A kind of preparation method and application of rosemary fermentation filtrate

Abstract

The invention provides a preparation method and application of rosemary fermentation filtrate. The invention firstly adopts ultra-low temperature means to quickly freeze rosemary stems and leaves, and adopts means such as high-speed grinding, ultra-high pressure nano homogenization and ultrafiltration to obtain rosemary homogenate filtrate; then the rosemary homogenate filtrate is pumped into a microbial fermentation process through a feed pump, rosemary is fermented and processed by microorganisms, and then subjected to multiple high-pressure homogenization, ultrasonic crushing, high-speed centrifugation, filtration and sterilization treatment steps to finally obtain rosemary fermentation filtrate. The whole process not only has mild reaction conditions and does not require additional addition of chemical reagents, but also reduces the large amount of loss and destruction of active ingredients in rosemary stems and leaves in the traditional high-temperature steaming and extraction process, and retains the original multiple natural active ingredients in the rosemary raw materials to the maximum extent, effectively solving the main defects of the traditional extraction method.

Description

Technical Field

[0001] The invention belongs to the technical field of cosmetics, and relates to a preparation method and application of rosemary fermentation filtrate, and in particular to a preparation method of rosemary fermentation filtrate with skin barrier repairing effect and application of the rosemary fermentation filtrate in cosmetics. Background Art

[0002] Rosemary is a natural spice with multiple biological activities such as antibacterial, anti-inflammatory, antioxidant, and antidepressant, and is widely used in the fields of food, cosmetics, spices, and medicine. Currently, common rosemary essential oils and hydrosol extracts generally use traditional high-temperature boiling water extraction, organic solvent extraction, supercritical extraction, and other processes. For example, patent CN103205314 discloses a method for preparing a rosemary extract, which uses supercritical CO2 fluid to separate the aroma components of rosemary, and further distills to obtain a rosemary extract. Patent CN103966027 discloses a method for preparing a rosemary compound extract, which crushes freeze-dried rosemary stems and extracts them with ethanol first, collects the rosemary ethanol extract after centrifugation, and uses supercritical CO2 to extract the precipitate after centrifugation to obtain a rosemary extract: finally, a rosemary compound extract is obtained after compounding.

[0003] However, the above-mentioned treatment method will not only seriously damage the effective active ingredients of rosemary, resulting in a low extraction rate, but also cause some organic solvent residues; under conditions such as light and heating, it is easy to volatilize and decompose and deteriorate, resulting in its low bioavailability, greatly limiting and weakening its huge application potential in the fields of cosmetics, skin care products, etc.

[0004] In addition, some patents disclose the use of biological fermentation methods to extract rosemary. For example, patent CN107951002 uses solid-state gradient temperature-variable fermentation and bioconversion technology of Polyporus umbellatus to improve the extraction efficiency and the flavor of rosemary extract. However, this method still has room for improvement in extraction efficiency, flavor improvement, and biological activity of the product. Summary of the invention

[0005] The present invention aims to solve the technical problems existing in the above-mentioned prior art and provide a preparation method and application of rosemary fermentation filtrate. The preparation method of the present invention firstly selects ultra-low temperature to quickly freeze rosemary stems and leaves, and then obtains rosemary homogenate filtrate by means of high-speed grinding, ultra-high pressure nano homogenization and ultrafiltration; then the rosemary homogenate filtrate is fed and pumped into the microbial fermentation process, and the rosemary is fermented and processed by microorganisms, and the rosemary is subjected to multiple high-pressure homogenization, ultrasonic crushing, high-speed centrifugation, filtration and sterilization treatment steps in sequence, and finally the rosemary fermentation filtrate is obtained.

[0006] The present invention provides a method for preparing rosemary fermentation filtrate, comprising the following steps:

[0007] S1) chopping fresh rosemary stems and leaves and freezing them at -180°C to -196°C for 10-20 minutes; then grinding and crushing with a grinder, filtering the obtained slurry powder through a 100-mesh filter / sieve to obtain rosemary fine slurry powder; keeping the temperature below 4°C during the grinding process; diluting the rosemary fine slurry powder with water and pumping it into a homogenizer to obtain rosemary fine slurry; centrifuging the rosemary fine slurry and filtering to obtain rosemary homogenized slurry filtrate;

[0008] S2) inoculating the microbial strain into the culture medium, taking samples every 2 hours to measure the absorbance at 600 nm, and then plotting a growth curve with the absorbance as the ordinate and the growth time as the abscissa; when OD600 is 9-12, preferably 10, obtaining a microbial fermentation seed liquid; the microbial strain is one of probiotic bacillus or yeast, and more preferably one of lactobacillus, pichia pastoris, bifidobacterium, saccharomyces cerevisiae, pasteurian yeast, candida, deep red yeast or rouxii yeast;

[0009] S3) transferring the microbial fermentation seed liquid to a fresh culture medium, culturing to the middle and late stages of the microbial logarithmic growth phase according to the growth curve drawn in S2, adding the rosemary homogenate filtrate as a feed to the microbial fermentation liquid within 2-3 hours, and continuing to culture until the OD600 stabilizes at 9-12, preferably 10, for 3-4 hours, to obtain the fermented rosemary fermentation stock liquid;

[0010] S4) The fermented rosemary fermentation liquid is subjected to high pressure homogenization, centrifugation, filtration or ultrasonic centrifugation treatment.

[0011] In step S1, it is further preferred to cut the fresh rosemary stems and leaves into small segments of 1-2 cm; during the grinding process, the heat generated by the crushing and grinding can be used to raise the temperature of the rosemary fine powder from a frozen state and maintain it at about 4°C, thereby gradually reviving and maintaining the activity of various natural ingredients in the rosemary fine powder.

