Preparation method of lactobacillus fermentation filtrate, product and application

Abstract

The invention relates to the technical field of biological fermentation, in particular to a preparation method of lactobacillus fermentation filtrate, a product and application. The preparation method of the lactobacillus fermentation filtrate specifically comprises the following steps: S1, inoculating lactobacillus into a culture medium, and carrying out continuous fermentation culture to obtain a fermentation liquid product I; s2, filtering the fermentation liquid product I, and separating lactobacillus to obtain a fermentation liquid product II; s3, carrying out antibacterial treatment on the fermentation liquid product II to obtain lactobacillus fermentation filtrate; the preparation raw materials of the culture medium at least comprise milk; the lactobacillus is lactobacillus paracasei. The lactobacillus paracasei is specifically adopted to ferment the milk, the fermented filtrate with a good skin care effect can be obtained, and the obtained fermented filtrate contains various amino acids and polysaccharides, can inject vitality to the skin, and has the remarkable effects of tightening, wrinkle resisting, moisturizing, relieving and the like.

Description

Technical Field

[0001] The invention relates to the technical field of biological fermentation, and in particular to a preparation method, product and application of lactobacillus fermentation filtrate. Background Art

[0002] Microbial fermentation originated in the food industry. In recent years, technical research has found that fermentation technology can produce products with skin care effects, making the combination of fermentation technology and cosmetics a hot topic in the cosmetics industry. With the promotion of the concept of "microecological skin care", the use of microbial fermentation to prepare cosmetic raw materials with skin care effects provides a new idea for the development of cosmetic raw materials.

[0003] Due to the special nature of cosmetics acting directly on the skin, existing microbial fermentation products are still difficult to achieve more effective, safer and more comprehensive effects. The current fermentation technology has gone through the traditional fermentation stage, engineering development stage, microbiology application stage, synthetic biology stage, and intelligent fermentation stage, and many fermentation products have been put into cosmetic formulas. However, there are still some problems with existing fermentation raw materials, such as (1) the fermentation process is complicated, the operation is cumbersome or time-consuming, the production efficiency is low, and it cannot meet the requirements of mass production; (2) the fermentation products are not stable enough, and adding them to skin care products may affect the appearance or the stability of the product, and the formula compatibility is poor; (3) the fermentation products obtained by the existing fermentation methods have low biological activity, are effective in conditioning and maintaining the skin, and it is difficult to achieve multiple functions at the same time.

[0004] Chinese patent CN112402455B discloses an application of yak milk fermented extract, which can reduce the ratio of Staphylococcus aureus / Staphylococcus epidermidis and promote skin proliferation by fermenting yak milk with Lactobacillus plantarum and centrifuging the fermented liquid. CN117431275B discloses a preparation method and application of lactobacillus fermented liquid, which promotes the synthesis of beneficial metabolites by combining Lactobacillus plantarum, Streptococcus thermophilus, Bifidobacterium adolescentis and Bifidobacterium longum as composite lactic acid bacteria liquid, and the obtained fermented product has anti-oxidation and whitening effects. However, none of these prior arts solves the above technical problems at the same time.

[0005] Under such circumstances, obtaining a fermentation filtrate with significant efficacy, comprehensive effects and good formula compatibility through a simple and easy fermentation method is a technical problem to be solved urgently in this field. Summary of the invention

[0006] The first aspect of the present invention provides a method for preparing a lactobacillus fermentation filtrate, which specifically comprises the following steps: S1. inoculating lactic acid bacteria into a culture medium, and continuously fermenting and culturing to obtain a fermentation broth product 1; S2. Filtering the fermentation broth product 1 to separate the lactic acid bacteria to obtain the fermentation broth product 2; S3. Perform antibacterial treatment on the fermentation liquid product 2 to obtain lactobacillus fermentation filtrate.

[0007] Preferably, the raw materials for preparing the culture medium include at least milk.

[0008] The present invention specifically selects lactic acid bacteria to ferment milk; lactic acid bacteria are bacteria that can ferment carbohydrates and convert them into lactic acid, and can be divided into the genera Lactobacillus, Streptococcus, Leuconostoc, Bifidobacterium and Pediococcus.

