Preparation method and related application of pearl protein fermentation liquor

Abstract

The invention discloses a preparation method and related application of pearl protein fermentation liquor, and relates to the field of cosmetics. The pearl protein enzymatic hydrolysate serves as a nitrogen source in the fermentation culture medium, the utilization rate of the pearl protein is increased, the pH value of the culture medium in the middle and later periods of fermentation can be stabilized by adding pearl powder and / or calcium carbonate into the fermentation culture medium of the probiotic fermented pearl protein, and high-density fermentation is achieved. The prepared fermentation liquor can be used as a raw material to be applied to preparation of cosmetics and / or skin care products, and has a remarkable beautifying effect.

Description

Technical Field

[0001] The present invention relates to the field of cosmetics, and in particular, to a preparation method of a pearl protein fermentation broth and related applications thereof. Background Art

[0002] Pearls and nacre have a long medicinal history in traditional Chinese medicine. Pearl powder and nacre powder are both considered to have effects such as calming the mind and relieving uneasiness, removing nebula and improving eyesight, and detoxifying and promoting granulation. For example, it is recorded in Compendium of Materia Medica that pearls can fade freckles and chloasma; modern medical research has also found that pearls contain a large number of active ingredients, which have effects such as antioxidant, enhancing skin activity, and clearing heat and detoxifying.

[0003] Existing processes for fermenting pearl powder with probiotics cannot effectively utilize the active substances in pearl powder, resulting in the fermentation broth being unable to exert its due effects.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of a pearl protein fermentation broth and related applications thereof.

[0006] The present invention is implemented as follows:

[0007] In a first aspect, an embodiment of the present invention provides a preparation method of a pearl protein fermentation broth, which includes: inoculating a seed solution of probiotics into a fermentation medium for culture to obtain a fermentation broth; wherein, the components of the fermentation medium include: a basal medium, a pH stabilizer, and a pearl protein hydrolysate; the pH stabilizer includes pearl powder and / or calcium carbonate, and the pearl protein hydrolysate is obtained by enzymatically hydrolyzing a pearl protein and / or a nacre protein solution.

[0008] In a second aspect, an embodiment of the present invention provides a pearl protein fermentation broth, which is prepared by the preparation method described in the foregoing embodiment.

[0009] In a third aspect, an embodiment of the present invention provides an application of the pearl protein fermentation broth described in the foregoing embodiment in the preparation of cosmetics and / or skin care products.

[0010] The present invention has the following beneficial effects:

[0011] Using the hydrolysate of pearl protein as a nitrogen source in the fermentation medium improves the utilization rate of pearl protein;

[0012] By adding pearl powder and / or calcium carbonate to the fermentation medium for fermenting pearl protein with probiotics, the pH value of the medium in the middle and late stages of fermentation can be stabilized, realizing high-density fermentation;

[0013] The prepared fermentation broth can be used as a raw material in the preparation of cosmetics and / or skin care products, and has beauty effects. Brief Description of the Drawings

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0015] Figure 1 Effect of the addition amount of different pearl powders (calcium carbonate) on the fermentation broth;

[0016] Figure 2 Effect of the enzymatic hydrolysate on the fermentation broth;

[0017] Figure 3 Effect of the enzymatic hydrolysate on the antioxidant activity of the fermentation broth;

[0018] Figure 4 Effect of the enzymatic hydrolysate on the cell activity of the fermentation broth;

[0019] Figure 5 Effect of the enzymatic hydrolysate on the expression of COL-1 in the fermentation broth. Detailed Embodiments

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0021] On the one hand, the embodiments of the present invention provide a method for preparing a pearl protein fermentation broth, which includes: inoculating a seed solution of probiotics into a fermentation medium for culture to obtain a fermentation broth;

[0022] Wherein, the components of the fermentation medium include a basal medium, a pH stabilizer, and a pearl protein enzymatic hydrolysate; the pH stabilizer includes pearl powder and / or calcium carbonate, and the pearl protein enzymatic hydrolysate is obtained by enzymatically hydrolyzing a pearl protein and / or nacre protein solution.

