CN113133951B

CN113133951B - Production method for extracting rice protein extract from cordyceps militaris fermentation product and application of rice protein extract

Info

Publication number: CN113133951B
Application number: CN202010062420.8A
Authority: CN
Inventors: 李志诚; 杨承琳; 洪维屏; 黄宏彰
Original assignee: KEYI CO LTD
Current assignee: KEYI CO LTD
Priority date: 2020-01-19
Filing date: 2020-01-19
Publication date: 2023-06-09
Anticipated expiration: 2040-01-19
Also published as: CN113133951A

Abstract

The invention discloses a production method for extracting rice protein extract from cordyceps militaris fermentation broth and application thereof, and the production method is mainly characterized by comprising the following steps: taking a cordyceps sinensis strain, and performing a solid state fermentation culture process by rice to obtain a fermentation culture; separating the fermentation culture to obtain cordyceps fruiting bodies and cordyceps byproducts; uniformly stirring and mixing the cordyceps byproduct and an aqueous solution, wherein the weight ratio of the cordyceps byproduct to the aqueous solution is between 0.5:10 and 1.5:10, extracting by a low-temperature ultrasonic cold extraction method, setting the temperature between 18 and 22 ℃ and setting the extraction time between 40 and 80 minutes, thereby obtaining a cordyceps rice fermentation extract; and the rice protein content of the cordyceps rice fermentation extract in every hundred grams weight is required to be between 2.5 and 8 grams.

Description

Production method for extracting rice protein extract from cordyceps militaris fermentation product and application of rice protein extract

Technical Field

The invention relates to a fermentation extraction production technology; in particular to a production method for extracting rice protein extract from cordyceps militaris fermentation broth and application thereof.

Background

With age, human skin gradually ages, and appearance changes such as wrinkles, spots, fine lines, rough skin, loose skin and the like occur, and problems such as dark skin, uneven skin color and the like are also caused. Therefore, anti-aging and whitening are key items of research and development of scientists, and related research and discussion of skin aging mechanism are gradually helped to find out methods for preventing and delaying skin aging along with the development of science.

The skin is the largest organ of human body, can be used as a barrier for protecting the inside of the body to prevent invasion of external bacteria, and has the functions of regulating body temperature, excreting and metabolizing wastes in the body, and the like. The skin is composed of epidermis layer, dermis layer and subcutaneous tissue, the epidermis is a protective layer outside the skin, it is a multi-layer squamous epithelium capable of continuous keratinization, structurally comprises stratum corneum, granular layer, acanthosis layer and basal layer, mainly comprises keratinocyte, melanocyte and langerhan's cell, while the dermis provides substantial support for the epidermis, comprises mastoid layer and reticular layer, it mainly comprises fibroblasts and collagen, besides enabling the skin to extend and shrink, it also can help wound healing, and the gaps in collagen fibers are elastic fibrin, which is an important component for providing skin elasticity.

However, during skin aging, elastin is broken or relaxed by the generation of free radicals and elastase, and the skin becomes thinner, skin elasticity is lowered, skin is relaxed, wrinkles are generated, and the like. The skin aging causes can be mainly divided into intrinsic physiological factors and extrinsic environmental factors; that is, in addition to physiological natural aging, the external environmental pressure is one of the causes of skin aging, including ultraviolet rays, air pollution, smoking, staying up, pressure, injury to free radicals … …, and the like. In addition to skin aging, the irradiation of ultraviolet rays can generate oxidation pressure on skin, and further cause melanin generation and precipitation, so that if the antioxidant capacity of the skin can be increased and free radicals can be removed by helping the skin, the purpose of whitening can be achieved by inhibiting melanin precipitation.

