Camellia fermented product as well as preparation method and application thereof

Abstract

The invention relates to a preparation method of a camellia fermentation product, which is characterized in that camellia is fermented by adopting a mode of firstly performing enzymolysis and then performing fermentation, in the fermentation step, a composite yeast strain fermentation mode is adopted, the camellia is fermented through the synergistic effect of two yeasts, and the obtained camellia fermentation product is rich in various active substances. Therefore, the fermented product has good comprehensive effects of repairing, relieving and controlling oil.

Description

Technical Field

[0001] The invention belongs to the technical field of plant fermentation and cosmetics, and in particular relates to a camellia fermentation product and a preparation method and application thereof. Background Art

[0002] Camellia japonica L. is a plant of the genus Camellia in the family Theaceae. It is native to China and was first recorded in the Compendium of Materia Medica. It is also known as camellia, small camellia, winter-resistant camellia, etc. Camellia prefers warm, humid and semi-shady environments, is afraid of high temperatures and avoids scorching sun. It is distributed from northwest Yunnan to the west of Guizhou, central Sichuan, northwest Hunan and northwest Guangxi. Camellia is rich in various biologically active ingredients such as protein, amino acids, saponins, reducing sugars, polysaccharides, polyphenols, flavonoids, anthocyanins and vitamins.

[0003] At present, camellia extract is usually extracted by organic solvent extraction. The camellia extract prepared by this method often has a low content of active substances, and polyols, ethyl acetate or alkanes are used as solvents in the extraction process. The prepared camellia extract is prone to organic solvent residues, which can easily cause skin irritation when used in cosmetics. In addition, the organic solvent extraction method is prone to increase the risk of the extraction process and cause environmental pollution. Therefore, seeking a new method for extracting active substances from camellia has become an urgent problem to be solved in the field. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing camellia fermented product. The method extracts active substances in camellia by enzymatic hydrolysis followed by fermentation. The prepared camellia fermented product has good comprehensive effects of repairing, soothing and oil control.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a method for preparing a camellia fermentation product, comprising the following steps:

[0007] (1) mixing camellia pollen with water, adding lysase for enzymolysis, and obtaining enzymolysis solution after inactivating the enzyme;

[0008] (2) adding culture medium material to the enzymatic hydrolyzate, mixing evenly, adding composite yeast seed liquid, and fermenting to obtain crude fermentation liquid;

[0009] (3) The crude fermentation liquid is sterilized and filtered in sequence to obtain the camellia fermentation product.

[0010] The present invention extracts active substances from camellia by enzymolysis followed by fermentation, and the prepared camellia ferment has good comprehensive effects of repair, soothing and oil control. The present invention uses the enzyme collapsin to hydrolyze camellia because it has a strong ability to digest cell walls, which helps to dissolve active substances in camellia cells. In addition, the collapsin used in the present invention can degrade saponins in camellia to a certain extent, and subsequently reduce the toxicity of camellia extracts.

[0011] As a preferred embodiment of the first aspect of the present invention, the amount of camellia pollen added is 1-3% of the mass of water.

[0012] As a preferred embodiment of the first aspect of the present invention, the amount of the collapsing enzyme added is 0.1-0.3% of the mass of the camellia pollen, which can be 0.1%, 0.2%, 0.3% or any value therebetween; the temperature of the enzymolysis is 35-45°C.

[0013] As a preferred embodiment of the first aspect of the present invention, the enzymolysis time is 6-10 hours. The enzymolysis time has a great influence on the final comprehensive effect of the camellia fermentation product. If the time is too short, the enzymolysis is not thorough and the camellia cell wall cannot be effectively degraded, and the intracellular active substances cannot be released; if the time is too long, the active substances are easily degraded and cannot play their functions. Therefore, the present invention has found through experiments that when the enzymolysis time is within the range of 6 to 10 hours, it can not only degrade the plant cell wall to release the intracellular active substances but also ensure the effectiveness of the active substances; preferably, the enzymolysis time can be 6 hours, 7 hours, 8 hours, 9 hours, 10 hours or any value therebetween.

[0014] As a preferred embodiment of the first aspect of the present invention, the temperature for inactivating the enzyme is 80-95° C., and the time for inactivating the enzyme is 15-30 min.

[0015] As a preferred embodiment of the first aspect of the present invention, the culture medium material in step (2) includes a carbon source, a nitrogen source, an alkaloid, a phosphate, an ammonium salt and an acidity regulator.

[0016] As a preferred embodiment of the first aspect of the present invention, the carbon source is at least one of sucrose, soybean oligosaccharides, and palatinose; preferably, the carbon source is at least two of sucrose, soybean oligosaccharides, and palatinose; most preferably, the carbon source is a composite carbon source composed of sucrose, soybean oligosaccharides, and palatinose. The results of the carbon source screening test of the present invention show that the use of at least one of sucrose, soybean oligosaccharides, and palatinose as the carbon source of the composite yeast strain of the present invention can improve the fermentation efficiency of the composite yeast strain and improve the comprehensive efficacy of the camellia fermentation product in repairing, soothing, and controlling oil, while the use of other carbon sources, such as glucose, has a poor effect, and the most preferred carbon source is a composite carbon source composed of sucrose, soybean oligosaccharides, and palatinose.

[0017] As a preferred embodiment of the first aspect of the present invention, the nitrogen source is beef extract and / or yeast extract.

[0018] As a preferred embodiment of the first aspect of the present invention, the ammonium salt is ammonium chloride and / or ammonium sulfate.

[0019] As a preferred embodiment of the first aspect of the present invention, the phosphate is at least one of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

[0020] As a preferred embodiment of the first aspect of the present invention, the alkaloid is betaine. The present invention selects betaine because it can be used as a regulator in the present invention to adjust the osmotic pressure and pH, provide a suitable growth environment for yeast, promote yeast metabolism of active substances, and improve the utilization rate of yeast for carbon sources.

[0021] As a preferred embodiment of the first aspect of the present invention, the acidity regulator is citric acid and / or malic acid; preferably, the acidity regulator is citric acid.