[0009] Preferably, the present invention selects Lactobacillus. Examples of Lactobacillus include Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus helveticus, Lactobacillus casei, and the like.

[0010] More preferably, the lactobacillus is Lactobacillus paracasei (Latin name: Lactobacillus paracasei).

[0011] Lactobacillus paracasei has a wide range of sources and can be extracted from plant surfaces, dairy products, meat products, wine, fermented dough and even sewage. In some preferred embodiments, the Lactobacillus paracasei is purified and separated from cheese, and is derived from Guangzhou Junyan Biotechnology Co., Ltd., with a biological deposit number of GDMCC NO: 64856, a biological deposit date of July 10, 2024, a biological deposit address of Guangdong Provincial Microbiological Culture Collection Center, and a depository unit of Guangdong Provincial Microbiological Culture Collection Center; the name of the biological material provided and the indicated identification characteristics are: Lacticaseibacillus paracasei DE001; The taxonomic name proposed in the deposit certificate is: Lacticaseibacillus paracasei .

[0012] The present invention specifically performs single purification and extraction on cheese, and the obtained Lactobacillus paracasei has significant fermentation activity and extremely strong survival ability, can stably survive at 25-38 DEG C, and maintain a high reproduction rate in a wide temperature range; the fermentation effect applied to milk is good, and only a short fermentation and cultivation time is required to obtain a fermentation filtrate with rich active ingredients and high skin care activity.

[0013] The present invention unexpectedly found that the Lactobacillus paracasei isolated from commercially available cheese grows well after being isolated on a plate, and the fermented liquid has a light lactic acid taste after fermentation, and is suitable as a fermentation raw material. The advantages of selecting Lactobacillus paracasei from a variety of bacterial species in the present invention are that it has a wide source and is safer and healthier to separate from food; and the lactic acid produced after fermentation can reduce the pH value of the skin surface and inhibit the growth of pathogens.

[0014] In some preferred embodiments, the raw materials for preparing the culture medium include, by mass percentage, 0.1-5% milk, 0.1-2% glucose, 0.01-1% yeast extract powder, and the balance made up of water.

[0015] The present invention preferably uses milk as the fermentation substrate, and the addition amount of 0.1-5% can be listed as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, and 5%.

[0016] The present invention prefers glucose as the carbon source / nitrogen source, and the addition amount of 0.1-2% can be listed as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, and 2%.

[0017] Further preferably, the raw materials for preparing the culture medium include, by mass percentage, 1% milk, 0.5% glucose, 0.1% yeast extract powder, and the balance made up of water.

[0018] In some preferred embodiments, the method for preparing the culture medium comprises: mixing the raw materials for preparing the culture medium according to the formula amount, adjusting the pH to 6.0±0.2, and sterilizing.

[0019] Preferably, the sterilization conditions are 105-130°C, 10-30 min; more preferably 121°C, 15 min.

[0020] In some preferred embodiments, the continuous fermentation culture conditions in step S1 are 20-40° C. for 15-30 hours.

[0021] More preferably, the continuous fermentation culture condition in step S1 is 25-38° C. for 16-24 hours.

[0022] More preferably, the continuous fermentation culture condition in step S1 is continuous fermentation at 37° C. for 20 h.

[0023] In some preferred embodiments, the antibacterial treatment in step S3 comprises: sterilizing the fermentation broth product 2 at 80-90° C. for 15-60 min; further preferably, adding a preservative after the sterilization treatment to obtain a lactobacillus fermentation filtrate.

[0024] More preferably, the sterilization condition is: sterilization at 85° C. for 30 min.

[0025] In some preferred embodiments, the preservative includes at least one of p-hydroxyacetophenone, 1,2-hexanediol, hydroxyphenyl ester, phenoxyethanol, and methylisothiazolinone.

[0026] Preferably, the preservative comprises p-hydroxyacetophenone and 1,2-hexanediol, and the mass ratio of p-hydroxyacetophenone to 1,2-hexanediol is (0.5-3):1; it can be listed as 0.5:1, 1:1, 1.5:1, 2:1, 3:1.

[0027] Further preferably, the mass ratio of p-hydroxyacetophenone to 1,2-hexanediol is 1:1.