The term "Cordyceps" refers to a complex of fungi growing on insects, and is a most common fungus in the world, including Cordyceps sinensis, cordyceps militaris, and Cordyceps sobolifera. Wherein the Cordyceps sinensis is hirsutella sinensis (Cordyceps sinensis), and the resultant dry composite of stroma and insect body is mainly produced in high altitude areas such as Qinghai, tibet, sichuan, etc. of China after parasitizing on larva of Lepidoptera bat. In Tibet, cordyceps sinensis is classified into 4 grades according to external color, body size and origin, which are yellow grass (first grade), red grass (second grade), white grass (third grade) and Ma Jibei (fourth grade). Because of the rare production area and yield of Cordyceps sinensis and the difficulty in artificial cultivation of fruiting body, the cost is very high, which causes the limitation of application. According to the maximum supplier of Tibetan budalae in 2014, cordyceps sinensis reaches 15 ten thousand RMB per kilogram, and therefore, the Cordyceps sinensis is processed by lead powder and sulfur by a Chinese zodiac, forged into first-class yellow grass products to obtain violence, and the risk of harm to body health caused by eating by mistake of eaters is increased. Therefore, many people are striving to find alternatives with the same efficacy as Cordyceps sinensis.

Cordyceps militaris mentioned in the previous paragraph is also called Cordyceps militaris or Cordyceps militaris, fungi are born in the body of the pupa, and the fruiting body of the Cordyceps militaris is similar to grass, namely Cordyceps militaris, the fruiting body of the Cordyceps militaris is named Cordyceps militaris, and fungi of the genus Cordyceps (Cordyceps) and the phylum Ascomycota, the class Pyrenomycetales (Pyrenomycetales), the order Hypojejunales (Hypojeales), the family Clavicitaceae (Clavicitaceae) are named as ascomycetes (Ascomycota) similar to Cordyceps sinensis (Cordyceps sinensis). Both are Cordyceps, but the species names are different. Regarding the earliest origin of Cordyceps sinensis, it is internationally recognized that in China, the earliest trace can be to the ecology of fungus parasites related to the "bacteria in bees" mentioned in the "unitary yang miscellaneous in the" formed by Tang dynasty (the first 863 year). The study of Cordyceps militaris is firstly recorded in the outline of Xinhua materia medica, and the efficacy of the Cordyceps militaris has the functions of 'sweet taste, flat nature, benefiting lung and kidney, replenishing essence, stopping bleeding and resolving phlegm'. The Chinese herb enters meridians and lung and kidney meridians, which are indicated for amnesia, insomnia, soreness and weakness of waist and knees, cough, dyspnea due to deficiency, cough with excessive phlegm and blood. The cordyceps militaris is found in the Jilin province of China for the first time in 1958, and is considered to be the same fungus as cordyceps sinensis after classification and identification, and the drug effect and pharmacological disease effect of the cordyceps militaris are similar to those of the cordyceps sinensis. However, due to the difference of parasitic bacteria, the contained 16 amino acids and Cordycepin (Cordycepin) are higher than those of Cordyceps sinensis, and the superoxide dismutase (SOD) with antioxidant activity is higher than that of Cordyceps sinensis, especially Cordyceps sinensis Polysaccharide (Polysaccharide), cordycepin (Cordycepin) and Adenosine (Adenosine) are several times to tens times higher than that of Cordyceps sinensis. Moreover, the cordyceps militaris can be economically produced in a large scale, and is not limited to a specific production place, so that the price is relatively high, and more people can be afforded the ability to use the cordyceps militaris, so that the cordyceps militaris can be selected as an option for health care.

The cultivation method of Cordyceps militaris can be classified into liquid submerged fermentation and solid fermentation, wherein liquid submerged fermentation can obtain more polysaccharide content, but only mycelium form can not form fruiting body. In general, beech (Beech wood), rice bran (Rice bran), wheat bran (white bran), white Rice (polishface), brown Rice (hulshface), wheat grain (white grains) and the like can be used as a culture medium for inducing the fruiting body of Cordyceps militaris by solid-state fermentation culture. At present, the solid cultivation mode can be divided into two modes of rice cultivation and host infection cultivation, wherein the rice cultivation method takes two modes of bag cultivation and bottle cultivation as main flows, the commonly used substrate mainly takes rice as main raw materials, different nutrition sources are additionally added for cultivation, the added nutrition materials comprise silkworm chrysalis powder, protein, vitamin B1 or sucrose and the like, the substrate to be cultivated is filled into a glass tank, and after high-temperature and high-pressure sterilization is completed, the fermented liquid strain is respectively added for cultivation, and the generation of sporophores is induced.

However, in terms of practical application, the Cordyceps culture technology is still insufficient in the amount of the finally obtained protein, so that no matter how effective the Cordyceps culture technology is in wrinkle resistance, whitening and the like, the Cordyceps culture technology is difficult to achieve remarkable effects if the Cordyceps culture technology is applied as a human care product.