[0022] As a preferred embodiment of the first aspect of the present invention, the amount of the carbon source added is 1.2-2.9% of the mass of the enzymatic hydrolyzate, which can be 1.2%, 1.5%, 1.8%, 2.0%, 2.2%, 2.5%, 2.6%, 2.8%, 2.9% or any value therebetween.

[0023] As a preferred embodiment of the first aspect of the present invention, the amount of the nitrogen source added is 1.5-2.5% of the mass of the enzymatic hydrolysate.

[0024] As a preferred embodiment of the first aspect of the present invention, the added amount of the alkaloid is 0.05-0.2% of the mass of the enzymatic hydrolyzate, which can be 0.05%, 0.08%, 0.1%, 0.12%, 0.15%, 0.18%, 0.2 or any value therebetween.

[0025] As a preferred embodiment of the first aspect of the present invention, the amount of phosphate added is 0.1-0.3% of the mass of the enzymatic hydrolyzate, and can be 0.1%, 0.2%, 0.3% or any value therebetween.

[0026] As a preferred embodiment of the first aspect of the present invention, the amount of ammonium salt added is 0.15-0.45% of the mass of the enzymatic hydrolyzate, which can be 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or any value therebetween.

[0027] As a preferred embodiment of the first aspect of the present invention, the amount of the acidity regulator added is 0.03-0.12% of the mass of the enzymatic hydrolyzate, which can be 0.03%, 0.05%, 0.07%, 0.09%, 0.12%, 0.15% or any value therebetween.

[0028] As a preferred embodiment of the first aspect of the present invention, the fermentation culture temperature is 25-30°C, the fermentation culture time is 60-90h, the fermentation culture rotation speed is 120-200rpm, and the amount of sterile air introduced during the fermentation culture is 0.3-0.5vvm.

[0029] As a preferred embodiment of the first aspect of the present invention, the composite yeast seed liquid in step (2) is a composite yeast seed liquid mixed with log yeast seed liquid and Candida krusei seed liquid. The present invention screens out the most suitable yeasts for fermenting camellia from a large number of yeasts through a large number of strain screening tests, which are log yeast and Candida krusei. The composite fermentation strains composed of the two have a synergistic effect on camellia fermentation, and can improve the comprehensive performance of camellia fermentation in repairing, soothing and oil control.

[0030] As a preferred embodiment of the first aspect of the present invention, the volume ratio of the log yeast seed liquid and the krusei candida seed liquid is 1: (1.5-3). The log yeast and krusei candida selected in the present invention have a synergistic effect on camellia fermentation, and the volume ratio of the seed liquid of the two also has a significant impact on the comprehensive performance of the final fermentation product. The present invention has a better effect by limiting the volume ratio of the two to the range of 1: (1.5-3) through experiments. Preferably, the volume ratio of the two can be 1: 1.5, 1: 1.8, 1: 1.9, 1: 2, 1: 2.2, 1: 2.4, 1: 2.6, 1: 2.8, 1: 3 or any value therebetween.

[0031] As a preferred embodiment of the first aspect of the present invention, the inoculation amount of the composite yeast seed solution is 4-8% of the volume of the enzymatic solution, preferably, it can be 4%, 5%, 6%, 7%, 8% or any value therebetween.

[0032] As a preferred embodiment of the first aspect of the present invention, the concentration of the yeast loggerhead seed solution or the yeast Candida krusei is 10 7 -10 9 CFU / mL; preferably, it can be 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL or any value in between.

[0033] As a preferred embodiment of the first aspect of the present invention, the sterilization temperature is 80-95° C., and the sterilization time is 15-30 min.

[0034] As a preferred embodiment of the first aspect of the present invention, the filtration is diatom filter filtration.

[0035] In a second aspect, the present invention provides a camellia fermented product produced by the preparation method of the first aspect. The fermented product of the present invention is non-irritating to the skin, has good comprehensive effects of repairing, soothing and oil control, and can be used in cosmetics as a plant active additive.

[0036] In a third aspect, the present invention provides a cosmetic composition comprising the camellia fermented product according to the second aspect.

[0037] As a preferred embodiment of the third aspect of the present invention, the amount of the camellia fermented product added to the cosmetic composition is 0.1%-80%.

[0038] In a fourth aspect, the present invention provides the use of Saccharomyces logeri and / or Candida krusei in preparing a camellia fermentation product.

[0039] In a fifth aspect, the present invention provides a culture medium for camellia fermentation, comprising the following components in mass fractions: 1-3% camellia pollen, 1.2-2.9% carbon source, 1.5-2.5% nitrogen source, 0.05-0.2% alkaloid, 0.1-0.3% phosphate, 0.15-0.45% ammonium salt, and 0.03-0.12% acidity regulator.

[0040] As a preferred embodiment of the fifth aspect of the present invention, the carbon source includes at least one of sucrose, soybean oligosaccharides and palatinose; preferably, the carbon source is at least two of sucrose, soybean oligosaccharides and palatinose; most preferably, the carbon source is a composite carbon source composed of sucrose, soybean oligosaccharides and palatinose.

[0041] As a preferred embodiment of the fifth aspect of the present invention, the composite carbon source comprises the following components in mass fractions: 1-2.3% sucrose, 0.1-0.3% soybean oligosaccharides and 0.1-0.3% palatinose.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) The present invention extracts camellia by enzymatic hydrolysis followed by fermentation. The obtained camellia fermented product is rich in various active substances, so that the fermented product has good repairing, soothing and oil-control effects.

[0044] (2) The preparation process of the camellia fermented product of the present invention avoids using polyols, ethyl acetate or alkanes as solvents, and the final product is non-irritating to the skin.

