 28°C for 5-7 days. When the fungus moss covered the eggplant bottle and produced a large number of spores, 500 ml of sterile physiological saline was used for elution, and the spore concentration was adjusted to 100 million CFU / mL, which was the Rhizopus oryzae seed solution.

[0067] The activated Rhizopus oryzae seed solution and Lactobacillus seed solution were mixed evenly in a ratio of 1:5. After mixing, 200 mL of the mixed seed solution was added to the fermentation solution A, stirred evenly, and put into a fermentation tank. The temperature was 28°C and the stirring was 200 r / h. The ventilation volume was 120 L / h in the first 8 hours, 240 L / h after 8 hours, and 360 L / h after 24 hours. After 48 hours, the fermentation was switched to an anaerobic environment for 28 hours to obtain fermentation solution B.

[0068] 4. The fermentation liquid B is filtered through a filter bag, sterilized, allowed to stand, centrifuged, and filtered through a membrane in sequence, and the filtrate is collected to obtain the ginseng fermentation filtrate.

[0069] In this embodiment, the mesh size of the filter bag for bag filtration is 200 mesh, and the filter is left to stand at 4°C for 30 days; the centrifugal speed is 6000 rpm, the centrifugal time can be 15 minutes, and the pore size of the membrane filtration filter is 0.22 μm.

[0070] The ginseng fermented liquid composition comprises: 2% ginseng fermented liquid, 49% 1,2-hexanediol and 49% 1,2-pentanediol.

[0071] Test Example 1 Anti-wrinkle effect

[0072] Experimental methods

[0073] Experimental animals were wild-type AB strain zebrafish.

[0074] Zebrafish age: 4 days post fertilization (4 dpf).

[0075] The number of experimental samples in each group was 30 (three biological replicates, N=3).

[0076] Adult fish rearing and breeding methods: follow the standard rearing and breeding methods of our laboratory, in line with the requirements of the international AAALAC certification (certification number: 001458).

[0077] Experimental principle: Skin growth, repair, nutrition, elasticity and tension are all related to collagen. Its loss will reduce skin smoothness and produce wrinkles. In tetrapods, type I collagen is a trimer, mainly composed of two al chains and one a2 chain, encoded by collala and colla2 genes, respectively, and performs collagen-related biological functions in connective tissue and bone. There are three type I collagen genes in zebrafish, encoding collala, collalb and colla2 of al(I), a2(I) and a3(I) chains respectively. Therefore, by detecting the relative expression of collala or (and) collalb or (and) colla2 genes, it can be shown whether the sample has anti-wrinkle effect.

[0078] 1. Randomly select zebrafish in a 6-well plate, 30 per well.

[0079] 2. Ginseng fermentation liquid composition was administered in water, and a normal control group was set up at the same time. The volume of each well was 3 mL. Three biological replicates

[0080] Incubate at 28°C in the dark for 24 hours.

[0081] 4. Extract total RNA from zebrafish in each experimental group, synthesize cDNA, and use q-PCR to detect the gene expression of β-actin and target genes.

[0082] 5. Use β-actin as the internal reference for gene expression and calculate the relative RNA expression of the target gene.

[0083] Relative RNA expression = 2 ΔΔC(t)

[0084]

[0085] ΔC(t)=C(t) 基因目的 -C(t) β-actin

[0086] If p<0.05 in statistical analysis, it was considered to be significantly different.

[0087]

[0088] Efficacy test column Figure 1 shown.

[0089] Test Example 2 Firming Effect

[0090] Zebrafish firming efficacy test method

[0091] 《T / ZHCA015-2022 Evaluation of the Firming Efficacy of Cosmetics by Relative Expression of Elastin Gene in Zebrafish Fry》

[0092] Experimental system: Wild-type AB strain zebrafish.

[0093] Zebrafish age: 4 days post fertilization (4dpf)

[0094] Sample size for each experiment: 30 (three biological replicates, N=3)

[0095] Adult fish rearing and breeding methods: follow the standard rearing and breeding methods of our laboratory, in line with the requirements of the international AAALAC certification (certification number: 001458).

