Wolfberry leavening with whitening effect and preparation method thereof

Abstract

According to the Chinese wolfberry fermented product with the whitening effect and the preparation method, the Chinese wolfberry extract is fermented through lactobacillus plantarum, the synergistic interaction effect is achieved, melanin cells can be effectively inhibited from generating melanin, generation of melanin in a zebra fish body is reduced, and the skin whitening effect is achieved. The method can be applied to the field of production of cosmetics such as cream, cream, water, milk and facial masks with whitening effects.

Description

Technical Field

[0001] The invention belongs to the technical field of beauty and skin care, and particularly relates to a wolfberry fermentation product with whitening effect and a preparation method thereof. The invention further provides application of the extract in preparing whitening cosmetics. Technical Background

[0002] As an important goal in beauty and skin care, whitening has attracted much attention from consumers. Fair skin is generally considered a symbol of health, youth and vitality, so consumers around the world are pursuing skin whitening effects. Especially in Asia, whitening products occupy a large share of the skin care market. This demand has driven the continuous innovation of whitening products and technologies to help inhibit melanin production and improve uneven skin tone.

[0003] Tyrosinase activity is a landmark indicator for evaluating whitening efficacy. Tyrosinase is a key rate-limiting enzyme in the synthesis of melanin, which can convert tyrosine into dihydroxyphenylalanine (DOPA) to produce melanin. The main function of melanin is to absorb ultraviolet rays and protect the skin from ultraviolet damage, but excessive melanin deposition can cause dull skin or spots. Inhibiting tyrosinase activity and reducing the production of melanin is the main mechanism of action of many whitening products.

[0004] Commonly used whitening agents on the market include vitamin C derivatives, arbutin, kojic acid, niacinamide, resorcinol, etc. These ingredients inhibit the production of melanin through different mechanisms to achieve whitening effects. However, they have certain drawbacks in practical applications. The production cost of some whitening ingredients is too high, which affects the widespread application of the ingredients and causes the prices of cosmetics containing such ingredients to remain high, limiting consumers' ability to purchase such products; secondly, vitamin C derivatives are easily oxidized, causing the product to fail during storage; and thirdly, due to differences in each person's skin quality, genetic factors, living environment, and melanin production levels, the effects of the same whitening agent on different individuals may vary greatly. Therefore, the development of whitening raw materials with low cost and better effect is in line with market demand.

[0005] Wolfberry (scientific name: Lycium barbarum) is a traditional Chinese medicinal material and health food that has been used in China for thousands of years. It belongs to the Solanaceae family and is mainly produced in Ningxia, Inner Mongolia and other regions of China. Wolfberry contains a variety of effective ingredients, such as wolfberry flavonoids, polysaccharides, alkaloids, etc., and has a variety of biological activities. The document "Preparation and Performance Evaluation of Lycium Barbarum Polysaccharide Facial Mask" discloses the in vitro tyrosinase activity of wolfberry polysaccharides. It reports that when the mass concentration of wolfberry polysaccharides is 2.5 g / L, the inhibition rate of tyrosinase activity reaches 51.1%. Summary of the invention

[0006] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a wolfberry fermented product with whitening effect for preparing whitening cosmetics.

[0007] Another object of the present invention is to provide a method for preparing a wolfberry fermented product having whitening effect, which is suitable for industrial production.

[0008] Another object of the present invention is to provide an application of wolfberry fermentation product with whitening effect.

[0009] The invention discloses a wolfberry fermented product with whitening effect, which can effectively inhibit melanocytes from producing melanin and reduce the production of melanin in zebrafish.

[0010] The invention discloses a wolfberry fermented product with whitening effect, wherein the extract is prepared by the following steps: (1) Add 6 to 10 times the amount of water to the dried wolfberry, heat to boiling state and start timing, keep heating at a slight boiling state for 0.5 to 1 hour, and obtain the first extract and the first medicinal residue; (2) Add 4 to 8 times the amount of water to the first medicinal residue, heat to boiling state and start timing, keep heating at a slight boiling state for 0.5 to 1 hour, and obtain the second extract and the second medicinal residue; (3) The two extracts were combined, and the extract filtrate was concentrated to a solid content of 2-4% at a temperature of 60-70°C and a vacuum degree of -0.07 MPa--0.09 MPa to obtain a first concentrated solution; (4) adding 3-8 g / L glucose, 3-8 g / L sodium acetate, 2-3.5 g / L potassium dihydrogen phosphate, 0.3-0.8 g / L magnesium sulfate, 1-3 g / L ammonium citrate, and 0.1-0.5 g / L manganese sulfate to the first concentrated solution, adjusting the pH to 5.5-7, and performing digestion to obtain a fermentation solution; (5) adding 1-5% of a lactobacillus culture solution to the fermented liquid, wherein the lactobacillus is Lactobacillus plantarum, numbered BN336503, and culturing the mixture under pressure at 25-30° C., a stirring speed of 200-400 rpm, a tank pressure of 0.035-0.065 MPa, and a ventilation volume of 1-2 vvm for 18-36 h; (6) After the fermentation is completed, the fermentation liquid is clarified by centrifugation to obtain a wolfberry fermentation product with whitening effect.

