A fermented rose extract, its preparation method, and its application in the preparation of skincare products.

Abstract

This invention discloses a fermented rose extract, its preparation method, and its application in the preparation of skincare products. The fermented rose extract is a fermentation product obtained by fermenting rose petals using *Bifidobacterium longum* ZJ1 as the strain. *Bifidobacterium longum* ZJ1 was deposited at the China Microbiological Culture Collection Center on June 26, 2019, with accession number CGMCC No. 18032. This invention screened multiple *Bifidobacterium longum* strains and found that the fermentation product of *Bifidobacterium longum* ZJ1 has an inhibitory effect on melanin. Fermenting rose petals with *Bifidobacterium longum* ZJ1 significantly enhances the dissolution and conversion of beneficial components in rose petals, exhibiting a synergistic effect with the probiotic fermentation product components in the fermentation filtrate.

Description

Technical Field

[0001] This invention relates to the field of skincare product preparation technology, specifically to a fermented rose extract, its preparation method, and its application in the preparation of skincare products. Background Technology

[0002] Bulgarian roses belong to the Rosaceae family and the Rosa genus. Their petals contain polysaccharides, terpenes, phenolic substances, and flavonoids. Bulgarian roses are an indispensable and very expensive raw material for making high-end perfumes, skincare cosmetics, hair care cosmetics, and beauty cosmetics. Studies have shown that rose extracts have significant inhibitory effects on bacteria, including Salmonella, Escherichia coli, Bacillus subtilis, and Bacillus thuringiensis; they also have cleansing, tightening, and lubricating effects on the skin; and can inhibit facial flushing caused by ultraviolet radiation from sunlight. Furthermore, they effectively inhibit melanin biosynthesis and promote epidermal moisturizing and barrier repair.

[0003] Prebiotics are substances that selectively stimulate the growth and reproduction of beneficial bacteria in the gut, enhancing their metabolism and promoting overall health. Over the past decade, scholars both domestically and internationally have conducted extensive research on prebiotics, discovering that they improve the gut microbiota, increase the number of beneficial bacteria, promote mineral absorption, regulate lipid metabolism, strengthen the immune system, and reduce the risk of cancer and cardiovascular diseases. Bifidobacterium longum is a type of probiotic and has been used in food and pharmaceuticals; however, there are no reports on the application of Bifidobacterium longum fermentation products in skincare products. Summary of the Invention

[0004] This invention, through screening multiple Bifidobacterium longum strains, discovered that the fermentation product of Bifidobacterium longum ZJ1 has the effect of inhibiting melanin. Fermenting rose petals with Bifidobacterium longum ZJ1 significantly enhances the dissolution and conversion of beneficial components in rose petals, and has a synergistic effect with the probiotic fermentation product components in the fermentation filtrate.

[0005] The present invention achieves the above objectives through the following technical solutions:

[0006] The primary objective of this invention is to provide a fermented rose extract, which is a fermentation product obtained by fermentation using *Bifidobacterium longum* ZJ1 as the strain and rose as the substrate. *Bifidobacterium longum* ZJ1 was deposited at the China Culture Collection Center on June 26, 2019, with accession number CGMCC No. 18032.

[0007] Furthermore, the rose is either rose petals or rose hydrosol.

[0008] Furthermore, the fermentation products include filtrate and lysate.

[0009] The second objective of this invention is to provide an application of the above-mentioned fermented rose extract in the preparation of whitening and anti-aging skin care products.

[0010] A third objective of this invention is to provide a method for preparing the above-mentioned fermented rose extract, comprising the following steps:

[0011] MRSL medium was prepared using rose as a substrate. A single colony of activated Bifidobacterium longum ZJ1 was picked and inoculated into the MRSL medium and cultured anaerobicly to obtain a primary seed culture. The primary seed culture was then inoculated into fresh MRSL medium and cultured anaerobically to obtain a seed culture.

[0012] The seed culture was inoculated into fresh MRSL medium, and after adjusting the pH to 5.7-6.7, anaerobic fermentation was carried out to obtain a Bifidobacterium longum ZJ1 rose petal fermentation broth, wherein the pH of the rose petal fermentation broth was 4.0-5.0.

[0013] Take the rose petal fermentation broth, centrifuge to obtain the supernatant, adjust the pH to 7.2-7.4, filter to remove bacteria and obtain the filtrate; take another rose petal fermentation broth, sonicate at low temperature, centrifuge to collect the supernatant, filter to remove bacteria and obtain the lysate; use the filtrate and lysate as fermented rose extract.

[0014] Furthermore, the MRSL medium comprises 20 g / L glucose, 10.0 g / L peptone, 8.0 g / L beef extract, 4.0 g / L yeast extract, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, 1.0 g / L Tween 80, and 0.5 g / L L-cysteine.