[0012] In step S1, fresh rosemary stems and leaves can be frozen in a short time under ultra-low temperature conditions, reducing the volatilization and destruction of various active ingredients in the fresh rosemary stems and leaves; crushing and grinding under low temperature and high-pressure nano homogenization are conducive to the full utilization of various nutrients in the microbial fermentation process. Compared with high-temperature extraction, this method reduces the large-scale loss and destruction of active ingredients in rosemary stems and leaves during high-temperature cooking.

[0013] In step S2, inoculation needs to be carried out under a sterile environment, and the culture medium should be sterilized by autoclaving to avoid introducing other strains and adding other additional chemical reagents during the inoculation process, thereby being conducive to ensuring that the source of nutrients in the rosemary fermented liquid is clear and well documented. In step S2, a growth curve is drawn to explore the optimal growth conditions of microbial strains, which is conducive to accurately controlling the various process parameters of microbial fermentation and obtaining maximum benefits. In general, the incubation time required for OD600 to reach 10 is 1-2 days.

[0014] In step S3, under normal circumstances, the culture time required for OD600 to reach 10 is 1-7 days, preferably 1-3 days, and more preferably 1-2 days.

[0015] In step S4, activated carbon or macroporous resin may be added for decolorization and deodorization according to different requirements of cosmetic manufacturers.

[0016] Preferably, the liquid-to-solid ratio of the rosemary fine slurry powder diluted with water in step S1 is 1:5; the rosemary fine slurry in step S1 is centrifuged and filtered with a 0.45 micron filter membrane, and the temperature during the centrifugal filtration is maintained below 10°C. Specifically, the rosemary can be crushed more evenly by using a high-pressure nano homogenizer for high-pressure homogenization and then filtering through a 0.45 micron filter membrane, which greatly improves the utilization rate of intracellular nutrients, thereby achieving full fermentation of microorganisms, effectively improving the yield of active substances, and facilitating the retention of multiple active ingredients of rosemary; after low-temperature centrifugation, filtering with a 0.45 micron filter membrane intercepts the miscellaneous bacteria carried in the rosemary raw material, and is conducive to the smooth progress of the subsequent fermentation process.

[0017] Preferably, the grinding condition of the grinder in step S1 is 5000 RPM for 3-5 min, with an interval time of 3-5 min, and a total of 3 times of grinding; the homogenizer in step S1 is a high-pressure nano homogenizer, and the homogenization condition is 30000 psi, 100 mL / min maximum flow rate; the machine used for centrifugation in step S1 is a high-speed freezer, and the centrifugation condition is 12000 rpm, 4°C centrifugation for 30 min.

[0018] Preferably, in step S2, the lactobacillus is preferably Lactobacillus plantarum of China Industrial Microorganism Culture Collection Administration Center, with a preservation number of CICC 23941; the bifidobacterium is preferably Bifidobacterium bifidum of China Industrial Microorganism Culture Collection Administration Center, with a preservation number of CICC 10395; the yeast is preferably Saccharomyces cerevisiae of China Industrial Microorganism Culture Collection Administration Center, with a preservation number of CICC 1001.

[0019] Preferably, in step S2, the volume ratio of the culture medium to the inoculation volume of the microbial strain is preferably 100:(1-5);

[0020] In step S2, different microbial strains are cultured under different conditions:

[0021] When the microbial strain is probiotic bacteria, the culture medium used in step S2 is MRS culture medium, and the culture conditions are anaerobic sealing, 37°C, 200-250rpm, preferably 220rpm shaking culture on a shaker;

[0022] When the microbial strain is yeast, the culture medium used in step S2 is YPD culture medium, and the culture conditions are ventilation air pressurization, the pressure value is 0.05-0.1 MPa, 28-32°C, 200-250rpm shaking culture on a shaker; preferably 30°C, 220rpm shaking culture on a shaker. It should be noted that the fermentation tank must be made airtight first, and then pressurized in real time by pumping in air. By pressurizing the fermentation tank with air, not only can the pressure tank maintain a certain pressure, but also the carbon dioxide produced by yeast fermentation can be discharged.

[0023] Preferably, in step S3, the volume ratio of the culture medium to the microbial fermentation seed liquid is 100: (1-5).

[0024] Preferably, the total amount of rosemary homogenate filtrate added in step S3 is 20-40% of the total volume of the fermentation liquid; preferably, the total amount of rosemary homogenate filtrate added is 30% of the total volume of the fermentation liquid. The total volume of the fermentation liquid refers to the total volume of the liquid after the rosemary homogenate filtrate is added. Although there will be a volume change during the fermentation process, it can be ignored.

[0025] Preferably, different microbial strains are cultured under different conditions in step S3:

[0026] When the microbial strain is probiotic bacteria, the culture medium used in step S3 is MRS culture medium, and the culture conditions are 36-38° C. and 200-250 rpm stirring fermentation;

[0027] When the microbial strain is yeast, the culture medium used in step S3 is YPD culture medium, and the culture conditions are 28-32° C. and 200-250 rpm stirring fermentation.

[0028] Preferably, the specific conditions of the high-pressure homogenization are 30,000 psi, 100 mL / min maximum flow rate; the centrifugation conditions are 0-10°C, 8,000-10,000 rpm high-speed centrifugation for 20-30 min; the filter membrane used for the filtration is 0.22 microns; the processing temperature of step S4 is kept below 10°C. Keeping the low temperature can avoid the occurrence of further enzymatic fermentation and affect the aroma and color of the product; by filtering and sterilizing, the decomposition and destruction of the rosemary fermentation filtrate by miscellaneous bacteria is avoided, ensuring that the rosemary fermentation filtrate does not deteriorate or stratify.