[0011] Preferably, 6 to 8 times of water is added to the dried wolfberry, and the temperature is heated to boiling state and the timing is started, and the heating is maintained at a slightly boiling state for 1 hour.

[0012] More preferably, add 6 times the amount of water to the dried wolfberry, heat to boiling state and start timing, and keep heating at a slight boiling state for 1 hour.

[0013] Preferably, 6 to 8 times of water is added to the first medicinal residue, and the temperature is heated to boiling state and the timing is started, and the heating is maintained at a slightly boiling state for 0.5 h.

[0014] More preferably, add 6 times the amount of water into the first medicinal residue, heat to boiling state and start timing, and keep heating at a slightly boiling state for 0.5h.

[0015] Preferably, the two extracts are combined, and the extract filtrate is concentrated to a solid content of 2-3% at a temperature of 63-68° C. and a vacuum degree of −0.05 MPa to −0.085 MPa.

[0016] More preferably, the two extracts are combined, and the extract filtrate is concentrated to a solid content of 2% at a temperature of 65°C and a vacuum degree of -0.08MPa. Preferably, 4-6 g / L glucose, 4-6 g / L sodium acetate, 2-3 g / L potassium dihydrogen phosphate, 0.5-0.8 g / L magnesium sulfate, 1.5-2.5 g / L ammonium citrate, and 0.2-0.3 g / L manganese sulfate are added to the first concentrated solution at a final concentration, and the pH is adjusted to 6-6.8 for actual elimination; More preferably, 5 g / L glucose, 5 g / L sodium acetate, 2.4 g / L potassium dihydrogen phosphate, 0.58 g / L magnesium sulfate, 2 g / L ammonium citrate, and 0.28 g / L manganese sulfate are added to the first concentrated solution to adjust the pH to 6.5 for actual elimination; Preferably, 2-4% of Lactobacillus culture is added to the fermented liquid, and cultured at 36-38° C., a stirring speed of 200-300 rpm, and a tank pressure of 0.04-0.06 MPa for 20-32 h; More preferably, 2% of Lactobacillus culture is added to the fermented liquid, and cultured at 37° C., a stirring speed of 220 rpm, and a tank pressure of 0.05 MPa for 24 h; The invention discloses a wolfberry fermented product with whitening effect, which is applied to the production of cosmetics with whitening effect such as ointment, cream, lotion, milk, facial mask, etc.

[0017] The positive effect of the present invention is that it provides a wolfberry fermentation product with whitening effect. The fermentation product is fermented on wolfberry extract by plant lactobacillus, which produces a synergistic effect, can effectively inhibit melanocytes from producing melanin, and reduce the production of melanin in zebrafish. The fermentation product can be applied to the production of creams, lotions, milks, facial masks and other cosmetics with whitening effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effects of various embodiments, comparative examples and positive drugs of the present invention on the production of melanin by melanocytes induced by α-MSH are shown in FIG. Figure 2The figure is a graph showing the effects of various embodiments, comparative examples and positive drugs of the present invention on the tyrosinase activity of melanocytes induced by α-MSH. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the present invention has no special requirements on the sources of the components of the Chinese medicine composition, and commercially available products known to those skilled in the art can be used.

