An antibacterial and antioxidant fermented product of camellia seed, its preparation method and application

Abstract

The present invention belongs to the technical field of microbial fermentation, and discloses an antibacterial and antioxidant fermented product of camellia seed, its preparation method and application. The raw materials for preparing the fermented product of camellia seed include: camellia seed extract, solvent, culture medium and fermentation strains; the fermentation strains include at least one of Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus pentosus and Lactobacillus casei. The fermented product of camellia seed obtained by fermenting the camellia seed extract with specific fermentation strains has significantly improved antibacterial effect, antioxidant activity and total polyphenol content compared with the unfermented camellia seed extract, and the irritation to the skin is significantly reduced. At the same time, some flavonoid components of the fermented product of camellia seed are also significantly increased, providing broad prospects for the application of camellia seed in cosmetics.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and particularly relates to an antibacterial and antioxidant fermented product of camellia seed, and a preparation method and application thereof. Background Art

[0002] The extract of camellia seed contains various active ingredients such as tea saponin, flavonoids, polyphenols, polysaccharides, etc.; among them, tea saponin is a natural non-ionic surfactant, which is a mixture with a structure similar to the oleanane type in pentacyclic triterpenoids, and is composed of sapogenin, glycoside and organic acid. It not only has good emulsifying, foaming, dispersing, penetrating, lubricating and other active effects, but also has functions such as anti-inflammatory, anti-cancer, antioxidant, antibacterial, hypoglycemic, liver protection and detoxification, weight loss, and itching relief.

[0003] At present, water or ethanol is mainly used as a solvent to extract camellia seeds to obtain camellia seed extract, but the active ingredients (such as flavonoids and polyphenols) in the camellia seed extract are less, the antibacterial and antioxidant effects are poor, and it has certain irritation. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. For this reason, the present invention provides an antibacterial and antioxidant fermented product of camellia seed, and a preparation method and application thereof. The content of active ingredients in the fermented product of camellia seed is higher, the antibacterial and antioxidant effects are stronger, and the irritation is significantly reduced.

[0005] The first aspect of the present invention provides a fermented product of camellia seed. The preparation raw materials of the fermented product of camellia seed include: camellia seed extract, solvent, culture medium and fermentation strains; the fermentation strains include at least one of Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus pentosus and Lactobacillus casei.

[0006] In some embodiments of the present invention, the mass ratio of the camellia seed extract, solvent and culture medium is 1:(5-20):(0.1-1).

[0007] In some embodiments of the present invention, the concentration of the fermentation strains is 10 5 -10 8 CFU / mL, preferably 10 5 -10 7 CFU / mL, more preferably 10 6 CFU / mL.

[0008] In some embodiments of the present invention, the volume of the fermentation strains accounts for 0.1-5% of the total volume after mixing the camellia seed extract, solvent and culture medium, preferably 0.5-2%, more preferably 1%.

[0009] In some embodiments of the present invention, the chemical components of the fermented camellia seed product include total polyphenols, and the content of the total polyphenols is > 3 mg / mL.

[0010] In some embodiments of the present invention, the solvent includes water.

[0011] In some embodiments of the present invention, the camellia seed extract is a camellia seed extract obtained by water extraction or alcohol extraction of camellia seeds, preferably a camellia seed extract obtained by water extraction of camellia seeds.

[0012] In some embodiments of the present invention, the culture medium in the preparation raw materials of the fermented camellia seed product is selected from MRS culture medium. The MRS culture medium includes casein peptone 5.0 - 15.0 g / L, beef extract powder 5.0 - 15.0 g / L, yeast extract powder 2.0 - 6.0 g / L, ammonium citrate 1.0 - 3.0 g / L, sodium acetate 4.0 - 6.0 g / L, magnesium sulfate 0.1 - 0.3 g / L, manganese sulfate 0.02 - 0.08 g / L, dipotassium hydrogen phosphate 1.0 - 3.0 g / L, glucose 10.0 - 30.0 g / L, Tween - 80 0.5 - 1.5 g / L, and pH 5.7 ± 0.2.

[0013] The second aspect of the present invention provides a preparation method of the fermented camellia seed product described in the first aspect of the present invention, including the following steps:

[0014] Mix the camellia seed extract, the solvent and the culture medium, sterilize, and then add a fermentation strain for fermentation;

[0015] After the fermentation is completed, perform centrifugation, collect the supernatant, and sterilize to obtain the fermented camellia seed product.

[0016] In some embodiments of the present invention, the sterilization is carried out by high - pressure steam sterilization; the temperature of the sterilization is 110 - 130 °C; and / or, the time of the sterilization is 10 - 30 min.

[0017] In some embodiments of the present invention, the temperature of the fermentation is 25 - 40 °C, preferably 30 - 40 °C, more preferably 37 °C; and / or, the time of the fermentation is 24 - 80 h, preferably 48 - 80 h, more preferably 72 h.

[0018] In some embodiments of the present invention, the rotation speed of the centrifugation is 4000 - 8000 rpm; and / or, the time of the centrifugation is 20 - 50 min.

[0019] In some embodiments of the present invention, the preparation method of the camellia seed extract includes the following steps:

[0020] Take camellia seeds, perform water extraction, alcohol precipitation, and spray drying to obtain the camellia seed extract.

[0021] In some embodiments of the present invention, the temperature of water extraction is 50 to 100 °C; and / or, the time of water extraction is 1 to 2 h.

[0022] In some embodiments of the present invention, the alcohol precipitation uses ethanol with a mass fraction of 75% or more, preferably an ethanol aqueous solution with a mass fraction of 75%.

[0023] In some embodiments of the present invention, the time of alcohol precipitation is 24 to 48 h.

[0024] The third aspect of the present invention provides a cosmetic, and the cosmetic comprises the camellia seed ferment of the first aspect of the present invention.