[0023] Embodiments of the present invention use enzymatically hydrolyzed pearls and / or nacre proteins as the main nitrogen source for fermentation, achieving efficient utilization of pearls and / or nacre scleroproteins by microorganisms. Secondly, the present application uses pearl powder and / or calcium carbonate to control the pH during fermentation to achieve high-density fermentation technology and enhance the efficacy. The active polypeptides in the incompletely hydrolyzed pearls / nacre proteins are retained in the later stage of fermentation and, together with other active substances in the fermentation broth, provide more excellent efficacy.

[0024] In some embodiments, the enzymes used for the enzymatic hydrolysis include any one or more of alkaline protease, neutral protease, acidic protease, trypsin, papain, bromelain, and bacterial protease.

[0025] In some embodiments, the enzymes used for the enzymatic hydrolysis include alkaline protease and / or acidic protease.

[0026] In some embodiments, the steps of the enzymatic hydrolysis include: first using alkaline protease for enzymatic hydrolysis, and then using acidic protease for enzymatic hydrolysis.

[0027] In some embodiments, the conditions for enzymatic hydrolysis using the alkaline protease include: 40 - 60°C, 1 - 5 h. The temperature can specifically be any one of 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60°C or the range between any two of them. The time can specifically be any one of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 h or the range between any two of them.

[0028] In some embodiments, when performing the enzymatic hydrolysis, the mass fraction of the alkaline protease in the enzymatic hydrolysis system is 0.01% - 1%, and can specifically be any one of 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1% or the range between any two of them.

[0029] In some embodiments, the conditions for enzymatic hydrolysis using the acidic protease include: 40 - 60°C, 1 - 5 h. The temperature can specifically be any one of 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, and 60°C or the range between any two of them. The time can specifically be any one of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 h or the range between any two of them.

[0030] In some embodiments, when performing the enzymatic hydrolysis, the mass fraction of the acid protease in the enzymatic hydrolysis system is 0.01% to 1%, specifically, it can be any one or the range between any two of 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% and 1%.

[0031] In some embodiments, the pH of the pearl protein and / or nacre protein solution is 7.5 to 8.5, specifically, it can be any one or the range between any two of 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4 and 8.5.

[0032] In some embodiments, in the pearl protein and / or nacre protein solution, the mass fraction of the pearl protein and / or nacre protein is 0.5% to 5%, specifically, it can be any one or the range between any two of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% and 5%.

[0033] In some embodiments, the pearl protein and / or nacre protein for performing the enzymatic hydrolysis is obtained by pretreating pearl powder and / or nacre powder, and the pretreatment includes: performing decalcification treatment on the pearl powder and / or nacre powder with an acid. Using an acid to perform acid decalcification on pearl powder and / or nacre powder can effectively enrich the protein therein, which is beneficial to repeatedly exert its efficacy.

[0034] In some embodiments, the acid includes any one or a combination of multiple of glacial acetic acid, lactic acid, dilute hydrochloric acid and citric acid.

[0035] In some embodiments, for every 100 g of pearl powder and / or nacre powder, any one or the range between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 mol of acid is added.

[0036] In some embodiments, when performing the decalcification treatment, an antifoaming agent is further added;

[0037] In some embodiments, the working concentration of the antifoaming agent is 0.01% to 0.5% (w / w); specifically, this working concentration can be any one or the range between any two of 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% and 0.5%.

[0038] In some embodiments, the antifoaming agent is polyoxyethylene polyoxypropylene pentaerythritol ether.

[0039] In some embodiments, the particle size of the pearl powder and / or nacre powder is 1-20 μm, specifically it can be any one or the range between any two of 1, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 μm.

[0040] In some embodiments, in the fermentation medium, the mass fraction of the pH stabilizer is 0.01%-1%, specifically it can be any one or the range between any two of 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%.

[0041] In some embodiments, the mass fraction of the pH stabilizer is 0.05%-0.5%, specifically it can be any one or the range between any two of 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%.

[0042] In some embodiments, the probiotic bacteria include Bifidobacterium adolescentis.

[0043] In some embodiments, the Bifidobacterium adolescentis includes: Bifidobacterium adolescentis ATCC 15703 strain.

[0044] In some embodiments, the culture conditions include: 20-50 °C, 24-48 h. This temperature can be any one or the range between any two of 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 and 50 °C; this time can be any one or the range between any two of 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 and 48 h.

[0045] In some embodiments, the culture also includes: maintaining the effective concentration of glucose in the fermentation medium to be any one or the range between any two of 0.5%, 1%, 1.5%, 2%, 2.5% and 3%.