[0045] (3) The present invention uses cleavage enzyme, log yeast seed liquid and krusei Candida seed liquid to extract camellia. Cracking enzyme has a strong ability to digest cell walls, which helps to dissolve active substances in camellia cells. In addition, the cleavage enzyme used in the present invention can degrade saponins in camellia to a certain extent, and subsequently reduce the toxicity of camellia extract. The experimental results of the present invention show that using cleavage enzyme for enzymolysis first and then fermentation can help reduce the irritation of camellia fermentation products, and the final product prepared is more suitable for cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 A schematic diagram of the camellia of the present invention;

[0047] Figure 2 This is a schematic diagram of preparing camellia fermentation product using the preparation method of Example 1 of the present invention. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with the examples. It should be noted that the following description is only for the purpose of explaining the present invention and is not intended to limit its content. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, if not otherwise specified, can all be obtained through commercial channels. Unless otherwise specified, the percentages mentioned in the present invention are all mass percentages.

[0049] The lyase used in the present invention is from Shanghai Yiji Industrial Co., Ltd.

[0050] The cellulase and pectinase used in the present invention are from Ningxia Xiasheng Industrial Group Co., Ltd.

[0051] The number of Saccharomyces logos used in the present invention is CICC 32888, and the number of Candida krusei is CICC 1683, which are from the China Industrial Microbiological Culture Collection Center.

[0052] The Saccharomyces cerevisiae used in the present invention is numbered BNCC 336054, and the Lactobacillus plantarum is numbered BNCC 194165, which are from Shangcheng Beina Chuanglian Biotechnology Co., Ltd. The Saccharomyces rouxii used in the present invention is numbered ACCC20287, which is from NTCC Type Culture Collection Center, and the Pichia fermentans is numbered BMZ339612, and the Kluyveromyces lactis is numbered B80850, which are from Ningbo Mingzhou Biotechnology Co., Ltd.

[0053] In the present invention, the cell concentration of the seed solution of each strain is 10 7 ~10 9 CFU / mL, the preparation methods of various sub-liquids can be prepared by the common seed liquid preparation methods in the prior art, and the present invention does not make any specific limitation on the preparation method of the seed liquid.

[0054] Example 1

[0055] This embodiment provides a method for preparing a camellia fermentation product, and the specific steps are as follows:

[0056] (1) Add 2% camellia pollen by mass into deionized water, then add 0.2% camellia pollen lyase by mass into deionized water, mix well, perform enzymolysis at 40° C. for 8 h, and after the enzymolysis is completed, inactivate the enzyme at 85° C. for 20 min to obtain an enzymolysis solution;

[0057] (2) Add 2% sucrose, 0.1% soybean oligosaccharide, 0.1% palatinose, 1% beef extract, 1% yeast extract, 0.3% ammonium chloride, 0.2% dipotassium hydrogen phosphate, 0.1% betaine and 0.08% citric acid to the enzymatic hydrolysate, mix well, sterilize at 115°C for 30 min, cool to 28°C, and add 6% of the enzymatic hydrolysate by volume of composite yeast seed liquid (the bacterial concentration of the log yeast seed liquid is about 10 8 CFU / mL, the bacterial concentration of Candida krusei seed solution is about 10 8 CFU / mL, the volume ratio of yeast loggerheads seed liquid to Candida krusei seed liquid was 1:2), the material temperature was maintained at 28°C, and the fermentation was carried out for 72 hours under the conditions of sterile air flow rate of 0.4vvm and rotation speed of 180rpm, and the crude fermentation liquid was obtained after the fermentation was completed;

[0058] (3) The crude fermentation liquid is sterilized by keeping it at 85°C for 20 minutes and then filtered through a diatom filter to obtain the filtrate as the camellia fermentation product.

[0059] The preparation method of camellia pollen is as follows: dry camellia is placed in a powder grinder and crushed, and the powder is passed through a 200-mesh sieve, and the lower layer powder obtained is camellia pollen (the camellia pollen used in the other embodiments and comparative examples is the same as that in embodiment 1).

[0060] Example 2

[0061] The difference between Example 2 and Example 1 is that the enzymatic hydrolysis time in step (1) is 6 hours, and the rest is the same as Example 1.

[0062] Example 3

[0063] The difference between Example 3 and Example 1 is that the enzymatic hydrolysis time in step (1) is 10 hours, and the rest is the same as Example 1.

[0064] Example 4

[0065] The difference between Example 4 and Example 1 is that the type and amount of the carbon source added in step (2) are different. The added carbon source is 2% sucrose and 0.2% soybean oligosaccharide. The rest is the same as Example 1.

[0066] Example 5

[0067] The difference between Example 5 and Example 1 is that the type and amount of the carbon source added in step (2) are different. The added carbon source is 2% sucrose and 0.2% palatinose. The rest is the same as Example 1.

[0068] Example 6

[0069] The difference between Example 6 and Example 1 is that the type of acidity regulator in step (2) is different, and malic acid is used as the acidity regulator. The rest is the same as Example 1.

[0070] Example 7

[0071] The difference between Example 7 and Example 1 is that the inoculation volume ratio of the yeast loggerheads seed solution and the Candida krusei seed solution in step (2) is different. In this example, the inoculation volume ratio of the yeast loggerheads seed solution and the Candida krusei seed solution is 1:1.5, and the rest is the same as Example 1.

[0072] Example 8

[0073] The difference between Example 8 and Example 1 is that the inoculation volume ratio of the yeast loggerheads seed solution and the Candida krusei seed solution in step (2) is different. In this example, the inoculation volume ratio of the yeast loggerheads seed solution and the Candida krusei seed solution is 1:3, and the rest is the same as Example 1.