[0029] The preparation method of rosemary fermentation filtrate disclosed in the present invention comprises the following steps: fully fermenting active components of rosemary homogenate filtrate by using microorganisms, and then sequentially subjecting the mixture to treatment steps such as rapid freezing, multiple high-pressure homogenization, high-speed centrifugation, filtration, etc., to obtain rosemary fermentation filtrate. Compared with traditional processes such as high-temperature boiling and water extraction, organic solvent extraction, etc., the rosemary fermentation filtrate of the present invention has high yield of active substances, strong activity, low production cost, and no organic solvent residue.

[0030] The present invention also provides use of the rosemary fermentation filtrate prepared by any of the above methods in preparing skin care products and cosmetics.

[0031] The present invention aims at at least this end. The present invention provides a preparation method of rosemary fermentation filtrate and an application example of the present invention in cosmetics. Microorganisms can be effectively used to perform biological fermentation on rosemary raw materials and reprocess biologically active components. The reaction conditions are mild and no additional chemical reagents need to be added. Various natural active components in the rosemary raw materials can be retained to the maximum extent. In addition, a large amount of active peptides, proteins, beta-glucans and other active substances unique to microorganisms are produced during the microbial fermentation process, which can significantly enhance the efficacy of the rosemary raw materials. Adding an appropriate amount of rosemary fermentation filtrate to cosmetics can effectively repair the barrier function of the skin, relieve sensitive skin problems such as redness, dry itching, tingling, and swelling, and restore the skin to a healthy state. The present invention has a good market prospect in the skin care product and cosmetics industries.

[0032] The preparation method and application of the rosemary fermentation filtrate of the present invention also include the use of HaCaT cell proliferation, migration experiment, skin barrier related protein gene expression detection, human irritation experiment and human body repair efficacy comprehensive evaluation and other technologies as efficacy detection means to ensure the quality and activity of the obtained rosemary fermentation filtrate. The rosemary fermentation liquid prepared by the present invention has a high content of active substances, strong activity, non-toxic and non-irritating, has a significant effect of repairing the skin barrier, and can significantly promote cell proliferation; the present invention also proposes an application method of the rosemary fermentation filtrate in cosmetics, and adding an appropriate amount of rosemary fermentation filtrate to cosmetics can repair the skin barrier, effectively alleviate the problems of sensitive skin such as redness, dry itching, tingling, redness and swelling, and keep the skin young and healthy.

[0033] Compared with the prior art, the invention is innovative in that:

[0034] 1. The preparation method of the rosemary fermentation filtrate has a simple process, can effectively utilize probiotic microorganisms to ferment rosemary raw materials and reprocess biologically active components, has mild reaction conditions, does not require the addition of additional chemical reagents, and a variety of natural active ingredients in the rosemary raw materials can be retained to the maximum extent. In addition, a large amount of active substances such as polypeptides, beta-glucans, and proteins unique to probiotic microorganisms are produced during the fermentation process of the probiotic microorganisms, which can significantly enhance the efficacy of the rosemary raw materials. The technical difficulty is small, energy-saving and labor-saving, and the production efficiency is high;

[0035] 2. The rosemary fermentation filtrate has a high content of active substances and strong activity, and has a significant effect on repairing the skin barrier. Adding this ingredient to cosmetics can promote the proliferation and migration of epidermal cells, repair the skin barrier, and effectively relieve sensitive skin problems such as redness, dryness, itching, tingling, and swelling, so as to keep the skin young and healthy.

[0036] 3. The rosemary fermentation filtrate is made from pure natural substances, with no additives, safe and non-irritating. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a technical roadmap for preparing rosemary fermentation filtrate of the present invention;

[0038] Figure 2 is the aroma sensory radar chart of Test Example 1 of the present invention;

[0039] Figure 3 is a comparison chart of moisture absorption rate of Test Example 2 of the present invention;

[0040] Figure 4 is a comparison chart of the moisture retention rate of Test Example 2 of the present invention; ↵

[0041] Figure 5 This is a graph showing the results of promoting HaCaT cell proliferation in Test Example 3 of the present invention;

[0042] Figure 6 ↵

[0043] Figure 7 This is a graph showing the effect of the gene expression of the skin barrier-related protein FLG in Test Example 5 of the present invention;

[0044] Figure 8 ↵

[0045] Fig. 9 This is a graph showing the effect of the skin barrier-related protein LOR gene expression level in Test Example 5 of the present invention;

[0046] Fig.10This is a graph showing the effect of the gene expression of the skin barrier-related protein CK-10 in Test Example 5 of the present invention. DETAILED DESCRIPTION

[0047] In the description of the present invention, it should be noted that, in the embodiments, those without specifying specific conditions, are carried out according to normal conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. Below in conjunction with the accompanying drawings and specific embodiments, the present invention is further described in detail, which is an explanation of the present invention rather than a limitation.

[0048] The embodiment of the present invention provides a preparation method of rosemary fermentation filtrate: firstly, ultra-low temperature is selected to quickly freeze rosemary stems and leaves, and then rosemary homogenate filtrate is obtained by means of high-speed grinding, ultra-high pressure nano homogenization and ultrafiltration; then the rosemary homogenate filtrate is pumped into microbial fermentation liquid through a feed pump, the active components of the rosemary homogenate filtrate are fully fermented by microorganisms, and then the rosemary fermentation filtrate is successively subjected to multiple high-pressure homogenization, ultrasonic crushing, high-speed centrifugation, filtration and sterilization treatment steps to finally obtain the rosemary fermentation filtrate.