[0028] The method for preparing lactobacillus fermentation filtrate of the present invention is simple, and only a single strain of bacteria is needed to ferment and culture milk, and multiple strains do not need to be compounded, and other substrates do not need to be introduced. The fermentation filtrate with high activity and good stability can be obtained in less than 1 day. The technology has strong operability, is convenient for mass production, and is highly practical; this is a level that the prior art has not yet achieved.

[0029] The second aspect of the present invention provides a lactobacillus fermentation filtrate prepared by the preparation method as described above.

[0030] On the one hand, the lactobacillus fermentation filtrate obtained by the present invention has significant firming and anti-wrinkle effects, and the inhibition rate of elastase activity reaches 15.20±1.52%. It can be seen that the filtrate of the present invention can effectively slow down the decomposition of elastin, effectively maintain skin elasticity, and has the effects of firming, anti-wrinkle, smoothing rough texture, and improving skin vitality; on the other hand, the lactobacillus fermentation filtrate obtained by the present invention has significant soothing and moisturizing effects, and the inhibition rate of hyaluronidase activity is as high as 41.07±4.48%. It can be seen that the filtrate of the present invention can effectively prevent hyaluronic acid from being specifically decomposed by hyaluronidase, and can inhibit skin inflammation and reduce the occurrence of allergic reactions, thereby achieving moisturizing and soothing effects and restoring healthy skin. On the third aspect, the lactobacillus fermentation filtrate obtained by the present invention has the advantages of transparency, stability, and strong formula compatibility, and is easy to add to various cosmetic products.

[0031] The third aspect of the present invention provides a use of the lactobacillus fermentation filtrate as described above in a skin care product, wherein the skin care product includes any one of a lotion, an essence, an emulsion, a cream, an ointment, and a facial mask.

[0032] In some preferred embodiments, the skin care product is a lotion; the amount of lactobacillus fermentation filtrate added to the lotion is 60wt% or more; more preferably, it is 75wt% or more, which has a significant soothing skin care effect.

[0033] The lactobacillus fermentation filtrate obtained by the present invention has excellent formula compatibility, can be used as a skin care raw material, and can be flexibly added to various skin care products. It is particularly suitable for lotions, and can give the products comprehensive effects such as firming, moisturizing, anti-wrinkle, and anti-aging. It acts deeply inside the skin, accelerates skin metabolism, and is easy to absorb. It has good application prospects in the field of cosmetics.

[0034] The cosmetic comprises 1-20% of skin care ingredients, 0-5% of auxiliary agents, and the balance is supplemented by lactobacillus fermentation filtrate.

[0035] The skin care ingredients can be listed as skin conditioners, emollients, moisturizers, etc. commonly used in the art; preferably, moisturizers.

[0036] As examples of the moisturizer, there are hyaluronic acid and its salt, C3-10 polyol, plant extract and the like.

[0037] Preferably, the hyaluronic acid and its salts are a combination of macromolecular hyaluronic acid and micromolecular hyaluronic acid.

[0038] Preferably, the C3-10 polyol includes at least one or more of glycerol, butylene glycol, D-panthenol, and 1,2-hexanediol.

[0039] Preferably, the plant extract includes at least one or more of Centella asiatica extract, orange flower extract, aloe vera extract, purslane extract, chamomile extract, and resurrection grass extract; more preferably, Centella asiatica extract and orange flower extract.

[0040] In some preferred embodiments, the mass ratio of Centella asiatica extract to orange flower extract is (10-30):1.

[0041] The auxiliary agent is not particularly limited, and can be selected from common auxiliary agents in the art as needed; for example, preservatives, thickeners, pH adjusters, flavors, etc.