Disclosure of Invention

The invention mainly aims to provide a production method for extracting rice protein extract from cordyceps militaris fermentation broth.

In order to achieve the above purpose, the invention adopts the following technical scheme:

a method for extracting rice protein extract from Cordyceps militaris fermentation product comprises: step one: taking a cordyceps sinensis strain, and performing a solid state fermentation culture process by rice to obtain a fermentation culture; step two: applying a separation means to the fermentation culture to obtain cordyceps fruiting bodies and cordyceps byproducts respectively, wherein the cordyceps byproducts are fermented cordyceps rice powder; step three: uniformly stirring and mixing the cordyceps byproduct and an aqueous solution, wherein the weight ratio of the cordyceps byproduct to the aqueous solution is between 0.5:10 and 1.5:10, extracting by a low-temperature ultrasonic cold extraction method, wherein the low-temperature setting condition of the low-temperature ultrasonic cold extraction method is between 18 ℃ and 22 ℃, and the extraction time setting condition is between 40 and 80 minutes, so as to obtain cordyceps rice fermentation extract; and the rice protein content contained in the fermented extract of cordyceps rice per hundred grams weight is required to be between 2.5 and 8 grams.

The production method disclosed by the invention can achieve the following main effects and advantages through the result that the protein amount which can be extracted per hundred grams of cordyceps sinensis rice is increased by more than 3 times compared with the water extraction mode:

firstly, activating human skin fibers and promoting fibroblast proliferation;

secondly, free radicals are effectively removed to reduce oxidative damage, and the potential of enhancing the oxidation resistance of the organism is provided;

thirdly, the loss of collagen is prevented, and the skin is increased in elasticity, so that the anti-wrinkle effect is achieved;

fourth, free radicals are removed, and oxidation damage is reduced, so that the antioxidation effect can be achieved;

fifthly, inhibiting the activity of elastase to achieve the anti-wrinkle effect;

sixth, inhibit melanin synthesis of melanoma cells, thereby achieving the whitening effect.

Another main object of the present invention is to provide a skin care product for human body.

a skin care product for human body comprises Cordyceps militaris rice fermented extract obtained by the method for preparing rice protein extract from Cordyceps militaris fermented extract, wherein the rice protein content of the Cordyceps militaris rice fermented extract is 2.5-8 g per hundred g; the fermented extract of Cordyceps rice is used as additive for preparing skin care product, and is present in an amount of 0.001-0.004% of the total weight of the whole composition; and the human skin care product has at least one of the following efficacy requirements: moisturizing, skin cell regenerating, anti-wrinkle, anti-aging, antioxidant, skin elasticity improving or whitening.

The present invention also provides a skin care composition.

a skin care composition comprising the fermented cordyceps rice extract obtained by the process for producing the fermented cordyceps militaris extract, wherein the fermented cordyceps rice extract contains 2.5 to 8 g of rice protein per hundred g of the fermented cordyceps rice extract; the fermented extract of Cordyceps sinensis rice is used as additive of skin care composition, and is present in an amount of 0.35% to 1.40% based on the total weight of the skin care composition; and the skin care composition has at least one of the following efficacy requirements: moisturizing, skin cell regenerating, anti-wrinkle, anti-aging, antioxidant, skin elasticity improving or whitening.

Drawings

FIG. 1 is a text block diagram showing steps of a production method according to a preferred embodiment of the present invention;

FIG. 2 is a graph showing the rice protein content extracted by the low temperature ultrasonic extraction method and the water extraction method according to the present invention;

FIG. 3 is a graph showing a cell proliferation assay performed 72 hours after treatment of dermal fibroblasts with rice protein in the test program of the present invention;

FIG. 4 is a graph showing the cell morphology test performed after treatment of dermal fibroblasts with rice protein in the test item of the present invention;

FIG. 5 is a graph showing the results of the anti-oxidative activity analysis of rice protein in the test item of the present invention;

FIG. 6 is a graph showing the results of the anti-oxidative activity analysis of rice protein in the test item of the present invention;

FIG. 7 is a graph showing the results of analysis of the elastase activity of rice protein in the test item of the present invention;

FIG. 8 is a graph showing the results of analysis of melanin synthesis in the inhibition of B16-F10 cells by rice protein in the test item of the present invention;

FIG. 9 is a graph showing the amount of melanin synthesis inhibited by alpha-MSH-induced B16-F10 cells in the test item of the present invention.