[0015] Furthermore, the inoculation amount of the primary seed solution is 2% by volume.

[0016] Furthermore, the inoculation amount of the seed liquid is 2%-5% by volume.

[0017] The beneficial effects of this invention are as follows: Many active ingredients in rose petals exist within the cells. When rose petals are fermented using fermentation bacteria, the microorganisms secrete various enzymes during fermentation, which can break down the cell walls and release effective ingredients such as flavonoids, polyphenols, and polysaccharides from within the cells, thereby significantly enhancing the anti-inflammatory, whitening, and free radical scavenging abilities of rose petals. In addition, fermentation can also degrade the large molecules of active substances in rose petals into smaller molecules, which is beneficial for the absorption of effective ingredients. Furthermore, compared with traditional organic reagent extraction, fermentation extraction is more environmentally friendly and safer.

[0018] (1) The rose petal fermentation extract of the present invention contains high levels of total phenols, free amino acids, flavonoids, anthocyanins, and has high antioxidant activity.

[0019] (2) The rose petal fermentation extract of the present invention significantly enhances the dissolution and transformation of beneficial components of rose petals through fermentation with Bifidobacterium longum ZJ1, and has a synergistic effect with the probiotic fermentation product components in the fermentation filtrate, thereby having significant antioxidant and whitening effects.

[0020] (3) The rose petal fermentation extract described in this invention can be used in a variety of cosmetics. It can effectively promote skin renewal, reduce melanin synthesis, whiten skin, effectively remove free radicals, improve the physiological function of dermal tissue, and has anti-aging effects. It is also highly safe.

[0021] (4) The rose petal fermentation extract of the present invention contains important secondary metabolites of rose such as citronellol, geraniol, phenylethyl alcohol and their esters, which have an aromatic odor and do not require the addition of fragrances or other chemical components, thus ensuring the safety of the product for the human body. Detailed Implementation

[0022] The present application will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0023] 1. Materials

[0024] The Bifidobacterium longum ZJ1, which was derived from the intestines of long-lived elderly people, was named Bifidobacterium longum ZJ1 and was deposited at the China Culture Collection Center for Microbial Cultures on June 26, 2019, with the accession number CGMCC No. 18032.

[0025] The Bifidobacterium longum ZJ1 used in the following examples was provided by Zhuoyuan Health Technology Co., Ltd.

[0026] The Bifidobacterium longum used in the following comparative examples was purchased from BNCC (Beina Biotechnology), with strain number BNCC232112 and batch number 221118.

[0027] The rose hydrosol used in the following examples is from Hefei Kadir Biotechnology Co., Ltd.

[0028] The rose petals used in the following examples are from Hefei Kadir Biotechnology Co., Ltd., and are Bulgarian Damask roses. The rose petals can be dried roses, fresh roses, or a combination of both.

[0029] The TPY solid culture medium formulation described in the following examples is as follows: per 1L of deionized water, it contains 5.0g glucose, 10.0g hydrolyzed casein, 5.0g soybean peptone, 2.0g yeast extract, 2.0g dipotassium hydrogen phosphate, 0.5g magnesium chloride, 0.25g zinc sulfate, 0.15g calcium chloride, 0.0001g ferric chloride, 1.0g Tween 80, 0.5g L-cysteine, and 20.0g agar, with a pH of 6.5 ± 0.1. It is then autoclaved at 121℃ for 15-20 minutes and ready for use.

[0030] The MRSL culture medium formulation described in the following examples is as follows: per 1L of deionized water, there are 20g of glucose, 10.0g of peptone, 8.0g of beef extract, 4.0g of yeast extract, 2.0g of dipotassium hydrogen phosphate, 2.0g of diammonium hydrogen citrate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.04g of manganese sulfate, 1.0g of Tween 80, and 0.5g of L-cysteine. The pH is 6.2 ± 0.5. The medium is then autoclaved at 118℃ for 15-20 minutes and ready for use.

[0031] 2. Method

[0032] 2.1 Preparation of Examples and Comparative Examples

[0033] Example 1

[0034] (1) Preparation of rose petal culture medium

[0035] By weight, 19 parts water are added to every 1 part of freshly collected Bulgarian rose petals, the mixture is blended in a blender, and then filtered through a 200-mesh filter to obtain rose petal filtrate.

[0036] MRSL medium was prepared using rose petal filtrate as a substrate, with the following ingredients added to each 1L of rose petal filtrate: 20g glucose, 10.0g peptone, 8.0g beef extract, 4.0g yeast extract, 2.0g dipotassium hydrogen phosphate, 2.0g diammonium hydrogen citrate, 5.0g sodium acetate, 0.2g magnesium sulfate, 0.04g manganese sulfate, 1.0g Tween 80, and 0.5g L-cysteine.

