Anti-inflammatory golden camellia fermentation liquor as well as preparation method and application thereof

Abstract

The invention relates to the technical field of cosmetics, and particularly discloses anti-inflammatory golden camellia fermentation liquor as well as a preparation method and application thereof. The preparation method of the anti-inflammatory golden camellia fermentation liquor comprises the following steps: inoculating lactic acid bacteria into a culture medium, and carrying out constant-temperature shaking culture to obtain a seed solution; camellia nitidissima is carefully selected, a cryoprotectant is added, a vacuum freeze drying technology is adopted for drying treatment, and a dried camellia nitidissima product is obtained; the dried golden camellia product is subjected to low-temperature impact superfine grinding and sieving, and golden camellia powder is obtained; adding 0.8-1.2% of golden camellia powder and the balance of deionized water into a fermentation tank, and sterilizing; inoculating the seed solution into a fermentation tank, and culturing at constant temperature to obtain a fermentation solution; and filtering the fermentation liquor to remove impurities, sterilizing, and adding a preservative to obtain the golden camellia fermentation liquor. The anti-inflammatory golden camellia fermentation liquor can be used for anti-inflammatory cosmetics; the preparation method has the advantage that the defect that the anti-inflammatory performance of the golden camellia fermentation liquor is still insufficient can be overcome.

Description

TECHNICAL FIELD

[0001] The application relates to the cosmetic technology field, in particular to an anti-inflammatory camellia chrysantha fermentation liquor and a preparation method and application thereof. BACKGROUND

[0002] Camellia chrysantha is a evergreen shrub or small tree of theaceae, is a unique national protected plant in China, is a world famous rare plant, and is extremely precious in resources, is praised as "plant giant panda" and "tea queen", contains dozens of amino acids such as theanine and threonine, and is rich in natural oil machine germanium, selenium, molybdenum, zinc, vanadium and other trace elements and potassium, calcium, magnesium and other macro elements, and camellia chrysantha is rich in active ingredients such as copper, polysaccharide and polyphenol, modern pharmacological studies show that the flowers and leaves of camellia chrysantha have good anti-inflammatory effect, however, camellia chrysantha is easily damaged in the drying process, leading to degradation of anti-inflammatory components such as polyphenol and flavonoid, thereby affecting the anti-inflammatory performance of camellia chrysantha. SUMMARY

[0003] In order to improve the anti-inflammatory performance of the camellia chrysantha fermentation liquor, the application provides an anti-inflammatory camellia chrysantha fermentation liquor and a preparation method and application thereof.

[0004] In a first aspect, the application provides a preparation method of an anti-inflammatory camellia chrysantha fermentation liquor, which adopts the following technical scheme:

[0005] A preparation method of an anti-inflammatory camellia chrysantha fermentation liquor, comprising the following steps:

[0006] S1: inoculating lactic acid bacteria into a culture medium, constant temperature oscillation culture, and obtaining a seed liquid;

[0007] S2: carefully selecting camellia chrysantha, adding a cryoprotective agent, and drying treatment by using a vacuum freeze-drying technology to obtain a camellia chrysantha dried product;

[0008] S3: carrying out low-temperature impact ultrafine grinding and sieving on the camellia chrysantha dried product to obtain camellia chrysantha powder;

[0009] S4: adding 0.8-1.2% camellia chrysantha powder and the rest deionized water into a fermentation tank for sterilization;

[0010] S5: inoculating the seed liquid into the fermentation tank for constant temperature culture to obtain a fermentation liquor;

[0011] S6: filtering and removing impurities, sterilizing the fermentation liquor, and adding a preservative to obtain a camellia chrysantha fermentation liquor.

[0012] Since the core anti-inflammatory active ingredients such as polyphenols and flavonoids in Camellia sinensis var. assamica are heat-sensitive substances, they are sensitive to heat, oxygen and mechanical force, and traditional processing can easily lead to their degradation. The vacuum freeze-drying technology is beneficial to solve the problem of component degradation in the drying process. The dried Camellia sinensis var. assamica has an intact cell structure, providing high-activity Camellia sinensis var. assamica dry products for subsequent processing. Ultrafine grinding opens the cell wall barrier at low temperature without damaging the structure of active ingredients, so that lactic acid bacteria can more efficiently utilize the nutrients in them during the fermentation stage, convert natural active ingredients into a form that is more easily absorbed by the skin, and improve the anti-inflammatory activity of Camellia sinensis var. assamica fermented liquid and its application effect in cosmetics.

[0013] Preferably, the vacuum freeze-drying conditions used in step S2 are: pre-freezing temperature of-25 to-35℃, pre-freezing time of 3-4h, and maximum heating temperature of the partition of 37-41℃.