[0049] In an embodiment of the present invention, the preparation method of YDP medium is as follows:

[0050] (1) Add 20.0 g peptone and 10.0 g yeast powder into 800 mL deionized water and stir evenly;

[0051] (2) Add 20.0 g glucose, 3.0 g potassium dihydrogen phosphate, and 1.5 g magnesium sulfate and stir evenly;

[0052] (3) Add ddH2O to make up to 1000 mL, adjust the pH to 5.8-6.0, sterilize by high pressure at 110-130°C for 15-30 min, and cool to room temperature to obtain the YPD medium.

[0053] In an embodiment of the present invention, the preparation method of MRS medium is as follows: (1) weigh 10.0 g peptone, 10.0 g beef extract, 5.0 g Yeast Extract (yeast powder), add to 800 mL ddH2O, and stir evenly;

[0054] (2) Add 20.0 g glucose, 5.0 g sodium acetate, 2.0 g potassium dihydrogen phosphate, 2.0 g ammonium citrate, 0.2 g MgSO4•7H2O, 0.05 g MnSO4•H2O, and 1.0 mL Tween 80 and stir well.

[0055] (3) Add ddH2O to make up to 1000 mL, adjust the pH to 6.2-6.5, sterilize under high pressure at 110-130°C for 15-30 min, and cool to room temperature to obtain the MRS medium.

[0056] The strains used in the present invention are purchased and are not preserved.

[0057] Example 1

[0058] S1, low temperature preparation of rosemary homogenate filtrate

[0059] Collect and weigh 1000 g of fresh rosemary stems and leaves, cut them into 1-2 cm small segments with scissors, put them into a freezing box, and then place them in a liquid nitrogen tank or a tunnel-type ultra-fast freezer, and quickly freeze them at -180°C to -196°C for 15 minutes.

[0060] Then, the frozen rosemary stem and leaf segments are crushed and ground using a water-cooled high-speed grinder and sieved to obtain rosemary fine slurry powder. The crushing and grinding conditions are: 5000 RPM crushing for 3-5 minutes, with an interval of 3-5 minutes in the middle, and crushing for a total of 3 times; this process can use the heat generated by grinding to raise the temperature of the rosemary fine slurry powder from the frozen state and maintain it at about 4°C; the sieving conditions are: after crushing and grinding, filter out through a 100-mesh filter / screen.

[0061] The filtered rosemary fine slurry powder was mixed with purified water at a solid-liquid ratio of 1:5 and diluted evenly, and then pumped into the Nanogenizer high-pressure nano-homogenizer for high-pressure homogenization to obtain rosemary fine slurry; the conditions for high-pressure homogenization were: 30,000 psi, 100 mL / min maximum flow rate. The obtained rosemary fine slurry was then centrifuged in a high-speed refrigerator at 12,000 rpm and 4°C for 30 minutes. After centrifugation, it was filtered with a 0.45 micron filter membrane to obtain a total of 5,500 mL of rosemary homogenate filtrate. The temperature of this process was always kept below 10°C.

[0062] S2, preparation of microbial fermentation seed solution

[0063] The strain used was Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), the deposit number is CICC 1001.

[0064] Under sterile conditions, inoculate brewer's yeast strains into the YPD medium after high-pressure sterilization, with the volume ratio of YPD medium to brewer's yeast being 100:1; seal and introduce air to pressurize, with the pressurization value being 0.1 MPa, and culture on a shaking table at 200-250 rpm at a temperature of 28-32°C for 1-2 days until OD600 = 10, to obtain a yeast seed solution;

[0065] In order to detect the change of OD value, during the culture process, 1 mL of bacterial solution was sampled every 2 hours to measure its absorbance at 600 nm, and then a growth curve was drawn with the absorbance as the ordinate and the growth time as the abscissa.

[0066] S3, Rosemary homogenate filtrate fed-batch microbial fermentation

[0067] Transfer an appropriate amount of yeast seed liquid to fresh YPD medium, with the inoculation volume ratio of medium to seed liquid being 100:1; according to the growth curve drawn by S2, culture in fresh medium to the middle and late logarithmic growth phase, use a pump to add rosemary homogenate filtrate to the microbial fermentation liquid within 2.5 h, with the final addition amount of rosemary homogenate filtrate being 30%, and then ferment at 28-32°C, 200-250rpm, and culture until OD600 stabilizes at 10 3-4 hours later.

[0068] S4, post-fermentation treatment

[0069] The fermented rosemary fermentation stock solution is subjected to high-pressure homogenization, centrifugation, filtration or ultrasonic centrifugation treatment.

[0070] The fermented rosemary fermentation liquid was placed in a tank and pumped into a Nanogenizer high-pressure nano-homogenizer to perform micro-jetting, impingement flow and high-pressure homogenization on the fermentation liquid; the specific conditions were: 30,000 psi, 100 mL / min maximum flow rate;

[0071] Then, the solid powder and the supernatant are obtained by centrifugation in a high-speed freezer. The centrifugation conditions are: 0-10°C, 8000-10000 rpm high-speed centrifugation for 20-30 min; the supernatant is the fermentation liquid with less impurities.

[0072] Finally, the fermentation liquid is filtered through a 0.22 micron filter membrane to obtain a rosemary fermentation filtrate with a high content of active substances. During the above treatment process, the temperature is always kept below 10°C.

[0073] The specific process is as follows Figure 1 shown.