[0074] Example 9

[0075] This embodiment provides a method for preparing a camellia fermentation product, comprising the following steps:

[0076] (1) Adding 1% camellia pollen by mass into deionized water, and then adding 0.3% camellia pollen by mass enzyme, mixing well, and performing enzymolysis at 45° C. for 6 h. After the enzymolysis is completed, the enzyme is inactivated at 95° C. for 15 min to obtain an enzymolysis solution;

[0077] (2) 1% sucrose, 0.3% soybean oligosaccharides, 0.2% palatinose, 1.2% beef extract, 1.3% yeast extract, 0.45% ammonium sulfate, 0.3% sodium dihydrogen phosphate, 0.05% betaine and 0.12% citric acid were added to the enzymatic hydrolysate, mixed evenly, sterilized at 115°C for 30 min, cooled to 30°C, and then added with 4% of the enzymatic hydrolysate by volume of composite yeast seed liquid (the bacterial concentration of the log yeast seed liquid is about 10 9 CFU / mL, the bacterial concentration of Candida krusei seed solution is about 10 9 CFU / mL, the volume ratio of yeast loggerheads seed liquid to Candida krusei seed liquid was 1:1.8), the material temperature was maintained at 30°C, the sterile air flow rate was 0.5vvm, the rotation speed was 120rpm°C, and the fermentation culture was carried out for 60h, and the crude fermentation liquid was obtained after the fermentation was completed;

[0078] (3) The crude fermentation broth was sterilized by keeping it at 95°C for 15 min and then filtered through a diatom filter to obtain the filtrate as the camellia fermentation product.

[0079] Example 10

[0080] (1) Add 3% camellia pollen by mass into deionized water, then add 0.1% camellia pollen lyase by mass into the water, mix well, perform enzymolysis at 35° C. for 10 h, and after the enzymolysis is completed, inactivate the enzyme at 80° C. for 30 min to obtain an enzymolysis solution;

[0081] (2) Add 2.3% sucrose, 0.1% soybean oligosaccharide, 0.3% palatinose, 0.7% beef extract, 0.8% yeast extract, 0.15% ammonium chloride, 0.1% disodium hydrogen phosphate, 0.2% betaine and 0.03% malic acid to the enzymatic hydrolysate, mix well, sterilize at 115°C for 30 min, cool to 25°C, and add 8% of the volume ratio of the enzymatic hydrolysate to the composite yeast seed solution (the bacterial concentration of the log yeast seed solution is about 10 7 CFU / mL, the bacterial concentration of Candida krusei seed solution is about 10 7 CFU / mL, the volume ratio of yeast loggerheads seed liquid to Candida krusei seed liquid was 1:2.5), the material temperature was maintained at 25°C, the sterile air flow rate was 0.3vvm, the rotation speed was 200rpm°C, and the fermentation culture was carried out for 90h, and the crude fermentation liquid was obtained after the fermentation was completed;

[0082] (3) The crude fermentation liquid is sterilized by keeping it at 80°C for 30 minutes and then filtered through a diatom filter. The filtrate obtained is the camellia fermentation product.

[0083] Comparative Example 1

[0084] The difference between Comparative Example 1 and Example 1 is that step (2) of Example 1 is deleted in Comparative Example 1, and the camellia is extracted only by enzymatic hydrolysis, and the rest is the same as Example 1, and the specific steps are as follows:

[0085] Add 2% camellia pollen by mass into deionized water, then add 0.2% catalytic enzyme by mass into camellia pollen, mix well, and perform enzymolysis at 40°C for 8 hours. After the enzymolysis is completed, inactivate the enzyme at 85°C for 20 minutes to obtain enzymolysis solution, filter the enzymolysis solution through a diatom filter, and the obtained filtrate is camellia extract.

[0086] Comparative Example 2

[0087] The difference between Comparative Example 2 and Example 1 is that step (1) of Example 1 is deleted in Comparative Example 2, and the camellia is extracted only by fermentation. The rest is the same as Example 1, and the specific steps are as follows:

[0088] (1) Add 2% camellia pollen, 2% sucrose, 0.1% soybean oligosaccharide, 0.1% palatinose, 1% beef extract, 1% yeast extract, 0.3% ammonium chloride, 0.2% dipotassium hydrogen phosphate, 0.1% betaine and 0.08% citric acid to deionized water, mix well, sterilize at 115°C for 30 min, cool to 28°C, and add 6% by volume of composite yeast seed liquid (the cell concentration of log yeast seed liquid is about 10 8 CFU / mL, the bacterial concentration of Candida krusei seed solution is about 10 8 CFU / mL, the volume ratio of yeast loggerheads seed liquid to Candida krusei seed liquid was 1:2), the material temperature was maintained at 28°C, the sterile air flow rate was 0.4vvm, the rotation speed was 180rpm, and the fermentation culture was carried out for 72h, and the crude fermentation liquid was obtained after the fermentation was completed;

[0089] (2) The crude fermentation broth was sterilized by keeping it at 85°C for 20 minutes and then filtered through a diatom filter to obtain the filtrate as the camellia fermentation product.

[0090] Comparative Example 3

[0091] The difference between Comparative Example 3 and Example 1 is that Comparative Example 3 uses a method of first fermentation and then enzymatic hydrolysis to extract camellia, and the remaining steps and parameters are the same as Example 1. The specific steps are as follows:

[0092] (1) Add 2% camellia pollen, 2% sucrose, 0.1% soybean oligosaccharide, 0.1% palatinose, 1% beef extract, 1% yeast extract, 0.3% ammonium chloride, 0.2% potassium dihydrogen phosphate, 0.1% betaine and 0.08% citric acid to deionized water, mix well, sterilize at 115°C for 30 min, cool to 28°C, and add 6% by volume of composite yeast seed liquid (the bacterial concentration of log yeast seed liquid is about 10 8 CFU / mL, the bacterial concentration of Candida krusei seed solution is about 10 8 CFU / mL, the volume ratio of yeast loggerheads seed liquid to Candida krusei seed liquid was 1:2), the material temperature was maintained at 28°C, the sterile air flow rate was 0.4vvm, the rotation speed was 180rpm, and the fermentation culture was carried out for 72h. After the fermentation, the fermentation was sterilized at 85°C for 20min to obtain a crude fermentation liquid;

[0093] (2) Add 0.2% of camellia pollen mass fraction of lysozyme to the crude fermentation liquid, mix well, and perform enzymolysis at 40° C. for 8 h. After the enzymolysis is completed, inactivate the enzyme at 85° C. for 20 min, filter through a diatom filter, and obtain a camellia fermentation product.

[0094] Comparative Example 4

[0095] The difference between Comparative Example 4 and Example 1 is that in Comparative Example 4, only the log yeast seed liquid is used for fermentation (the addition amount of the log yeast seed liquid is 6%), and the remaining steps and parameters are the same as in Example 1.