[0025] In some embodiments of the present invention, the content of the camellia seed ferment in the cosmetic is 0.05 to 20 wt%, preferably 0.1 to 10 wt%, and more preferably 0.5 to 5 wt%.

[0026] In some embodiments of the present invention, the cosmetic is shampoo, body wash, essence or facial cleanser.

[0027] In some embodiments of the present invention, the cosmetic is a shampoo; the components of the shampoo further include at least one of sodium lauryl polyether sulfate, cocamidopropyl betaine, cocamide MEA / glycerin, sodium lauroamphoacetate, cocamide MEA, C14-16 olefin sulfonate, sodium methyl cocoyl taurate, guar hydroxypropyltrimonium chloride, PPG-3 octyl ether, cetyl alcohol, sorbitan octanoate, allantoin, carbohydrate isomers, citric acid, sodium citrate, phenoxyethanol, disodium EDTA and (daily use) fragrance.

[0028] In some embodiments of the present invention, the shampoo is composed of the following components by mass fraction: 0.5 to 3% of the camellia seed ferment of the first aspect of the present invention, 12 to 20% of sodium lauryl polyether sulfate, 3 to 14% of cocamidopropyl betaine, 0.05 to 2% of cocamide MEA / glycerin, 0.05 to 5% of sodium lauroamphoacetate, 0.01 to 2% of cocamide MEA, 1 to 5% of C14-16 olefin sulfonate, 0.01 to 3% of sodium methyl cocoyl taurate, 0.05 to 0.7% of guar hydroxypropyltrimonium chloride, 0.05 to 2% of PPG-3 octyl ether, 0.1 to 0.3% of cetyl alcohol, 0.05 to 0.3% of sorbitan octanoate, 0.05 to 0.5% of allantoin, 0.01 to 0.5% of carbohydrate isomers, 0.05 to 0.3% of citric acid, 0.05 to 0.3% of sodium citrate, 0.1 to 0.5% of phenoxyethanol, 0.05 to 2% of disodium EDTA, 0.1 to 0.5% of (daily use) fragrance and the balance of water.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] The present invention uses the extract of camellia seed as raw material, and the fermented product of camellia seed obtained by fermenting with specific fermentation strains. Compared with the unfermented extract of camellia seed, the antibacterial effect, antioxidant activity and total polyphenol content of the fermented product of camellia seed are significantly improved, and the irritation to the skin is significantly reduced. At the same time, the content of some flavonoid components (camelliaside B, astragalin, kaempferol-3-O-rutinoside, kaempferol-7-O-neohesperidoside, kaempferol 3-O-sambubioside) in the fermented product of camellia seed also increases significantly, providing a broad prospect for the application of camellia seed in cosmetics.

[0031] The present invention uses the microbial fermentation technology to ferment the extract of camellia seed, and carry out biotransformation, which improves the active ingredients. At the same time, the fermented product of camellia seed prepared after fermentation has stronger biological activity, lower irritation and higher safety, and efficiently utilizes natural plant resources. Specific Embodiments

[0032] In order to make the technical solutions described in the present invention clearer and more understandable to those skilled in the art, the following examples are listed for illustration. It should be noted that the following examples do not limit the scope of protection required by the present invention.

[0033] The raw materials, reagents or devices used in the present invention can be obtained from conventional commercial channels or can be obtained by existing known methods without special instructions.

[0034] The specific formulations of the culture media required for the examples, comparative examples and test examples in the present invention are as follows:

[0035] Tryptic Soy Broth TSB (for culturing Staphylococcus aureus and Escherichia coli): Tryptone 15.0 g / L, Soybean Papain Hydrolysate 5.0 g / L, Sodium Chloride 5.0 g / L, Agar 15 g / L, pH 7.3 ± 0.2;

[0036] Clostridium Enrichment Medium (for culturing Propionibacterium acnes): Beef Extract Powder 10.0 g / L, Peptone 10.0 g / L, Yeast Extract Powder 3.0 g / L, Soluble Starch 1.0 g / L, Glucose 5.0 g / L, Cysteine Hydrochloride 0.5 g / L, Sodium Chloride 5.0 g / L, Sodium Acetate 3.0 g / L, Agar 0.5 g / L, pH 6.8 ± 0.2;

[0037] Leeming-Notman Medium Base (for the culture of Malassezia furfur): Peptone 10.0 g / L, Glucose 5.0 g / L, Yeast Extract Powder 1.0 g / L, Bile Salts 4.0 g / L, Glycerol 1.0 g / L, Glycerol Monostearate 0.5 g / L, Tween-60 0.5 mL, Whole Milk Powder 10.0 g / L, Chloramphenicol 0.05 g / L, Agar 12.0 g / L, pH 5.6 ± 0.2;

[0038] MRS Medium (for the culture of Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus pentosus, Lactobacillus casei and Lactococcus lactis): Casein Enzymatic Digest 10.0 g / L, Beef Extract Powder 10.0 g / L, Yeast Extract Powder 4.0 g / L, Ammonium Citrate 2.0 g / L, Sodium Acetate 5.0 g / L, Magnesium Sulfate 0.2 g / L, Manganese Sulfate 0.05 g / L, Dipotassium Hydrogen Phosphate 2.0 g / L, Glucose 20.0 g / L, Tween-80 1.0 g / L, pH 5.7 ± 0.2;

[0039] Potato Dextrose Broth (PDB) (for the culture of Saccharomyces cerevisiae): Potato 300.0 g / L, Glucose 20.0 g / L, pH 5.6 ± 0.2.

[0040] Example 1

[0041] An oil-tea camellia seed ferment, the preparation raw materials of which include: oil-tea camellia seed extract, pure water, medium and fermentation strains; wherein, the fermentation strains are selected from Lactobacillus salivarius.