[0074] Example 2

[0075] The difference between this embodiment and embodiment 1 is that in S3, the final addition amount of rosemary homogenate filtrate is 50%, and the other parts are the same as in embodiment 1.

[0076] Example 3

[0077] The difference between this embodiment and embodiment 1 is that in S3, the final addition amount of rosemary homogenate filtrate is 10%, and the other parts are the same as in embodiment 1.

[0078] Comparative Example 1

[0079] 1000 g of fresh rosemary stems and leaves were cut into pieces, mixed with purified water at a solid-liquid ratio of 1:5, added into a Soxhlet extractor, and refluxed in a high-temperature water bath for 3-8 h to obtain rosemary volatile oil.

[0080] Comparative Example 2

[0081] The difference between this comparative example and Example 1 is that S1 and S2 are lacking, only S3 and S4 are operated, and pure water is used instead of rosemary homogenate filtrate to obtain the microbial fermentation liquid.

[0082] Comparative Example 3

[0083] The difference between this comparative example and Example 1 is that in S1, the fresh rosemary stems and leaves are not rapidly frozen at -180°C to -196°C, but are directly chopped and ground and sieved, and S1 uses rosemary homogenate filtrate obtained by water extraction at 0~-10°C throughout the process.

[0084] Comparative Example 4

[0085] The difference between this comparative example and Example 1 is that in S1, the fresh rosemary stems and leaves are not rapidly frozen at -180°C to -196°C, but are directly crushed, ground and sieved after being chopped; and ethanol is used instead of purified water during the dilution process, and S1 uses rosemary homogenate filtrate obtained by extraction with ethanol at 0~-10°C throughout the process.

[0086] Comparative Example 5

[0087] The difference between this comparative example and Example 1 is that after the fresh rosemary stems and leaves are rapidly frozen at -180°C to -196°C, ethanol is used instead of pure water in the dilution process, and S1 uses rosemary homogenate filtrate obtained by extraction with ethanol at 0~-10°C throughout the process.

[0088] The compositions in Examples 1-3 and Comparative Examples 1-5 were subjected to an aroma sensory evaluation, and efficacy tests related to moisturizing, soothing, and skin repairing.

[0089] Specifically, several compositions in Examples 1-3 and Comparative Examples 1-5 were taken for aroma sensory evaluation, water absorption and moisturizing, soothing, human keratinocyte proliferation experiments, tests, and cell experiments such as inflammatory factors and inflammatory mediator detection, to detect and evaluate the water absorption and moisturizing, soothing, and skin repair-related effects of the rosemary fermentation liquid filtrate.

[0090] Test Example 1. Aroma sensory evaluation

[0091] Specifically, take an appropriate amount of filtrate or essential oil of Examples 1-3 and Comparative Examples 1-5, recruit 40 volunteers with rich experience in smelling fragrance, smell the samples of Examples 1-3 and Comparative Examples 1-5, and then perform aroma description evaluation and analysis;

[0092] (1) 40 volunteers with rich experience in smelling fragrance smelled the samples of Examples 1-3 and Comparative Examples 1-5 respectively, rested for 30 minutes, and then performed aroma description and evaluation;

[0093] (2) using the quantitative description profile method (5-point scale) (0 = no aroma; 1 = very weak aroma, just can be smelled; 2 = weak aroma; 3 = moderate and stable aroma; 4 = strong and prominent aroma; 5 = the strongest aroma);

[0094] (3) Produce corresponding aroma sensory radar chart.

[0095] like Figure 2 As shown, there are certain differences in the aroma and flavor profiles of Examples 1-3 and Comparative Examples 1-5. In Comparative Example 1, the aroma with the strongest intensity is rosemary, followed by green aroma and sweet aroma; in Comparative Example 2, the aroma with the strongest intensity is fermentation aroma, followed by sour aroma; in Comparative Example 3, the aroma with the strongest intensity is green aroma, and the intensity of other aroma indexes is relatively weak; in Comparative Example 4, the aroma with the strongest intensity is fermentation aroma, and the intensity of other aroma indexes is relatively weak; the aroma profile of Comparative Example 5 is very similar to that of Comparative Example 4, but the intensity of aroma indexes is stronger than that of Comparative Example 4;

[0096] The aroma profiles of Examples 1-3 are very similar, but the intensity of each aroma index of Example 3 is the weakest. The individual aroma indexes of Examples 1 and 2 are slightly different, but the overall scores are the same; the strongest aromas in Example 1 are fermentation aroma and sour aroma, followed by rosemary, in addition to green aroma and sweet aroma. It can be seen that Example 1 uses less materials than Example 2 and has better effects than Example 3; the aroma levels of Example 1 are richer and more intense than those of Comparative Examples 1-5.

[0097] Test Example 2. Determination of hygroscopicity and moisture retention

[0098] Accurately measure 1.0 mL of the filtrate or essential oil of Examples 1-3 and Comparative Examples 1-5 in a watch glass, place it in a desiccator (saturated ammonium sulfate solution, stored in a constant temperature water bath at 25°C) with a relative humidity of 86% to absorb moisture for 72 hours, weigh it every 12 hours and record it, and set two reference samples, glycerin and hyaluronic acid (HA), to calculate the moisture absorption rate.

[0099] After weighing the above moisture-absorbed sample, continue to put it into a sealed desiccator (take out a saturated ammonium sulfate solution with a relative humidity of 86%), leave it for 72 hours, weigh and record its weight every 12 hours, and then calculate the moisture retention rate.