[0037] (2) Preparation of seed liquid

[0038] Take a glycerol cryopreservation tube of *Bifidobacterium longum* ZJ1 stored at -80℃, thaw it at room temperature, and then, under aseptic conditions, use an inoculation loop to pick up the bacteria and streak it onto a TPY solid medium plate. Incubate at 37℃ in an anaerobic incubator (or anaerobic culture bag) for 24-28 hours. Observe the colony morphology on the plate to confirm that it is *Bifidobacterium longum* and uncontaminated. In a clean bench, pick a single colony from the activated TPY solid medium plate and inoculate it into MRSL medium. Incubate at 37℃ in an anaerobic incubator (or anaerobic culture bag) at 100-120 rpm (or let it stand) for 12-16 hours to obtain a primary seed culture. Then, inoculate the primary seed culture into fresh MRSL medium at an inoculation rate of 2.0% (v / v) and incubate at 37℃ in an anaerobic incubator (or anaerobic culture bag) at 100-120 rpm (or let it stand) for 12-16 hours. When OD... 600 Seed liquid is obtained when the concentration reaches 2.5-3.5.

[0039] (3) Fermentation

[0040] Inoculate the seed culture into MRSL medium at an inoculation rate of 2%-5% (v / v), adjust the pH to 5.7-6.7, and anaerobic ferment at 37℃ for 16-24 hours to obtain the fermentation broth of Bifidobacterium longum ZJ1 rose petals. At this time, the pH of the fermentation broth is 4.0-5.0.

[0041] (4) Preparation of rose petal fermentation product filtrate

[0042] Take the fermentation broth of Bifidobacterium longum ZJ1 rose petals, centrifuge at 10000g for 25 minutes at 4℃, collect the supernatant, adjust the pH to 7.2-7.4 to obtain the fermentation filtrate, filter it with a disposable filter with a pore size of 0.22μm to remove bacteria, and then store it in a refrigerator at -20℃ for later use.

[0043] (5) Preparation of rose petal fermentation product lysate

[0044] Take the fermentation broth of Bifidobacterium longum ZJ1 rose petals and sonicate it at low temperature 4-5 times. The sonication procedure is the same for each cycle: sonicate for 4 seconds, then sonicate again for 5 seconds as one cycle, and sonicate for 25-30 cycles. The sonication power range is 150-225W. Then centrifuge at 10000g for 20 minutes at 4℃, collect the supernatant to obtain the lysate, filter it with a disposable filter with a pore size of 0.22μm to remove bacteria, and then store it in a refrigerator at -20℃ for later use.

[0045] Example 2

[0046] (1) Preparation of rose hydrosol culture medium

[0047] MRSL medium was prepared using Bulgarian rose hydrosol (obtained by rose distillation) as a substrate. The following ingredients were added to each 1 L of hydrosol: 20 g glucose, 10.0 g peptone, 8.0 g beef extract, 4.0 g yeast extract, 2.0 g dipotassium hydrogen phosphate, 2.0 g diammonium hydrogen citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1.0 g Tween 80, and 0.5 g L-cysteine.

[0048] (2) Preparation of seed liquid

[0049] Take a glycerol cryopreservation tube of *Bifidobacterium longum* ZJ1 stored at -80℃, thaw it at room temperature, and then, under aseptic conditions, use an inoculation loop to pick up the bacteria and streak it onto a TPY solid medium plate. Incubate at 37℃ in an anaerobic incubator (or anaerobic culture bag) for 24-28 hours. Observe the colony morphology on the plate to confirm that it is *Bifidobacterium longum* and uncontaminated. In a clean bench, pick a single colony from the activated TPY solid medium plate and inoculate it into MRSL medium. Incubate at 37℃ in an anaerobic incubator (or anaerobic culture bag) at 100-120 rpm (or let it stand) for 12-16 hours to obtain a primary seed culture. Then, inoculate the primary seed culture into fresh MRSL medium at an inoculation rate of 2.0% (v / v) and incubate at 37℃ in an anaerobic incubator (or anaerobic culture bag) at 100-120 rpm (or let it stand) for 12-16 hours. When OD... 600 Seed liquid is obtained when the concentration reaches 2.5-3.5.

[0050] (3) Fermentation

[0051] Inoculate the seed culture into MRSL medium at an inoculation rate of 2%-5% (v / v), adjust the pH to 5.7-6.7, and anaerobic ferment at 37℃ for 16-24 hours to obtain the fermentation broth of Bifidobacterium longum ZJ1 rose hydrosol. At this time, the pH of the fermentation broth is 4.0-5.0.