[0020] Embodiment 1: A fermented wolfberry extract with whitening effect, the extract is prepared by the following steps: (1) Add 6 times the amount of water to the dried wolfberry, heat to boiling state and start timing, keep heating at a slight boiling state for 1 hour, and obtain the first extract and the first medicinal residue; (2) Add 6 times the amount of water to the first medicinal residue, heat to boiling state and start timing, keep heating at a slight boiling state for 0.5 h, and obtain the second extract and the second medicinal residue; (3) The two extracts were combined, and the extract filtrate was concentrated to a solid content of 2-4% at a temperature of 65°C and a vacuum degree of -0.07 MPa to -0.09 MPa to obtain a first concentrated solution; (4) adding 5 g / L glucose, 5 g / L sodium acetate, 2.4 g / L potassium dihydrogen phosphate, 0.58 g / L magnesium sulfate, 2 g / L ammonium citrate, and 0.28 g / L manganese sulfate to the first concentrated solution, adjusting the pH to 6.5, and performing digestion to obtain a fermentation solution; (5) adding 2% of a lactobacillus culture solution to the fermented liquid, wherein the lactobacillus is Lactobacillus plantarum, numbered BN336503, and culturing the mixture at 37° C., a stirring speed of 220 pm, and a tank pressure of 0.05 MPa for 24 h; (6) After the fermentation is completed, the fermentation liquid is clarified by centrifugation and diluted into an aqueous solution with a solid content of 1%, thereby obtaining a wolfberry fermentation product with whitening effect.

[0021] Embodiment 2: A fermented wolfberry extract with whitening effect, the extract is prepared by the following steps: (1) Add 8 times the amount of water to the dried wolfberry, heat to boiling state and start timing, keep heating at a slight boiling state for 0.5 h, and obtain the first extract and the first medicinal residue; (2) Add 4 times the amount of water to the first medicinal residue, heat to boiling state and start timing, keep heating at a slight boiling state for 0.5 h, and obtain the second extract and the second medicinal residue; (3) The two extracts were combined, and the extract filtrate was concentrated to a solid content of 2% at a temperature of 60°C and a vacuum degree of -0.07 MPa to obtain a first concentrated solution; (4) adding 3 g / L glucose, 3 g / L sodium acetate, 3.5 g / L potassium dihydrogen phosphate, 0.3 g / L magnesium sulfate, 1 g / L ammonium citrate, and 0.1 g / L manganese sulfate to the first concentrated solution, adjusting the pH to 5.5, and performing digestion to obtain a fermentation solution; (5) adding 1% of a lactobacillus culture solution to the fermented liquid, wherein the lactobacillus is Lactobacillus plantarum, numbered BN336503, and culturing the mixture at 35° C., a stirring speed of 200 rpm, and a tank pressure of 0.035 MPa for 18 h; (6) After the fermentation is completed, the fermentation liquid is clarified by centrifugation and diluted into an aqueous solution with a solid content of 1%, thereby obtaining a wolfberry fermentation product with whitening effect.

[0022] Example 3: A fermented wolfberry extract with whitening effect, the extract is prepared by the following steps: (1) Add 10 times the amount of water to the dried wolfberry, heat to boiling state and start timing, keep heating at a slight boiling state for 1 hour, and obtain the first extract and the first medicinal residue; (2) Add 8 times the amount of water to the first medicinal residue, heat to boiling state and start timing, keep heating at a slight boiling state for 1 hour, and obtain the second extract and the second medicinal residue; (3) The two extracts were combined, and the extract filtrate was concentrated to a solid content of 4% at a temperature of 70°C and a vacuum degree of -0.09 MPa to obtain a first concentrated solution; (4) adding 8 g / L glucose, 8 g / L sodium acetate, 3.5 g / L potassium dihydrogen phosphate, 0.8 g / L magnesium sulfate, 3 g / L ammonium citrate, and 0.5 g / L manganese sulfate to the first concentrated solution, adjusting the pH to 7, and performing digestion to obtain a fermentation solution; (5) adding 5% of a lactobacillus culture solution to the fermented liquid, wherein the lactobacillus is Lactobacillus plantarum, numbered BN336503, and culturing the mixture at 39° C. and a stirring speed of 400 rpm for 36 h; (6) After the fermentation is completed, the fermentation liquid is clarified by centrifugation and diluted into an aqueous solution with a solid content of 1%, thereby obtaining a wolfberry fermentation product with whitening effect.