[0042] Beneficial effects: The present invention provides a preparation method of lactobacillus fermentation filtrate and its product and application, which have the following advantages: (1) The present invention specifically uses Lactobacillus paracasei to ferment milk, thereby obtaining a fermentation filtrate with good skin care effects. The obtained fermentation filtrate contains a variety of amino acids and polysaccharides, which can inject vitality into the skin and improve skin problems; (2) The present invention specifically purifies and extracts cheese, and the obtained Lactobacillus paracasei has significant fermentation activity and extremely strong survival ability. It can survive stably at 25-38°C and maintain a high reproduction rate in a wide temperature range. It has a good fermentation effect when applied to milk, and only needs a short fermentation and cultivation time to obtain a fermentation filtrate with rich active ingredients and high skin care activity. (3) The lactobacillus fermentation filtrate obtained by the present invention has significant firming and anti-wrinkle effects, and the inhibition rate of elastase activity reaches 15.20±1.52%. It can be seen that the filtrate of the present invention can effectively slow down the decomposition of elastin, effectively maintain skin elasticity, and has the effects of firming, anti-wrinkle, smoothing rough texture, and enhancing skin vitality; (4) The lactobacillus fermentation filtrate obtained by the present invention has significant soothing and moisturizing effects, and the inhibition rate of hyaluronidase activity is as high as 41.07±4.48%. It can be seen that the filtrate of the present invention can effectively prevent hyaluronic acid from being specifically decomposed by hyaluronidase, and can inhibit skin inflammation and reduce the occurrence of allergic reactions, thereby achieving moisturizing and soothing effects and restoring healthy skin; (5) The lactobacillus fermentation filtrate obtained by the present invention is clear, transparent, stable and safe, and has excellent formula compatibility. It can be used as a skin care raw material and flexibly added to various skin care products, especially suitable for lotions, which can give the products comprehensive effects such as firming, moisturizing, anti-wrinkle and anti-aging. It acts deeply inside the skin, accelerates skin metabolism, and is easy to absorb. It has good application prospects in the field of cosmetics. (6) The method for preparing lactobacillus fermentation filtrate of the present invention is simple and convenient. It only requires a single strain of bacteria to ferment milk. It does not require the compounding of multiple strains of bacteria or the introduction of other substrates. It only takes less than 1 day of fermentation to obtain a fermentation filtrate with high activity and good stability. The technology is highly operable, convenient for mass production, and highly practical. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 . Appearance of the fermentation filtrate prepared in Comparative Example 1; Figure 2 . Appearance of the fermentation filtrate prepared in Example 1; Figure 3 . Test results of amino acids in lactobacillus fermentation filtrate in Example 1; Figure 4 . Test results of crude polysaccharides in lactobacillus fermentation filtrate in Example 1; Figure 5. VISIA test results of user 1 using the lotion of Example 1; Figure 6 . VISIA test results of user 2 using the lotion of Example 1; Figure 7 . VISIA test results of user 3 using the lotion of Example 1; Figure 8 .VISIA test results of user 4 using the lotion of Example 1. DETAILED DESCRIPTION

[0044] Example 1. This embodiment provides a method for preparing a lactobacillus fermentation filtrate, a product thereof, and an application thereof.

[0045] The preparation method of the lactobacillus fermentation filtrate specifically comprises the following steps: S1. inoculating lactic acid bacteria into the culture medium, and continuously fermenting and culturing at 37° C. for 20 h to obtain a fermentation broth product 1; S2. Filtering the fermentation broth product 1 to separate lactobacilli to obtain the fermentation broth product 2; S3. Perform antibacterial treatment on the fermentation liquid product 2 to obtain lactobacillus fermentation filtrate.

[0046] The lactobacillus in step S1 is specifically a lactobacillus seed liquid, and the preparation step of the lactobacillus seed liquid includes: mixing 2.5wt% MRS broth + water (T0 100%) to obtain a seed liquid culture medium, sterilizing it at 121°C for 15min; then inoculating 2wt% of the lactobacillus strain on the plate into the seed liquid culture medium, facultatively anaerobic culture, and culturing it at 37°C and 200rpm for 16h. The OD value of the lactobacillus seed liquid is 2.063.

[0047] The lactobacillus is Lactobacillus casei (Latin name: Lactobacillus casei), which is obtained by single purification and separation from cheese; the method for obtaining the lactobacillus is as follows: (1) Preparation of culture medium MRS liquid culture medium: 10 g peptone, 5 g beef extract, 4 g yeast powder, 20 g glucose, 1 mL Tween 80, 2 g K2HPO4·7H2O, 3.02 g anhydrous sodium acetate, 2 g triammonium citrate, 0.05 g MnSO4·4H2O, 0.2 g MgSO4·7H2O, 15 g agar, and 1 000 mL distilled water. Adjust the culture medium to pH 6.2 and sterilize at 121 ℃ for 15 min.

