Bifidobacterium adecentis milk ferment, products containing the same, and production and use thereof

Abstract

The present invention provides a Bifidobacterium adecentis milk ferment, a product containing the same, and its production and use. The method for producing the Bifidobacterium adecentis milk ferment includes the following steps. Inoculate Bifidobacterium adecentis into a fermentation substrate, ferment and culture for 20 to 36 hours, sterilize, and centrifuge to collect the supernatant to complete. Among them, the fermentation substrate contains milk and does not add a saccharide carbon source. This application can perform fermentation treatment on milk by a specific type of microorganism, and at the same time has an antioxidant effect, improves the cell elastin content, improves the cell lysosome activity, promotes cell migration, has high safety in use, has no irritating effect on the skin, can improve the skin barrier effect, has good stability, is easy to store, and has a simple manufacturing technology and energy-saving environmental protection. The Bifidobacterium adecentis milk ferment has very good application prospects in the field of cosmetics.

Description

Technical Field

[0001] The present invention belongs to the field of cosmetics, and particularly relates to Bifidobacterium adolescentis milk ferment, products containing the same, and their production and use.

Background Art

[0002] Milk is the oldest natural beverage, containing nutrients such as rich calcium, vitamin D, protein, etc., which can promote the growth and development of the human body, promote brain development, promote digestion, protect the gastrointestinal tract, prevent constipation, improve immunity, and have effects such as beauty, and is widely applied in the food field. It has also been reported in the field of cosmetics and is used, for example, during face washing, face packs, and bathing. Milk contains rich high-molecular proteins, and long-term use is likely to cause allergies, especially for people with sensitive skin. In addition, milk is rich in fats, which are dispersed in the emulsion as fine particulate fat globules and are in an emulsified state. Long-term use of milk may cause the fats contained therein to clog pores and easily induce folliculitis. Milk has poor stability. When stored at room temperature for one week, obvious stratification and cotton-like substances appear, and phenomena such as an unpleasant smell occur, so it cannot be said to be a cosmetic that can be stored for a long time.

[0003] With the improvement of living standards and the development of science and technology, cosmetics have already become necessities in people's lives. Cosmetic raw materials can be divided into two types: synthetic and natural extraction. Among them, synthetic raw materials have obvious effects with certain efficacy, but the side effects are also relatively obvious, which can cause skin irritation, damage the skin barrier, and cause the skin to become sensitive skin, and the manufacturing cost is relatively high. Compared with synthetic raw materials, natural extracts have improved safety and significantly reduced manufacturing costs. Cosmetic raw materials containing natural active ingredients derived from plants have become the research focus of this field. However, due to the complex components in natural extracts, if the extraction technology is not appropriate, it will not only affect the effects of active ingredients, but also may cause allergic reactions on the skin.

[0004] Therefore, in this field, it is necessary to develop a method for efficiently extracting active ingredients in milk, and to promote that the obtained active ingredients have ideal cosmetic effects, have high use safety, no irritation to the skin, and expand the application fields of milk.

Summary of the Invention

[0005] The technical problem to be solved by this application is to overcome the deficiencies of the prior art and provide Bifidobacterium adecentis milk ferment, products containing the same, and their production and use. By fermenting milk with specific types of microorganisms, not only can the application fields of milk be expanded, but at the same time, it has antioxidant effects, can improve the cell elastin content, improve the cell lysosome activity, promote cell migration, has high use safety, no irritating effect on the skin, can improve the skin barrier function, has good stability, is easy to store, has a simple manufacturing technology and is energy-saving and environmentally friendly. Bifidobacterium adecentis milk ferment has very good application prospects in the cosmetics field.

[0006] This application adopts the following technical solutions to solve the above technical problems. This application provides a method for producing Bifidobacterium adecentis milk ferment, including the following steps. Inoculate Bifidobacterium adecentis into the fermentation substrate, after 20 - 36 hours of fermentation culture, sterilize, and centrifuge to collect the supernatant to complete. Among them, the fermentation substrate contains milk and does not add a saccharide carbon source.

[0007] Not adding the saccharide carbon source means that in addition to the milk, it is not necessary to add the saccharide carbon sources commonly used in this field, such as glucose, sucrose, or starch. During the research and development process, when adding a saccharide carbon source additionally, it provides sufficient nutrition for the inoculum, but has a profound impact on the performance of the final product. For example, the antioxidant ability decreases, the irritation to the skin increases, and the use safety is poor.

[0008] In some embodiments, the fermentation substrate may also contain a nitrogen source. The nitrogen source used in this application is a nitrogen source commonly used in this field added in addition to the milk.

[0009] Here, the nitrogen source may include L-cysteine hydrochloride. Here, the mass percentage of the nitrogen source occupying the milk is 0.01% - 0.05%, preferably 0.03% - 0.05%.

[0010] In some embodiments, the fermentation substrate includes a sterilization operation as conventional in this field before use. Here, the conditions and methods of the sterilization may be conventional conditions and methods in the industry, and generally, it is the high-temperature sterilization method.

[0011] When performing the sterilization on the fermentation substrate by adopting the high-temperature sterilization method, the temperature of the sterilization may be a conventional temperature in the industry for such an operation, preferably 95 - 100 °C.

[0012] When performing the sterilization on the fermentation substrate by adopting the high-temperature sterilization method, the time of the sterilization may be a conventional temperature in the industry for such an operation, preferably 15 - 35 minutes, more preferably 30 minutes.

[0013] Normally in this field, after the sterilization operation, a cooling operation may further be included, and generally, it can be cooled to room temperature.

[0014] In some embodiments, the Bifidobacterium adecentis may include Bifidobacterium adecentis of model number BBF-06 manufactured by "Shandong Zhongke Jiayi Biological Engineering Co., Ltd." and / or Bifidobacterium adecentis of model number SF-B40 manufactured by "Shandong Sunflower Biological Engineering Co., Ltd.".

[0015] In some embodiments, the Bifidobacterium adlescentis can be added in the form of a Bifidobacterium adlescentis solution as commonly used in this field. The viable count in the Bifidobacterium adlescentis solution is 10 6 ~10 10 CFU / mL, preferably 10 7 ~10 9 CFU / mL.