[0046] In some embodiments, the components and their ratios of the basal medium can adopt the relevant components and their ratios of the conventional fermentation medium in the art.

[0047] In some embodiments, the fermentation medium comprises: the pearl protein hydrolysate, yeast peptone with a mass fraction of 0.1% - 1%, glucose with a mass fraction of 0.5% - 3%, the pH stabilizer with a mass fraction of 0.01% - 1%, Tween 80 with a mass fraction of 0.01% - 1%, resin azure with a mass fraction of 0.01% - 1%, L-cysteine hydrochloride with a mass fraction of 0.01% - 0.5%, magnesium sulfate with a mass fraction of 0.0001% - 0.005%, dipotassium hydrogen phosphate with a mass fraction of 0.0001% - 0.05%, and potassium dihydrogen phosphate with a mass fraction of 0.0001% - 0.05%.

[0048] In some embodiments, the mass fraction of yeast peptone can be any one of 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1% or within the range between any two of them. The mass fraction of glucose can be any one of 0.5%, 1%, 1.5%, 2%, 2.5%, 3% or within the range between any two of them. The mass fraction of the pH stabilizer can be any one of 0.01%, 0.05%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1% or within the range between any two of them. The mass fraction of Tween 80 can be any one of 0.01%, 0.05%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1% or within the range between any two of them. The mass fraction of resin azure can be any one of 0.01%, 0.05%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1% or within the range between any two of them. The mass fraction of L-cysteine hydrochloride can be any one of 0.01%, 0.05%, 0.1%, 0.2%, 0.4%, 0.5% or within the range between any two of them. The mass fraction of magnesium sulfate can be any one of 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.4%, 0.5% or within the range between any two of them. The mass fraction of dipotassium hydrogen phosphate can be any one of 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.4%, 0.5% or within the range between any two of them. The mass fraction of potassium dihydrogen phosphate can be any one of 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.2%, 0.4%, 0.5% or within the range between any two of them.

[0049] In some embodiments, the pH of the fermentation medium is 6 - 7, and specifically can be any one of 6, 6.2, 6.4, 6.6, 6.8, 7 or within the range between any two of them.

[0050] In some embodiments, the seed liquid is obtained by inoculating the probiotic bacteria into a seed culture medium and culturing them.

[0051] In some embodiments, the seed culture medium comprises: water, yeast extract powder with a mass fraction of 0.1% - 5%, yeast peptone with a mass fraction of 0.1% - 5%, glucose with a mass fraction of 0.01% - 0.5%, Tween 80 with a mass fraction of 0.01% - 0.5%, resin azurin with a mass fraction of 0.01% - 0.5%, L-cysteine hydrochloride with a mass fraction of 0.01% - 0.5%, magnesium sulfate with a mass fraction of 0.0001% - 0.01%, dipotassium hydrogen phosphate with a mass fraction of 0.0001% - 0.01%, and potassium dihydrogen phosphate with a mass fraction of 0.0001% - 0.01%.

[0052] In some embodiments, the pH of the seed culture medium is 6 - 7, specifically any one of 6, 6.2, 6.4, 6.6, 6.8, and 7 or the range between any two of them.

[0053] In some embodiments, the preparation method further comprises: performing cell wall breaking centrifugation on the fermentation broth to obtain a supernatant.

[0054] In some embodiments, the preparation method further comprises filtering the supernatant.

[0055] On the other hand, an embodiment of the present invention provides a pearl protein fermentation broth, which is obtained by the preparation method described in any of the foregoing embodiments.

[0056] In addition, an embodiment of the present invention also provides the application of the pearl protein fermentation broth described in any of the foregoing embodiments in the preparation of cosmetics and / or skin care products.

[0057] In some embodiments, the cosmetics and / or skin care products have at least one of the following effects: antioxidant, sunscreen, anti-aging, whitening, freckle removal, and anti-wrinkle.

[0058] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0059] Example 1

[0060] (1) Pearl pretreatment:

[0061] The pearls are crushed to a diameter of 4 μm. Take 100 g of pearl powder, and use 1 L of 20% (w / w) glacial acetic acid and 1 g of polyoxyethylene polyoxypropylene pentaerythritol ether (antifoaming agent) to perform decalcification treatment on the pearls. After the reaction is complete, centrifuge to collect the undissolved solid insoluble matter, wash it 2 - 3 times with clear water and centrifuge to collect the solid insoluble matter, and dry it at 50 °C to obtain pearl protein.