[0048] MRS solid medium: Add 2 g agar to 100 mL liquid medium and sterilize at 121°C for 15 min.

[0049] Calcium plate medium: Add 1 g of calcium carbonate to 100 mL of solid culture medium and sterilize at 121°C for 15 min.

[0050] (2) Initial screening of lactic acid bacteria 10 g of commercial cheese samples were placed in 100 mL of MRS liquid medium and enriched at 37 °C. The enriched samples were diluted with 0.9% saline in gradients, spread on appropriate gradient plates, and cultured at 37 °C for 48 h. Single colonies were picked and separated and purified by plate streaking to obtain single colonies with consistent morphology.

[0051] (3) Rescreening of lactic acid bacteria Calcium plate experiment: After the initially screened strains are activated, they are inoculated into calcium plate culture medium using the three-point method and cultured at 37°C for 24 h to observe whether a calcium dissolution zone is produced.

[0052] Catalase test: Pick one loop of pure culture and place it on a glass slide. Mix it with 3% H2O2 aqueous solution. If bubbles are produced, it is positive, otherwise it is negative.

[0053] (4) Molecular biological identification of bacterial species based on 16S rDNA The sample (positive product in step (3)) was sent to Shanghai Bioengineering Co., Ltd. for sequencing and identification. The obtained gene sequence was compared by Blast on the NCBI gene bank, and the sequence of the strain with the highest similarity was selected.

[0054] The lactobacillus is derived from Guangzhou Junyan Biotechnology Co., Ltd., and its biological collection number is GDMCC NO: 64856.

[0055] Calculated by mass percentage, the raw materials for preparing the culture medium include: 1% milk, 0.5% glucose, 0.1% yeast extract powder, and the balance is supplemented by water.

[0056] The preparation method of the culture medium comprises: mixing the raw materials for preparing the culture medium according to the formula amount, adjusting the pH to 6.0±0.2, and sterilizing at 121° C. for 15 minutes.

[0057] Note: Sodium hydroxide is used to increase pH and lactic acid is used to decrease pH.

[0058] The antibacterial treatment in step S3 includes: sterilizing the fermentation liquid product II at 85° C. for 30 minutes; and then adding a preservative to obtain a lactobacillus fermentation filtrate.

[0059] The preservatives are p-hydroxyacetophenone and 1,2-hexanediol, and the added amounts are 0.5 wt % of p-hydroxyacetophenone and 0.5 wt % of 1,2-hexanediol based on the total mass of the culture medium.

[0060] The lactobacillus fermentation filtrate prepared by the preparation method as described above is applied to lotion, and the formula of the lotion is shown in Table 1 below; wherein, the molecular weight of the large molecular hyaluronic acid is 2000 KDa, which is sourced from Dongying Foster Bioengineering Co., Ltd.; the molecular weight of the small molecular hyaluronic acid is 100 KDa, which is sourced from Shandong Tiansheng Biotechnology Co., Ltd.; the orange blossom hydrosol is sourced from Quzhou Zhanhong Biotechnology Co., Ltd., and the asiatica glycoside is sourced from Shanghai Jiakai Biotechnology Co., Ltd.

[0061] Table 1

[0062] The preparation method of the lotion is: a. Mix the lactobacillus fermentation filtrate, 1,3-butanediol, small molecule hyaluronic acid, large molecule hyaluronic acid, p-hydroxyacetophenone and glycerin, raise the temperature to 85°C while stirring, keep the temperature for 15 minutes until the materials are completely dissolved, and then start to cool down; b. Mix and pre-dissolve orange blossom hydrosol and Centella asiatica glycoside and set aside; c. When the material in step a is cooled to 45°C, a premix of D-panthenol, 1,2-hexanediol, neroli hydrosol and asiatica glycoside is mixed in sequence under stirring and stirred evenly to obtain a soothing lotion containing lactobacillus fermentation filtrate.