[0096] Comparative Example 5

[0097] The difference between Comparative Example 5 and Example 1 is that in Comparative Example 5, only Candida krusei seed liquid is used for fermentation (the addition amount of Candida krusei seed liquid is 6%), and the remaining steps and parameters are the same as in Example 1.

[0098] Comparative Example 6

[0099] The difference between Comparative Example 6 and Example 1 is that in Comparative Example 6, the volume ratio of the log yeast seed solution and the Candida krusei seed solution in Example 1 is changed to 1:1 (the addition amount of the composite yeast seed solution is 6%), and the remaining steps and parameters are the same as in Example 1.

[0100] Comparative Example 7

[0101] The difference between Comparative Example 7 and Example 1 is that in Comparative Example 7, the composite yeast seed solution in Example 1 is replaced with a brewer's yeast seed solution (the cell concentration of the brewer's yeast seed solution is about 10 8 CFU / mL), the addition amount was the same as that of the composite seed solution, and the remaining steps and parameters were the same as those in Example 1.

[0102] Comparative Example 8

[0103] The difference between Comparative Example 8 and Example 1 is that in Comparative Example 8, the composite yeast seed liquid in Example 1 is replaced with Lactobacillus plantarum seed liquid (the bacterial concentration of Lactobacillus plantarum seed liquid is about 10 8 CFU / mL, the addition amount was the same as that of the composite seed liquid), the fermentation temperature was changed to 37°C, and the remaining steps and parameters were the same as in Example 1.

[0104] Comparative Example 9

[0105] The difference between Comparative Example 9 and Example 1 is that in Comparative Example 9, the composite yeast seed solution in Example 1 is replaced by a composite yeast seed solution composed of a combination of a log yeast seed solution and a rouxii yeast seed solution (the bacterial cell concentrations of the log yeast seed solution and the rouxii yeast seed solution are both 10 8 CFU / mL, the volume ratio of yeast loggerheads seed liquid to yeast rouxii seed liquid was 1:2, and the total inoculation volume ratio was 6%), and the remaining steps and parameters were the same as those in Example 1.

[0106] Comparative Example 10

[0107] The difference between Comparative Example 10 and Example 1 is that in Comparative Example 10, the composite yeast seed solution in Example 1 is replaced by a composite yeast seed solution composed of a log yeast seed solution and a fermentation Pichia pastoris seed solution (the bacterial cell concentrations of the log yeast seed solution and the fermentation Pichia pastoris seed solution are both 10 8 CFU / mL, the volume ratio of yeast loggerheads seed liquid and Pichia pastoris seed liquid was 1:2, and the total inoculation volume ratio was 6%), and the remaining steps and parameters were the same as those in Example 1.

[0108] Comparative Example 11

[0109] The difference between Comparative Example 11 and Example 1 is that in Comparative Example 11, the composite yeast seed liquid in Example 1 is replaced by a composite yeast seed liquid composed of log yeast seed liquid and lactic acid yeast seed liquid (the bacterial concentration of log yeast seed liquid and lactic acid yeast seed liquid is 10 8 CFU / mL, the volume ratio of logger yeast seed liquid and lactic acid yeast seed liquid was 1:2, and the total inoculation volume ratio was 6%), and the remaining steps and parameters were the same as those in Example 1.

[0110] Comparative Example 12

[0111] The difference between Comparative Example 12 and Example 1 is that in Comparative Example 12, the composite yeast seed solution in Example 1 is replaced by a composite yeast seed solution composed of a Saccharomyces rouxii seed solution and a Candida krusei seed solution (the bacterial cell concentrations of the Saccharomyces rouxii seed solution and the Candida krusei seed solution are both 10 8CFU / mL, the volume ratio of Saccharomyces rouxii seed solution to Candida krusei seed solution was 1:2, and the total inoculation volume ratio was 6%), and the remaining steps and parameters were the same as those in Example 1.

[0112] Comparative Example 13

[0113] The difference between Comparative Example 13 and Example 1 is that in Comparative Example 13, the composite yeast seed solution in Example 1 is replaced by a composite yeast seed solution composed of a Pichia yeast seed solution and a Candida krusei seed solution (the bacterial concentrations of the Pichia yeast seed solution and the Candida krusei seed solution are both 10 8 CFU / mL, the volume ratio of Pichia pastoris seed solution to Candida krusei seed solution was 1:2, and the total inoculation volume ratio was 6%), and the remaining steps and parameters were the same as those in Example 1.

[0114] Comparative Example 14

[0115] The difference between Comparative Example 14 and Example 1 is that in Comparative Example 14, the composite yeast seed solution in Example 1 is changed to a composite yeast seed solution composed of lactic acid yeast seed solution and Candida krusei seed solution (the bacterial concentrations of the lactic acid yeast seed solution and the Candida krusei seed solution are both 10 8 CFU / mL, the volume ratio of lactic acid yeast seed liquid to Candida krusei seed liquid was 1:2, and the total inoculation volume ratio was 6%), and the remaining steps and parameters were the same as those in Example 1.

[0116] Comparative Example 15

[0117] The difference between Comparative Example 15 and Example 1 is that in Comparative Example 15, the composite yeast seed liquid in Example 1 is replaced by a composite yeast seed liquid composed of a brewer's yeast seed liquid and a lactic acid yeast seed liquid (the cell concentrations of the brewer's yeast seed liquid and the lactic acid yeast seed liquid are both 10 8 CFU / mL, the volume ratio of cerevisiae seed liquid to lactic acid yeast seed liquid was 1:2, and the total inoculation volume ratio was 6%), and the remaining steps and parameters were the same as those in Example 1.

[0118] Comparative Example 16

[0119] The difference between Comparative Example 16 and Example 1 is that in Comparative Example 16, the lyase in Example 1 is replaced by cellulase and pectinase (the amount of cellulase added is 0.1%, and the amount of pectinase added is 0.1%), and the remaining steps and parameters are the same as in Example 1.