[0052] (4) Preparation of rose hydrosol fermentation product filtrate

[0053] Take the fermentation broth of Bifidobacterium longum ZJ1 rose hydrosol, centrifuge at 10000g for 25 minutes at 4℃, take the supernatant, adjust the pH to 7.2-7.4 to obtain the fermentation filtrate, filter it with a disposable filter with a pore size of 0.22μm to remove bacteria, and then store it in a refrigerator at -20℃ for later use.

[0054] (5) Preparation of rose hydrosol fermentation product lysate

[0055] Take the fermentation broth of Bifidobacterium longum ZJ1 rose hydrosol and sonicate it at low temperature 4-5 times. The sonication procedure is the same for each cycle: sonicate for 4 seconds, then sonicate again for 5 seconds as one cycle, and sonicate for 25-30 cycles. The sonication power range is 150-225W. Then centrifuge at 10000g for 20 minutes at 4℃, collect the supernatant to obtain the lysate, filter it with a disposable filter with a pore size of 0.22μm to remove bacteria, and then store it in a refrigerator at -20℃ for later use.

[0056] Comparative Example 1

[0057] (1) Preparation of rose petal culture medium

[0058] By weight, 19 parts water are added to every 1 part of freshly collected Bulgarian rose petals, the mixture is blended in a blender, and then filtered through a 200-mesh filter to obtain rose petal filtrate.

[0059] MRSL medium was prepared using rose petal filtrate as a substrate, with the following ingredients added to each 1L of rose petal filtrate: 20g glucose, 10.0g peptone, 8.0g beef extract, 4.0g yeast extract, 2.0g dipotassium hydrogen phosphate, 2.0g diammonium hydrogen citrate, 5.0g sodium acetate, 0.2g magnesium sulfate, 0.04g manganese sulfate, 1.0g Tween 80, and 0.5g L-cysteine.

[0060] (2) Preparation of unfermented rose petal filtrate

[0061] Without inoculating the rose petal culture medium for fermentation, the rose petal filtrate with added MRSL medium was centrifuged at 10000g for 25 minutes at 4℃. The supernatant was collected, and the pH was adjusted to 7.2-7.4 to obtain the filtrate. The filtrate was then filtered through a disposable filter with a pore size of 0.22μm for sterilization and stored at -20℃ for later use.

[0062] Comparative Example 2

[0063] (1) Preparation of rose hydrosol culture medium

[0064] MRSL medium was prepared using Bulgarian rose hydrosol (obtained by rose distillation) as a substrate. Each 1L of hydrosol contained 20g glucose, 10.0g peptone, 8.0g beef extract, 4.0g yeast extract, 2.0g dipotassium hydrogen phosphate, 2.0g diammonium hydrogen citrate, 5.0g sodium acetate, 0.2g magnesium sulfate, 0.04g manganese sulfate, 1.0g Tween 80, and 0.5g L-cysteine.

[0065] (2) Preparation of unfermented rose hydrosol filtrate

[0066] Without inoculating the rose hydrosol culture medium for fermentation, the rose hydrosol with added MRSL medium was centrifuged at 10000g for 25 minutes at 4℃. The supernatant was collected, and the pH was adjusted to 7.2-7.4 to obtain the filtrate. The filtrate was then filtered through a disposable filter with a pore size of 0.22μm for sterilization and stored at -20℃ for later use.

[0067] Comparative Example 3

[0068] (1) Preparation of culture medium

[0069] Prepare MRSL culture medium without adding rose hydrosol or rose filtrate as substrates. The MRSL culture medium contains 20 g / L glucose, 10.0 g / L peptone, 8.0 g / L beef extract, 4.0 g / L yeast extract, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, 1.0 g / L Tween 80, and 0.5 g / L L-cysteine.

[0070] (2) Preparation of seed liquid

[0071] Take a glycerol cryopreservation tube of *Bifidobacterium longum* ZJ1 stored at -80℃, thaw it at room temperature, and then, under aseptic conditions, use an inoculation loop to pick up the bacteria and streak it onto a TPY solid medium plate. Incubate at 37℃ in an anaerobic incubator (or anaerobic culture bag) for 24-28 hours. Observe the colony morphology on the plate to confirm that it is *Bifidobacterium longum* and uncontaminated. In a clean bench, pick a single colony from the activated TPY solid medium plate and inoculate it into MRSL medium. Incubate at 37℃ in an anaerobic incubator (or anaerobic culture bag) at 100-120 rpm (or let it stand) for 12-16 hours to obtain a primary seed culture. Then, inoculate the primary seed culture into fresh MRSL medium at an inoculation rate of 2.0% (v / v) and incubate at 37℃ in an anaerobic incubator (or anaerobic culture bag) at 100-120 rpm (or let it stand) for 12-16 hours. When OD... 600Seed liquid is obtained when the concentration reaches 2.5-3.5.