[0016] In some embodiments, the number of Bifidobacterium adlescentis inoculated into the fermentation substrate per unit volume may be normal in this field, preferably 10 5 ~10 9 CFU / mL, more preferably 10 6 ~10 8 CFU / mL.

[0017] In some embodiments, the conditions and methods of the fermentation culture are normal in this field and can generally be statically cultured in a constant temperature incubator.

[0018] In some embodiments, the time of the fermentation culture is preferably 24 to 30 hours. In some embodiments, the temperature of the fermentation culture may be 37 to 43 °C. In some embodiments, the conditions and methods of the sterilization are normal in this field and are generally high-temperature sterilization.

[0019] When performing the sterilization by adopting the high-temperature sterilization method, the temperature may be normal in the industry for such an operation, preferably 95 to 100 °C.

[0020] When performing the sterilization by adopting the high-temperature sterilization method, the sterilization time may be the normal time for such an operation in the industry, preferably 20 to 40 minutes, more preferably 30 minutes.

[0021] In some embodiments, the rotational speed of the centrifugation may be the conventional rotational speed for such operations in the art, preferably 4000 - 8000 rpm, more preferably 4000 - 6000 rpm, for example, 4800 rpm.

[0022] In some embodiments, the radius of the centrifugation may be the conventional radius for such operations in the art, preferably 8 - 15 cm.

[0023] In some embodiments, the time of the centrifugation may be the conventional time for such operations in the art, preferably 10 - 40 minutes, more preferably 20 - 40 minutes, for example, 30 minutes.

[0024] In some embodiments, after the centrifugation operation, it may further include filtration and an operation of recovering the filtrate. Here, the pore size of the filter membrane used for the filtration may be 0.22 - 0.8 μm, preferably 0.22 - 0.45 μm.

[0025] In a preferred embodiment, after the filtration operation, it may further include secondary sterilization and / or an operation of mixing with a preservative. Here, the method of the secondary sterilization is the high-temperature sterilization method commonly used in this field.

[0026] When performing the secondary sterilization using the high-temperature sterilization method, the temperature of the secondary sterilization may be the conventional temperature for such operations in the art, preferably 95 - 100 °C.

[0027] When performing the secondary sterilization using the high-temperature sterilization method, the time of the secondary sterilization may be the conventional time for such operations in the art, preferably 20 - 40 minutes, more preferably 30 - 40 minutes.

[0028] The mixing temperature in the mixing step with the preservative may be a conventional temperature for such operations in the industry, preferably 50 to 80 °C, more preferably 70 to 80 °C.

[0029] Here, the preservative can contain p-hydroxyacetophenone and / or 1,2-hexanediol as is conventional in the industry.

[0030] When the preservative contains the p-hydroxyacetophenone and the 1,2-hexanediol, the mass percentage of the p-hydroxyacetophenone in the filtrate obtained by the filtration is 0.2% to 0.6%, and the mass percentage of the 1,2-hexanediol in the filtrate obtained by the filtration is 0.5% to 2%. Preferably, the mass percentage of the p-hydroxyacetophenone in the filtrate obtained by the filtration is 0.2 to 0.5%, and the mass percentage of the 1,2-hexanediol in the filtrate obtained by the filtration is 0.5 to 1%.

[0031] The present invention further provides a milk ferment of Bifidobacterium adecentis, which is produced by the method for producing a milk ferment of Bifidobacterium adecentis as described above.

[0032] The present invention further provides the use of the Bifidobacterium adecentis milk ferment as described above as a product as it is, as an additive or as a base material in the production of a topical skin preparation.

[0033] In some embodiments, the Bifidobacterium adecentis milk ferment can be at least one of an antioxidant active ingredient, a skin barrier activity improving ingredient, an anti-aging active ingredient, and a relaxation active ingredient in the topical skin preparation.

[0034] Here, the antioxidant active ingredient may be an antioxidant active ingredient having DPPH radical scavenging ability and / or hydroxyl radical scavenging ability. Here, the skin barrier activity improving component may be a skin barrier activity improving component having a moisturizing and hydrating effect. Here, the anti-aging activity component may be an anti-aging activity component that improves the skin elastin content. Here, the relaxation activity component may be a relaxation activity component that suppresses the hemolysis of red blood cells by SDS.

[0035] The present invention further provides a topical skin preparation containing the above-mentioned Bifidobacterium adecentis milk ferment.

[0036] In some embodiments, the topical skin preparation includes, but is not limited to, conventional creams, masks, essences, or toners in the art.

[0037] In some examples, the topical skin preparation can further contain at least one of active ingredients, preservatives, thickeners, oils and fats, emulsifiers, and solvents commonly used in this field. Here, the active ingredient can contain at least one of a moisturizing active ingredient, a whitening active ingredient, an anti-inflammatory active ingredient, an anti-allergy active ingredient, and an antioxidant active ingredient. Here, the preservative can contain preservatives commonly used in this field, preferably including butanediol and / or phenoxyethanol. Here, the thickener can contain thickeners commonly used in this field, preferably including carbomer. Here, the oil and fat can contain oils and fats commonly used in this field, preferably including at least one of jojoba oil, polydimethylsiloxane, squalane, and cetanol. Here, the emulsifier can contain emulsifiers commonly used in this field, preferably including polysorbate-20 and / or sorbitan isostearate. Here, the solvent can contain solvents commonly used in this field, preferably deionized water. Here, the mass percentage of the preservative in the topical skin preparation may be 12% to 15%, preferably 13.03%. Here, the mass percentage of the thickening agent in the topical skin preparation may be 0.5% to 1%, preferably 0.6%. Here, the mass percentage of the oil and fat in the topical skin preparation may be 4% to 10%, preferably 5% to 6%. Here, the mass percentage of the emulsifier in the topical skin preparation may be 1% to 3%, preferably 1.5% to 1.6%. Here, the amount of the solvent used can supplement the total amount of the topical skin preparation to 100% as commonly practiced in the art.