[0014] Setting the pre-freezing temperature between-25 and-35℃ is beneficial to form a large number of small and uniform ice crystals, reduce the mechanical damage of local large ice crystals to cell walls and cell membranes, and thus retain the anti-inflammatory active ingredients in the cells. The low temperature of-25 to-35℃ is beneficial to inactivate polyphenol oxidase, so that it is not easy to oxidize and decompose tea polyphenols, and reduce the loss of active ingredients caused by enzymatic hydrolysis. Setting the pre-freezing time to 3-4h is beneficial to convert free water and bound water in Camellia sinensis var. assamica tissue into ice crystals. Setting the maximum heating temperature of the partition to 37-41℃ provides sufficient heat energy to drive the sublimation of ice crystals. Gentle heating is beneficial to prevent the active ingredients from being damaged due to high temperature, and maximizes the retention of their anti-inflammatory activity.

[0015] Preferably, the cryoprotective agent is mesoporous silica loaded with trehalose modified by modified polyethylene glycol, and the modified polyethylene glycol is spermidine grafted polyethylene glycol.

[0016] The porous structure of mesoporous silica is beneficial to efficiently load trehalose. The silicon hydroxyl groups on the surface of mesoporous silica form hydrogen bonds with trehalose, so that trehalose is not easy to crystallize during freeze-drying. During freeze-drying, trehalose is released from the pore channels of mesoporous silica, wraps the cells of Camellia sinensis var. assamica, forms an amorphous glass state, and makes the molecules of polyphenols and flavonoids not easy to diffuse and oxidize. The hydroxyl groups of trehalose can combine with the phospholipids of the cell membrane to replace the hydrogen bond network of water molecules, so that they are not easy to be dehydrated and damaged.

[0017] Spermidine is a natural polyamine that can help scavenge free radicals and reduce the oxidative degradation of polyphenols. By linking spermidine to polyethylene glycol, it can be evenly distributed in the intercellular space of L. japonica and improve the stability and safety of the cryoprotectant in the system. Spermidine can also inhibit the release of inflammatory factors, giving L. japonica certain anti-inflammatory activity and forming a synergistic system with L. japonica's anti-inflammatory effect. The three components form a synergistic system of carrier-potentiation-protection, significantly reducing the loss of active ingredients due to ice crystal damage and oxidative degradation during freeze-drying, and improving the retention rate and stability of anti-inflammatory ingredients in the fermentation broth.

[0018] Preferably, the cryoprotectant comprises the following raw materials: 8-12 ml of modified polyethylene glycol modified mesoporous silica and 9-11 ml of trehalose solution.

[0019] Preferably, the preparation method of the cryoprotectant is: mix 8-12 ml of modified polyethylene glycol modified mesoporous silica and 9-11 ml of trehalose solution, ultrasonic dispersion, oscillation for 22-26 h, centrifugation, and freeze-drying.

[0020] The pore channels of the mesoporous silica are fully loaded with trehalose, and the excess trehalose does not easily block the pore channels, forming a homogeneous protection system. The rigid skeleton of the modified polyethylene glycol modified mesoporous silica helps resist mechanical stress during freeze-drying, preventing cell collapse. The flexible glass state of trehalose can encapsulate active ingredients, preventing ice crystal growth and oxidative damage.

[0021] Preferably, the modified polyethylene glycol modified mesoporous silica comprises the following raw materials: 45-55 mL of mesoporous silica, 0.8-1.2 g of spermidine, 2-2.4 g of di-tert-butyl dicarbonate, 18-22 mL of acetonitrile solution, 1.5-2.5 g of carboxylated polyethylene glycol, 25-35 mL of N,N-dimethylformamide, 2-2.8 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-1.8 g of N-hydroxysuccinimide, 0.3-0.7 mL of triethylamine, 9-11 mL of trifluoroacetic acid, and 9-11 mL of dichloromethane.

[0022] Preferably, the preparation method of the modified polyethylene glycol modified mesoporous silica is as follows:

[0023] S1: weigh 0.8-1.2 g spermidine and 2-2.4 g di-tert-butyl dicarbonate into 18-22 mL acetonitrile solution, stir for 10-14 h to obtain a mixed solution, weigh 1.5-2.5 g carboxylated polyethylene glycol into 25-35 mL N,N-dimethylformamide, add 2-2.8 g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-1.8 g N-hydroxysuccinimide, stir for 1-3 h to obtain an activated polyethylene glycol solution, add the mixed solution into the activated polyethylene glycol solution, then add 0.3-0.7 mL triethylamine, stir for 22-26 h, then add 9-11 mL trifluoroacetic acid and 9-11 mL dichloromethane, stir for 3-5 h to obtain modified polyethylene glycol;

[0024] S2: stir 45-55 mL mesoporous silica with 45-55 mL modified polyethylene glycol at 25-35℃ for 28-32 min, then stir at 110-130℃ for 85-95 min, centrifuge, and wash with ionized water for 2-4 times to obtain mesoporous silica modified with modified polyethylene glycol.