[0120] Comparative Example 17

[0121] The difference between Comparative Example 17 and Example 1 is that in Comparative Example 17, the camellia pollen in Example 1 is replaced with camellia leaf powder, and the remaining steps and parameters are the same as those in Example 1; the preparation method of the camellia leaf powder is: placing the dried camellia leaves in a powder grinder and crushing them, passing through a 200-mesh sieve, and the obtained lower layer powder is the camellia leaf powder.

[0122] Comparative Example 18

[0123] The difference between Comparative Example 18 and Example 1 is that in Comparative Example 18, the carbon sources 2% sucrose, 0.1% soybean oligosaccharides, and 0.1% palatinose in Example 1 are replaced with 2.2% glucose, and the remaining steps and parameters are the same as in Example 1.

[0124] Experimental Example 1 Irritation Test

[0125] The irritation test is carried out according to "SN / T 2329-2009 Chicken chorioallantoic membrane test for eye irritation and corrosion of cosmetics".

[0126] Test samples: the camellia fermented products or camellia extracts of Examples 1-10 and Comparative Examples 1-18.

[0127] Test procedure: the purchased 0-day-old eggs were placed in an incubator and cultured for 9 days (the temperature of the incubator was 37.5°C and the relative humidity was 60%), chicken embryos with good vascular development were selected, the air chamber position was marked on the eggshell surface, and the eggs needed for the sample to be tested were distributed (six chicken embryos were used for one sample, three chicken embryos were used for the negative control, i.e., 0.9wt% sodium chloride solution, and three chicken embryos for the positive control, i.e., 1wt% SDS solution, were used).

[0128] Use a tool to remove the marked eggshell part, use a pipette to drip 0.9% sodium chloride solution (normal saline) to moisten the egg membrane, pour out the sodium chloride solution, carefully remove the inner membrane with tweezers to ensure that the blood vessels are not damaged, observe the chicken embryo for no obvious bleeding or turbidity, and then judge that it can be used for the experiment; take 300 μL of the test substance and cover it on the chorioallantoic membrane, rinse the test substance on the membrane with normal saline after 3 minutes, the rinsing time is 30 seconds, immediately observe the reaction of the chorioallantoic membrane, take pictures to record the state of the chicken embryo, end the observation, record the IS value of each egg and calculate the average IS value to obtain the result.

[0129] Table 1 Irritation rating table

[0130] Stimulus score IS<1 1≤IS<5 5≤IS<9 IS ≥ 10 Irritation classification Non-irritating Mild irritation Moderate irritation Strong irritant / corrosive

[0131] Table 2 Irritation test results

[0132]

[0133]

[0134] The test results show that the camellia fermented products of the embodiments of the present invention are not eye irritating and are suitable for preparing and applying in mild and non-irritating cosmetics. Comparative Examples 1-3, the results of Comparative Examples 1, 2, 3, and 16 show that the necessary enzymatic hydrolysis and fermentation steps, the order of enzymatic hydrolysis and fermentation, and the type of enzyme will affect the irritation of the camellia fermented products. The present invention uses a crash enzyme for enzymatic hydrolysis before the fermentation of the camellia. The crash enzyme not only has a strong degradation effect on the cell wall of the camellia, but also has a certain degradation effect on the saponin substances of the camellia, which can reduce the irritation of the camellia. The present invention uses a crash enzyme to enzymatically hydrolyze the camellia first, and then uses a composite yeast strain for fermentation, and the irritation of the final product can be greatly reduced. The enzymatic hydrolysis time of Examples 1 and 3 is longer than that of Example 2, and the IS values ​​of Examples 1 and 3 are lower, indicating that the enzymatic hydrolysis time also affects the irritation of the final product, but the enzymatic hydrolysis time cannot be too long, which easily leads to the degradation of the active substance. Therefore, the preferred enzymatic hydrolysis time of the present invention is 6 to 10 hours.

[0135] Experimental Example 2 Repair Performance Test

[0136] Test principle: Silent information regulator Sirtuin 1 (SIRT1) is a group of type III protein deacetylases, which is considered to be a new anti-aging protein involved in the regulation of cell aging and inflammation. SIRT1 levels and activity are reduced in cell aging caused by oxidative stress. SIRT1-mediated anti-inflammatory aging mechanisms are related to inflammation, premature cell aging, telomere loss, aging-related secretory phenotypes and regulation of DNA damage response. SIRT1 activation has the potential to attenuate cellular oxidative aging by downregulating oxidative stress-mediated cell aging.

[0137] In this experiment, H2O2-induced L929 cells were used as an in vitro oxidative damage cell model. RNA was extracted from the cells using an RNA extraction kit and reverse transcribed into cDNA. Finally, fluorescent quantitative PCR was performed to detect the expression level of the silent information regulator (SIRT1) of the cell gene, thereby evaluating the repair effect of the test sample.

[0138] The higher the expression level of SIRT1, the faster the cell oxidation is reduced and the better the repair effect of the test sample.

[0139] Test sample: the camellia fermented products or camellia extracts of Examples 1-10 and Comparative Examples 1-18 were prepared into a solution with a mass concentration of 1% using H-DMEM culture medium.

[0140] Test steps: Add 2×10 5L929 cells were added with H-DMEM culture medium (referred to as culture medium) and cultured for 24 hours at 37°C, 5% CO2, and saturated humidity. The 6-well plate was removed, the original culture medium in the well was discarded, and the test sample group and the model group were added with culture medium containing 0.1 μg / mL H2O2 solution as a stimulus for 1 hour, and then the H2O2 solution was removed. The negative control group was not induced by adding H2O2 solution; the test sample group was added with the test sample, and the negative control group and the model group were added with culture medium, and cultured in the incubator for 24 hours. The cells in each group were broken and RNA was collected, reverse transcribed into cDNA, and the expression of SIRT1 gene was determined by fluorescence quantitative PCR. Each group of samples was tested in parallel 4 times. The model group was used as a reference, and the significant difference of each test sample was calculated by SPSS statistical analysis software. The analysis of significant differences is shown in the following table.