[0072] (3) Fermentation

[0073] Inoculate the seed culture into MRSL medium at an inoculation rate of 2%-5% (v / v), adjust the pH to 5.7-6.7, and anaerobic ferment at 37℃ for 16-24 hours to obtain the fermentation broth of Bifidobacterium longum ZJ1, at which point the pH of the fermentation broth is 4.0-5.0.

[0074] (4) Preparation of fermentation product filtrate

[0075] Take the fermentation broth of Bifidobacterium longum ZJ1, centrifuge at 10000g for 25 minutes at 4℃, take the supernatant, adjust the pH to 7.2-7.4 to obtain the fermentation filtrate, filter it with a disposable filter with a pore size of 0.22μm to remove bacteria, and then store it in a refrigerator at -20℃ for later use.

[0076] (5) Preparation of fermentation product lysate

[0077] Take the fermentation broth of Bifidobacterium longum ZJ1 and sonicate it at low temperature 4-5 times. The sonication procedure is the same for each cycle: sonicate for 4 seconds, then sonicate again for 5 seconds as one cycle, and sonicate for 25-30 cycles. The sonication power range is 150-225W. Then centrifuge at 10000g for 20 minutes at 4℃. Collect the supernatant to obtain the lysate, filter it with a disposable filter with a pore size of 0.22μm to remove bacteria, and then store it in a refrigerator at -20℃ for later use.

[0078] Comparative Example 4

[0079] (1) Preparation of culture medium, same as comparative example 3.

[0080] (2) Preparation of seed liquid

[0081] The seed culture was prepared using the same method as in Comparative Example 3; however, Bifidobacterium longum ZJ1 in Comparative Example 3 was replaced with Bifidobacterium longum BNCC.

[0082] (3)-(5) Same as Comparative Example 3.

[0083] Comparative Example 5

[0084] (1) The preparation of rose petal culture medium is the same as in Example 1.

[0085] (2) Preparation of seed liquid

[0086] The preparation method of the seed liquid is the same as that in Example 1; the Bifidobacterium longum ZJ1 in Example 1 is replaced with Bifidobacterium longum BNCC232112, hereinafter referred to as BNCC.

[0087] (3)-(5) Same as Example 1.

[0088] Comparative Example 6

[0089] (1) Preparation of rose hydrosol culture medium, same as in Example 2.

[0090] (2) Preparation of seed liquid

[0091] The preparation method of the seed liquid is the same as in Example 2; except that Bifidobacterium longum ZJ1 in Example 2 is replaced with Bifidobacterium longum BNCC.

[0092] (4)-(5) Same as Example 2.

[0093] Comparative Example 7

[0094] (1) The preparation of rose petal culture medium is the same as in Example 1.

[0095] (2) Preparation of seed liquid

[0096] The preparation method of the seed liquid is the same as that in Example 1; except that Bifidobacterium longum ZJ1 in Example 1 is replaced with Lactobacillus plantarum (L. plantarum), hereinafter referred to as LP.

[0097] (3)-(5) Same as Example 1.

[0098] Comparative Example 8

[0099] (1) The preparation of rose petal culture medium is the same as in Example 1.

[0100] (2) Preparation of seed liquid

[0101] The preparation method of the seed liquid is the same as that in Example 1; except that Bifidobacterium longum ZJ1 in Example 1 is replaced with Lactobacillus acidophilus (L.acidophilus, NCFM), hereinafter referred to as LA.

[0102] (3)-(5) Same as Example 1.

[0103] The specific preparation methods, raw materials, and products of Examples 1-2 and Comparative Examples 1-8 are shown in Table 1:

[0104] Table 1 shows the raw materials and products prepared in Examples 1-2 and Comparative Examples 1-8.

[0105]

[0106]

[0107] 2.2 Content test of effective components of each product

[0108] The effective component content of the products from Examples 1-2 and Comparative Examples 1-8 was tested: the protein detection method followed GB5009.5-2010; the flavonoid detection method followed GB / T 5009.124-2003; and the total phenol detection method followed GB / T8313-2008. Details are shown in Table 2.

[0109] Table 2 Content of active ingredients in each product

[0110]

[0111]

[0112] The following can be derived from the table above:

[0113] 1. Comparative Examples 1 and 2: The rose petal filtrate and rose hydrosol contained certain amounts of total phenols, flavonoids, and proteins. Compared with unfermented rose petals and rose hydrosol, the fermentation products of rose petals and rose hydrosol fermented with Bifidobacterium longum ZJ1 contained significantly higher levels of total phenols, flavonoids, and proteins.

[0114] 2. Comparative Examples 3 and 4 show that fermentation with Bifidobacterium longum ZJ1 and Bifidobacterium longum BNCC using MRSL medium alone resulted in lower levels of total phenols, flavonoids, and proteins.