[0038] In some embodiments, the mass % of the Bifidobacterium adolescentis milk ferment in the topical skin preparation may be 5 to 99%, preferably 60 to 99%.

[0039] In some embodiments, the room temperature generally means 15 to 40 °C.

[0040] Based on the common knowledge in the industry, the above preferred conditions can be arbitrarily combined to obtain each preferred example of the present application.

[0041] The reagents and raw materials used in the present application are commercially available.

[0042] The positive progressive effect of the present invention is that the Bifidobacterium adolescentis milk ferment obtained in the present invention has ideal antioxidant performance, can increase the cell elastin content, enhance the cell lysosome activity, and promote cell migration. It has high safety, no irritating effect on the skin, and can enhance the skin barrier function. It has excellent stability, is easy to store, has a simple manufacturing process, mild fermentation conditions, low energy consumption, saves costs, has zero burden on the skin, has high safety, meets the needs of the functionality and safety of modern topical skin preparations, and can be widely applied in the field of topical skin preparations.

Brief Description of the Drawings

[0043] This application can be better understood with reference to the following description in conjunction with the accompanying drawings. The accompanying drawings are included in this specification together with the following detailed description, form a part of this specification, further illustrate the preferred embodiments of this application, and are used to explain the principles and advantages of this application.

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Mode for Carrying Out the Invention

[0044] Hereinafter, the present application will be further described by way of examples, but the present application is not limited to the scope of the examples for explaining the present application. For the experimental methods for which specific conditions are not specified in the following examples, follow the normal methods and conditions or select according to the product manuals.

[0045] The experimental methods used in the following examples are normal methods unless otherwise specified.

[0046] The Bifidobacterium adecentis in Example 1 below was purchased from "Shandong Zhongke Jiayi Biological Engineering Co., Ltd.", and the model number is BBF-06.

[0047] The Bifidobacterium adecentis in Example 3 below was purchased from "Shandong Sunflower Biological Engineering Co., Ltd.", and the model number is SF-B40.

[0048] The Bifidobacterium lactis in Comparative Example 3 below was purchased from "Zhengzhou Hehe Biological Engineering Technology Co., Ltd.", and the product number is HH-BA68.

[0049] Example 1 (1) Sterilize 300 g of milk in a high-temperature sterilization kettle at 95 °C for 30 minutes, and cool it to room temperature after sterilization to obtain a fermentation substrate. (2) Prepare the Bifidobacterium adolescentis with product number BBF-06 produced by "Shandong Zhongke Jiayi Biotechnology Co., Ltd." into a Bifidobacterium adolescentis bacterial liquid with a viable count of 10 10 CFU / mL. Inoculate 3 mL of the Bifidobacterium adolescentis bacterial liquid into the fermentation substrate obtained in step (1), and perform static fermentation culture in a constant temperature incubator at 37°C for 24 hours. After fermentation, transfer the material to a high-pressure steam sterilizer and sterilize it under the condition of 95°C for 30 minutes. After sterilization, cool it to room temperature, centrifuge it for 30 minutes under the conditions of a rotation speed of 4800 rpm and a centrifugal radius of 15 cm, collect the supernatant, and further filter the supernatant with a 0.22 μm filter membrane to remove Bifidobacterium adolescentis and impurities. Sterilize the filtered filtrate for the second time and sterilize it under the condition of 95°C for 30 minutes. After the second sterilization is completed, when the system temperature drops to 75°C, add the preservatives p-hydroxyacetophenone and 1,2-hexanediol to the system, where the mass percentage of p-hydroxyacetophenone in the filtrate is 0.5%, and the mass percentage of 1,2-hexanediol in the filtrate is 0.5%, to obtain Bifidobacterium adolescentis milk ferment.

[0050] Example 2 Compared with Example 1, the difference is only that the fermentation substrate is different. In step (1), 300 g of milk is mixed with 0.15 g of L-cysteine hydrochloride, sterilized in a high-temperature sterilizer at 95°C for 30 minutes, cooled to room temperature after sterilization to obtain the fermentation substrate, and the other condition parameters are the same as those in Example 1, to obtain Bifidobacterium adolescentis milk ferment.

[0051] Example 3 Compared with Example 1, the difference is only that the fermentation inoculum is different. The fermentation inoculum is purchased from "Shandong Sunflower Biotechnology Co., Ltd." and is Bifidobacterium adolescentis with product number SF-B40. The other condition parameters are the same as those in Example 1, to obtain Bifidobacterium adolescentis milk ferment.

[0052] Comparative Example 1 Compared with Example 1, the difference is only that the fermentation substrate is an equal amount of soybean extract, and the other condition parameters are the same as those in Example 1, and a soybean ferment is obtained. The method for producing the soybean extract is as follows. Weigh soybeans and soak them for 8 hours, then boil them under the condition of 100°C, mix them according to the mass ratio of soybeans to water of 1:10, beat them, add monosaccharide (glucose) with a mass ratio of 0.5%, sterilize them under the condition of 100°C for 30 minutes, and obtain a fermentation substrate.

[0053] Comparative Example 2 Compared with Example 1, the difference is only the difference in the fermentation substrate. In step (1), 300 g of milk and 1.5 g of glucose are mixed, sterilized in a high-temperature sterilization kettle at 100°C for 30 minutes, cooled to room temperature after sterilization, and a fermentation substrate is obtained. The other condition parameters are the same as those in Example 1, and a Bifidobacterium adolescentis / milk glucose ferment is obtained.

[0054] Comparative Example 3 Compared with Example 1, the difference is only the difference in the fermentation inoculum. The fermentation inoculum is Bifidobacterium lactis with the product number HH-BA68 purchased from "Zhengzhou Hehe Biological Engineering Technology Co., Ltd.", and the other condition parameters are the same as those in Example 1, and a Bifidobacterium lactis / milk ferment is obtained.

[0055] Comparative Example 4 Compared with Example 1, the difference is only that no fermentation treatment is performed, and it is the fermentation substrate obtained in step (1) of Example 1.

[0056] Comparative Example 5 Compared with Example 1, the difference is only that in step (2), the fermentation time is 7 hours, and the other condition parameters are the same as those in Example 1, and a Bifidobacterium adolescentis milk ferment is obtained.