[0063] Comparative Example 1 Specific implementation method is the same as Example 1, except that the milk in the culture medium is replaced by skimmed milk powder.

[0064] Comparative Example 2 Specific implementation method is the same as Example 1, except that the lactobacillus in step S1 is specifically lactobacillus seed liquid, and the preparation step of the lactobacillus seed liquid includes: mixing 5wt% MRS broth + water (T0 100%) to obtain a seed liquid culture medium, treating it at 121°C for 15 minutes for sterilization; then inoculating 2wt% of the lactobacillus strain on the plate into the seed liquid culture medium, facultative anaerobic culture, and culturing it at 37°C and 200rpm for 16 hours. The OD value of the lactobacillus seed liquid is 2.485.

[0065] Performance Testing Physical and Chemical Test Appearance The lactobacillus fermentation filtrate of Example 1 (see Figure 2 ): clear and transparent; and the fermentation filtrate has a milky aroma.

[0066] The lactobacillus fermentation filtrate of Comparative Example 1 (see Figure 1): Slightly turbid, esters float on the upper layer of the filtrate and are not used.

[0067] 1.2 Content test The lactobacillus fermentation filtrate of Example 1 was tested for components, and the test results are shown in Figure 3 , Figure 4 .Depend on Figure 3 , Figure 4 It can be seen that the lactobacillus fermentation filtrate prepared by the present invention contains a variety of amino acids and polysaccharides and is rich in active substances.

[0068] Efficacy test 2.1 Firming and anti-wrinkle efficacy (characterized by elastase inhibition rate) Detection test method: refer to Table 2 below to design sample addition groups, and use 96 microplates for the test. The positive control is prepared with Tris-HCl buffer at pH = 8, and diluted by half to a series of concentrations to verify the test system. The test substance is diluted with Tris-HCl buffer at pH = 8 to form multi-level concentration samples. AAAPAN is the color substrate N-succinyl-alanine-alanine-alanine-p-nitroaniline.

[0069] According to the sample grouping, a sample group (T), a sample background group (To), an enzyme reaction group (C) and a solvent background group (Co) were set up, and a positive control group (epigallocatechin gallate, EGCG) was set up to be treated the same as the sample group; 3 parallels were set up for each group, and the experimental data were averaged. The sample group was the lactobacillus fermentation filtrate prepared in Example 1, and the liquid addition conditions of each group of samples are shown in Table 2 below (+ indicates addition, - indicates no addition).

[0070] Table 2

[0071] After adding liquid to the sample, the sample was immediately tested using an ELISA reader with the following parameters set: slow oscillation for 10 seconds, measuring absorbance at OD410nm, and exporting the file after the instrument completed the reading; the calculation formula for the inhibition rate of elastase activity is as follows.

[0072]

[0073] The test results are shown in Table 3 below. PC corresponds to the sample results of the positive control group, and Sample corresponds to the sample results of the lactobacillus fermentation filtrate of Example 1.

[0074] Table 3

[0075] It can be seen from the data in Table 3 that the lactobacillus fermentation filtrate of Example 1 has an inhibitory effect on the decomposition of AAAPAN catalyzed by elastase, with an inhibition rate of 15.20±1.52%, and the data have a significant difference (p=0.00067). It can be seen that the lactobacillus fermentation filtrate prepared by the method described in Example 1 can achieve firming and anti-wrinkle effects by inhibiting the activity of elastase, and improve skin elasticity and vitality.

[0076] 2.2 Soothing and moisturizing effects (characterized by hyaluronidase inhibition rate) Detection test method: refer to Table 4 below to design sample addition groups, and use 96-well plates for the test. The positive control is prepared with Tris-HCl buffer at pH = 8, and diluted by half to a series of concentrations to verify the test system. The test substance is diluted with Tris-HCl buffer at pH = 8 to form multi-level concentration samples. DNS is a colorimetric reagent, dinitrosalicylic acid.

[0077] According to the sample grouping, a sample group (T), a sample background group (To), an enzyme reaction group (C) and a solvent background group (Co) were set up, wherein the positive control group (dexamethasone) was treated in the same manner as the sample group; 3 parallels were set up for each group, and the experimental data were averaged. Among them, the sample group was the lactobacillus fermentation filtrate prepared in Example 1, and the liquid addition conditions of each group of samples were shown in Table 4 below (+ indicates addition, - indicates no addition).