[0023] Comparative Example 1: A wolfberry extract, which is prepared by the following steps: (1) Add 6 times the amount of water to the dried wolfberry, heat to boiling state and start timing, keep heating at a slight boiling state for 1 hour, and obtain the first extract and the first medicinal residue; (2) Add 6 times the amount of water to the first medicinal residue, heat to boiling state and start timing, keep heating at a slight boiling state for 0.5 h, and obtain the second extract and the second medicinal residue; (3) combining the two extracts, and concentrating the extract filtrate to a solid content of 1% at a temperature of 65° C. and a vacuum degree of −0.07 MPa to −0.09 MPa to obtain a wolfberry extract; Comparative Example 2: A wolfberry extract, which is prepared by the following steps: (1) Adding 5 g / L glucose, 5 g / L sodium acetate, 2.4 g / L potassium dihydrogen phosphate, 0.58 g / L magnesium sulfate, 2 g / L ammonium citrate, and 0.28 g / L manganese sulfate to water at a final concentration, adjusting the pH to 6.5, and performing digestion to obtain a fermentation liquid; (2) adding 2% of a lactobacillus culture solution to the fermented liquid, wherein the lactobacillus is Lactobacillus plantarum, numbered BN336503, and culturing the mixture at 37° C., a stirring speed of 220 pm, and a tank pressure of 0.05 MPa for 24 h; (3) After the fermentation is completed, the fermentation liquid is clarified by centrifugation and diluted into an aqueous solution with a solid content of 1%, thereby obtaining a wolfberry fermentation product with whitening effect.

[0024] Comparative Example 3: A fermented wolfberry extract with whitening effect, the extract is prepared by the following steps: (1) Add 6 times the amount of water to the dried wolfberry, heat to boiling state and start timing, keep heating at a slight boiling state for 1 hour, and obtain the first extract and the first medicinal residue; (2) Add 6 times the amount of water to the first medicinal residue, heat to boiling state and start timing, keep heating at a slight boiling state for 0.5 h, and obtain the second extract and the second medicinal residue; (3) The two extracts were combined, and the extract filtrate was concentrated to a solid content of 2-4% at a temperature of 65°C and a vacuum degree of -0.07 MPa to -0.09 MPa to obtain a first concentrated solution; (4) adding 5 g / L glucose, 5 g / L sodium acetate, 2.4 g / L potassium dihydrogen phosphate, 0.58 g / L magnesium sulfate, 2 g / L ammonium citrate, and 0.28 g / L manganese sulfate to the first concentrated solution, adjusting the pH to 6.5, and performing digestion to obtain a fermentation solution; (5) adding 2% of a Lactobacillus culture solution to the fermented liquid, wherein the Lactobacillus is fermented Lactobacillus, numbered BN194390, and culturing the mixture at 37° C., a stirring speed of 220 pm, and a tank pressure of 0.05 MPa for 24 h; (6) After the fermentation is completed, the fermentation liquid is clarified by centrifugation and diluted into an aqueous solution with a solid content of 1%, thereby obtaining a wolfberry fermentation product with whitening effect.

[0025] Test Example 1

[0026] Melanocytes were seeded in 6-well plates and 2 × 10 5cells, add 2 mL of DMEM complete medium. The culture plate was placed in an incubator at 37°C and 5% CO2 for 24 hours to allow the cells to adhere to the wall and grow. After 24 hours, the culture medium was replaced with fresh medium, and α-MSH (final concentration 10 μM) was added to the model group, example group, comparative group and positive drug group to induce melanin production, wherein the example and comparative group added 5% of the fermentation product or extract by volume, and the positive drug group added 20 μM nicotinamide. Each group of cells continued to be cultured for 48 hours. After the culture was completed, the cells were gently washed twice with PBS buffer to prepare for subsequent detection.

[0027] (1) Melanin content detection The cells were collected, the cell pellets were separated by centrifugation, 200 μL of 1 M NaOH solution (containing 10% DMSO) was added, and the mixture was incubated in an 80°C water bath for 1 hour to fully dissolve the melanin. The absorbance of melanin was measured at a wavelength of 405 nm using an ELISA reader, and the melanin content of each group of cells was calculated.

[0028] (2) Tyrosinase activity detection Use the tyrosinase activity detection kit according to the kit instructions. Add substrate (L-DOPA) to each group of cell lysates and incubate at 37°C for 30 minutes. Measure the absorbance of the reaction solution at a wavelength of 490 nm and calculate the tyrosinase activity. The results are as follows: Figure 1 , Figure 2 As shown, after irradiation with a-MSH, the tyrosinase activity of melanocytes and the secretion of melanin are significantly increased. Examples 1 to 3 have a significant inhibitory effect on the formation of melanin and the increase of tyrosinase activity. Although nicotinamide and comparative examples 1 to 3 have similar effects, their effects are lower than those of the example group at the same concentration.