[0057] Effect Example 1: DPPH Radical Scavenging Experiment DPPH is a type of organic radical synthesized early, often used to evaluate the hydrogen supply ability of antioxidants. It is very stable in organic solvents, purple in color, and has a characteristic absorption peak at 517 nm. When it encounters a radical scavenger, the unpaired electron of DPPH pairs up, causing it to fade, that is, the absorbance value at the maximum absorption wavelength decreases. Therefore, by measuring the change in absorbance value, the DPPH radical scavenging effect of the sample can be evaluated.

[0058] The specific experimental steps of the DPPH radical scavenging experiment are as follows. (1) Mix an equal volume (1 mL) of the measurement solution uniformly with a 2×10 -4 mol / L DPPH solution (Tube A1). (2) Mix an equal volume (1 mL) of absolute ethanol (solvent waiting to be measured) uniformly with a 2×10 -4 mol / L DPPH solution (Tube A2). (3) Mix an equal volume (1 mL) of absolute ethanol uniformly with the solution waiting to be measured (Tube A3). (4) After a 30-minute light-shielded reaction, measure the absorbance values of Tubes A1, A2, and A3 at 517 nm. The calculation formula for the scavenging rate is: scavenging rate = [(A2 + A3) - A1] / A2 × 100%.

[0059] In this experiment, DPPH radical scavenging experiments were conducted on the products manufactured in Examples 1 - 3 and Comparative Examples 1 - 3. The solution waiting to be measured was the product manufactured in the above-mentioned examples and comparative examples, and the results are shown in Table 1 and Figure 1. From the results of JPEG2025519668000002.jpg41170, it was found that the DPPH radical scavenging rates of the products obtained in Examples 1, 2, and 3 were all higher than those of the products obtained in Comparative Examples 1 - 3.

[0060] Effect Example 2: Hydroxyl radical scavenging rate Hydroxyl radical is the most chemically active radical among reactive oxygen species. It can react with almost any biopolymer in living cells, has an extremely fast reaction rate, and is the radical that causes the greatest harm to organisms. When salicylic acid is added to the reaction system, hydroxyl radicals can be effectively captured, and colored product 2,3-dihydroxybenzoic acid is generated. The colored product has a strong absorption peak at 510 nm.

[0061] If a sample with the function of eliminating hydroxyl radicals is added to the system and the ability of the sample to capture hydroxyl radicals is greater than that of salicylic acid, hydroxyl radicals can be immediately eliminated, thus reducing the production amount of the colored product and decreasing the absorbance. Therefore, by adopting the fixed reaction time method, measuring the absorbance of the reaction solution containing the sample to be measured at 510 nm and comparing it with the blank solution, the elimination effect of the sample on hydroxyl radicals can be measured.

[0062] The specific experimental steps of the hydroxyl radical elimination experiment are as follows. A1: Sequentially add 2 mL of 6 mmol / L FeSO4, 2 mL of 6 mmol / L H2O2 (here, H2O2 is added last to start the whole reaction), and 2 mL of the sample to be measured to a test tube, shake evenly, and let it stand at room temperature for 15 minutes. Then add 2 mL of 6 mmol / L salicylic acid, shake evenly, take it out after heating in a 37°C water bath for 30 minutes, measure its absorbance, and record it as A1. A2: Sequentially add 2 mL of 6 mmol / L FeSO4, 2 mL of 6 mmol / L H2O2, and 2 mL of the sample to be measured to a test tube, shake evenly, let it stand at room temperature for 15 minutes, then add 2 mL of deionized water, shake evenly, take it out after heating in a 37°C water bath for 30 minutes, measure its absorbance, and record it as A2. A3: Sequentially add 2 mL of 6 mmol / L FeSO4, 2 mL of deionized water, and 2 mL of 6 mmol / L H2O2 to a test tube, shake well, let stand at room temperature for 15 minutes, then add 2 mL of 6 mmol / L salicylic acid, shake well, take out after heating in a 37°C water bath for 30 minutes, measure its absorbance, and denote it as A3. Hydroxyl radical scavenging rate = [(A3 + A2) - A1] / A3 × 100%.

[0063] Measure the hydroxyl radical scavenging rates of the products obtained in the above examples and comparative examples, and show the results in Table 2 and Figure 2. JPEG2025519668000003.jpg38170 From the results, the hydroxyl radical scavenging rates of the products obtained in Examples 1 to 3 were all higher than those of the products obtained in Comparative Example 1 and Comparative Example 2.

[0064] Effect Example 3: Erythrocyte hemolysis stimulation experiment 1. Preparation of erythrocyte suspension (RBC) 1.1. Washing Dilute fresh sheep blood with PBS solution at a dilution ratio of 2:5 in a 50 mL centrifuge tube. Gently invert the centrifuge tube up and down to thoroughly mix the blood and PBS. Then, centrifuge at 1000×g for 10 minutes at room temperature, discard the upper supernatant (the pale yellow leukocyte layer of the supernatant), and then add PBS buffer and repeat this 1 - 2 times to remove a large amount of leukocytes, plasma, and yellow fragments. 1.2. Preparation of erythrocyte suspension (RBC) Transfer the blood cell precipitate in the centrifuge tube to a new EP tube with a disposable straw, take 1 mL of RBC and precipitate it in a 50 mL EP tube, add 49 mL of PBS buffer for dilution, and then gently shake to mix evenly. Put 250 μL of RBC into a 1.5 mL EP tube, add 750 μL of distilled water to make up to 1 mL, centrifuge at a speed of 10000×g for 1 minute to stop the culture, take the upper supernatant after centrifugation, and measure the OD value at 540 nm. Measure the OD value between 0.5 - 0.8 at a wavelength of 540 nm. 2. Erythrocyte hemolysis stimulation experiment Prepare according to Table 3 above. After the addition of the sample is completed, place it in a shaker, rotate the shaker at 180 rpm, incubate at 37 °C for 60 minutes, and then take it out. Centrifuge at 10,000 g for 1 minute to stop the reaction. Record the photo, aspirate 200 μL of the supernatant into a 96-well plate, and measure the absorbance at 540 nm. Erythrocyte hemolysis rate = (OD sample group - OD blank group) / (OD positive group - OD blank group) * 100% Measure the erythrocyte hemolysis rate of the products obtained from the above-mentioned examples and comparative examples in Table 4 and Figure 3. JPEG2025519668000005.jpg39170 The erythrocyte hemolysis rate can, to a certain extent, test the irritation of the product. From the experimental results, the erythrocyte hemolysis rates of the products manufactured in Examples 1 to 3 are lower than those of the products manufactured in Comparative Examples 1 to 3, indicating that the products manufactured in Examples 1 to 3 are milder and safer.