[0025] Spermidine is efficiently coupled with carboxylated polyethylene glycol after being protected by di-tert-butyl dicarbonate, which significantly reduces oxidative damage during the freeze-drying process. Mesoporous silica is covalently bonded with modified polyethylene glycol to form a nanocarrier with good biocompatibility and high loading capacity, which can efficiently load trehalose.

[0026] Preferably, in the step S3, the ultrafine grinding is performed by using a low-temperature impact mill, and the specific conditions are as follows: the temperature of the grinding cavity is 16-17℃, the frequency of the main machine is 25-35 Hz, the frequency of the air blower is 25-35 Hz, and the frequency of the air classifier is 10-30 Hz.

[0027] Due to the low-temperature environment combined with liquid nitrogen refrigeration, thermal damage is eliminated, which improves the retention rate of heat-sensitive components such as polyphenols and flavonoids. The frequency settings of the main machine and the air blower can provide appropriate impact force and air flow intensity, achieving efficient grinding and preventing material aggregation. The frequency of the air classifier can control the uniformity of the particle size distribution of the powder, which improves the specific surface area of the Camellia japonica powder, and is beneficial to the decomposition of components and the dissolution of active components by lactic acid bacteria in the subsequent fermentation process, thereby further enhancing the anti-inflammatory effect of the fermentation liquor.

[0028] In a second aspect, the present application provides an anti-inflammatory Camellia japonica fermentation liquor, which adopts the following technical solution:

[0029] An anti-inflammatory Camellia japonica fermentation liquor is prepared by the above preparation method.

[0030] In a third aspect, the present application provides an anti-inflammatory cosmetic, which adopts the following technical solution:

[0031] An anti-inflammatory cosmetic containing the above-mentioned L. japonica fermentation broth as the main active ingredient or the only active ingredient.

[0032] In summary, the present application has the following beneficial effects:

[0033] 1. The vacuum freeze-drying technology is beneficial to solve the degradation problem of components in the drying process. The cell structure of L. japonica after drying is complete, providing high-activity L. japonica dried products for subsequent processing. Ultrafine grinding opens the cell wall barrier at low temperature without damaging the structure of active ingredients, so that lactic acid bacteria in the fermentation stage can more efficiently utilize the nutrients in the L. japonica, convert the natural active ingredients into a form that is more easily absorbed by the skin, and improve the anti-inflammatory activity of the L. japonica fermentation broth and its application effect in cosmetics.

[0034] 2. The pre-freezing temperature is set between -25 and -35℃, which is beneficial to form a large number of small and uniform ice crystals, reduce the mechanical damage of local large ice crystals to cell walls and cell membranes, and thus retain the anti-inflammatory active ingredients in the cells. The low temperature of -25 to -35℃ is beneficial to inactivate polyphenol oxidase, so that it is not easy to oxidize and decompose tea polyphenols, and reduces the loss of active ingredients caused by enzymatic hydrolysis. The pre-freezing time is set to 3-4h, which is beneficial to the conversion of free water and bound water in L. japonica tissue into ice crystals. The maximum heating temperature of the baffle is set to 37-41℃, which provides sufficient heat energy to drive the sublimation of ice crystals. Gentle heating is beneficial to the active ingredients not being easily damaged by high temperature, and maximizes the retention of their anti-inflammatory activity.

[0035] 3. The porous structure of mesoporous silica is beneficial to efficiently load trehalose. The silicon hydroxyl groups on the surface of mesoporous silica form hydrogen bonds with trehalose, so that trehalose is not easy to crystallize during freeze-drying. During freeze-drying, trehalose is released from the mesoporous silica pores, wraps the L. japonica cells, forms an amorphous glass state, and makes it difficult for polyphenols and flavonoids to diffuse and oxidize. The hydroxyl groups of trehalose can combine with the phospholipids of the cell membrane, replacing the hydrogen bond network of water molecules, so that they are not easy to be dehydrated and damaged.

[0036] Spermidine is a natural polyamine that is beneficial to scavenge free radicals and reduce the oxidative degradation of polyphenols. By connecting spermidine with polyethylene glycol, it is beneficial to uniformly distribute in the intercellular space of L. japonica, improve the stability and safety of the cryoprotective agent in the system, and inhibit the release of inflammatory factors, so that it can endow L. japonica with certain anti-inflammatory activity, thereby forming a synergistic system of carrier-potentiation-protection with L. japonica, significantly reducing the loss of active ingredients caused by ice crystal damage and oxidative degradation during freeze-drying of L. japonica, and improving the retention rate and stability of anti-inflammatory components in the fermentation broth. BRIEF DESCRIPTION OF DRAWINGS

[0037] Pre-freezing temperature / °CTypical figure of protein expression of Example 1, normal control group and model control group. DETAILED DESCRIPTION

[0038] The present application is further described in detail below in conjunction with Examples 1-12 and Comparative Examples 1-4.