[0141] Table 3: Analysis of significant differences in repair performance

[0142] p>0.05 p≤0.05 p≤0.01 p≤0.001 No statistically significant difference There is a statistical difference There is a statistically significant difference There is a statistically significant difference

[0143] Table 4: Repair performance test results

[0144]

[0145]

[0146] The test results show that: compared with the model control group, the camellia fermented products of the embodiments of the present invention can increase the relative expression of SIRT1 gene in cells, indicating that the camellia fermented products of the embodiments of the present invention have a good repair effect. The relative expression of SIRT1 gene in some camellia fermented products in the comparative example is equivalent to that in the model control group, indicating that they do not have the effect of repair.

[0147] Experimental Example 3 Soothing Performance Test

[0148] Experimental principle: Inflammatory pain caused by skin sensitivity is a reaction produced under the stimulation of various inflammatory and pain-causing factors. The mitochondrial function of skin cells is impaired, lysosomes and lipid membranes are destroyed, and tissue and organ damage is aggravated, so the pain is caused. Prostaglandins (PGs) are a class of strong inflammatory mediators. Among them, prostaglandin E2 (PGE2) has a stronger inflammatory effect, which can induce chemotaxis and aggregation of inflammatory cells, dilate capillaries to increase their permeability, promote inflammatory exudation, cause fever and increase the inflammatory and pain-causing effects of other inflammatory mediators, induce explosive inflammatory cascade reactions, and further enhance and amplify inflammatory reactions. In this experiment, SDS-induced mouse fibroblasts L929 were used as an in vitro sensitive cell model, and the relative expression of PGE2 gene was measured to evaluate whether the test sample has a soothing effect. If the expression of the inflammatory factor PGE2 gene decreases after the test sample is used, it indicates that the test sample can reduce the inflammatory factor PGE2 in the cell, and the test sample has a soothing and anti-allergic effect.

[0149] Experimental samples: the camellia fermented products or camellia extracts of Examples 1-10 and Comparative Examples 1-18 were prepared into solutions with a mass concentration of 0.5% using H-DMEM culture medium.

[0150] Experimental steps: L929 cells were seeded in 6-well plates (2×10 5 cells), added H-DMEM culture medium, and cultured for 24 hours at 37°C, 5% CO2, and saturated humidity. After the culture was completed, the culture medium was aspirated and discarded, 1 mL of H-DMEM culture medium (referred to as culture medium) was added to the blank control group, and 1 mL of 1% SDS solution was added to the model group and the test sample group to induce modeling for 1 hour, and the culture medium was aspirated and discarded. Subsequently, 1 mL of culture medium was added to the blank control group and the model group, and 1 mL of the corresponding sample solution was added to each test sample group, and culture was continued for 24 hours. After the culture was completed, the expression of PGE2 mRNA was detected by real-time qPCR. The relative expression multiple of PGE2 gene in the blank control group was F=1, and 2 -ΔΔCT The F value of the model group and each sample was calculated by the method. Each group of samples was tested 4 times in parallel. The model group was used as a reference, and the significant difference of the test sample group was calculated by SPSS statistical analysis software. The test results are shown in the following table.

[0151] Table 5: Soothing performance test results

[0152]

[0153]

[0154] By comparing the blank control group and the model group, it can be seen that the relative expression of PGE2 gene in the cells of the model group increased after induction with SDS solution, indicating that cellular inflammation is occurring. After induction with SDS solution, the test sample group was added with the test sample and cultured for 24 hours. The relative expression of PGE2 gene in some test sample group cells decreased, indicating that these test samples can reduce cellular inflammation and show a soothing effect. This shows that the camellia fermented products of the embodiments of the present invention all have a good soothing effect, while the control group does not have a soothing effect.

[0155] Experimental Example 4 Oil Control Performance Test

[0156] Experimental principle: Excessive secretion of sebaceous glands, imbalance in sebum composition, and lipid peroxidation are considered to be the main factors causing skin problems in oily skin. Sebum secretion is related to the activity of 5α-reductase in the sebaceous glands. 5α-reductase can convert testosterone into dihydrotestosterone (DHT). DHT binds to the receptor AR, increases the number of active sebaceous glands, and stimulates the sebaceous glands to secrete oil. In this experiment, human sebaceous gland cells SZ95 were selected for the construction of an in vitro acne model. The sebum secretion process of human skin was simulated by the oil secretion of SZ95 cells. Nile red dye was used to perform fluorescent staining on neutral lipids. The oil content of sebaceous gland cells was qualitatively and quantitatively detected by fluorescence microscopy and fluorescence quantification. The lower the fluorescence intensity, the better the oil control performance, thereby evaluating whether the experimental samples have the effect of inhibiting oil secretion.

[0157] Experimental samples: the camellia fermented products or camellia extracts of Examples 1-10 and Comparative Examples 1-18, prepared into a solution with a mass concentration of 0.5% using H-DMEM culture medium; 100 ng / mL minoxidil.

[0158] Experimental steps: SZ95 cells in the logarithmic phase were plated in a 24-well culture plate (2×10 4 cells), added H-DMEM culture medium (referred to as culture medium) and cultured at 37°C, 5% CO2, saturated humidity for 24h; the culture medium was discarded, and 0.5mL 10μM testosterone culture medium, 0.5mL 10μM testosterone sample solution, and 0.5mL 100ng / mL minoxidil culture medium were added to the model group, experimental sample group, and minoxidil group to induce modeling for 24 hours, and the culture medium was discarded. 0.5mL PBS solution was added to each well for washing, and then 3mM Nile red (source leaf biology) diluted with PBS was stained, incubated at 37°C in the dark for 10min, and after washing with PBS, the fluorescence intensity of Nile red was detected using a multifunctional microplate reader and photographed. The fluorescence gray value was converted by Image J to evaluate the relative fluorescence intensity of each group. Each group of samples was tested in parallel 4 times, and the model group was used as a reference. The significant difference of each group of test samples was calculated by SPSS statistical analysis software. The test results are shown in the following table.