[0065] Effect Example 4: Erythrocyte hemolysis inhibition rate Measure the erythrocyte hemolysis inhibition rate of the products manufactured by the above-mentioned examples and comparative examples. The measurement method is as follows. I. Experimental materials and reagents 1. Prepare a PBS buffer solution with a pH value of 7.4, prepare it with pure water, and store it at 4 °C. 2. Prepare a 1% SDS solution Weigh 0.5 g of SDS powder and dissolve it in 50 mL of PBS buffer solution. When using, dilute the 1% SDS solution to 0.1% SDS with PBS buffer solution for subsequent use. II. Experimental plan 1. Preparation of erythrocyte suspension (RBC) 1.1. Washing Dilute fresh sheep blood with PBS solution at a dilution ratio of 2:5 in a 50 mL centrifuge tube. Gently invert the centrifuge tube up and down to mix the blood and PBS thoroughly. Then, centrifuge at 1000 × g for 10 minutes at room temperature, discard the upper supernatant (the pale yellow leukocyte layer of the supernatant), and then add PBS buffer solution and repeat this 1 - 2 times to remove a large amount of leukocytes, plasma, and yellow fragments. 1.2. Preparation of Red Blood Cell Suspension (RBC) Use a disposable straw to transfer the blood cell precipitate in the centrifuge tube to a new EP tube, take 1 mL of RBC and precipitate it in a 50 mL EP tube, add 49 mL of PBS buffer solution for dilution, and then gently shake to mix evenly. Put 250 μL of RBC into a 1.5 mL EP tube, add 750 μL of distilled water to make up to 1 mL, centrifuge at a speed of 10000×g for 1 minute to stop the culture. After centrifugation, take the upper supernatant and measure the OD value at 540 nm. Measure the OD value between 0.5 and 0.8 at a wavelength of 540 nm.

[0066] Conduct experiments according to Table 5, and the specific process is as follows. JPEG2025519668000006.jpg34170

[0067] Put 4 groups of samples (numbers 2 - 5) into the incubator, culture at 37°C at 180 rpm for 60 minutes, then take out, centrifuge at 10000×g for 1 minute to stop the reaction. Record the photo, and then aspirate 200 μL of the supernatant into a 96-well plate, measure the absorbance at 540 nm, calculate the sample hemolysis inhibition rate %, and refer to Table 6 and Figure 4 for the results. 1) Hemolysis rate of the model group = (OD_model - OD_blank) / (OD_model control - OD_blank) * 100% 2) Hemolysis rate of the model group = (OD_sample group - OD_sample control) / (OD_model control - OD_blank) * 100% 3) Sample hemolysis inhibition rate = (Hemolysis rate of the model group - Hemolysis rate of the sample treatment group) / Hemolysis rate of the model group * 100%. JPEG2025519668000007.jpg39170

[0068] Effect Example 5 1×10 6 fibroblasts per well were inoculated into a 6-well plate and cultured for 24 hours. Aspirate and discard the original medium, add serum-free DMEM respectively, and treat for 24 hours. After sample treatment, aspirate and discard the original medium, add 1 mL of PBS, and the cells are irradiated with UVA at 7 J / cm2 Exposed to 2 , the blank control group was not treated. After 40 minutes of treatment, DMEM (125 μL of the sample waiting to be measured, 875 μL of DMEM) containing Examples 1 - 3 and Comparative Examples 1 - 3 was added respectively, and the treatment was continued with overnight culture. Then, the supernatant and cells were collected respectively. After washing the cells with PBS, 200 μL of lysis solution (containing PMSF with a final concentration of 1 mM) was added, pipetted evenly, centrifuged at 10,000 rpm for 5 minutes at 4°C to collect the cell supernatant, and then the elastin content in fibroblasts was measured.

[0069] After the fibroblasts obtained products by the above - mentioned Examples and Comparative Examples for treatment, the elastin content in the cells was measured, and the results are shown in Table 7 and Figure 5. According to the results of JPEG2025519668000008.jpg48170, compared with the Comparative Examples, the products obtained in Examples 1 - 3 are beneficial for promoting elastin secretion in fibroblasts and have a more ideal repair effect on damaged fibroblasts.

[0070] Effect Example 6 The protein content in the products obtained in Comparative Example 4, Comparative Example 5 and Example 1 was measured using the color qualitative method. The darker the purple color, the higher the protein content, and the measurement results are shown in Figure 6. Measurement was carried out using the "Bairuiji Biology" BCA kit. The specific measurement process is as follows. (1) Prepare the BCA working solution with reagent A: reagent B in a ratio of 50:1. (2) Add 20 μL of standard products with different concentrations (0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg / mL) and the sample waiting to be measured to a 96 - well plate respectively. The sample waiting to be measured in the blank group is PBS buffer, and the sample waiting to be measured in the experimental group is the product prepared in Example 1, Comparative Example 4 or Comparative Example 5. (3) Add 200 μL of the prepared BCA working solution to each well, incubate at 37°C for 30 minutes, and then measure the OD562nm value. (4) Based on the standard curve formula, the protein concentration in the sample (y = 0.0008x + 0.016, R 2Calculate ( = 0.996). In Figure 6, from left to right, there are blank control, Comparative Example 4, Comparative Example 5 and Example 1. The darker the purple color, the higher the protein content in the product. From Figure 6, it is proved that after fermentation, the product obtained in Example 1 is significantly lighter in color and the protein content has significantly decreased compared with the products obtained in Comparative Example 4 and Comparative Example 5.