[0042] The preparation method of the oil-tea camellia seed ferment of this example includes the following steps:

[0043] Mix the oil-tea camellia seed extract, pure water and medium according to a mass ratio of 1:10:0.54 to obtain a mixture;

[0044] Sterilize the mixture at 121 °C by high-pressure steam for 20 min. After cooling, inoculate Lactobacillus salivarius, mix evenly, and ferment at 37 °C for 72 h to obtain a fermentation broth; wherein, the concentration of Lactobacillus salivarius is 10 6 CFU / mL, and the volume of Lactobacillus salivarius accounts for 1% of the volume of the mixture;

[0045] Centrifuge the fermentation broth at 5000 rpm for 20 min, collect the supernatant, and sterilize it at 121 °C by high-pressure steam for 20 min to obtain the oil-tea camellia seed ferment.

[0046] The oil-tea camellia seed extract of this example is obtained by mixing oil-tea camellia seeds with water, heating and extracting at 100 °C for 1.5 h, repeating the extraction 3 times, then performing alcohol precipitation with 75% ethanol, and spray drying.

[0047] Example 2

[0048] The difference from Example 1 is only that the fermentation strain used in Example 2 is selected from Lactobacillus fermentum.

[0049] Example 3

[0050] The difference from Example 1 is only that the fermentation strain used in Example 3 is selected from Lactobacillus rhamnosus.

[0051] Example 4

[0052] The difference from Example 1 is only that the fermentation strain used in Example 4 is selected from Lactobacillus pentosus.

[0053] Example 5

[0054] The difference from Example 1 is only that the fermentation strain used in Example 5 is selected from Lactobacillus casei.

[0055] Example 6

[0056] The difference from Example 1 is only that the fermentation strains used in Example 6 are selected from Lactobacillus salivarius and Lactobacillus rhamnosus; wherein, the total concentration of Lactobacillus salivarius and Lactobacillus rhamnosus is 10 6 CFU / mL, the total volume of Lactobacillus salivarius and Lactobacillus rhamnosus accounts for 1% of the volume of the mixture, and the volume ratio of Lactobacillus salivarius to Lactobacillus rhamnosus is 1:1.

[0057] Example 7

[0058] The difference from Example 1 is only that the fermentation strains used in Example 7 are selected from Lactobacillus fermentum and Lactobacillus pentosus; wherein, the total concentration of Lactobacillus fermentum and Lactobacillus pentosus is 10 6 CFU / mL, the total volume of Lactobacillus fermentum and Lactobacillus pentosus accounts for 1% of the volume of the mixture, and the volume ratio of Lactobacillus fermentum to Lactobacillus pentosus is 1:1.

[0059] Example 8

[0060] The difference from Example 1 is only that the fermentation strains used in Example 8 are selected from Lactobacillus fermentum, Lactobacillus pentosus and Lactobacillus rhamnosus; wherein, the total concentration of Lactobacillus fermentum, Lactobacillus pentosus and Lactobacillus rhamnosus is 10 6 CFU / mL, the total volume of Lactobacillus fermentum, Lactobacillus pentosus and Lactobacillus rhamnosus accounts for 1% of the volume of the mixture, and the volume ratio of Lactobacillus fermentum, Lactobacillus pentosus and Lactobacillus rhamnosus is 1:1:1.

[0061] Example 9

[0062] The difference from Example 1 is only that the fermentation strains used in Example 9 are selected from Lactobacillus fermentum, Lactobacillus pentosus, Lactobacillus rhamnosus, and Lactobacillus salivarius; among them, the total concentration of Lactobacillus fermentum, Lactobacillus pentosus, Lactobacillus rhamnosus, and Lactobacillus salivarius is 10 6 CFU / mL, the total volume of Lactobacillus fermentum, Lactobacillus pentosus, Lactobacillus rhamnosus, and Lactobacillus salivarius accounts for 1% of the volume of the mixture, and the volume ratio of Lactobacillus fermentum, Lactobacillus pentosus, Lactobacillus rhamnosus, and Lactobacillus salivarius is 1:1:1:1.

[0063] Example 10

[0064] The difference from Example 1 is only that the fermentation strains used in Example 10 are selected from Lactobacillus fermentum, Lactobacillus pentosus, Lactobacillus rhamnosus, Lactobacillus salivarius, and Lactobacillus casei; among them, the total concentration of Lactobacillus fermentum, Lactobacillus pentosus, Lactobacillus rhamnosus, Lactobacillus salivarius, and Lactobacillus casei is 10 6 CFU / mL, the total volume of Lactobacillus fermentum, Lactobacillus pentosus, Lactobacillus rhamnosus, Lactobacillus salivarius, and Lactobacillus casei accounts for 1% of the volume of the mixture, and the volume ratio of Lactobacillus fermentum, Lactobacillus pentosus, Lactobacillus rhamnosus, Lactobacillus salivarius, and Lactobacillus casei is 1:1:1:1:1.

[0065] Comparative Example 1

[0066] The difference from Example 1 is only that the fermentation strain used in Comparative Example 1 is selected from Saccharomyces cerevisiae.

[0067] Comparative Example 2

[0068] The difference from Example 1 is only that the fermentation strain used in Comparative Example 2 is selected from Lactococcus lactis.

[0069] Comparative Example 3

[0070] Comparative Example 3 provides an aqueous solution of camellia seed extract, which is prepared by mixing camellia seed extract and pure water in a mass ratio of 1:10, and sterilizing at 121°C with high-pressure steam for 20 min to obtain the aqueous solution of camellia seed extract.

[0071] The preparation method of the camellia seed extract is the same as that in Example 1.

[0072] Comparative Example 4

[0073] Comparative Example 4 provides a Saccharomyces cerevisiae fermentation broth, and the preparation method is as follows:

[0074] The culture medium is sterilized at 121°C with high-pressure steam for 20 min, cooled and then inoculated with Saccharomyces cerevisiae, mixed evenly, fermented at 37°C for 72 h, centrifuged at 5000 rpm for 20 min, and the supernatant is collected to obtain the Saccharomyces cerevisiae fermentation broth; among them, the concentration of Saccharomyces cerevisiae is 10 6CFU / mL, and the volume of Saccharomyces cerevisiae accounts for 1% of the volume of the culture medium.