Detailed Description

Referring to fig. 1, a preferred embodiment of the method for extracting rice protein extract from Cordyceps militaris fermentation product and its application of the present invention is shown, but these embodiments are only for illustrative purposes, and the present invention is not limited to this structure.

The method comprises the following steps: taking a cordyceps sinensis strain, and performing a solid state fermentation culture process by rice to obtain a fermentation culture; step two: applying a separation means to the fermentation culture to obtain a cordyceps fruiting body and a cordyceps byproduct, respectively, wherein the cordyceps byproduct is fermented cordyceps rice powder; step three: uniformly stirring and mixing Cordyceps byproduct (lyophilized Cordyceps rice powder) and an aqueous solution, wherein the weight ratio of the two is 0.5:10 to 1.5:10, extracting by a low-temperature ultrasonic cold extraction method, wherein the low-temperature setting condition of the low-temperature ultrasonic cold extraction method is 18-22 ℃, and the extraction time setting condition is 40-80 minutes, thereby obtaining a fermented extract of Cordyceps rice; and wherein the rice protein content of the fermented Cordyceps rice extract is 2.5-8 g per hundred g.

Wherein the Cordyceps is Cordyceps militaris, cordyceps sinensis or Cordyceps cicadae.

Wherein, in the third step, the water solution which is used for stirring and mixing with the cordyceps byproduct adopts ultrapure water (ddH 2O for short).

Wherein the rice is organic rice or five cereal ferment containing rice.

Wherein, the cordyceps fruiting body obtained in the second step is a material of medical or health-care food.

Through the technical characteristics, the production method for extracting the rice protein extract from the cordyceps militaris fermentation product is further illustrated by the following specific measurement and activity analysis, and the efficacy requirements of the invention are supported by objective left evidence:

protein concentration determination of rice protein: BCA is an abbreviation for bicinchoninic acid (i.e., bicinchoninic acid) and is commonly used to detect total protein in a sample by the principle that the protein (protein) will bind Cu in alkaline solution ²⁺ Reduction of ions to Cu ⁺ Monovalent copper ions in turn form soluble chelates with BCA, which chelates exhibit a maximum absorption peak at 562 nm. Taking 25 mu l of rice protein sample or standard substance into 96-well plates, adding 200 mu l of Working Solution (Working Solution) into each 96-well plate, sealing and uniformly mixing; standing at 37deg.C for 30 min, placing 96-well plate at room temperature, measuring absorbance at 562nm with enzyme immunoassay reader, and establishing standard curve with the measured value of standard substance to calculate protein concentration of sample. The test uses traditional water extraction mode and new low-temperature ultrasonic extraction mode to extract rice protein respectively, and after extraction, samples are taken respectively to measure protein concentration. As shown in table one below:

if the conventional water extraction method is used, 1.677 g of protein (namely, rice protein-1 in the table above) can be extracted per 100 g of Cordyceps sinensis rice, and the low-temperature ultrasonic extraction method of the present invention is used for rice protein extraction, 5.722 g of protein (namely, rice protein-2 in the table above) can be extracted per 100 g of Cordyceps sinensis rice. Accordingly, the content of the rice protein extractable by the low-temperature ultrasonic extraction method is 3.4 times that of the rice protein extractable by the traditional water extraction method, and the result is shown in FIG. 2.

Cell culture: WS1 (BCRC 60300) is a dermal fibroblast isolated from human skin, B16-F10 (BCRC 60031) is a melanoma cell isolated from mouse skin, both of which are purchased from Taiwan biological resource conservation and research center (Bioresource Collection and Research Center, BCRC for short; new bamboo in Taiwan, china). The culture medium of WS1 cells is a culture medium (Minimum Essential Medium, abbreviated as MEM) containing 10% (v/v) of fetal bovine serum and penicillin/streptomycin (100 IU/50. Mu.g/ml); the culture medium of B16-F10 cells is a culture medium (DMEM) containing 10% (v/v) of fetal bovine serum, sodium bicarbonate (1.5 g/L) and glucose (4.5 g/L); the culture was carried out at 37℃in a cell incubator containing 5% carbon dioxide. In the secondary culture, after cells are rinsed by phosphate buffer physiological saline (Phosphate buffered saline, PBS for short), a cell separation reagent Trypsin (Trypsin-EDTA) is added to act at 37 ℃, fresh culture medium is added to uniformly mix, centrifugation is carried out for 5 minutes at 1500rpm, cell counting is carried out, and then the cells are planted into each culture dish according to the required proportion.