[0159] Table 6: Oil control performance test results

[0160]

[0161] The test results show that compared with the model group, after using minoxidil or the camellia fermented product of the embodiment of the present invention, the fluorescence intensity decreases, indicating that the camellia fermented product of the embodiment of the present invention has an oil control effect. Minoxidil has been proven to have an oil control effect, and it is used as a positive control group in this experiment to verify the validity of the results of Experimental Example 4. The relative expression of the fluorescence intensity of some camellia fermented products in the comparative example is higher than that of the model group, indicating that it does not have the effect of oil control.

[0162] The above test results show that the repair effect of Examples 1-10 is better than that of Comparative Examples 1-3, indicating that the camellia fermented product prepared by the steps of enzymolysis followed by fermentation has a good comprehensive effect of repair, soothing and oil control. Only enzymolysis (Comparative Example 1), only fermentation (Comparative Example 2) or the order of fermentation and enzymolysis is reversed (Comparative Example 3), the repair, soothing and oil control effects of the final product are all deteriorated. The repair, soothing and oil control effects of Examples 1-10 are all better than those of Comparative Examples 4-15, indicating that the fermented strains and the volume ratio of the composite strains accessed during the fermentation process have a great influence on the comprehensive performance of the final product. The present invention uses Loger yeast and Candida russii to ferment the enzymolyzed camellia, and the obtained camellia fermented product has the best comprehensive effect of repair, soothing and oil control, which indicates that Loger yeast and Candida russii have a synergistic effect and can improve the comprehensive effect of camellia fermented product. Comparative Examples 4-5 use Log Saccharomyces or Candida Russicaria to ferment Camellia hydrolysate, Comparative Example 6 adjusts the ratio of Log Saccharomyces and Candida Russicaria, Comparative Examples 7-8 replace the composite yeast strains of the present invention with other strains to ferment Camellia, Comparative Examples 9-14 combine Log Saccharomyces and Candida Russicaria with other yeasts for composite strain fermentation, and Comparative Example 15 replaces the composite yeast strains of Log Saccharomyces and Candida Russicaria with composite strains of other yeast strains, but the results show that the repair, soothing and oil control comprehensive effects of the final Camellia fermentation products of Comparative Examples 4-15 are all poor. Among them, Comparative Example 8 uses plant lactobacillus for fermentation, although its irritation is low, the comprehensive effect of the Camellia fermentation product prepared by plant lactobacillus is poor. The comprehensive effect of Example 1-10 is better than that of Comparative Example 16, indicating that the type of enzyme used for enzymolysis has a great influence on the repair effect of the final product, therefore, the present invention preferably uses the collapse enzyme for enzymolysis. The comprehensive effects of repair, soothing and oil control of Examples 1-10 are better than those of Comparative Example 17, indicating that the type of plant has an impact on the repair effect of the final product. Although the final product has no eye irritation when the camellia powder is replaced with camellia powder, the repair, soothing and oil control effects of the final product are deteriorated. The comprehensive effects of repair, soothing and oil control of Examples 1-10 are all better than those of Comparative Example 18. It can be seen that the carbon source of the fermentation medium has a significant impact on the composite yeast strain fermentation of camellia, affecting the comprehensive effects of repair, soothing and oil control of the final camellia fermentation product. Therefore, the carbon source used for composite yeast strain fermentation of the present invention is preferably a composite carbon source of sucrose, soybean oligosaccharides and palatinose.

[0163] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a camellia fermented product, characterized in that: The steps include: (1) mixing camellia pollen with water, adding lysase for enzymolysis, and obtaining enzymolysis solution after inactivating the enzyme; (2) adding culture medium material to the enzymatic hydrolyzate, mixing evenly, and then adding composite yeast seed liquid to carry out fermentation culture to obtain a crude fermentation liquid; (3) The crude fermentation liquid is sterilized and filtered in sequence to obtain the camellia fermentation product.

2. The preparation method according to claim 1, characterized in that In step (1), the amount of the degrading enzyme added is 0.1-0.3% of the mass of the camellia pollen, the temperature of the enzymolysis is 35-45° C., and the time of the enzymolysis is 6-10 h.

3. The preparation method according to claim 1, characterized in that: The culture medium materials in step (2) include a carbon source, a nitrogen source, an alkaloid, a phosphate, an ammonium salt and an acidity regulator; Preferably, the carbon source is at least one of sucrose, soybean oligosaccharides, and palatinose; the nitrogen source is beef extract and / or yeast extract; the ammonium salt is ammonium chloride and / or ammonium sulfate; the phosphate is at least one of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate; the alkaloid is betaine; and the acidity regulator is citric acid and / or malic acid; Preferably, the carbon source is a composite carbon source composed of sucrose, soybean oligosaccharides and palatinose.

4. The preparation method according to claim 1, characterized in that: The fermentation temperature of step (2) is 25-30°C, the fermentation time is 60-90h, the fermentation speed is 120-200rpm, and the amount of sterile air introduced during the fermentation is 0.3-0.5vvm.

5. The preparation method according to claim 1, characterized in that: The composite yeast seed liquid in step (2) is a mixture of Saccharomyces logos seed liquid and Candida krusei seed liquid.

6. The preparation method according to claim 5, characterized in that: The volume ratio of the yeast loggerheads seed solution to the Candida krusei seed solution is 1:(1.5-3).

7. A camellia fermented product produced by the production method according to any one of claims 1 to 6.

8. A cosmetic composition, characterized in that It comprises the camellia fermented product as claimed in claim 7.

9. Use of Saccharomyces logeri and / or Candida krusei in preparing camellia fermentation products.

10. A culture medium for camellia fermentation, characterized in that: The invention comprises the following components in mass fractions: 1-3% of camellia pollen, 1.2-2.9% of carbon source, 1.5-2.5% of nitrogen source, 0.05-0.2% of alkaloid, 0.1-0.3% of phosphate, 0.15-0.45% of ammonium salt and 0.03-0.12% of acidity regulator. Preferably, the carbon source comprises the following components in mass fractions: 1-2.3% sucrose, 0.1-0.3% soybean oligosaccharides and 0.1-0.3% palatinose.

Images