[0075] Comparative Example 5

[0076] Comparative Example 5 provides a Lactococcus lactis fermentation broth, and the preparation method is as follows:

[0077] The culture medium was sterilized at 121 °C under high-pressure steam for 20 min, cooled and then inoculated with Lactococcus lactis, mixed evenly, fermented at 37 °C for 72 h, centrifuged at 5000 rpm for 20 min, and the supernatant was collected to obtain the Lactococcus lactis fermentation broth; among them, the concentration of Lactococcus lactis was 10 6 CFU / mL, and the volume of Lactococcus lactis accounts for 1% of the volume of the culture medium.

[0078] Comparative Example 6

[0079] Comparative Example 6 provides a Lactobacillus salivarius fermentation broth, and the preparation method is as follows:

[0080] The culture medium was sterilized at 121 °C under high-pressure steam for 20 min, cooled and then inoculated with Lactobacillus salivarius, mixed evenly, fermented at 37 °C for 72 h, centrifuged at 5000 rpm for 20 min, and the supernatant was collected to obtain the Lactobacillus salivarius fermentation broth; among them, the concentration of Lactobacillus salivarius was 10 6 CFU / mL, and the volume of Lactobacillus salivarius accounts for 1% of the volume of the culture medium.

[0081] Comparative Example 7

[0082] Comparative Example 7 provides a Lactobacillus fermentum fermentation broth, and the preparation method is as follows:

[0083] The culture medium was sterilized at 121 °C under high-pressure steam for 20 min, cooled and then inoculated with Lactobacillus fermentum, mixed evenly, fermented at 37 °C for 72 h, centrifuged at 5000 rpm for 20 min, and the supernatant was collected to obtain the Lactobacillus fermentum fermentation broth; among them, the concentration of Lactobacillus fermentum was 10 6 CFU / mL, and the volume of Lactobacillus fermentum accounts for 1% of the volume of the culture medium.

[0084] Comparative Example 8

[0085] Comparative Example 8 provides a Lactobacillus rhamnosus fermentation broth, and the preparation method is as follows:

[0086] The culture medium was sterilized at 121 °C under high-pressure steam for 20 min, cooled and then inoculated with Lactobacillus rhamnosus, mixed evenly, fermented at 37 °C for 72 h, centrifuged at 5000 rpm for 20 min, and the supernatant was collected to obtain the Lactobacillus rhamnosus fermentation broth; among them, the concentration of Lactobacillus rhamnosus was 10 6 CFU / mL, and the volume of Lactobacillus rhamnosus accounts for 1% of the volume of the culture medium.

[0087] Comparative Example 9

[0088] Comparative Example 9 provides a Lactobacillus pentosus fermentation broth, and the preparation method is as follows:

[0089] The culture medium was autoclaved at 121 °C for 20 min, cooled and then inoculated with Lactobacillus pentosus, mixed evenly, fermented at 37 °C for 72 h, centrifuged at 5000 rpm for 20 min, and the supernatant was collected to obtain the Lactobacillus pentosus fermentation broth; among them, the concentration of Lactobacillus pentosus was 10 6 CFU / mL, and the volume of Lactobacillus pentosus accounted for 1% of the volume of the culture medium.

[0090] Comparative Example 10

[0091] Comparative Example 10 provides a Lactobacillus casei fermentation broth, and the preparation method is as follows:

[0092] The culture medium was autoclaved at 121 °C for 20 min, cooled and then inoculated with Lactobacillus casei, mixed evenly, fermented at 37 °C for 72 h, centrifuged at 5000 rpm for 20 min, and the supernatant was collected to obtain the Lactobacillus casei fermentation broth; among them, the concentration of Lactobacillus casei was 10 6 CFU / mL, and the volume of Lactobacillus casei accounted for 1% of the volume of the culture medium.

[0093] Test Example 1 Determination of DPPH Free Radical Scavenging Ability

[0094] In this test example, the camellia seed fermented products prepared in Examples 1 to 10 and Comparative Examples 1 to 2, and the aqueous solution of camellia seed extract prepared in Comparative Example 3 were used as samples to detect the free radical scavenging ability.

[0095] IC 50 value represents the sample concentration required to scavenge half of the DPPH free radicals. The lower the IC 50 value, the better the scavenging effect on DPPH free radicals.

[0096] Experimental method: Each sample was diluted 100 times with double-distilled water to prepare a sample solution. 10, 20, 30, 40, and 50 μL of 5 gradients of each sample solution were placed in a 96-well plate, and then deionized water was added to make up the volume to 50 μL. Then, 100 μL of 0.05 mg / mL DPPH (prepared with absolute ethanol) solution was added respectively. After mixing, it was incubated in the dark for 30 min. The absorbance was measured with an enzyme-labeling instrument at a wavelength of 519 nm. Free radical scavenging rate (%) = [1 - (A1 - A2) / A0] × 100, where A0 is the absorbance of only the DPPH solution, A1 is the absorbance of the DPPH and sample mixture, and A2 is the absorbance of only the sample solution.