[0100] Hygroscopicity and moisture retention test results are as follows Figure 3 , Figure 4It shows that Examples 1-2 and Comparative Example 5 all have certain moisture absorption and moisturizing properties, and the performances of the three are almost the same. Although they are not as good as HA in moisture absorption and moisturizing properties, they are significantly enhanced compared with Example 3, Comparative Examples 1-4 and glycerol.

[0101] Test Example 3. HaCaT cell proliferation test

[0102] Test method: The filtrates or essential oils prepared in Examples 1-3 and Comparative Examples 1-5 were diluted to 10% (v / v) with DMEM basal medium as the test solution. HaCaT cells in the logarithmic growth phase were cultured at 1×10 5 cells / mL, 100μL / well was inoculated in a 96-well cell culture plate, and cultured at 37℃ in a 5% CO2 incubator for 24h; the old culture medium was discarded, washed once with PBS solution, and then 100μL of the test solution was added to each well of the cell solution, fetal bovine serum (10% FBS) was added to the positive control group, and an equal volume of DMEM basal culture medium was added to the blank group, and cultured at 37℃ for 48h; 10μL CCK8 solution was added to each well, and incubated at 37℃ in the dark for 2h; 630nm was used as the reference wavelength, and the absorbance was measured at 450nm, and the measurement results were recorded. The specific test results are shown in Figure 5 .

[0103] , where: The absorbance of the sample group is As ,450nm- As ,630nm; the absorbance of the blank group was A 0 ,450nm- A 0 ,630nm.

[0104] Depend on Figure 5 It can be seen that the filtrates or essential oils obtained in Examples 1-3 and Comparative Examples 1-5 at a concentration of 10% are non-toxic to HaCaT cells and have different degrees of cell proliferation promoting effects; Examples 1-3 have a significant promoting effect on HaCaT cell proliferation, and the promoting effects of Examples 1 and 2 are the most significant, which is almost the same as the positive control group 10% fetal bovine serum. Comparative Examples 1-5 have a weaker effect on HaCaT cell proliferation, and the proliferation effect is slightly higher than that of the blank group. The cell proliferation effect of Comparative Example 5 compared with Example 1 and Comparative Example 4 compared with Comparative Example 3 are both reduced, because the residual organic solvent ethanol in the extraction process has an adverse effect on HaCaT cell proliferation.

[0105] Test Example 4. HaCaT cell migration assay

[0106] Test method: The filtrates or essential oils prepared in Examples 1-3 and Comparative Examples 1-5 were diluted to 10% (v / v) with DMEM basal medium as the test solution. HaCaT cells in the logarithmic growth phase were cultured at 5×10 4 cells / mL, 2 mL / well were inoculated in 6-well cell culture plates, cultured in a 37℃, 5% CO2 incubator for 24-48 h, and the cell morphology was observed under an inverted microscope; when a sparse HaCaT monolayer was formed at the bottom of the 6-well cell culture plate, the old culture medium was discarded, and the cells were washed once with PBS solution. A cross scratch was made in the center of the cells along the ruler with the tip of a micropipette to obtain four blank areas without cells in the upper, lower, left and right areas, and points were marked on the cross scratches in the four quadrants; then the cells were washed twice with PBS to remove cell debris; then 2 mL / well of the test solution was added to each well of the cells, fetal bovine serum (10% FBS) was added to the positive control group, and an equal volume of DMEM basal culture medium was added to the blank group, and the cells were cultured at a constant temperature of 37℃ for 48 h; the closure of the scratch area at the same position was observed under a microscope at 0 h, 12 h and 24 h after the scratching, and photographed and recorded. The migration rate was calculated as follows: the blank area of ​​the scratch area measured at 12 h or 24 h was subtracted from the blank area of ​​the scratch area at 0 h, and the difference was divided by 0 The blank area was measured after 12 h or 24 h, and the obtained percentage was the cell migration rate after 12 h or 24 h. Two parallel wells were set in each group of the scratch test, and the experiment was repeated three times independently. The specific test results are shown in Figure 6 .

[0107] Depend on Figure 6 It can be seen that Examples 1-2 have a significant promoting effect on HaCaT cell migration. The promoting migration effect of Example 2 is more significant after about 12 hours, and the promoting migration effect of Example 1 is most significant after 24 hours. The promoting migration effect of Example 1 is the most significant overall, and the promoting migration effect is slightly higher than that of the positive control group 10% fetal bovine serum; Comparative Examples 1-5 have weak or almost no promoting migration effect on HaCaT cells, and the effect is slightly higher than that of the blank group but the difference is not much.

[0108] Test Example 5. Detection of skin barrier-related protein gene expression

[0109] Test method: The filtrates or essential oils prepared in Examples 1-3 and Comparative Examples 1-5 were diluted to 10% (v / v) with DMEM basal medium as test solutions; then the following operations were performed: HaCaT cells with good growth status were taken at 1×10 6The density of cells / mL was 2 mL / well, and 6-well plates (37°C, 5% CO2, 24h) were plated. After observing the cell adhesion, the cells were gently rinsed once or twice with 1 mL PBS. Replace with 2 mL of each test solution; DMEM complete medium was used as a blank control, and DMEM complete medium containing WY-14643 (50 μM) was used as a positive control; after culturing under the above conditions (37°C, 5% CO2, 24h), total RNA was extracted, and cDNA was synthesized using a cDNA reverse transcription kit. Finally, RT-PCR was used to detect the expression of skin barrier-related protein genes (filament aggregate protein, FLG; transmembrane protein 1, CLDN 1; loricrin, LOR; cytokeratin-10, CK-10). The detection steps refer to the instructions of the kit. Adopt The data were processed by the qPCR experimental data processing method. For specific experimental results, see Figure 7-Figure 10 .