[0029] Test Example 2

[0030] Zebrafish embryos (6-8 hours post fertilization, hpf) were incubated for 72 hours with 2% volume ratio of the wolfberry fermented product described in Example 1 and the control group with the same volume fraction of Angel Yeast Extract. After 72 h of incubation, the live embryos were anesthetized in tricaine methanesulfonic acid solution (0.02%). To determine the tyrosinase activity and melanin content, zebrafish embryos were placed in a centrifuge tube and killed at -80°C for 20 min, and then the dead embryos were sonicated in RIPA lysis buffer for 15 min. Then, the lysate was centrifuged at 12000×g at 4°C for 30 min, and the supernatant was collected to determine the protein concentration. The supernatant (80 μg protein each) and L-DOPA (5 mM, 80 μL) were added to a 96-well microplate and then incubated at 37°C for 25 min. The absorbance was measured at 492 nm using a microplate reader to detect the tyrosinase activity. After precipitation in 50 μL of 1 N sodium hydroxide (containing 10% DMSO), the metal bath was placed at 80°C for 1 hour, and the absorbance at 405 nm was read to determine the melanin content level. The test results are shown in Table 1 below: Table 1. Comparison of the activity of wolfberry fermentation products and commercially available whitening fermentation products

[0031] Conclusion: Lycium barbarum fermentation product has significant whitening effect, can effectively inhibit the production of melanin by melanocytes, and reduce the production of melanin in zebrafish. Its efficacy value is equivalent to that of Angel Yeast Essence, and can be used in cosmetics with whitening effect such as ointment, cream, lotion, milk, mask, etc.

Claims

1. A fermented wolfberry product with whitening effect, characterized in that Prepared by the following steps: (1) Add 6 to 10 times the amount of water to the dried wolfberry, heat to boiling state and start timing, keep heating at a slight boiling state for 0.5 to 1 hour, and obtain the first extract and the first medicinal residue; (2) Add 4 to 8 times the amount of water to the first medicinal residue, heat to boiling state and start timing, keep heating at a slight boiling state for 0.5 to 1 hour, and obtain the second extract and the second medicinal residue; (3) The two extracts were combined, and the extract filtrate was concentrated to a solid content of 2-4% at a temperature of 60-70°C and a vacuum degree of -0.07 MPa--0.09 MPa to obtain a first concentrated solution; (4) adding 3-8 g / L glucose, 3-8 g / L sodium acetate, 2-3.5 g / L potassium dihydrogen phosphate, 0.3-0.8 g / L magnesium sulfate, 1-3 g / L ammonium citrate, and 0.1-0.5 g / L manganese sulfate to the first concentrated solution, adjusting the pH to 5.5-7, and performing digestion to obtain a fermentation solution; (5) adding 1-5% of a lactobacillus culture solution to the fermented liquid, wherein the lactobacillus is Lactobacillus plantarum, numbered BN336503, and culturing the mixture under pressure at 25-30° C., a stirring speed of 200-400 rpm, a tank pressure of 0.035-0.065 MPa, and a ventilation volume of 1-2 vvm for 18-36 h; (6) After the fermentation is completed, the fermentation liquid is clarified by centrifugation to obtain a wolfberry fermentation product with whitening effect.

2. The wolfberry fermented product with whitening effect according to claim 1, characterized in that: In step 1, add 6 times the amount of water to the dried wolfberries, heat to boiling state and start timing, and keep heating at a slight boiling state for 1 hour.

3. The wolfberry fermented product with whitening effect according to claim 1, characterized in that: In step 2, add 6 times the amount of water to the first medicinal residue, heat to boiling state and start timing, and keep heating at a slight boiling state for 0.5 h.

4. The wolfberry fermented product with whitening effect according to claim 1, characterized in that: In step 3, the two extracts are combined, and the extract filtrate is concentrated to a solid content of 2% at a temperature of 65° C. and a vacuum degree of −0.08 MPa.

5. The wolfberry fermented product with whitening effect according to claim 1, characterized in that: In step 4, 5 g / L glucose, 5 g / L sodium acetate, 2.4 g / L potassium dihydrogen phosphate, 0.58 g / L magnesium sulfate, 2 g / L ammonium citrate, and 0.28 g / L manganese sulfate were added to the first concentrated solution at a final concentration, and the pH was adjusted to 6.5 for actual elimination.

6. The wolfberry fermented product with whitening effect according to claim 1, characterized in that: In step 5, 2% of Lactobacillus culture was added to the fermented liquid, and the mixture was cultured at 37° C., a stirring speed of 220 rpm, and a tank pressure of 0.05 MPa for 24 h.

7. A method comprising preparing a whitening ointment, cream, lotion, milk, or facial mask cosmetic containing the wolfberry fermented product having whitening effect prepared by the method of claims 1 to 6.

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