[0097] IC50 value calculation: Calculate the IC using SPSS software50 Value

[0098] The experimental results are shown in Table 1

[0099] Table 1 IC of scavenging DPPH free radicals 50 Value (unit: mg / mL)

[0100] Example 1 45.6 Example 2 42.5 Example 3 42.9 Example 4 41.3 Example 5 41.7 Example 6 40.1 Example 7 39.7 Example 8 30.8 Example 9 34.3 Example 10 41.8 Comparative Example 1 60.5 Comparative Example 2 61.9 Comparative Example 3 60.4

[0101] As can be seen from the results in Table 1, after the camellia seed extracts of Examples 1-10 were fermented with specific fermentation strains (one or more of Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus pentosus, and Lactobacillus casei), the IC of the camellia seed fermentates obtained for scavenging DPPH free radicals 50 Values were all lower than those of Comparative Examples 1-3. After Comparative Example 1 and Comparative Example 2 were fermented with Saccharomyces cerevisiae and Lactococcus lactis respectively, the IC of scavenging DPPH free radicals 50 Value was higher than that before fermentation (Comparative Example 3). This shows that after the camellia seed extract of the present invention is fermented with specific fermentation strains, the ability to scavenge DPPH free radicals can be significantly improved

[0102] Test Example 2 Detection of total antioxidant capacity by FRAP method

[0103] In this test example, the camellia seed fermentates prepared from Examples 1-10 and Comparative Examples 1-2, and the aqueous solution of the camellia seed extract prepared from Comparative Example 3 were used as samples to detect the total antioxidant value

[0104] Detection method: Prepare FeSO4 standard solutions with concentration gradients of 0, 0.2, 0.5, 0.8, 1, 1.2, and 1.5 mmol / L. Measure the absorbance at 593 nm with an enzyme-linked immunosorbent assay (ELISA) reader, draw a standard curve with the FeSO4 concentration as the abscissa, and obtain the standard linear equation. Dilute each sample 50 times with double-distilled water to prepare a sample solution. Take 200 μL of the prepared sample solution, add 3 mL of FRAP working solution and mix well. Each sample has 3 replicates, incubate at 37 °C for 15 min, and measure the absorbance A at 593 nm with an ELISA reader. Substitute the A value into the standard linear equation to calculate the FRAP value. The larger the FRAP value, the stronger the antioxidant activity. Preparation of FRAP solution: Mix 10 mmol / L TPTZ solution, 20 mmol / L FeCl3·6H2O solution, and acetate buffer solution with pH = 3.6 and 300 mmol / L in a ratio of 10:1:1, and prepare it for immediate use. The test results are shown in Table 2

[0105] Table 2 Total antioxidant values (unit: mmol / L)

[0106]

[0107]

[0108] As can be seen from the results in Table 2, after the camellia seed extracts in Examples 1 to 10 were fermented with specific fermentation strains (one or more of Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus pentosus, and Lactobacillus casei), the total antioxidant capacity of the obtained fermented camellia seeds was significantly improved compared with that before fermentation (Comparative Example 3). After the fermentations of Comparative Example 1 and Comparative Example 2 with Saccharomyces cerevisiae and Lactococcus lactis respectively, the total antioxidant value remained basically unchanged compared with that before fermentation (Comparative Example 3). This shows that after the camellia seed extract is fermented with specific fermentation strains in the present invention, the antioxidant capacity can be significantly improved.

[0109] Test Example 3 Detection of Total Polyphenol Content

[0110] In this test example, the fermented camellia seeds prepared in Examples 1 to 10 and Comparative Examples 1 to 2, and the aqueous solution of camellia seed extract prepared in Comparative Example 3 were used as samples to detect the total polyphenol content. The concentration of total polyphenols in the samples was calculated from the absorbance values of the samples, and the total polyphenol content in the samples was expressed as the content of gallic acid.

[0111] Detection method: Prepare 0.5 mL of standard gallic acid (prepared with 95% ethanol) solutions with concentration gradients of 0, 0.002, 0.004, 0.010, 0.020, 0.030, and 0.040 mg / mL. Add 1.5 mL of 0.1 mmol / L Folin-Ciocalteu reagent, mix well, and let stand for 5 min. Then add 1 mL of 7.5% sodium carbonate solution, mix well, and place in the dark at room temperature for 30 min. Using double-distilled water as the blank, measure the absorbance of the solution at a wavelength of 765 nm. Taking the absorbance as the ordinate and the gallic acid concentration as the abscissa, plot the standard curve and obtain the standard linear equation.

[0112] Determination of total polyphenol content in samples: Dilute each sample 100 times with double-distilled water to prepare a sample solution. Take 0.5 mL of the prepared sample solution, add 1.5 mL of 0.1 mmol / L Folin-Ciocalteu reagent, mix well, and let stand for 5 min. Then add 1 mL of 7.5% sodium carbonate solution, mix well, and place in the dark at room temperature for 30 min. Using the corresponding solvent as the blank solution, measure the absorbance at a wavelength of 765 nm. Substitute the absorbance into the standard linear equation to calculate the total polyphenol content in the samples. The test results are shown in Table 3.

[0113] Table 3 Total Polyphenol Content (Unit: mg / mL)

[0114]

[0115]

[0116] As can be seen from the results in Table 3, after the camellia seed extracts in Examples 1 to 10 were fermented by specific fermentation strains (one or several of Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus pentosus, and Lactobacillus casei), the content of total polyphenols was significantly increased compared with that before fermentation (Comparative Example 3). However, after the extracts in Comparative Example 1 and Comparative Example 2 were fermented by Saccharomyces cerevisiae and Lactococcus lactis respectively, the content of total polyphenols decreased compared with that before fermentation (Comparative Example 3). This indicates that after the camellia seed extracts are fermented by specific fermentation strains in the present invention, the antioxidant capacity can be significantly improved.

[0117] Test Example 4 Antibacterial Effect

[0118] In this test example, the antibacterial effects of the camellia seed fermented products prepared in Examples 1 to 10 and Comparative Examples 1 to 2, the aqueous solution of the camellia seed extract prepared in Comparative Example 3, and the bacterial fermentation broths prepared in Comparative Examples 4 to 10 were detected.