The principle of WST-1 cell proliferation assay is that NADH granular dehydrogenase on the granular electron transfer chain of living cells can reduce WST-1 medicine to orange formazan product (formazan) in the presence of electron coupling carrier 1-Methoxy-5-methylphenazine dimethyl sulfate (1-Methoxy PMS) and the survival rate of cells can be obtained by measuring the light absorption value of 450 nm. 5X 103 cells were seeded into 96-well cell culture dishes, respectively, rice proteins at different concentrations were treated, and after culturing at 37℃for 72 hours, the cell culture medium was removed and a 2- (4-Iodophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazolium solution (WST-1, manufacturer Dojindo, japan Tonic Chemie) was added, and after culturing at 37℃for 2 hours, absorbance at two wavelengths of 450nm and 620nm was measured with an enzyme immunoassay reader. Cell viability of WS1 dermal fibroblasts was analyzed by WST-1 assay.

Regarding anti-wrinkle test: the experiment is to use human skin fibroblast WS1 cell strain to conduct cell proliferation assay by WST-1 proliferation assay and further conduct cell proliferation morphological detection. WS1 human skin fibroblast viability was measured by WST-1 cell proliferation assay and the effect of the extract components of the present invention on skin cell regeneration and anti-wrinkle activity was combined by cell morphology detection. As shown in fig. 3 and 4, the results indicate that the rice protein has the effect of activating WS1 human skin fibroblast proliferation.

Test for antioxidant Activity: the experiment uses the diphenyl/radical assay (DPPH assay) and vitamin C (vit. C) as a standard control to perform antioxidant effect assays. In order to take the extracts with different concentrations of 0.5 and mL, 0.125mL 0.5 mM DPPH (2, 2-diphenyl-1-picryl) is added and uniformly mixed, after the extracts react for 30 minutes at room temperature in the dark, the enzyme immunoassay reader wavelength 517 nm is used for determination, the DPPH ethanol solution adopted in the experiment is purple, and has a strong light absorption value under 517 nm, if the DPPH ethanol solution is combined with a sample, the light absorption value is reduced, thereby judging the capability of the sample for removing DPPH free radicals, and the lower the measured light absorption value is, the stronger the capability of capturing DPPH free radicals is represented. The results were obtained from three independent experiments. As shown in FIG. 5 and FIG. 6, the concentration of the rice protein increases, the free radical scavenging rate also increases, and the free radicals can be effectively scavenged to reduce oxidative damage, so that the rice protein has the potential of enhancing the antioxidant capacity of the organism.

Regarding the inhibition elastase activity assay: the present experiment was conducted by using the principle that elastase (elastase) catalyzes the production of p-nitroaniline (p-nitroaniline) from N-succinic acid- (L-alanine) 3-p-nitroaniline (N-Succinyl-Ala-Ala-Ala-p-nitroaniline), and measuring the production of p-nitroaniline in a substrate at a wavelength of 405 nm. The results were obtained from three independent experiments. As shown in FIG. 7, it is pointed out that 50. Mu.g/mL of rice protein can significantly inhibit elastase (elastase) activity, prevent collagen loss, and increase skin elasticity, thereby achieving anti-wrinkle effect.