[0039] Raw materials

[0040] The lactic acid bacteria used in the present application is Lactobacillus casei SM003, which is preserved in the China General Microbiological Culture Collection Center on February 10, 2023, and the preservation number is CGMCC NO. 26537.

[0041] Camellia sinensis var. assamica Guangxi Fangchenggang Changlong Camellia sinensis Plantation; deionized water CAS: 7732-18-5; glucose Jinan Dehao Chemical Co., Ltd.; proteose peptone CAS: 73049-73-7; yeast powder Hefei Tannun Biological Technology Co., Ltd.; gallic acid Shandong Hongjiu Biological Technology Co., Ltd.; dipotassium hydrogen phosphate CAS: 7758-11-4; magnesium sulfate Hubei Kewode Chemical Co., Ltd.; manganese sulfate Hebei Kolongduo Biological Technology Co., Ltd.; trehalose CAS: 99-20-7; spermidine CAS: 124-20-9; di-tert-butyl dicarbonate CAS: 24424-99-5; acetonitrile CAS: 75-05-8; carboxylated polyethylene glycol Guangzhou Carbon Hydrotech Co., Ltd.; N,N-dimethylformamide CAS: 68-12-2; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride CAS: 25952-53-8; N-hydroxysuccinimide Shanghai Covalent Chemical Technology Co., Ltd.; triethylamine CAS: 121-44-8; trifluoroacetic acid CAS: 76-05-1; dichloromethane CAS: 75-09-2.

[0042] Example 1

[0043] The present embodiment provides an anti-inflammatory Camellia sinensis fermentation liquor, and the preparation method thereof comprises the following steps:

[0044] S1: 1 g of spermidine and 2.2 g of di-tert-butyl dicarbonate were weighed and dissolved in 20 mL of acetonitrile solution, and stirred for 12 h to obtain a mixed solution. 2 g of carboxylated polyethylene glycol was weighed and dissolved in 30 mL of N,N-dimethylformamide, and 2.4 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1.4 g of N-hydroxysuccinimide were added, and stirred for 2 h to obtain an activated polyethylene glycol solution. The mixed solution was added to the activated polyethylene glycol solution, followed by the addition of 0.5 mL of triethylamine, and stirred for 24 h. Then 10 mL of trifluoroacetic acid and 10 mL of dichloromethane were added, and stirred for 4 h to obtain a modified polyethylene glycol;

[0045] S2: 50 mL mesoporous silica was stirred with 50 mL modified polyethylene glycol at 30°C for 30 min, then stirred at 120°C for 90 min, centrifuged, and washed with ionized water three times to obtain mesoporous silica modified with modified polyethylene glycol;

[0046] S3: 10 ml of mesoporous silica modified with modified polyethylene glycol and 10 ml of trehalose solution were mixed, ultrasonically dispersed, oscillated for 24 h, centrifuged, and freeze-dried to obtain a cryoprotectant;

[0047] S4: Lactobacillus casei SM003 was inoculated into a culture medium and incubated at 37°C for 24 h to obtain a seed solution;

[0048] S5: Camellia nitidissima was carefully selected, the cryoprotectant was added, and vacuum freeze-drying technology was used for drying treatment to obtain Camellia nitidissima dry products;

[0049] S6: The Camellia nitidissima dry products were subjected to low-temperature impact grinding and ultra-fine grinding and sieving to obtain Camellia nitidissima powder;

[0050] S7: 1% Camellia nitidissima powder, 3% glucose, 2% protein peptone, 1.25% yeast powder, 1.5% gallic acid, 0.3% potassium phosphate dibasic, 0.3% magnesium sulfate, 0.2% manganese sulfate, and the rest deionized water were added to a fermentation tank, sterilized at 121°C for 20 min, and then cooled to 24°C;

[0051] S8: The seed solution was inoculated into the fermentation tank at an inoculation amount of 6.66%, and incubated at a constant temperature for 26 h to obtain a fermentation broth;

[0052] S9: The fermentation broth was filtered, sterilized, and preservative was added to obtain a Camellia nitidissima fermentation broth.

[0053] The vacuum freeze-drying conditions were: pre-freezing temperature of -30°C, pre-freezing time of 3.5 h, and maximum heating temperature of the baffle of 39°C;

[0054] The low-temperature impact grinding and ultra-fine grinding conditions were: grinding cavity temperature of 16.5°C, main machine frequency of 30 Hz, air blower frequency of 30 Hz, and air classifier frequency of 20 Hz.

[0055] Examples 2-3

[0056] The difference between Example 1 and Examples 2-3 is that the vacuum freeze-drying conditions are different, as shown in Table 1.

[0057] Table 1: Vacuum freeze-drying conditions in Examples 1-3

[0058] Pre-freezing time / h Maximum heating temperature of the partition / °C Example 1 Example 4 -30 -35 -25 Example 5 3.5 3 4 Temperature of the pulverizing chamber / °C 39 37 41

[0059] Example 4 - Example 5

[0060] The difference from Example 1 is that the conditions for the supermicronization by the cryomill are different, as shown in Table 2.