[0115] 3. As can be seen from Comparative Examples 5 and 6, the total phenol, flavonoid, and protein content of rose petals and rose hydrosol fermented with Bifidobacterium longum BNCC is not as high as that in Examples 1 and 2.

[0116] 4. As can be seen from Comparative Examples 7 and 8 and Example 1, the total phenol, flavonoid, and protein content of rose fermentation products using other fermentation strains is not as high as that in Example 1.

[0117] Therefore, it is concluded that the rose fermentation product fermented with Bifidobacterium longum ZJ1 contains extremely high levels of total phenols, flavonoids, and proteins, which greatly enhances the activity of the product and makes it possible for its use in topical skin compositions.

[0118] 2.3 Whitening Efficacy Test

[0119] 2.3.1 Test of Inhibition of Tyrosinase Activity

[0120] Tyrosinase is a copper-containing metalloenzyme widely found in mammals, plants, and microorganisms, playing a vital physiological role in organisms. It is also associated with conditions such as freckles and age spots caused by excessive melanin deposition. The reaction involving tyrosinase in melanin synthesis proceeds as follows: in a phosphoric acid solution at pH 6.8, tyrosinase catalyzes the conversion of tyrosine to dopa (monophenolase activity), followed by the conversion of dopa to dopaquinone (diphenolase activity). Both steps are catalyzed by tyrosinase, demonstrating its unique dual catalytic function. Therefore, in vitro detection of a analyte's inhibitory effect on tyrosinase activity can be used to assess whether the analyte has melanin-inhibiting activity. Many whitening and spot-removing products on the market achieve their whitening effect by inhibiting tyrosinase; therefore, the strength of tyrosinase inhibition is a key indicator for evaluating whitening cosmetics.

[0121] Using the above-described examples, comparative samples, and 25 μg / ml kojic acid solution as samples, the inhibitory effect of each sample on tyrosinase activity was detected, specifically including the following steps:

[0122] Take a 96-well plate and add each sample and substance according to the sample addition information of experimental group T and experimental group T0 in Table 3 to form each experimental group;

[0123] At the same time, each substance was added according to the sample addition information of control group C and control group C0 in Table 3 to serve as a control group;

[0124] When the substrate is tyrosine, the concentration of the substrate solution is 0.5 mg / ml. After reacting for 60 min, the UV absorbance of the reaction solution is detected at 475 nm, and the UV absorbance of the control group reacting for the same time is also detected. The inhibition rate of tyrosinase activity in the experimental group is calculated.

[0125] When the substrate is levodopa, the concentration of the substrate solution is 1.0 mg / ml. After reacting for 30 min, the UV absorbance of the reaction solution is detected at 475 nm, and the UV absorbance of the control group reacting for the same time is also detected. The inhibition rate of tyrosinase activity in the experimental group is calculated.

[0126] Table 3. Sampling Information for Each Group

[0127]

[0128] Calculation of IC50 for tyrosinase activity inhibition rate:

[0129]

[0130] In the formula:

[0131] T—Absorbance of the sample tube, i.e., the absorbance of the solution after the sample reacts with tyrosinase;

[0132] T0—Sample background absorbance;

[0133] C—Absorbance of the enzyme reaction tube, i.e., the absorbance of the reaction between tyrosinase and substrate when no sample is added;

[0134] C0 — Solvent background absorbance.

[0135] The half-maximal inhibitory concentration (IC50) of tyrosinase activity for each sample was calculated using SPSS. The lower the concentration, the better the inhibitory effect.

[0136] The IC50 values ​​for tyrosinase activity inhibition of the products of Examples 1-2 and Comparative Examples 1-8 are shown in Table 4:

[0137] Table 4. IC50 values ​​of in vitro tyrosinase activity inhibition for each product.

[0138]

[0139] As can be seen from the table above, Comparative Examples 1 and 2 demonstrate that rose petals and rose hydrosol themselves have a certain inhibitory effect on tyrosinase activity, which is consistent with existing technical knowledge. Comparative Example 3 shows that Bifidobacterium longum ZJ1 also has a certain inhibitory effect on tyrosinase activity, which has not been reported before. Moreover, as can be seen from Examples 1 and 2, the ZJ1 / rose fermentation product filtrate and lysate have a better inhibitory effect on tyrosinase activity than the ZJ1 fermentation product alone. Furthermore, compared with the BNCC rose fermentation product filtrate and lysate and rose petal fermentation products of other fermentation strains, the use of Bifidobacterium longum ZJ1 to ferment roses significantly improves the inhibitory effect on tyrosine monophenolase and diphenolase.