Measurement of melanin synthesis amount: the experiment is carried out by inoculating B16-F10 cells into a 6-hole tray with melanoma cell strain B16-F10, culturing for 24 hours, adding melanocyte stimulating hormone (alpha-melanocyte-stimulating hormones, alpha-MSH for short) and rice protein with different concentrations, culturing for 48 hours, washing with phosphate buffer, adding trypsin to digest cells, centrifugally collecting, adding 1N NaOH, reacting at 80 ℃ for 30 minutes, and measuring the absorbance at 450nm wavelength with an enzyme immunoassay reader, wherein the results are obtained from three independent experiments. As shown in FIG. 8, it was revealed that the melanin content in B16-F10 melanoma cells could be greatly reduced after 48 hours of culture with 40. Mu.g/mL of rice protein; as shown in FIG. 9, after the rice protein of 20. Mu.g/mL is added for 48 hours, the melanin content synthesized by the melanoma cells induced by alpha-MSH can be effectively reduced, so that the whitening effect is achieved. In view of the above, the podandlor rice protein can reduce oxidative damage by scavenging free radicals, thereby achieving the antioxidation effect; and the anti-wrinkle effect can be achieved by promoting proliferation of fibroblasts and inhibiting elastase activity; in addition, the whitening effect can be achieved by inhibiting melanin synthesis of melanoma cells.

The invention relates to a production method for extracting rice protein extract from cordyceps militaris fermentation broth, which can be used as one of skin care products (such as face cream, emulsion and the like) or skin care compositions for human body in practical application, wherein the rice protein content contained in each hundred gram weight ratio of cordyceps militaris fermentation broth is between 2.5 and 8 grams; the fermented extract of Cordyceps rice is used as additive composition for preparing skin care product or skin care composition, and is present in an amount of 0.001-0.004% of the total weight of the whole composition of the skin care product, or 0.35-1.40% of the total weight of the skin care composition; and the human skin care product or the skin care composition has at least one of the following efficacy requirements: moisturizing, skin cell regenerating, anti-wrinkle, anti-aging, antioxidant, skin elasticity improving or whitening.

The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A production method for extracting rice protein extract from cordyceps militaris fermentation broth is characterized by comprising the following steps:

step one: taking a cordyceps sinensis strain, and performing a solid state fermentation culture process by rice to obtain a fermentation culture;

step two: applying a separation means to the fermentation culture to obtain a cordyceps fruiting body and a cordyceps byproduct, respectively, wherein the cordyceps byproduct is fermented cordyceps rice powder;

step three: uniformly stirring and mixing the cordyceps byproduct and an aqueous solution, wherein the weight ratio of the cordyceps byproduct to the aqueous solution is between 0.5:10 and 1.5:10, extracting by a low-temperature ultrasonic cold extraction method, wherein the low-temperature setting condition of the low-temperature ultrasonic cold extraction method is between 18 ℃ and 22 ℃, and the extraction time setting condition is between 40 and 80 minutes, thereby obtaining a cordyceps rice fermentation extract; and wherein the rice protein content of the fermented Cordyceps rice extract is 2.5-8 g per hundred g.

2. The method for producing rice protein extract from Cordyceps militaris fermentation broth of claim 1, wherein the Cordyceps militaris is Cordyceps militaris, cordyceps sinensis or Cordyceps cicadae.

3. The method for producing rice protein extract from Cordyceps militaris fermentation broth of claim 2, wherein the aqueous solution in step three is mixed with Cordyceps militaris by-product by stirring, and ultrapure water is used.

4. A process for the production of rice protein extract from Cordyceps militaris fermentation broth according to claim 3, wherein the rice is organic rice or five cereal fermentation broth containing rice.

5. The method for producing rice protein extract from Cordyceps militaris fermentation broth of claim 4, wherein the Cordyceps militaris fruiting body obtained in the second step is a material for pharmaceutical or health food.

6. A skin care product for human body comprising the fermented extract of chinese caterpillar fungus rice obtained by the production process according to any one of claims 1 to 5, wherein the fermented extract of chinese caterpillar fungus rice comprises rice protein in an amount of 2.5 to 8 g per hundred g by weight; the fermented extract of Cordyceps rice is used as additive for preparing skin care product, and is present in an amount of 0.001-0.004% of the total weight of the whole composition.

7. A skin care composition comprising the fermented extract of chinese caterpillar fungus rice obtained by the production process according to any one of claims 1 to 5, wherein the fermented extract of chinese caterpillar fungus rice has a rice protein content of 2.5 to 8 g per hundred g by weight; the fermented extract of Cordyceps sinensis rice is used as an additive of the skin care composition, and is present in an amount of 0.35% to 1.40% by weight based on the total weight of the skin care composition.