[0061] Table 2 Table of the conditions for the supermicronization by the cryomill in Example 1 and Examples 4 - 5

[0062] Frequency of the main machine / Hz Frequency of the air blower / Hz Frequency of the air classifier / Hz Example 1 16.5 16 17 Example 10 30 35 25 Example 11 30 25 35 Mesoporous silica 20 30 10

[0063] Example 6

[0064] The difference from Example 1 is that the cryoprotective agent is mesoporous silica modified with polyethylene glycol loaded with trehalose.

[0065] Example 7

[0066] The difference from Example 1 is that the cryoprotective agent is trehalose.

[0067] Example 8 - Example 9

[0068] The difference from Example 1 is that the amounts of the components of the cryoprotective agent are different, as shown in Table 3.

[0069] Table 3 Table of the amounts of the components of the cryoprotective agent in Example 1 and Examples 8 - 9 (ml)

[0070]

[0071]

[0072] Example 10 - Example 11

[0073] The difference from Example 1 is that the amounts of the components of the mesoporous silica modified with modified polyethylene glycol are different, as shown in Table 4.

[0074] Table 4 Table of the amounts of the components of the mesoporous silica modified with modified polyethylene glycol in Example 1 and Examples 10 - 11 (ml)

[0075] Modified polyethylene glycol Figure 1 NF-κB / β-actin ratio Effect (%) 50 45 55 p value 50 55 45

[0076] Example 11 - Example 12

[0077] The difference from Example 1 is that the amounts of the components of the modified polyethylene glycol are different, as shown in Table 5.

[0078] Table 5 Table of the amounts of the components of the modified polyethylene glycol in Example 1 and Examples 11 - 12

[0079]

[0080]

[0081] Comparative Example 1

[0082] The difference from Example 1 is that the conditions of vacuum freeze-drying are as follows: pre-freezing temperature is -10℃, pre-freezing time is 2h, and the maximum heating temperature of the shelf is 50℃.

[0083] Comparative Example 2

[0084] The difference from Example 1 is that the hot air drying technology is used to dry the Jintea flowers, and the preheating temperature is 70℃.

[0085] Comparative Example 3

[0086] The difference from Example 1 is that the conditions of ultrafine grinding by the low-temperature impact mill are as follows: the grinding cavity temperature is 25℃, the main machine frequency is 10Hz, the air blower frequency is 40Hz, and the air classifier frequency is 5Hz.

[0087] Comparative Example 4

[0088] The difference from Example 1 is that no cryoprotectant is added.

[0089] Performance detection test

[0090] I. Anti-inflammatory performance

[0091] Three samples are taken from each of Examples 1-12 and Comparative Examples 1-4, and mouse monocyte macrophages (RAW264.7) are inoculated into 6-well plates (6x10 5 cells / well), incubated at 37℃, 5% CO2 for 24h, and then the normal control group is replaced with fresh culture medium, the model control group is replaced with fresh culture medium containing LPS, the sample group is added with fresh culture medium containing the sample, and the positive control group is added with fresh culture medium containing LPS and dexamethasone, and then incubated at 37℃, 5% CO2 for 24h. Total RNA is extracted from each experimental group, cDNA is synthesized, and the gene expression of β-actin and the target gene is detected by q-PCR. The cells are lysed by RIPA lysis buffer, and the protein is extracted. The expression amount of NF-κB protein is detected by Western Blot method.

[0092] The test data are shown in Tables 6-7 and Test result .

[0093] Table 6 Anti-inflammatory performance test table of Example 1 and normal and model control groups

[0094] Example 1 Significant Normal control group Significant Model control group 0.487 61 ＜0.001 NF-κB / β-actin ratio Effect (%) 0.772 / ＜0.05 p value Test result 1.25 / - -

[0095] Table 7 Anti-inflammatory performance test table of Example 2-Example 12 and Comparative Example 1-Comparative Example 4

[0096] Example 2 Significant Example 3 Significant Example 4 0.492 60 ＜0.001 Significant Example 5 0.505 59 ＜0.001 Significant Example 6 0.510 58 ＜0.001 Significant Example 7 0.495 60 ＜0.001 Significant Example 8 0.520 56 ＜0.001 Significant Example 9 0.550 53 ＜0.001 Significant Example 10 0.500 59 ＜0.001 Significant Example 11 0.501 59 ＜0.001 Significant Example 12 0.505 58 ＜0.001 Significant Comparative Example 1 0.497 60 ＜0.001 General Comparative Example 2 0.493 60 ＜0.001 General Comparative Example 3 0.700 42 ＜0.05 General Comparative Example 4 0.820 32 ＜0.05 General Figure 1 0.750 38 ＜0.05 ​ ​ 0.850 30 ＜0.05 ​

[0097] In combination with Example 1 and the normal, model control group, and in combination with Table 6 and ​ It can be seen that the NF-κB / β-actin ratio and the protein expression amount in Example 1 are obviously reduced, which reveals that Example 1 has an anti-inflammatory effect.