[0096] Principle and method: Elastin is the main component of elastic fibers in skin tissue, which can provide structural support for the skin and protect the skin from aging and sagging. Elastin is composed of two types of short brain segments arranged alternately. Elnl and eln2 are responsible for encoding different peptide segments of elastin. The two genes are jointly responsible for regulating the expression level of elastin, making the skin firm and elastic. Zebrafish have elastin (eln1.eln2) genes similar to those of humans. Therefore, by detecting the relative expression of eln1 or (and) en2 genes, it can be shown whether the sample has a firming effect.

[0097] Experimental steps: 1. Randomly select zebrafish in a 6-well plate, 30 per well.

[0098] 2. The water-soluble sample was administered and a normal control group was set up at the same time. The volume of each well was 3 mL. Three biological replicates were performed.

[0099] 3.3, incubate at 28°C in the dark for 24 h.

[0100] 4. Extract total RNA from zebrafish in each experimental group, synthesize cDNA, and use q-PCR to detect the gene expression of B-actin and target genes.

[0101] Relative RNA expression = 2 ΔΔC(t)

[0102]

[0103] ΔC(t)=C(t) 基因目的 -C(t) β-actin

[0104] If p<0.05 in statistical analysis, it was considered to be significantly different.

[0105]

[0106] Efficacy test bar chart Figure 2 shown.

[0107] From the above experimental results, it can be seen that the first enzymatic saccharification by amylase and amyloglucosidase, and then the secondary fermentation by Rhizopus oryzae and Lactobacillus mixture, the combined use of the two fermentations can significantly improve the loss of skin collagen and elastin and has a synergistic effect in improving fine lines, wrinkles, dry skin, reduced elasticity, excess hair, and thinning skin.

[0108] The beneficial effects of the present invention are as follows: the combination of two fermentations can synergistically enhance the body's cellular immunity and humoral immunity, promote the expression of the collalb gene and elnl gene, and thus help resist wrinkles and tighten the skin. Therefore, the first enzymatic saccharification by amylase and amyloglucosidase, followed by the secondary fermentation of a mixed solution of Rhizopus oryzae and lactobacillus, has broad prospects in extracting ginseng components. The research of the present invention provides new ideas for the development of future beauty and skin care products.

[0109] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A method for preparing ginseng fermentation filtrate, characterized in that: The steps include: (1) obtaining a fermentation raw material, using ginseng as the raw material, and crushing and puffing the raw material to obtain the fermentation raw material; (2) primary fermentation, wherein the amount of the fermentation raw material added is 0.5-2% of the total mass of the fermentation liquid; enzymatic hydrolysis is performed with amylase and amyloglucosidase to obtain an enzymatic hydrolyzate A; the amount of the amylase added is 1-10 KNU / g, and the amount of the amyloglucosidase added is 10-40 U / g; (3) secondary fermentation, mixing the activated Rhizopus oryzae seed solution and the Lactobacillus seed solution in a ratio of Rhizopus oryzae seed solution: Lactobacillus seed solution = 1:5 to obtain a mixed solution of Rhizopus oryzae and Lactobacillus, adding the mixed solution in an amount of 0.1 to 2% of the total mass of the fermentation solution to the enzymatic hydrolyzate A, first aerobically fermenting for 45 to 48 hours, and then anaerobic fermenting for 28 to 35 hours to obtain a fermentation solution B; (4) The fermentation liquid B is filtered through a membrane, and the filtrate is collected to obtain the ginseng fermentation filtrate.

2. The preparation method according to claim 1, characterized in that: The ginseng comprises one or more compositions such as ginseng root, ginseng leaf, ginseng stem or ginseng root.

3. A ginseng fermentation filtrate, characterized in that: The ginseng fermentation filtrate is prepared by the preparation method according to any one of claims 1-2.

4. A ginseng fermented liquid composition, characterized in that: include: The ginseng fermentation filtrate according to claim 3; 1,2-Hexanediol; and 1,2-Pentanediol.

5. The ginseng fermented liquid composition according to claim 4, characterized in that: The ginseng fermentation filtrate accounts for 1-5% of the total mass of the ginseng fermentation composition.

6. Use of the ginseng fermented liquid composition as claimed in claims 4-5 in preparing beauty and skin care products.

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