[0110] Specifically, HaCaT cells in the logarithmic growth phase were obtained, trypsinized, and then prepared into a cell suspension using DMEM complete medium containing 10% fetal bovine serum and 1% double antibody. The cell concentration was 1×10 6 / mL, inoculated in 6-well plates, 2mL per well, and cultured in a constant temperature incubator at 37°C and 5% CO2. After 24 hours of adaptive culture, the original culture medium was poured out and the cells were gently rinsed once or twice with 1mL PBS. Then, the filtrate or essential oil obtained in the above Examples 1-3 and Comparative Examples 1-2 was diluted to 10% (v / v) with DMEM basal culture medium as the test solution, 2 mL of each test solution was drawn and added to the wells of the 6-well plate; DMEM complete culture medium was used as a blank control, and DMEM complete culture medium containing WY-14643 (50 μM) was used as a positive control; the 6-well plate was returned to the constant temperature incubator and cultured for 24 hours under the above conditions (37°C, 5% CO2).

[0111] After 24 hours, the 6-well cell culture plate was taken out of the incubator and the cell RNA was extracted according to the instructions of the TRNzol Universal Total RNA Extraction Reagent. RNA reverse transcriptase was then taken and mixed with the RNA product according to the proportion, and cDNA was synthesized using a PCR instrument. The specific cDNA operation process is shown in Table 1.

[0112]

[0113] The cDNA was diluted 10 times with RNase-Free ddH2O and used as a template. The upper and lower primers of skin barrier-related protein genes: Filaggrin, Claudin 1, Loricrin, and CK-10 were taken. The primer sequences are shown in Table 2.

[0114]

[0115] Mix the enzyme solution, upper primer, and lower primer at a ratio of 5 μL:0.5 μL:0.5 μL, then add 4 μL of cDNA and 6 μL of premix solution to a 96-well plate in sequence, and perform qPCR reaction according to the steps in Table 3 below.

[0116]

[0117] qPCR experimental data processing formula (using Law):

[0118] A=2^(Cq internal reference - Cq gene): A represents the gene expression level of the blank control, and Cq is the original value.

[0119] B=2^(Cq internal reference - Cq gene); where B represents the gene expression level of the test sample and Cq is the original value.

[0120] Relative expression of sample gene = average value of B / average value of A

[0121] The data were analyzed using the tested samples as the horizontal axis and the relative gene expression as the vertical axis using data processing software (prism) to obtain the graph. For specific test results, see Figure 7-Figure 10 .

[0122] Depend on Figure 7-Figure 10 It can be seen that the filtrates or essential oils prepared in Examples 1-3 of the present invention and Comparative Examples 1-5 promoted the expression of skin barrier-related protein genes (FLG, CLDN 1, LOR, CK-10) to varying degrees, and the rosemary fermentation filtrate prepared in Example 1 had the most significant comprehensive promotion effect, which was higher than the positive control group at the same level. It can be seen that the rosemary fermentation filtrate prepared in Example 1 of the present invention can promote the efficient expression of skin barrier-related genes, thereby exerting its effect of repairing the skin barrier.

[0123] Test Example 6

[0124] Based on the above efficacy evaluation results, the rosemary fermentation filtrate described in Example 1 and Comparative Example 5 was selected and added to basic formulas such as cosmetic essences, essences, and essence creams at an addition amount of 10% to prepare a rosemary fermentation liquid filtrate with water absorption, moisturizing, and skin repair related effects. The filtrate can be used as an additive for applying cosmetic repair essences, repair lotions, repair toners, repair creams, and repair masks, and human safety and repair efficacy tests were carried out.

[0125] (1) Human patch test

[0126] Experimental method: 40-50 volunteers were selected to fill in a voluntary informed consent form. The test samples obtained according to the methods of Example 1 and Comparative Example 5 were added to the basic cosmetic formula at a mass content of 10% to prepare an essence. The obtained essence composition was: 77.15% water, 10% rosemary fermentation filtrate, 5% glycerol, 4% 1,3-butanediol, 2% 1,2-pentanediol, 0.5% panthenol, 0.5% squalane, 0.5% p-hydroxyacetophenone, 0.3% ammonium acryloyldimethyltaurate / VP copolymer, 0.05% disodium EDTA, etc. Then take about 0.020~0.025ml of essence and apply it to the small chamber of the spot tester, and apply it to the back or curved forearm of the subject with special tape. Remove the spot tester after 24 hours. If there is any remaining sample, gently wipe it off with a tissue. Observe the skin reaction 0.5 hour, 24 hours, and 48 hours after removing the spot tester, and record the results according to the skin reaction grading standards in the "Technical Specification for Safety of Cosmetics" (2015).

[0127] The results of the human skin patch test in Comparative Example 5 showed that 5 out of 40 people had grade 1 or above reactions, indicating that the residual organic solvent ethanol in the rosemary extraction process in Comparative Example 5 had an adverse effect on the skin; at the same time, the results of the human skin patch test in Example 1 showed that 0 out of 48 people had grade 1 or above reactions, indicating that the rosemary fermentation filtrate of Example 1 has satisfactory safety when added to cosmetics.