[0098] In combination with Example 1 and Comparative Example 1 and in combination with Table 6-Table 7, it can be seen that, compared with Example 1, the NF-κB / β-actin ratio in Comparative Example 1 is obviously increased, the action percentage value of Comparative Example 1 is obviously reduced, the p value of Comparative Example 1 is <0.05, and the detection result is general, which shows that the anti-inflammatory performance of the Camellia ptilota fermentation liquor is affected by the vacuum freeze-drying condition, and when the vacuum freeze-drying condition is far beyond the set range, the anti-inflammatory performance of the Camellia ptilota is obviously reduced.

[0099] The reason is that setting the pre-freezing temperature between-25 and-35℃ is beneficial to form a large number of small and uniform ice crystals, reduce the mechanical damage of local large ice crystals to the cell wall and cell membrane, and thus retain the anti-inflammatory active ingredients in the cells. The low temperature of-25 to-35℃ is beneficial to passivate polyphenol oxidase, so that it is not easy to oxidize and decompose tea polyphenols, and reduce the loss of active ingredients caused by enzymolysis. Setting the pre-freezing time to 3-4h is beneficial to the conversion of free water and bound water in the Camellia ptilota tissue into ice crystals. Setting the maximum heating temperature of the partition plate to 37-41℃ provides sufficient heat energy to drive the sublimation of ice crystals. Mild heating is beneficial to the active ingredients not being easy to cause structural damage due to high temperature, and maximizes the retention of their anti-inflammatory activity.

[0100] In combination with Example 1 and Comparative Example 2 and in combination with Table 6-Table 7, it can be seen that, compared with Example 1, the NF-κB / β-actin ratio in Comparative Example 2 is obviously increased, the action percentage value of Comparative Example 2 is obviously reduced, the p value of Comparative Example 2 is <0.05, and the detection result is general, which shows that, compared with using the hot air drying technology, using the vacuum freeze-drying technology can effectively improve the anti-inflammatory performance of the Camellia ptilota fermentation liquor.

[0101] The reason is that the vacuum freeze-drying technology can solve the problem of degradation of components in the drying process, and the dried camellia sinensis cell structure is complete, providing high-activity camellia sinensis dry products for subsequent processing. The ultrafine grinding opens the cell wall barrier at low temperature without damaging the structure of active ingredients, so that the lactic acid bacteria in the fermentation stage can more efficiently utilize the nutrients in the cell wall, convert the natural active ingredients into a form that is more easily absorbed by the skin, and improve the anti-inflammatory activity of the camellia sinensis fermentation liquor and the application effect in cosmetics.

[0102] As can be seen from the combination of Example 1 and Comparative Example 3 and Tables 6-7, the NF-κB / β-actin ratio in Comparative Example 3 is significantly increased compared to Example 1, the action percentage of Comparative Example 3 is significantly reduced, and the p value of Comparative Example 3 is <0.05, and the test result is general, which shows that the conditions of ultrafine grinding by low-temperature impact mill affect the anti-inflammatory performance of camellia sinensis fermentation liquor, and when the conditions of ultrafine grinding by low-temperature impact mill are far beyond the set range, the anti-inflammatory performance of camellia sinensis is significantly reduced.

[0103] The reason is that the low-temperature environment combined with liquid nitrogen refrigeration can eliminate thermal damage and improve the retention rate of heat-sensitive components such as polyphenols and flavonoids. The frequency setting of the main machine and the induced draft fan can provide appropriate impact force and air flow intensity to achieve efficient grinding and prevent material aggregation. The frequency of the air classifier can control the uniform distribution of the particle size of the powder, improve the specific surface area of the camellia sinensis powder, and facilitate the decomposition and dissolution of active ingredients by lactic acid bacteria in the subsequent fermentation process, thereby further enhancing the anti-inflammatory effect of the fermentation liquor.

[0104] As can be seen from the combination of Example 1 and Comparative Example 4 and Tables 6-7, the NF-κB / β-actin ratio in Comparative Example 4 is significantly increased compared to Example 1, the action percentage of Comparative Example 4 is significantly reduced, and the p value of Comparative Example 4 is <0.05, and the test result is general, which shows that compared to not adding a cryoprotective agent, adding a cryoprotective agent can effectively improve the anti-inflammatory performance of camellia sinensis fermentation liquor.