[0062] (2) Pearl protease hydrolysis:

[0063] Disperse 20 g of pearl protein in 1 L of an aqueous solution containing 0.1% (w / w) Tween 80, disperse thoroughly for 30 min, adjust the dispersion to pH 8.0 with 4 M NaOH, then add Solarbio B8360 alkaline protease to the system so that the working concentration of alkaline protease in the system is 0.1% (w / w), enzymatically hydrolyze at 50 °C for 2 h, then add Solarbio B8410 acidic protease to the system so that the working concentration of acidic protease in the system is 0.1% (w / w), and enzymatically hydrolyze at 50 °C for 2 h for standby.

[0064] (3) Bifidobacterium adolescentis fermentation:

[0065] 3.1 Related culture media:

[0066] Table 1 Material information

[0067] Serial number Material name 1 Yeast extract powder 2 Yeast peptone 3 Glucose 4 Pearl powder 5 Tween 80 6 L-cysteine hydrochloride 7 Magnesium sulfate 8 Dipotassium hydrogen phosphate 9 Potassium dihydrogen phosphate 10 Resazurin 11 Sodium hydroxide 12 Defoamer 13 Alkaline protease 14 Acid protease

[0068] Modified MRS seed culture medium: 1% (w / w) yeast extract powder, 2% (w / w) yeast peptone, 0.2% (w / w) glucose, 0.1% (w / w) Tween 80, 0.1% (w / w) resazurin, 0.05% (w / w) L-cysteine hydrochloride, 0.002% (w / w) magnesium sulfate, 0.002% (w / w) dipotassium hydrogen phosphate, 0.002% (w / w) potassium dihydrogen phosphate, made up to the preset volume with purified water, pH 6.5.

[0069] Fermentation culture medium: 0.5% (w / w) yeast peptone, 2% (w / w) glucose (add glucose mother liquor after sterilization until the preset concentration), 0.2% (w / w) pearl powder, 0.1% (w / w) Tween 80, 0.1% (w / w) resazurin, 0.05% (w / w) L-cysteine hydrochloride, 0.002% (w / w) magnesium sulfate, 0.005% (w / w) dipotassium hydrogen phosphate, 0.005% (w / w) potassium dihydrogen phosphate, made up to the preset volume with pearl protease hydrolysate (nitrogen source), pH 6.5.

[0070] Glucose mother liquor: 50% (w / w) anhydrous glucose, made up to the preset volume with purified water.

[0071] After the above solution was prepared (the pearl powder was weighed separately), 100 mL was taken and dispensed into 250 mL two-way anaerobic culture flasks. The bottom air inlet was sealed with a stopcock clip and heated by microwave. After heating, nitrogen was introduced from the bottom until the culture medium changed from blue to light yellow or other light colors, then pearl powder was added and nitrogen purging continued for 10 - 15 min. Tighten the bottle cap, seal the air inlet and outlet with a stopcock clip, and sterilize in an autoclave at 121 °C for 20 min. After sterilization, transfer it to an anaerobic workstation and add the glucose stock solution from the feeding port until the preset concentration is reached and set aside.

[0072] 3.2 Strain activation

[0073] In the anaerobic workstation, pipette the glycerol stock (Bifidobacterium adolescentis ATCC 15703 strain) and inoculate it into the modified MRS seed medium at an inoculation amount of 0.5% (v / v), and culture at 37 °C and 80 rpm for 28 h.

[0074] 3.3 Fermentation culture

[0075] In the anaerobic workstation, use a sterile syringe to pipette the glucose stock solution and supplement glucose from the silicone stopper at the top of the anaerobic culture flask to make the final concentration of glucose in the fermentation medium 2%, shake well and set aside.

[0076] Use a sterile syringe to pipette the cultured seed liquid and inoculate the strain into the fermentation medium from the silicone stopper at the top of the anaerobic culture flask at an inoculation amount of 5% (v / v), and culture at 37 °C and 80 rpm for 36 h.