[0071] Effect Example 7 Stability Experimental method: The products obtained in Example 1, Comparative Example 4 and Comparative Example 5 were separately placed in 15 mL transparent glass bottles (the state of the products is shown in Figure 7). Each product was placed at room temperature, 60 °C, -20 °C and under light irradiation for one week, and the resistance of the products to high temperature, low temperature and light irradiation was observed, that is, the stable state of the products was considered. Refer to Figure 8 for the situation of the products. Result analysis: The products obtained in Comparative Example 4 and Comparative Example 5 are uniform milky white suspensions, both with the smell of fresh milk. The product obtained in Example 1 presents a light yellow transparent liquid, with a weak fermentation smell and no smell of fresh milk. After placing the above products for one week and observing the product form, the products obtained in Comparative Example 4 and Comparative Example 5 are in an unstable state, and a layering phenomenon occurs under room temperature, light irradiation and 60 °C, and white foam adheres to the bottle wall, especially significant at high temperature. The product obtained in Example 1 is relatively stable. After placing the above products for one week and observing the product form, under the condition of low temperature -20 °C, there are a large number of large white cotton-like precipitates in the products obtained in Comparative Example 4 and Comparative Example 5, and they cannot be redissolved at room temperature. Only a small amount of fine precipitates precipitate in the product obtained in Example 1, and the fine precipitates can be uniformly shaken and redissolved at room temperature.

[0072] As a result, milk is an unstable system containing suspensions, and phenomena such as layering and protein macromolecule aggregation precipitation are likely to occur during the storage process. The product obtained after fermenting milk by adopting the specific fermentation method of the present application significantly overcomes the above defects, and as the fermentation time is extended, the product is more stable. The product of Example 1 is less affected by light and temperature and is a relatively stable product system.

[0073] Effect Example 8 After the products prepared in Example 1, Comparative Example 4, and Comparative Example 5 were prepared, the pH values were measured, and the pH values of these products after one-week storage were also measured. The correlation results are shown in Table 8. JPEG2025519668000009.jpg45170 Weak acidity is crucial for skin properties. The pH value of healthy skin is about 4.2 - 6.5, with an average pH value of about 5.5, showing a weakly acidic state. In this state, the skin can maintain the balance of natural water and oil, has strong resistance, and is not prone to bacterial reproduction. The pH value on the skin surface affects the lipid components and hydration of the stratum corneum. Usually, appropriate adjustments are required to keep the skin's pH value within a reasonable range. As a result, the pH value of the product obtained in Example 1 is significantly lower than the pH values of the products obtained in Comparative Example 4 and Comparative Example 5, which is more beneficial for maintaining the slightly acidic environment of the skin.

[0074] Effect Example 9 Questionnaire survey: 3 types of products, number of people: 10 Using the products obtained in Example 1 and Comparative Examples 4 - 5 above as test products, then applying the test products to the inner side of the arm, comparing the products with relatively favorable states, scents, skin feelings, and absorption degrees, and the results of the population statistics are shown in Table 9 and Figure 9. From the figure, 100% of the people like the state of the product obtained in Example 1, 80% of the people like the scent of the product obtained in Example 1, and at the same time, 90% of the people think that the product obtained in Example 1 has relatively good skin feeling and absorption degree. JPEG2025519668000010.jpg31170

[0075] Effect Example 10 Application Formulation Manufacture a facial cream containing the product manufactured in Example 1 above, and its composition is shown in Table 10. JPEG2025519668000011.jpg61170

[0076] Effect Example 11 Improvement of Skin Barrier Effect 14 healthy adults were screened. According to the requirements, samples (the facial cream obtained in Example 10 of the effect) were used to test the moisture content and transepidermal water loss rate of the test area on the inner side of the forearm, and the skin barrier improvement effect was compared before using the sample (T0), 30 minutes after using the sample, and 1 hour later. Intra-group control: Compare the changes in the moisture content and transepidermal water loss rate of the test area at different time points before and after using the sample. Inter-group control: Using the experimental group and the blank control (the difference from Example 10 of the effect is only that the product obtained in Example 1 is replaced with an equal amount of water), compare the difference in the changes in the moisture content and transepidermal water outflow rate of the test area at different time points. 1) Volunteers should not apply any products on the arms two days and on the day before the test, and should not wash or touch the test site with water 1 - 3 hours before the visit. After coming to the laboratory, sit quietly in the thermostatic and humidity-controlled room for 20 minutes, and should not drink water and beverages during the test period. 2) Mark the test areas on the inner sides of the left and right arms with a skin marker for the arms. The area of the area is 3×3 cm 2 and the interval between the test areas is at least 1 cm. The sample application area and the blank control area are randomly distributed in the test areas, and ensure that the positions of all samples and blank areas reach statistical balance. 3) After the volunteers rested for 20 minutes, collect the transepidermal water loss rate baseline value (T0) data for each test area on the inner side of the arm, and test each area once. Clean each test area with a dry paper towel, collect the skin stratum corneum water content baseline value (T0) data, and perform 3 parallel tests for each area and take the average value. 4) Apply the sample to the corresponding test area according to the requirements. After 30 minutes, collect the data of the epidermal water loss rate and the skin moisture content value for each test area, and create relevant records, and record them as T30min. 5) After 1 hour, collect the data of the transepidermal water loss rate value and the skin water content value for each test area, and create relevant records, and record them as T1h. Refer to Table 11 for the test results of skin moisture content, and calculate the change values of skin moisture content at 30 minutes and 1 hour respectively compared with that at 0 minute. Refer to Table 11 and Figure 10 for the calculation results. JPEG2025519668000012.jpg33170 After using the facial cream obtained in Effect Example 10, the moisture content of the skin increased significantly (P = 0.001 < 0.05). Compared with the blank area, after using the facial cream obtained in Effect Example 10 for 30 minutes and 1 hour, the moisture content increased by 62.40% and 68.81% respectively (see the following calculation formula). The results indicate that the facial cream (Effect Embodiment 10) containing the product obtained in Example 1 has an ideal moisturizing effect.