[0140] 2.3.2 Test of the ability to inhibit melanin synthesis in melanocytes

[0141] Mouse melanoma cells B16-F10 are a commonly used cell model for studying melanin production. Melanocytes can synthesize and secrete melanin, which is an amino acid derivative. When melanin reacts with sodium hydroxide, it produces a water-soluble compound. The melanin content is calculated by detecting the ultraviolet absorbance at 405 nm.

[0142] Melanocyte-stimulating hormone (α-MSH) primarily activates tyrosinase and promotes its synthesis, thereby promoting melanin production and darkening skin and hair color. This study evaluated the skin-lightening activity of mouse melanoma cells B16-F10 by calculating the difference in melanin secreted by these cells compared to those in the sample group.

[0143] The inhibitory effects of the above products on melanin synthesis in B16-F10 cells were investigated separately, and the specific steps are as follows:

[0144] a. First, the effect of each product on the proliferation of melanoma cells B16-F10 was detected, and the concentration that had no effect on the proliferation of B16-F10 cells was selected for subsequent experiments;

[0145] b. Take a 6-well plate and add 2 ml of B16-F10 cell suspension to each well to make the cell density 4 × 10⁻⁶. 4 Cells / well were collected and incubated in a 5% CO2 incubator for 24 hours. The supernatant was then discarded. According to the sample addition information in Table 5, 2 ml of solution was added to each well, which were designated as blank control group, model group, positive control group, and experimental group, respectively. The cells were then incubated in a 5% CO2 incubator at 37°C for 72 hours (based on the drug administration time). The supernatant was then discarded.

[0146] c. Add samples again according to Table 5, then add 2 ml of DMEM complete culture medium to each well, and continue to incubate at 37°C for 48-72 h in an incubator containing 5% CO2 until the cell fusion rate reaches more than 90% under a microscope;

[0147] d. Then discard the culture supernatant, wash each well twice with 1 ml of sterile PBS buffer; discard the PBS buffer, add 200 μl of trypsin solution to each well for 3 min; add 1 ml of PBS buffer to each well and pipette the digested cells, and aspirate the cell suspension into a 1.5 ml EP tube, centrifuge at 300 g for 5 min, and discard the supernatant; add 150 μl of melanin extraction solution to each tube, and place in a 90℃ water bath for 1 h to completely dissolve the melanin.

[0148] e. Pipette 100 μl of solution from each EP tube into a 96-well plate, using melanin extract as a zero control. Measure the absorbance at 405 nm using a microplate reader, and record the OD value. 405 The size of the value indicates the amount of melanin. The melanin synthesis inhibition rate is calculated using the following formula:

[0149]

[0150] In the formula: T is the absorbance of the model group, positive control group or experimental group; C is the absorbance of the blank control group; C0 is the absorbance of the melanin extract.

[0151] Table 5. Sampling Information for Well Plates

[0152]

[0153] Note: Three parallel samples are set up for each group.

[0154] The effects of each product on melanin content in B16-F10 cells are shown in Table 6:

[0155] Table 6. Inhibition rate of each product on B16-F10 melanin

[0156]

[0157]

[0158] As shown in the table above, both rose petals and rose hydrosol alone exhibit varying degrees of melanin inhibition in B16-F10 cells. The ZJ1 / rose fermentation product filtrate and lysate showed significantly higher melanin inhibition rates compared to ZJ1 fermentation product alone, BNCC rose fermentation product filtrate and lysate, and rose petal fermentation products from conventional fermentation strains. 2.5 wt% ZJ1 lysate and fermentation filtrate reduced melanin synthesis in B16-F10 cells by 87% ± 0% and 79% ± 2%, respectively. At the same concentration, ZJ1 rose fermentation lysate and fermentation filtrate showed superior melanin inhibition compared to ZJ1 lysate and fermentation filtrate alone.

[0159] 2.4 Antioxidant efficacy test

[0160] Free radical scavengers or antioxidants can provide electrons and inhibit oxidation. By absorbing free radicals while protecting cells from attack, they can eliminate free radicals, reduce wrinkles, and thus slow down the skin aging process. The finished products, semi-finished products, and raw materials of cosmetics can be tested to determine whether they have the ability to eliminate free radicals and the strength of that ability.

[0161] (1) ABTS free radical scavenging ability test

[0162] ABTS reacts with potassium persulfate to form green ABTS+. Substances with antioxidant properties can scavenge ABTS+. ABTS+ has maximum absorption at 734 nm; by measuring the absorbance, the magnitude of the absorbance value can quantitatively determine the free radical scavenging ability of the reactant. That is, the lower the absorbance value, the stronger the ability of the antioxidant to scavenge ABTS free radicals.