[0077] 3.4 Post-treatment of the fermentation broth

[0078] Use a high-pressure homogenizer to break the cell wall of the cultured fermentation broth. After cell wall breaking, centrifuge at 10000 rpm for 10 min to collect the supernatant, and filter through a 0.22 μm filter membrane to obtain the final product.

[0079] Example 2 verified the effect of the addition amount of pearl powder on the product.

[0080] Based on the method for preparing the fermentation broth in Example 1, multiple experimental groups were set up. The difference between different experimental groups was only the content of pearl powder in the fermentation medium, which were 0%, 0.05%, 0.10%, 0.15%, 0.20% (the same as in Example 1), 0.25% and 0.30% (w / w) respectively. The culture method was the same as in Example 1.

[0081] After the culture was completed, 5 mL of the fermentation broth was taken from each group and the pH value at the end of fermentation was measured using a pH meter; 1 mL of the fermentation broth was taken from each group and gradually diluted until the OD measured by the ultraviolet spectrophotometer 600When the value is between 0.2 and 0.8, record the reading and multiply it by the corresponding dilution factor to obtain the OD at the end of fermentation. 600 value.

[0082] The results are shown in Figure 1 . The results show that after adding pearl powder, the pH at the end of the fermentation broth is effectively controlled, the bacterial concentration is significantly increased, and high-density fermentation is achieved.

[0083] Example 3 Effect of Pearl Protein on Fermentation

[0084] Based on the method for preparing the fermentation broth in Example 1, multiple experimental groups were set up. The difference between different experimental groups was only the nitrogen source in the fermentation medium. Purified water (negative control), unhydrolyzed pearl protein suspension, and pearl protein hydrolysate (Example 1) were used as nitrogen sources for fermentation, and the three groups were labeled as purified water, unhydrolyzed, and hydrolyzed respectively.

[0085] After the cultivation was completed, 1 mL of the fermentation broth was taken from each group and gradually diluted until the OD 600 value measured by the ultraviolet spectrophotometer was between 0.2 and 0.8. Record the reading and multiply it by the corresponding dilution factor to obtain the OD 600 value at the end of fermentation.

[0086] The results are shown in Figure 2 . The results show that adding hydrolyzed pearl protein to the fermentation medium can significantly improve the strain propagation efficiency.

[0087] Example 4 Determination of DPPH Free Radical Scavenging Rate

[0088] Experimental Method

[0089] Preparation of DPPH working solution: Accurately weigh 1,1-diphenyl-2-picrylhydrazyl (DPPH), add purified water to a final concentration of 2×10 -4 mol / L, sonicate for 10 min, and invert several times during the period until it is evenly mixed.

[0090] Centrifuge the three groups of fermentation broths in Example 3 at 12,000 rpm, collect the supernatant, and dilute them to 10% (v / v) with purified water respectively; after dilution, mix them evenly with the DPPH working solution at a ratio of 1:1, and label them as purified water, unhydrolyzed, and hydrolyzed; place the three groups of samples in a 25 °C incubator for reaction for 30 min; after the reaction is completed, centrifuge at 12,000 rpm for 1 min; pipette 200 μL of the supernatant into a 96-well plate and measure the absorbance at 517 nm respectively; calculate the scavenging rate according to the following formula: (control group - experimental group) / (control group - absorbance of blank well) = scavenging rate.

[0091] The results are shown in Figure 3. As can be seen from the results, adding pearls and / or nacre proteins to the fermentation medium can significantly improve the free radical scavenging rate and provide excellent antioxidant activity.

[0092] Example 5 Cytotoxicity Assay

[0093] Experimental Method

[0094] Cell Pretreatment: Take out HDF cells from the incubator, discard the old medium, add 10 mL of 0.01 M PBS to wash once, and discard the 0.01 M PBS completely. Add 1 mL of trypsin, place it in the incubator for digestion for about 1 - 2 min. After digestion, add 3 mL of FM complete medium to terminate digestion, pipette the cells, transfer them to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant after centrifugation, and add 1 mL of FM complete medium to pipette and mix well.

[0095] Cell Seeding: Adjust the cell density to 5×10 4 cells / mL, seed them into a 96 - well plate, 100 μL per well. Place it in the incubator for culturing for 24 h.