[0119] Propionibacterium acnes is a skin commensal bacterium that can cause skin problems such as acne and pimples; Malassezia furfur is a resident lipophilic yeast in human skin and mucous membranes, which participates in the occurrence and development of various skin diseases and can cause skin problems such as Malassezia folliculitis and seborrheic dermatitis; Staphylococcus aureus is also a common skin pathogen, which usually can cause skin papules or pustules; Staphylococcus aureus can also cause skin diseases such as folliculitis, suppurative acne, and hordeolum. The Oxford cup punching method was used to analyze the antibacterial effects of the samples against Escherichia coli (ATCC8739), Staphylococcus aureus (ATCC6538), Malassezia furfur (ATCC44344), and Propionibacterium acnes (ATCC11827).

[0120] Take the activated Escherichia coli and make it into a bacterial suspension with PBS at a concentration of 5×10 6 CFU / mL. Add the bacteria and the culture medium into the petri dish and mix well. Use forceps to place a sterile Oxford cup on the surface of the culture medium to punch holes. After solidification, add 200 μL of the sample to be tested with a pipette gun. Each sample has 3 replicates. After culturing at 37°C ± 1°C for 2 d, use a vernier caliper to measure the diameter of the antibacterial circle, and evaluate the antibacterial effect based on the diameter of the antibacterial circle. The operation steps for Escherichia coli are the same as those for Staphylococcus aureus. Propionibacterium acnes is cultured under anaerobic conditions at 37°C for 2 d, and Malassezia furfur is cultured at 30°C for 3 d.

[0121] The negative control sample (PBS) should have no antibacterial ring; if the diameter of the antibacterial ring of the test sample > 7 mm, it is judged to have antibacterial effect; if the diameter of the antibacterial ring ≤ 7 mm, it is judged to have no antibacterial effect. If all 3 parallel samples have antibacterial effects, it is judged to be qualified. The antibacterial results are shown in Table 4.

[0122] Table 4 Diameter of Antibacterial Circle (unit: mm)

[0123]

[0124]

[0125] As can be seen from the results in Table 4, the aqueous solution of the camellia seed extract prepared in Comparative Example 3 has no antibacterial effect on Escherichia coli, Staphylococcus aureus and Propionibacterium acnes. After the camellia seed extracts of Examples 1 to 10 are fermented by specific fermentation strains (one or more of Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus pentosus and Lactobacillus casei), they have antibacterial effects on Escherichia coli, Staphylococcus aureus and Propionibacterium acnes, and the diameter of the antibacterial zone is larger than that of the fermentation broth of the strains, and the antibacterial effect is stronger. After being fermented by Saccharomyces cerevisiae and Lactococcus lactis respectively, Comparative Example 1 and Comparative Example 2 have no antibacterial effect on Escherichia coli, Staphylococcus aureus and Propionibacterium acnes;

[0126] After the camellia seed extracts of Examples 1 to 10 are fermented by specific fermentation strains (one or more of Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus pentosus and Lactobacillus casei), the diameter of the antibacterial zone against Malassezia furfur is larger than that of the aqueous solution of the camellia seed extract prepared in Comparative Example 3, and the antibacterial effect is stronger. After being fermented by Saccharomyces cerevisiae and Lactococcus lactis respectively, Comparative Example 1 and Comparative Example 2 have a smaller diameter of the antibacterial zone against Malassezia furfur than that of the aqueous solution of the camellia seed extract prepared in Comparative Example 3, and the antibacterial effect is reduced.

[0127] Test Example 5 Verification of Biofilm Removal

[0128] In this test example, the camellia seed fermented products prepared in Examples 1 to 10 and Comparative Examples 1 to 2, and the aqueous solution of the camellia seed extract prepared in Comparative Example 3 are used as samples.

[0129] The activated Staphylococcus aureus is diluted to a concentration of 1.0×10 6 CFU / mL with a culture medium. 200 μL of the bacterial suspension is added to each well of a 96-well plate and incubated at a constant temperature and humidity of 37°C ± 1°C for 24 h. The culture medium is aspirated. After diluting the sample 5 times with the culture medium, 200 μL is taken and added to the 96-well plate, and incubated at a constant temperature and humidity of 37°C ± 1°C for 24 h. The supernatant is aspirated, washed 3 times with PBS, 100 μL of XTT (dimethylthiazolyl diphenyl tetrazolium bromide) dye solution is added, and incubated at 37°C ± 1°C for 2 h. The OD value is measured at a wavelength of 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader. The culture medium is used as the blank control group, and the experiment is repeated 3 times.

[0130] Calculation of biofilm removal rate: Removal rate (%) = [1 - (OD 样品 - OD 溶剂 ) / (OD 空白菌 - OD 溶剂 )] × 100;

[0131] The results are shown in Table 5.

[0132] Table 5 Biofilm clearance rate (unit: %)

[0133] Example 1 60.6 Example 2 63.3 Example 3 62.7 Example 4 66.1 Example 5 64.2 Example 6 67.3 Example 7 67.9 Example 8 68.3 Example 9 68.0 Example 10 69.72 Comparative Example 1 18.2 Comparative Example 2 19.9 Comparative Example 3 20.2

[0134] As can be seen from the results in Table 5, after the camellia seed extracts of Examples 1-10 were fermented with specific fermentation strains (one or several of Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus pentosus, and Lactobacillus casei), the clearance efficiency of the biofilm was significantly improved compared with that of Comparative Example 3. However, after the fermentations of Comparative Examples 1 and 2 with Saccharomyces cerevisiae and Lactococcus lactis respectively, the clearance efficiency of the biofilm decreased instead.

[0135] Test Example 6 Chicken embryo chorioallantoic membrane experiment for eye irritation / corrosion of cosmetics

[0136] In this test example, the irritations of the camellia seed fermentates prepared in Examples 1-10 and Comparative Examples 1-2, as well as the aqueous solution of the camellia seed extract prepared in Comparative Example 3, were detected. Since tea saponin is irritating to eyes and skin, the irritation of the camellia seed extract before and after fermentation to the skin was evaluated by the chicken embryo chorioallantoic membrane experiment in this test example.