[0163] (2) DPPH free radical scavenging ability test

[0164] DPPH ethanol solution is purple-red and has a maximum absorption wavelength at 519 nm. When a free radical scavenger is added, DPPH free radicals are eliminated, the solution gradually becomes lighter in color, and the absorbance at 519 nm decreases accordingly. The lighter the solution color and the lower its absorbance value, the better the free radical scavenger is at eliminating DPPH free radicals.

[0165] (3) Hydroxyl radical scavenging ability test

[0166] H2O2 / Fe 2+ Hydroxyl radicals are generated through the Fenton reaction. Salicylic acid can effectively capture the generated hydroxyl radicals and react with them to form the colored substance 2,3-dihydroxybenzoic acid, which has a maximum absorption peak at 510 nm. After adding a substance with scavenging ability, the colored substance will be reduced, thus lowering the absorbance at 510 nm. Therefore, the ability of the sample to scavenge hydroxyl radicals can be judged based on the absorbance value.

[0167] The above methods can be used to calculate the free radical scavenging rate of a sample and its half-maximal scavenging concentration (IC50). By comparing the free radical scavenging abilities of different samples, it can be seen that the smaller the IC50 value, the stronger the free radical scavenging ability of the sample, thus allowing for a comparison of the free radical scavenging abilities of different finished cosmetic products, semi-finished products, or raw materials. The results are shown in Table 7.

[0168] Table 7. IC50 values ​​of ABTS radical, DPPH radical, and hydroxyl radical scavenging abilities of each product.

[0169]

[0170] As shown in the table above, all co-fermentation products exhibited certain antioxidant effects in the three free radical scavenging capacity tests. The rose petal co-fermentation product showed the best effect, followed by rose hydrosol, both superior to the original ZJ1 fermentation product. This indicates that the rose co-fermentation product possesses strong antioxidant capabilities, can scavenge free radicals, promote cell metabolism, enhance cell vitality, improve the structure and function of the body, and increase the body's vitality, thereby delaying cell aging and exerting its anti-aging effect.

[0171] In practical use, the fermented rose extract of this invention can be added at 2.5-5 wt% to cosmetics, skin care products, or pharmaceuticals used for whitening and anti-aging, such as toners, lotions, creams, masks, serums, foundations, liquid foundations, concealers, sunscreens, oral liquids, granules, tablets, pills, powders, capsules, or drop pills. Excipients can also be added to cosmetics, skin care products, or pharmaceuticals, such as oils, surfactants, polyols, moisturizers, defoamers, thickeners, preservatives, antioxidants, pH adjusters, lubricants, emulsifiers, or binders.

[0172] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The application of a fermented rose extract in the preparation of whitening and anti-aging skincare products, characterized in that, The fermented rose extract is prepared using Bifidobacterium longum (Bifidobacterium longum) Bifidobacterium longum The fermentation product obtained by using ZJ1 as a strain and rose as a substrate; The Bifidobacterium longum ( Bifidobacterium longum ZJ1 was deposited at the China Culture Collection Center for Microbial Cultures on June 26, 2019, with accession number CGMCC No. 18032; The rose in question is a rose petal; The fermentation products include filtrate and lysate; The fermented rose extract is used to inhibit tyrosine monophenolase and diphenolase, inhibit melanin synthesis in melanocytes, and increase antioxidant capacity. The preparation method of the fermented rose extract includes the following steps: MRSL medium was prepared using rose as a substrate, and activated Bifidobacterium longum was picked. Bifidobacterium longum A single colony of ZJ1 was inoculated into the MRSL medium and cultured anaerobically to obtain a primary seed culture; the primary seed culture was then inoculated into fresh MRSL medium and cultured anaerobically to obtain a seed culture. The seed culture was inoculated into fresh MRSL medium, and after adjusting the pH to 5.7-6.7, anaerobic fermentation was carried out to obtain Bifidobacterium longum (Bifidobacterium longum). Bifidobacterium longum ZJ1 rose petal fermentation broth, wherein the pH of the rose petal fermentation broth is 4.0-5.0; Take the rose petal fermentation broth, centrifuge to obtain the supernatant, adjust the pH to 7.2-7.4, filter to remove bacteria and obtain the filtrate; take another rose petal fermentation broth, sonicate at low temperature, centrifuge to collect the supernatant, filter to remove bacteria and obtain the lysate; use the filtrate and lysate as fermented rose extract.

2. The application according to claim 1, characterized in that, The MRSL medium consists of 20 g / L glucose, 10.0 g / L peptone, 8.0 g / L beef extract, 4.0 g / L yeast extract, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium hydrogen citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, 1.0 g / L Tween 80, and 0.5 g / L L-cysteine.

3. The application according to claim 1, characterized in that, The inoculation amount of the primary seed solution is 2% by volume.

4. The application according to claim 1, characterized in that, The inoculation amount of the seed liquid is 2%-5% by volume.

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