[0096] Sample - Treated Cells: Centrifuge the test sample (Example 1) at 12000 rpm for 10 min, take the supernatant and dilute it to the maximum concentration with 0.01 M PBS, vortex and mix well, then filter and sterilize it through a 0.22 μm filter membrane. After sterilization, dilute it to other preset concentrations (blank, 0.10%, 0.50%, 1%, 5%, and 10%, % is volume fraction) with sterile 0.01 M PBS respectively. Take out the cells, add the above - mentioned different - concentration samples, and add an equal volume of 0.01 M PBS to the blank group, and place them in the incubator for culturing for 24 h.

[0097] Result Detection: Dilute the CCK8 reagent with FM complete medium at a ratio of 1:10, and mix well. Aspirate the old medium in the 96 - well cell culture plate, add 100 μL of the diluted CCK8 reagent to each well, and place it in the incubator for culturing for 1.5 h. After completion, use an enzyme - linked immunosorbent assay (ELISA) reader, set the dual - wavelength, Lm1 as 450 and Lm2 as 650 for detection; calculate the average absorbance values of the blank group and the control group, denoted as Ab and Ac respectively. The calculation formula for cell viability is: Cell Viability (%)=(Absorbance - Ab) / (Ac - Ab)×100%.

[0098] The results are shown in Figure 4 .

[0099] As can be seen from the results, there is no toxicity to cells at a concentration of 10% for the fermentation product filtrate.

[0100] Example 6 COL1 Promoting Expression Experiment

[0101] Experimental Method

[0102] Cell pretreatment: Take out HDF cells from the incubator, discard the old culture medium, add 10 mL of 0.01 M PBS to wash once, and discard the 0.01 M PBS completely. Add 1 mL of trypsin, place it in the incubator for digestion for about 2 min. After digestion, add 3 mL of FM complete medium to terminate digestion, pipette and collect the cells, transfer them to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant after centrifugation, and add 1 mL of FM complete medium to pipette and mix evenly.

[0103] Cell seeding: Adjust the cell density to 5×10 4 cells / mL, seed into a 6-well plate, 2 mL per well. Place it in the incubator and culture for 24 h. Sample treatment of cells: Dilute the test samples (blank, purified water, unenzymolyzed and enzymolyzed fermentation broth, same as Example 3) to the maximum concentration with FM complete medium, vortex and mix evenly, then filter and sterilize with a 0.22 μm filter membrane. After sterilization, dilute them to other preset concentrations (1%, v / v) respectively. Take out the cells, add 2 mL of the blank, purified water, unenzymolyzed and enzymolyzed fermentation broth diluted to the preset concentration above, and add an equal volume of FM complete medium to the blank group, place it in the incubator and culture for 24 h.

[0104] mRNA extraction and reverse transcription: Use Takara mRNA extraction kit and reverse transcription kit to obtain the cDNA groups of cells in the blank group and sample groups.

[0105] Fluorescent quantitative PCR: Use the corresponding primers to detect the expression level of COL1 by fluorescent quantitative PCR.

[0106] The results are shown in Figure 5 .

[0107] It can be seen from the results that the fermentation broth obtained by fermenting with added enzymolyzed pearls and / or nacre proteins significantly increases the effect of promoting COL1 expression.

[0108] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a pearl protein fermentation broth, characterized in that, It includes: Inoculating the seed liquid of probiotics into a fermentation medium for cultivation to obtain a fermentation broth; Among them, the components of the fermentation medium include: a basal medium, a pH stabilizer, and a pearl protein hydrolysate; the pH stabilizer includes pearl powder and / or calcium carbonate, and the pearl protein hydrolysate is obtained by enzymatically hydrolyzing a pearl protein and / or nacre protein solution.

2. The preparation method according to claim 1, characterized in that, The enzyme used for the enzymatic hydrolysis includes any one or more of alkaline protease, neutral protease, acidic protease, trypsin, papain, bromelain, and bacterial protease; Optionally, the enzyme used for the enzymatic hydrolysis includes alkaline protease and / or acidic protease; Optionally, the steps of the enzymatic hydrolysis include: first performing enzymatic hydrolysis with alkaline protease, and then performing enzymatic hydrolysis with acidic protease; Optionally, the conditions for the enzymatic hydrolysis using the alkaline protease include: 40 - 60°C, 1 - 5 h; Optionally, when performing the enzymatic hydrolysis, the mass fraction of the alkaline protease in the enzymatic hydrolysis system is 0.01% - 1%; Optionally, the conditions for the enzymatic hydrolysis using the acidic protease include: 40 - 60°C, 1 - 5 h; Optionally, when performing the enzymatic hydrolysis, the mass fraction of the acidic protease in the enzymatic hydrolysis system is 0.01% - 1%; Optionally, the pH of the pearl protein and / or nacre protein solution is 7.5 - 8.5; Optionally, in the pearl protein and / or nacre protein solution, the mass fraction of the pearl protein and / or nacre protein is 0.5% - 5%.