[0077] The calculation formula for the increase rate of moisture content at different time points compared with the blank area is as follows. Change rate on the sample side = (Average test value at time Tx on the sample side - Average test value at time T0 on the sample side) / Average test value at time T0 on the sample side × 100% Change rate on the blank side = (Average test value at time Tx on the blank side - Average test value at time T0 on the blank side) / Average test value at time T0 on the blank side × 100% Increase rate of moisture content at different time points = Change rate on the sample side - Change rate on the blank side Here, T0 is before product use, and Tx is x hours after product use.

[0078] The test results of skin transdermal water loss rate are shown in Table 12, and calculate the change values of skin transdermal water outflow rate at 30 minutes and 1 hour respectively compared with that at 0 minute. The calculation results are shown in Table 12 and Figure 11. After using the facial cream manufactured in Effect Example 10 for 30 minutes and 1 hour, compared with the blank control area, the TEWL decreased significantly (P < 0.05). Compared with the blank area, after using the facial cream manufactured in Effect Example 10 for 30 minutes and 1 hour, the skin transdermal water loss rate decreased by 18.10% and 17.93% respectively (the calculation formula is the same as that for the water content improvement rate). This shows that the facial cream product containing the Bifidobacterium adecentis milk ferment produced in Example 1 has an ideal water retention effect. From the above experiments, it can be seen that the facial cream manufactured in Effect Example 10 has an ideal water replenishment and water retention effect, that is, it has a good skin barrier improvement effect.

[0079] Effect Example 12 Measurement of Lysosome Activity Lysosomes contain various hydrolytic enzymes and can decompose macromolecular substances. The lysosome activity and quantity indirectly reflect the cell's metabolic renewal ability. The higher the lysosome activity, the stronger the cell's metabolic ability. Under physiological pH conditions, the net charge of neutral red dye is almost 0, and it enters the cell by penetrating the cell membrane in a non-ion passive diffusion manner. The proton gradient in lysosomes makes the pH value in lysosomes lower than that in the cytoplasm, thereby charging neutral red and accumulating it in lysosomes, thereby detecting the lysosome body activity.

[0080] Test method: Spread skin fibroblasts on a 96-well plate and place 8000 cells in each well. After the cells adhere to the wall, set up a blank control group and a sample group. Add serum-free culture medium to the blank control group, and add serum-free culture medium containing 1.25% sample (products manufactured according to Examples 1 to 3 and Comparative Examples 3 to 4 above) to the sample group. After treatment for 24 hours, discard the cell culture medium, replace it with 200 μL of fresh serum-free culture medium, add 20 μL of neutral red and incubate for 2 hours. Then, remove the culture medium containing neutral red, wash twice with PBS, add 200 μL of neutral red detection decomposition solution, dissolve on a room temperature shaker for 10 minutes, and measure the A540 of each well. With the blank control as a reference, calculate the lysosome activity of the sample group. Lysosome activity % = (A540 of the sample group / A540 of the blank) × 100%. The results are shown in Table 13 and Figure 12. JPEG2025519668000014.jpg39170 The results indicated the following: Compared with the blank control group, the products obtained in Examples 1 to 3 of the present application significantly increased the lysosome activity after treating the cells. After treating the cells with the samples of Comparative Example 3 and Comparative Example 4, not only did it not improve the lysosome activity, but it also decreased the lysosome activity. Therefore, the products obtained in Examples 1 to 3 can promote the catabolism of lysosome nutrients and promote the cell metabolism renewal ability.

[0081] Effect Example 13 Cell migration experiment Spread skin fibroblasts on a 12-well plate and culture overnight in an incubator. Then, remove the culture medium, draw a line with a sterile gun head, and take a photo to record the state before treatment (marked as 0 hours in the figure). After that, add serum-free culture medium containing 1.25% sample (products manufactured according to Examples 1 to 3 and Comparative Example 4 above). After treatment for 24 hours, take a photo to record (marked as 24 hours in the figure). The results are shown in Figure 13. From the results of Fig. 13, after treating cells with the products prepared using Examples 1 to 3 of the present application, cell migration was significantly promoted. In particular, the product manufactured in Example 2 had the optimal effect of promoting cell migration. After treating cells with the product manufactured in Comparative Example 4, there was no significant cell migration phenomenon.

[0082] Finally, it should be noted that the term "comprising", "containing" or any other variation thereof is intended to include non-exclusive inclusion, such that a process, method, article or apparatus of a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article or apparatus.

[0083] As described above, although the present application has been disclosed by the description of specific embodiments, those skilled in the art should understand that various modifications, improvements or equivalents of the present application can be designed within the spirit and scope of the appended claims. These changes, improvements or equivalents should also be regarded as being included within the scope claimed in the present application.

Claims

1. A method for producing Bifidobacterium adolescentis fermented milk, comprising: The method includes the steps of inoculating Bifidobacterium adolescentis into a fermentation substrate, fermenting for 20 to 36 hours, sterilizing, and centrifuging the supernatant to complete the process; The method for producing Bifidobacterium adolescentis fermented milk is characterized in that the fermentation substrate contains milk and no sugar carbon source is added.

2. The fermentation substrate further comprises a nitrogen source; Preferably, the nitrogen source comprises L-cysteine ​​hydrochloride; The method for producing Bifidobacterium adolescentis fermented milk product according to claim 1, characterized in that the mass percentage of the nitrogen source in the milk is preferably 0.01% to 0.05%, more preferably 0.03% to 0.05%.

3. The fermentation substrate includes a sterilization procedure before use; Preferably, the method of sterilization is a high temperature sterilization method, When the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization temperature is 95 to 100°C; When the high-temperature sterilization method is used to sterilize the fermentation substrate, the sterilization time is 15 to 35 minutes, preferably 30 minutes; The method for producing Bifidobacterium adolescentis fermented milk product according to claim 1, further comprising a cooling operation to room temperature, preferably after the sterilization operation.