[0128] (2) Skin barrier repair effect face detection experiment

[0129] Test method: 35 volunteers with skin barrier problems such as slight facial damage, redness, swelling, redness, dryness, tingling, itching, desquamation and tightness were selected, half of them were male and half were female, aged 20-60 years old, and they filled in the voluntary informed consent form, then cleaned their faces with cleansing products, sat quietly in a laboratory at a temperature of 21±1℃ and 50±10% RH for at least 20 minutes and remained relaxed; the researchers evaluated the acne grade of the subjects and counted the number of acne, papules and closed comedones using VISIA and Corneometer The CM825 and TMHex instruments were used to measure the skin moisture content, transepidermal water loss (TEWL), skin surface pH, skin erythema index, skin brightness, smoothness and other indicators of the test parts of the subjects, which were calculated as the basic values. The products obtained according to the methods of Example 1 and Comparative Example 5 were used, and the essence was prepared according to the formula in the human patch test in this section (1). The subjects used it for 28 consecutive days, once in the morning and once in the evening after cleansing their faces. After 14 and 28 days, the subjects were revisited, and the staff checked the subjects' product usage and collected the remaining products. When the subjects were tested, they first cleaned their faces with a cleansing product, sat quietly in a laboratory at a temperature of 21±1°C and 50±10% RH for at least 20 minutes, and remained relaxed. The subjects completed the product use questionnaire during the rest period. The researchers were required to first conduct an intuitive assessment of the skin condition of the subjects, and then operate the VISIA, CorneometerCM825 and TMHex instruments to measure the relevant indicators of the test parts of the subjects; analyze the data and draw conclusions.

[0130] The test results show that after 28 days of using the product containing the rosemary fermentation filtrate of Example 1, the improvement rate of skin barrier conditions (skin moisture content, transepidermal water loss (TEWL), skin surface pH, skin erythema index, skin brightness, smoothness and other indicators) can generally reach about 10-20% (obtained according to the average of the measured values ​​after product use - the average of the basal values ​​before product use); and after 28 days of using the product containing the rosemary fermentation filtrate of Comparative Example 5, although the skin barrier conditions of most volunteers were improved, some volunteers experienced adverse reactions such as facial redness. Therefore, Example 1 has the best comprehensive effect.

[0131] In summary, the rosemary fermentation filtrate with repairing effect described in the present invention is highly active, non-toxic and non-irritating, and has significant repairing effect; the application method in cosmetics, adding an appropriate amount of rosemary fermentation filtrate to cosmetics can repair the skin barrier, effectively alleviate sensitive skin problems such as redness, dry itching, tingling, redness and swelling, restore the skin to a young and healthy state, and achieve the effect of skin repair.

[0132] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing rosemary fermentation filtrate, characterized in that, For the following steps: S1) chopping fresh rosemary stems and leaves and freezing them at -180°C to -196°C for 10-20 minutes; then grinding and crushing with a grinder, filtering the obtained slurry powder through a 100-mesh filter / sieve to obtain rosemary fine slurry powder; keeping the temperature below 4°C during the grinding process; diluting the rosemary fine slurry powder with water and pumping it into a homogenizer to obtain rosemary fine slurry, with a solid-liquid ratio of 1:5; centrifuging the rosemary fine slurry and filtering it with a 0.45-micron filter membrane, keeping the temperature below 10°C during the centrifugal filtration process, to obtain rosemary homogenized slurry filtrate; S2) inoculating the microbial strain into the culture medium, taking samples every 2 hours to measure the absorbance at 600 nm, and then plotting a growth curve with the absorbance as the ordinate and the growth time as the abscissa; when the OD600 is 9-12, obtaining a microbial fermentation seed liquid; the microbial strain is Saccharomyces cerevisiae; S3) transferring the microbial fermentation seed liquid to a fresh culture medium, culturing to the middle and late stages of the microbial logarithmic growth phase according to the growth curve drawn in S2, adding the rosemary homogenate filtrate to the microbial fermentation liquid within 2-3 hours, and continuing to culture until the OD600 stabilizes at 9-12, 3-4 hours later, to obtain the fermented rosemary fermentation stock liquid; the microbial strain is Saccharomyces cerevisiae; S4) performing high pressure homogenization, centrifugation, filtration or ultrasonic centrifugation treatment on the fermented rosemary fermentation stock solution; The brewer's yeast is the brewer's yeast of China Industrial Microbiological Culture Collection Administration Center, with the collection number of CICC 1001; The grinding conditions of the grinder in step S1 are 5000 rpm for 3-5 min, with an interval of 3-5 min, and a total of 3 times of grinding; the homogenizer in step S1 is a high-pressure nano homogenizer, and the homogenization conditions are 30000 psi, 100 mL / min maximum flow rate; the centrifugal machine in step S1 is a high-speed freezer, and the centrifugal conditions are 12000 rpm, 4°C for 30 min; In step S2, the volume ratio of the culture medium to the inoculation volume of the microbial strain is 100: (1-5); The culture medium used in step S2 is YPD culture medium, and the culture conditions are air pressurization, the pressure value is 0.05-0.1 MPa, 28-32° C., 200-250 rpm shaking culture on a shaker; In step S3, the volume ratio of the culture medium to the microbial fermentation seed liquid is 100: (1-5); The total amount of rosemary homogenate filtrate added in step S3 is 20-40% of the total volume of the fermentation liquid; The culture medium used in step S3 is YPD culture medium, and the culture conditions are 28-32° C. and 200-250 rpm stirring fermentation.

2. The preparation method according to claim 1, characterized in that: The specific conditions of the high-pressure homogenization in step S4 are 30,000 psi, 100 mL / min maximum flow rate; the centrifugation conditions are 0-10°C, 8,000-10,000 rpm high-speed centrifugation for 20-30 min; the filter membrane used for filtration is 0.22 microns; the processing temperature of step S4 is maintained below 10°C.

3. Use of the rosemary fermentation filtrate prepared according to any one of claims 1-2 in the preparation of cosmetics.

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