3. The preparation method according to claim 2, wherein The pearl protein and / or nacre protein for performing the enzymatic hydrolysis is obtained by pre-treating pearl powder and / or nacre powder, and the pre-treatment includes: performing decalcification treatment on the pearl powder and / or nacre powder with an acid; Optionally, the acid includes any one or a combination of acetic acid, lactic acid, dilute hydrochloric acid, and citric acid; Optionally, for every 100 g of pearl powder and / or nacre powder, 1 - 10 mol of acid is added; Optionally, when performing the decalcification treatment, an antifoaming agent is also added; Optionally, the mass percentage of the antifoaming agent is 0.01% - 0.5%; Optionally, the antifoaming agent is polyoxyethylene polyoxypropylene pentaerythritol ether; Optionally, the particle size of the pearl powder and / or nacre powder is 1 - 20 μm.

4. The preparation method according to claim 1, characterized in that, In the fermentation medium, the mass fraction of the pH stabilizer is 0.01% - 1%; Optionally, the mass fraction of the pH stabilizer is 0.05% - 0.5%.

5. The preparation method according to claim 1, wherein The probiotics include Bifidobacterium adolescentis; Optionally, the Bifidobacterium adolescentis includes: Bifidobacterium adolescentis ATCC15703 strain.

6. The preparation method according to claim 1, wherein The conditions for the cultivation include: 20 - 50°C, 24 - 48 h; Optionally, the cultivation further includes: maintaining the effective concentration of glucose in the fermentation medium at 0.5% - 3%.

7. The preparation method according to claim 1, wherein The fermentation medium includes: the pearl protein hydrolysate, yeast peptone with a mass fraction of 0.1% - 1%, glucose with a mass fraction of 0.5% - 3%, the pH stabilizer with a mass fraction of 0.01% - 1%, Tween 80 with a mass fraction of 0.01% - 1%, resin azure with a mass fraction of 0.01% - 1%, L-cysteine hydrochloride with a mass fraction of 0.01% - 0.5%, magnesium sulfate with a mass fraction of 0.0001% - 0.005%, dipotassium hydrogen phosphate with a mass fraction of 0.0001% - 0.05, and potassium dihydrogen phosphate with a mass fraction of 0.0001% - 0.05%; Optionally, the pH of the fermentation medium is 6 - 7; Optionally, the seed liquid is obtained by inoculating the probiotic bacteria into a seed medium for cultivation; Optionally, the seed medium includes: water, yeast extract powder with a mass fraction of 0.1% - 5%, yeast peptone with a mass fraction of 0.1% - 5%, glucose with a mass fraction of 0.01% - 0.5%, Tween 80 with a mass fraction of 0.01% - 0.5%, resin azure with a mass fraction of 0.01% - 0.5%, L-cysteine hydrochloride with a mass fraction of 0.01% - 0.5%, magnesium sulfate with a mass fraction of 0.0001% - 0.01%, dipotassium hydrogen phosphate with a mass fraction of 0.0001% - 0.01, and potassium dihydrogen phosphate with a mass fraction of 0.0001% - 0.01%; Optionally, the pH of the seed medium is 6 - 7.

8. The preparation method according to any one of claims 1 to 7, characterized in that, The preparation method further includes: performing cell wall breaking centrifugation on the fermentation broth to obtain a supernatant; Optionally, the preparation method further includes filtering the supernatant.

9. A pearl protein fermentation broth, characterized in that, It is prepared by the preparation method according to any one of claims 1 - 8.

10. Use of the pearl protein fermentation broth according to claim 9 in the preparation of cosmetics and / or skin care products; Optionally, the cosmetics and / or skin care products have at least one of the following effects: antioxidant, sunscreen, anti-aging, whitening, freckle removal, and anti-wrinkle.

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