[0137] Experimental method: Select 9-day-old chicken embryos with a mass of 50-60 g, mark the position of the air chamber on the eggshell surface, use forceps to peel off part of the eggshell, expose the white egg membrane, use a pipette to drop 0.9% physiological saline to moisten the egg membrane, use forceps to remove the inner membrane, take 0.3 mL of the sample and directly apply it to the chorioallantoic membrane (CAM), ensuring that the coverage area reaches at least 50%. Immediately observe the reaction of the CAM until 5 min and stop, record the observation results and make a judgment. Each sample is repeated 3 times, and physiological saline is used as a negative control. Among them, IS < 1 means no irritation, 1 ≤ IS < 5 means mild irritation, and 5 ≤ IS < 10 means moderate irritation. The experimental results are shown in Table 6.

[0138] Table 6 IS values of chicken embryo chorioallantoic membrane experiment for eye irritation / corrosion of cosmetics

[0139] Example 1 4.12 Example 2 4.06 Example 3 3.43 Example 4 3.94 Example 5 3.63 Example 6 3.51 Example 7 3.07 Example 8 3.47 Example 9 3.03 Example 10 3.08 Comparative Example 1 4.86 Comparative Example 2 4.78 Comparative Example 3 4.76

[0140] As can be seen from the results in Table 6, after the camellia seed extracts of Examples 1-10 were fermented with specific fermentation strains (one or several of Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus pentosus, and Lactobacillus casei), the irritation to the chicken embryo chorioallantois decreased compared with that of Comparative Example 3. However, after the fermentations of Comparative Examples 1 and 2 with Saccharomyces cerevisiae and Lactococcus lactis respectively, the irritation to the skin increased compared with that of Comparative Example 3.

[0141] Test Example 7 Component analysis

[0142] Detection method: The components, namely camelliaside B, astragalin, kaempferol-3-O-rutinoside, kaempferol-7-O-neohesperidoside, and kaempferol 3-O-sambubioside, in the fermented camellia seeds prepared in Examples 1 to 10, Comparative Examples 1 to 2, and the aqueous solution of camellia seed extract prepared in Comparative Example 3 were detected using an ultra-high performance liquid chromatography-linear ion trap-electrostatic field orbitrap high-resolution mass spectrometry system (UPLC-LTQ-Orbitrap-MS).

[0143] Chromatographic conditions: The chromatographic column was ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm); mobile phase A was 0.1% formic acid aqueous solution; mobile phase B was acetonitrile solution containing 0.1% formic acid. Gradient elution program: 0 - 2 min, 5% B; 2 - 42 min, 5% - 95% B; 42 - 47 min, 95% B; 47 - 47.1 min, 95% - 5% B; 47.1 - 50 min, 5% B. The flow rate was 0.3 mL / min; the injection volume was 5 μL; the column temperature was 40°C.

[0144] Mass spectrometry conditions were: Thermo electrospray ionization source HESI-II; positive and negative ion detection modes; scanning mode: Full MS-ddms 2 ,Full MS resolution: 70000, ddms 2 ,resolution: 17500; scanning range was: 100 - 1500; spray voltage: 3 kV (+) / 2.5 kV (−); ion transfer tube temperature: 320°C; auxiliary temperature: 350°C; sheath gas: 35 arb; auxiliary gas: 10 arb; automatic gain control number Full MS: 1e 6 ,MS / MS: 2e 5 ; maximum injection time Full MS: 100 ms, MS / MS: 50 ms.

[0145] The results are shown in Table 7.

[0146] Table 7 Results of component content determination

[0147]

[0148]

[0149] As can be seen from the results in Table 7, after the camellia seed extracts of Examples 1 to 10 were fermented with specific fermentation strains (one or more of Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus pentosus, and Lactobacillus casei), the contents of camellioside B, astragalin, kaempferol-3-O-rutinoside, kaempferol-7-O-neohesperidoside, and kaempferol 3-O-sambubioside were all significantly increased compared to Comparative Example 3 before fermentation; after Comparative Example 1 was fermented with Saccharomyces cerevisiae, the contents of astragalin and kaempferol-7-O-neohesperidoside were significantly decreased compared to before fermentation; after Comparative Example 2 was fermented with Lactococcus lactis, the contents of kaempferol-3-O-rutinoside and kaempferol 3-O-sambubioside were significantly decreased compared to before fermentation.

[0150] Application Example 1

[0151] A shampoo, and its specific formula is shown in Table 8; the specific preparation method of the shampoo can be prepared according to the preparation method of conventional shampoo.

[0152] Table 8 Shampoo Formula

[0153] Component Mass fraction Deionized water The balance Sodium lauryl polyether sulfate 12.0 Cocamidopropyl betaine 4.0 Cocamide MEA / glycerol 0.1 Sodium lauroamphoacetate 0.1 Cocamide MEA 0.2 Sodium C14-16 olefin sulfonate 2.0 Sodium methyl cocoyl taurate 0.2 Guar hydroxypropyltrimonium chloride 0.05 PPG-3 octyl ether 0.1 Cetyl alcohol 0.1 Sorbitan octanoate 0.2 Allantoin 0.05 Carbohydrate isomers 0.01 Camellia seed ferment prepared in Example 1 2.0 Citric acid 0.2 Sodium citrate 0.2 Phenoxyethanol 0.4 Disodium EDTA 0.1 (Daily use) Fragrance 0.5

[0154] Application Example 2

[0155] The difference from Application Example 1 is only that in Application Example 2, the camellia seed ferment prepared in Example 1 is replaced with the camellia seed ferment prepared in Example 2 in equal amount.

[0156] Application Example 3

[0157] The difference from Application Example 1 is only that in Application Example 3, the camellia seed ferment prepared in Example 1 is replaced with the camellia seed ferment prepared in Example 3 in equal amount.