[0078] Table 4

[0079] After the test tube has cooled, it is tested using an ELISA instrument with the following parameters set: OD540nm absorbance measurement, and the file is exported after the instrument completes the reading; the calculation formula for the hyaluronidase activity inhibition rate is as follows.

[0080]

[0081] The test results are shown in Table 5 below. PC corresponds to the sample results of the positive control group, and Sample corresponds to the sample results of the lactobacillus fermentation filtrate of Example 1.

[0082] Table 5

[0083] It can be seen from the data in Table 5 that the lactobacillus fermentation filtrate of Example 1 has an inhibitory effect on the decomposition of hyaluronic acid by hyaluronidase, with an inhibition rate of 41.07±4.48%, and the data have a significant difference (p=0.0154). It can be seen that the lactobacillus fermentation filtrate prepared by the method described in Example 1 can achieve soothing and moisturizing effects by inhibiting the activity of hyaluronidase, and repair and nourish the skin.

[0084] 3. Results of VISIA skin test on lotion Test method: 4 users were randomly selected to try the lotion of Example 1, using it once in the morning and once in the evening for a period of time, and the facial skin changes at the beginning and after use were recorded. User 1 used it from 2023.3.2 to 2023.3.31, user 2 used it from 2023.3.2 to 2023.3.31, user 3 used it from 2023.3.2 to 2023.3.31, and user 4 used it from 2023.3.2 to 2023.3.14; VISIA results are shown in Table 1. Figure 5-8 .from Figure 5-8 It can be seen that after using the lotion containing lactobacillus fermentation filtrate provided by the present invention, the distribution of blue areas (characterizing red blood streaks) on the skin can be reduced within just one month of use, indicating that the lotion has an obvious soothing effect.

Claims

1. A method for preparing a lactobacillus fermentation filtrate, characterized in that: The specific steps include: S1. inoculating lactic acid bacteria into a culture medium, and continuously fermenting and culturing to obtain a fermentation broth product 1; S2. Filtering the fermentation broth product 1 to separate lactobacilli to obtain the fermentation broth product 2; S3. Performing antibacterial treatment on the fermentation broth product II to obtain a lactobacillus fermentation filtrate; The raw materials for preparing the culture medium at least include milk; The lactobacillus is Lactobacillus paracasei.

2. The method for preparing the lactobacillus fermentation filtrate according to claim 1, characterized in that: The Lactobacillus paracasei is purified and separated from cheese.

3. The method for preparing the lactobacillus fermentation filtrate according to claim 1, characterized in that: Calculated by mass percentage, the raw materials for preparing the culture medium include: 0.1-5% milk, 0.1-2% glucose, 0.01-1% yeast extract powder, and the balance is supplemented by water.

4. The method for preparing the lactobacillus fermentation filtrate according to claim 1, characterized in that: The continuous fermentation culture condition in the step S1 is 20-40° C. for 15-30 hours.

5. The method for preparing lactobacillus fermentation filtrate according to claim 1, characterized in that: The antibacterial treatment in step S3 includes: sterilizing the fermentation broth product 2 at 80-90° C. for 15-60 minutes.

6. The method for preparing the lactobacillus fermentation filtrate according to claim 5, characterized in that: In the step S3, a preservative is added after heat preservation and sterilization to obtain a lactobacillus fermentation filtrate.

7. The method for preparing lactobacillus fermentation filtrate according to claim 6, characterized in that: The preservative comprises at least one of p-hydroxyacetophenone, 1,2-hexanediol, hydroxyphenyl ester, phenoxyethanol and methylisothiazolinone.

8. A lactobacillus fermentation filtrate prepared according to the preparation method according to claim 1.

9. Use of the lactobacillus fermentation filtrate according to claim 8 in skin care products, characterized in that: The skin care products include any one of lotion, essence, emulsion, cream, ointment and facial mask.

10. A use of the lactobacillus fermentation filtrate according to claim 9, characterized in that: The skin care product is a lotion; the amount of lactobacillus fermentation filtrate added to the lotion is more than 60wt%.