4. The method for producing Bifidobacterium adolescentis fermented milk product satisfies at least one of the following conditions: The Bifidobacterium adolescentis includes Bifidobacterium adolescentis model number BBF-06 manufactured by "Shandong Zhongke Jiayi Bioengineering Co., Ltd." and / or Bifidobacterium adolescentis model number SF-B40 manufactured by "Shandong Sunflower Bioengineering Co., Ltd."; The Bifidobacterium adolescentis is added in the form of a Bifidobacterium adolescentis liquid, and the viable cell count of the Bifidobacterium adolescentis liquid is 10 ６ ~10 １０ CFU / mL, preferably 10 ７ ~10 ９ CFU / mL, The number of Bifidobacterium adolescentis inoculated into the fermentation substrate per unit volume is 10 ５ ~10 ９ CFU / mL, preferably 10 ６ ~10 ８ The method for producing Bifidobacterium adolescentis fermented milk product according to claim 1, characterized in that the Bifidobacterium adolescentis fermented milk product is fermented in an amount of 1000 CFU / mL.

5. The method for producing Bifidobacterium adolescentis fermented milk product satisfies at least one of the following conditions: The fermentation culture is carried out in a constant temperature incubator, The fermentation culture time is 24 to 30 hours, The temperature of the fermentation culture is 37 to 43°C; The sterilization method is a high-temperature sterilization method. When the high-temperature sterilization method is used for the sterilization, the sterilization temperature is 95 to 100°C. When the high-temperature sterilization method is used for the sterilization, the sterilization time is 20 to 40 minutes, preferably 30 minutes. The rotation speed of the centrifuge is 4000 to 8000 rpm, preferably 4000 to 6000 rpm; The radius of the centrifuge is between 8 and 15 cm; The method for producing Bifidobacterium adolescentis fermented milk according to any one of claims 1 to 4, characterized in that the centrifugation time is 10 to 40 minutes, preferably 20 to 40 minutes.

6. The method further comprises the steps of filtering and recovering the filtrate after the centrifugation step, Preferably, the pore size of the filtration membrane used for the filtration is 0.22 to 0.8 μm, preferably 0.22 to 0.45 μm; The method for producing Bifidobacterium adolescentis milk fermentation product according to claim 1, further comprising a secondary sterilization and / or mixing with a preservative, preferably after the filtration.

7. The method for producing Bifidobacterium adolescentis fermented milk product satisfies at least one of the following conditions: The secondary sterilization method is a high-temperature sterilization method, When the secondary sterilization is performed using the high-temperature sterilization method, the temperature of the secondary sterilization is 95 to 100° C., When the secondary sterilization is performed using the high-temperature sterilization method, the secondary sterilization time is 20 to 40 minutes, preferably 30 to 40 minutes; The mixing temperature in the mixing step with the preservative is 50 to 80°C, preferably 70 to 80°C. The preservative comprises p-hydroxyacetophenone and / or 1,2-hexanediol; The method for producing a Bifidobacterium adolescentis fermented milk product according to claim 6, characterized in that, when the preservatives include the p-hydroxyacetophenone and the 1,2-hexanediol, the mass percentage of the p-hydroxyacetophenone in the filtrate obtained by the filtration is 0.2% to 0.6%, and the mass percentage of the 1,2-hexanediol in the filtrate obtained by the filtration is 0.5% to 2%, preferably, the mass percentage of the p-hydroxyacetophenone in the filtrate obtained by the filtration is 0.2 to 0.5%, and the mass percentage of the 1,2-hexanediol in the filtrate obtained by the filtration is 0.5 to 1%.

8. Bifidobacterium adolescentis milk fermentation product, A Bifidobacterium adolescentis milk fermentation product, characterized in that it is produced by the method for producing Bifidobacterium adolescentis milk fermentation product according to any one of claims 1 to 7.

9. The Bifidobacterium adolescentis milk fermentation product according to claim 8 is used as a product as it is, as an additive or as a base material in the manufacture of a skin external preparation, Preferably, the Bifidobacterium adolescentis fermented milk product is at least one of an antioxidant active ingredient, a skin barrier activity improving ingredient, an anti-aging active ingredient, and a relaxing active ingredient in the skin topical preparation; More preferably, the antioxidant active ingredient is an antioxidant active ingredient having a DPPH radical scavenging ability and / or a hydroxyl radical scavenging ability. More preferably, the skin barrier activity improving ingredient is a skin barrier activity improving ingredient having a hydrating and moisturizing effect, More preferably, the anti-aging active is an anti-aging active that increases skin elastin content, More preferably, the relaxing active ingredient has a relaxing active ingredient that inhibits hemolysis of red blood cells by SDS.

10. Use of the Bifidobacterium adolescentis fermented milk product as described in claim 8 as a product as it is, as an additive or as a base material in the manufacture of a skin topical preparation.

10. A skin topical preparation comprising: The Bifidobacterium adolescentis milk fermentation product according to claim 8, Preferably, the skin topical preparation further comprises at least one of an active ingredient, a preservative, a thickener, an oil, an emulsifier, and a solvent, More preferably, the active ingredient comprises at least one of a moisturizing active ingredient, a whitening active ingredient, an anti-inflammatory active ingredient, an anti-allergic active ingredient, and an antioxidant active ingredient; More preferably, the preservative comprises butanediol and / or phenoxyethanol; More preferably, the thickener comprises a carbomer; More preferably, the oil comprises at least one of jojoba oil, polydimethylsiloxane, squalane and cetanol; More preferably, the emulsifier comprises polysorbate-20 and / or sorbitan isostearate; More preferably, the solvent comprises deionized water; More preferably, the mass percentage of the preservative in the topical skin preparation is 12% to 15%, and even more preferably 13.03%; More preferably, the mass percentage of the thickener in the topical skin preparation is 0.5% to 1%, and even more preferably 0.6%. More preferably, the mass percentage of the oil or fat in the external skin preparation is 4% to 10%, and even more preferably 5% to 6%. More preferably, the mass percentage of the emulsifier in the topical skin preparation is 1% to 3%, and even more preferably 1.5% to 1.6%. Preferably, the skin preparation comprises a face cream, a mask, an essence or a toner, Preferably, the mass percentage of the Bifidobacterium adolescentis fermented milk product in the topical skin preparation is 5 to 99%, more preferably 60 to 99%.

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