[0158] Application Example 4

[0159] The difference from Application Example 1 is only that in Application Example 4, the camellia seed ferment prepared in Example 1 is replaced with the camellia seed ferment prepared in Example 4 in equal amount.

[0160] Application Example 5

[0161] The difference from Application Example 1 is only that in Application Example 5, the camellia seed ferment prepared in Example 1 is replaced with the camellia seed ferment prepared in Example 5 in equal amount.

[0162] Application Example 6

[0163] The difference from Application Example 1 is only that in Application Example 6, the camellia seed ferment prepared in Example 1 is replaced with the camellia seed ferment prepared in Example 6 in equal amount.

[0164] Application Example 7

[0165] The difference from Application Example 1 is only that in Application Example 7, the fermented camellia seed product prepared in Example 1 is replaced with the fermented camellia seed product prepared in Example 7 in equal amount.

[0166] Application Example 8

[0167] The difference from Application Example 1 is only that in Application Example 8, the fermented camellia seed product prepared in Example 1 is replaced with the fermented camellia seed product prepared in Example 8 in equal amount.

[0168] Application Example 9

[0169] The difference from Application Example 1 is only that in Application Example 9, the fermented camellia seed product prepared in Example 1 is replaced with the fermented camellia seed product prepared in Example 9 in equal amount.

[0170] Application Example 10

[0171] The difference from Application Example 1 is only that in Application Example 10, the fermented camellia seed product prepared in Example 1 is replaced with the fermented camellia seed product prepared in Example 10 in equal amount.

[0172] Comparative Application Example 1

[0173] The difference from Application Example 1 is only that in Comparative Application Example 1, the fermented camellia seed product prepared in Example 1 is replaced with the fermented camellia seed product prepared in Comparative Example 1 in equal amount.

[0174] Comparative Application Example 2

[0175] The difference from Application Example 1 is only that in Comparative Application Example 2, the fermented camellia seed product prepared in Example 1 is replaced with the fermented camellia seed product prepared in Comparative Example 2 in equal amount.

[0176] Comparative Application Example 3

[0177] The difference from Application Example 1 is only that in Comparative Application Example 3, the fermented camellia seed product prepared in Example 1 is replaced with the aqueous solution of camellia seed extract prepared in Comparative Example 3 in equal amount.

[0178] Blank Control Group

[0179] The difference from Application Example 1 is only that in the Blank Control Group, the fermented camellia seed product prepared in Example 1 is replaced with deionized water in equal amount.

[0180] Test Example 8 Shampoo Efficacy Test

[0181] The shampoo of the above Application Examples 1 - 10, Comparative Application Examples 1 - 3 and Blank Control Group was evaluated for its oil control and anti-dandruff efficacy.

[0182] 140 volunteers aged 20 to 45 years old, with an equal number of men and women, were selected as subjects. They were divided into 14 groups and used the shampoos of Application Examples 1 to 10, Comparative Application Examples 1 to 3, and the blank control group three times a week. They filled out a trial evaluation form according to the sensory evaluation. Statistics were conducted on the 7th day, 14th day, and 28th day. The volunteers evaluated the oil control and anti-dandruff effects of the samples during the use process; among them, the highest score of 5 indicates a significant effect, and the lowest score of 0 indicates no effect. The scoring results are shown in Table 9.

[0183] Table 9 Scoring of the Oil Control and Anti-dandruff Effects of Shampoos

[0184]

[0185]

[0186] From the results in Table 9, it can be seen that after the volunteers used the shampoos of Application Examples 1 to 10, the oil control and anti-dandruff effects of the scalp were significantly improved, and the scores on the 7th day, 14th day, and 28th day were always higher than those of the shampoos of Comparative Examples 1 to 3 and the blank control group. Since the oil control and anti-dandruff effects of the scalp are related to the tea saponin in the camellia seed extract, it shows that after the camellia seed extract of the present invention is fermented by a specific strain, the tea saponin increases, thereby improving both the oil control and anti-dandruff effects of the scalp.

[0187] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art can make various equivalent variations or substitutions without departing from the spirit of the present invention, and these equivalent variations or substitutions are all included within the scope defined by the claims of this application.

Claims

1. An oil-tea camellia seed ferment, characterized in that The preparation raw materials of the camellia seed ferment include: camellia seed extract, solvent, culture medium and fermentation strains; the fermentation strains are at least one of Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus rhamnosus and Lactobacillus pentosus; The mass ratio of the camellia seed extract, solvent and culture medium is 1:(5-20):(0.1-1); The concentration of the fermentation strain is 10 5 ~10 8 CFU / mL; The volume of the fermentation strains accounts for 0.1-5% of the total volume after mixing the camellia seed extract, solvent and culture medium; The camellia seed extract is prepared by a preparation method including the following steps: Take camellia seeds, perform water extraction, alcohol precipitation and spray drying to obtain camellia seed extract.

2. The fermented camellia seed product according to claim 1, wherein The chemical composition of the camellia seed ferment includes total polyphenols, and the content of the total polyphenols > 3 mg / mL.

3. The preparation method of the fermented camellia seed as claimed in claim 1 or 2, characterized in that It includes the following steps: Mix the camellia seed extract, solvent and culture medium, sterilize, and then add fermentation strains for fermentation; After the fermentation ends, perform centrifugation, collect the supernatant, and sterilize to obtain the camellia seed ferment.

4. The preparation method according to claim 3, characterized in that, The temperature of the fermentation is 25-40 °C; and / or, the time of the fermentation is 24-80 h.

5. A cosmetic, characterized in that, It includes the camellia seed ferment described in claim 1 or 2.

6. The cosmetic according to claim 5, wherein The content of the camellia seed ferment is 0.05-20 wt%.

7. The cosmetic according to claim 5, wherein The cosmetic is shampoo, body wash, essence or facial cleanser.

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