[0105] The reason is that the porous structure of mesoporous silica is beneficial to efficient loading of trehalose, and the silicon hydroxyl groups on the surface of mesoporous silica form hydrogen bonds with trehalose, making it difficult for trehalose to crystallize during freeze-drying. During freeze-drying, trehalose is released from the mesoporous silica pores, wraps the camellia sinensis cells, forms an amorphous glass state, and makes it difficult for polyphenols and flavonoids to diffuse and oxidize. The hydroxyl groups of trehalose can combine with the phospholipids of the cell membrane, replacing the hydrogen bond network of water molecules, making it difficult for them to be dehydrated and damaged.

[0106] Spermidine is a natural polyamine that can help scavenge free radicals and reduce the oxidative degradation of polyphenols. By linking spermidine to polyethylene glycol, it can be evenly distributed in the intercellular space of L. japonica, improving the stability and safety of the cryoprotectant in the system. Spermidine can also inhibit the release of inflammatory factors, allowing it to impart certain anti-inflammatory activity to L. japonica, thereby forming a synergistic system of carrier-potentiation-protection that significantly reduces the loss of active ingredients due to ice crystal damage and oxidative degradation during freeze-drying, and helps to improve the retention rate and stability of anti-inflammatory ingredients in the fermentation broth.

[0107] As can be seen from the combination of Example 1 and Examples 2-3 and Tables 6-7, compared with Example 1, the NF-κB / β-actin ratio in Examples 2 and 3 increased, the percentage of action in Examples 2 and 3 decreased, and the p value and test results in Examples 2 and 3 were both <0.001 and significant, indicating that the conditions of vacuum freeze-drying affect the anti-inflammatory performance of L. japonica fermentation broth, and thus the conditions of vacuum freeze-drying in Example 1 are optimal.

[0108] As can be seen from the combination of Example 1 and Examples 4-5 and Tables 6-7, compared with Example 1, the NF-κB / β-actin ratio in Examples 4 and 5 increased, the percentage of action in Examples 4 and 5 decreased, and the p value and test results in Examples 4 and 5 were both <0.001 and significant, indicating that the conditions of low-temperature impact grinding for ultra-fine grinding affect the anti-inflammatory performance of L. japonica fermentation broth, and thus the conditions of low-temperature impact grinding for ultra-fine grinding in Example 1 are optimal.

[0109] As can be seen from the combination of Example 1 and Example 6 and Tables 6-7, compared with Example 1, the NF-κB / β-actin ratio in Example 6 increased, the percentage of action in Example 6 decreased, and the p value and test results in Example 6 were both <0.001 and significant, indicating that compared with adding polyethylene glycol-modified mesoporous silica loaded with trehalose, adding modified polyethylene glycol-modified mesoporous silica loaded with trehalose as a cryoprotectant to prepare L. japonica fermentation broth has better anti-inflammatory performance.

[0110] It can be seen from the combination of Example 1 and Example 7 and in combination with Tables 6-7 that, relative to Example 1, the NF-κB / β-actin ratio in Example 7 is increased, the effect percentage of Example 7 is reduced, and the p value and detection result of Example 7 are both <0.001 and significant, thus indicating that, relative to the addition of trehalose as a cryoprotectant alone, the Camellia reticulata fermented liquid prepared by adding the modified mesoporous silica loaded with trehalose as a cryoprotectant has better anti-inflammatory performance.

[0111] It can be seen from the combination of Example 1 and Example 8-Example 9 and in combination with Tables 6-7 that, relative to Example 1, the NF-κB / β-actin ratio in Example 8 and Example 9 is increased, the effect percentage of Example 8 and Example 9 is reduced, and the p value and detection result of Example 8 and Example 9 are both <0.001 and significant, thus indicating that the addition amount of the components of the cryoprotectant affects the anti-inflammatory performance of the Camellia reticulata fermented liquid, and the addition amount of the components of the cryoprotectant in Example 1 is optimal.

[0112] It can be seen from the combination of Example 1 and Example 10-Example 11 and in combination with Tables 6-7 that, relative to Example 1, the NF-κB / β-actin ratio in Example 10 and Example 11 is increased, the effect percentage of Example 10 and Example 11 is reduced, and the p value and detection result of Example 10 and Example 11 are both <0.001 and significant, thus indicating that the addition amount of the components of the modified mesoporous silica affects the anti-inflammatory performance of the Camellia reticulata fermented liquid, and the addition amount of the components of the modified mesoporous silica in Example 1 is optimal.

[0113] It can be seen from the combination of Example 1 and Example 11-Example 12 and in combination with Tables 6-7 that, relative to Example 1, the NF-κB / β-actin ratio in Example 11 and Example 12 is increased, the effect percentage of Example 11 and Example 12 is reduced, and the p value and detection result of Example 11 and Example 12 are both <0.001 and significant, thus indicating that the addition amount of the components of the modified polyethylene glycol affects the anti-inflammatory performance of the Camellia reticulata fermented liquid, and the addition amount of the components of the modified polyethylene glycol in Example 1 is optimal.

[0114] The specific embodiments are merely illustrative of the present application, and are not intended to limit the present application, and those skilled in the art can make modifications to the embodiments without creative contribution after reading the present specification, but as long as the modifications are within the scope of the claims of the present application, they are protected by the Patent Law.

Claims

1. A method for preparing an anti-inflammatory Camellia reticulata Lindl. fermentation broth, characterized in that, The method comprises the following steps: S1: inoculate lactic acid bacteria into culture medium, and perform constant-temperature vibration culture to obtain seed liquid; S2: carefully select Camellia nitidissima, add a freezing protective agent, and perform drying treatment by using a vacuum freeze-drying technology to obtain Camellia nitidissima dried products; S3: perform low-temperature impact ultrafine grinding and sieving on the Camellia nitidissima dried products to obtain Camellia nitidissima powder; S4: add 0.8-1.2% Camellia nitidissima powder and the rest deionized water into a fermentation tank, and perform sterilization; S5: inoculate the seed liquid into the fermentation tank, and perform constant-temperature culture to obtain fermentation liquid; S6: perform impurity removal and sterilization on the fermentation liquid, and add a preservative to obtain Camellia nitidissima fermentation liquid.

2. The preparation method of the anti-inflammatory Camellia fermentation liquor according to claim 1, characterized in that, The vacuum freeze-drying condition used in the step S2 is that the pre-freezing temperature is-25 to-35℃, the pre-freezing time is 3-4h, and the maximum heating temperature of the baffle is 37-41℃.

3. The preparation method of the anti-inflammatory camellia fermentation liquor according to claim 1, characterized in that: The freezing protective agent is mesoporous silica loaded with trehalose modified by modified polyethylene glycol, and the modified polyethylene glycol is spermidine grafted polyethylene glycol.

4. The method for preparing an anti-inflammatory Camellia chrysanthemi fermented liquid according to claim 3, characterized in that, The freezing protective agent comprises the following raw materials: 8-12ml modified polyethylene glycol modified mesoporous silica and 9-11ml trehalose solution.

5. The method for preparing an anti-inflammatory Camellia chrysanthemi fermented liquid according to claim 4, characterized in that, The preparation method of the freezing protective agent is that 8-12ml modified polyethylene glycol modified mesoporous silica and 9-11ml trehalose solution are mixed, ultrasonic dispersion is performed, vibration is performed for 22-26h, centrifugation is performed, and freeze-drying is performed.

6. The method for preparing an anti-inflammatory Camellia chrysanthemi fermented liquid according to claim 5, characterized in that, The modified polyethylene glycol modified mesoporous silica comprises the following raw materials: 45-55mL mesoporous silica, 0.8-1.2g spermidine, 2-2.4g di-tert-butyl dicarbonate, 18-22mL acetonitrile solution, 1.5-2.5g carboxylated polyethylene glycol, 25-35mL N,N-dimethylformamide, 2-2.8g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1-1.8g N-hydroxysuccinimide, 0.3-0.7mL triethylamine, 9-11mL trifluoroacetic acid, and 9-11mL dichloromethane.

7. The method for preparing an anti-inflammatory Camellia chrysanthemi fermented liquid according to claim 6, characterized in that, The preparation method of the modified polyethylene glycol modified mesoporous silica is as follows: S1: 0.8-1.2g spermidine and 2-2.4g di-tert-butyl dicarbonate are dissolved in 18-22mL acetonitrile solution, stirring reaction is performed for 10-14h to obtain a mixed solution, 1.5-2.5g carboxylated polyethylene glycol is dissolved in 25-35mL N,N-dimethylformamide, 2-2.8g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-1.8g N-hydroxysuccinimide are added, stirring reaction is performed for 1-3h to obtain an activated polyethylene glycol solution, the mixed solution is added to the activated polyethylene glycol solution, then 0.3-0.7mL triethylamine is added, stirring reaction is performed for 22-26h, and then 9-11mL trifluoroacetic acid and 9-11mL dichloromethane are added, stirring reaction is performed for 3-5h to obtain modified polyethylene glycol. S2: 45-55 mL mesoporous silica is stirred with 45-55 mL modified polyethylene glycol at 25-35 °C for 28-32 min, then stirred at 110-130 °C for 85-95 min, centrifuged, and washed with ionized water 2-4 times to obtain mesoporous silica modified with modified polyethylene glycol.

8. The method for preparing an anti-inflammatory Camellia chrysanthemi fermented liquid according to claim 1, characterized in that, In the step S3, the low-temperature impact mill is used for ultrafine grinding, and the specific conditions are as follows: the temperature of the grinding cavity is 16-17 °C, the frequency of the main machine is 25-35 Hz, the frequency of the air blower is 25-35 Hz, and the frequency of the air classifier is 10-30 Hz.

9. An anti-inflammatory camellia fermentation liquor prepared by the preparation method of any one of claims 1-8.

10. An anti-inflammatory cosmetic containing the camellia fermentation liquor of claim 9 as the main active ingredient or the only active ingredient.

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