CN113413351B

CN113413351B - Fermented liquid and fermented polypeptide with whitening and anti-aging effects, and preparation methods and applications thereof

Info

Publication number: CN113413351B
Application number: CN202110802646.1A
Authority: CN
Inventors: 张冬生; 周安; 吴成亮; 高先亭; 刘玉梅
Original assignee: Hefei Kadier Biotechnology Co ltd
Current assignee: Hefei Kadier Biotechnology Co ltd
Priority date: 2021-07-15
Filing date: 2021-07-15
Publication date: 2023-10-20
Anticipated expiration: 2041-07-15
Also published as: CN113413351A

Abstract

The application relates to a fermentation liquor with whitening and anti-aging effects, a fermentation polypeptide and a preparation method and application thereof, wherein the fermentation liquor is prepared by taking natural plants with whitening and anti-aging effects as raw materials, firstly carrying out primary fermentation on the raw materials by lactobacillus plantarum, and then carrying out secondary fermentation on primary fermentation products by saccharomyces cerevisiae, and the fermentation polypeptide is prepared by taking proteins in the fermentation liquor as raw materials through enzymolysis. According to the application, the mixed rose and rhizoma polygonati powder are subjected to lactobacillus plantarum fermentation and ultrasound, and then subjected to saccharomyces cerevisiae low-temperature fermentation and ultrasound again, so that the obtained fermentation liquor has a strong antioxidation effect, the polypeptide prepared by enzymolysis of fermentation liquor protein has an inhibition effect on tyrosinase, in addition, the preparation processes of the fermentation liquor and the fermentation polypeptide are simple and easy to obtain, and no additive or organic reagent is used for whitening and anti-aging cosmetics, so that the healthy microecology of skin can be improved.

Description

Fermented liquid and fermented polypeptide with whitening and anti-aging effects, and preparation methods and applications thereof

Technical Field

The application belongs to the technical field of microbial fermentation and biology, and particularly relates to fermentation liquor with whitening and anti-aging effects, a fermentation polypeptide, a preparation method and application thereof.

Background

The health effect of the probiotics is that the Russian scientist Mctchnikoff firstly proposes that the probiotics have the effects of prolonging the service life and preventing putrefying bacteria, and treating intestinal disorder, resisting tumor and the like in medicine, and the probiotics are widely applied to medical care and food due to the advantages of no toxicity, no residue, no pollution and the like. The Ministry of agriculture of China publishes 6 probiotics, namely yeast, lactobacillus, bifidobacterium, streptococcus faecalis, DM423 bacillus cereus and SA38 bacillus cereus, and the probiotics have various biological effects and generate beneficial metabolites such as organic acid; and the probiotics produce multiple nutritional active ingredients such as volatile acid, vitamin, folic acid, nicotinic acid, pantothenic acid and the like. On the other hand, the probiotics improves the macrophage activity, further enhances the organism immunity function, and the protease-producing probiotics broiler chickens promote the growth and development of immune organs. The probiotic preparation has the effects of resisting radiation, resisting fatigue and the like through a white rat experiment. Yeast is mainly a yeast single cell protein and is therefore also known as a single cell protein. Yeast as fungus culture contains a large amount of effective protein and beneficial bacteria for growth, inhibits pathogenic microorganism reproduction, improves organism immunity and disease resistance, has rich nutrition, is rich in crude protein, B vitamins, minerals, digestive enzymes, growth promoting factors and complete amino acids, and is widely applied to medicine health care and industrial production. The yeast can be divided into nutritional yeast and functional yeast, wherein the nutritional yeast is mainly a product obtained by fermenting beer yeast or baker's yeast and then performing enzymolysis treatment and spray drying, and the product contains abundant proteins, small peptides, nucleotides, vitamins and the like. The functional yeast is mainly yeast derivatives, active dry yeast and the like, and is rich in yeast cell wall polysaccharide, yeast selenium, yeast chromium and other nutrient substances. Lactobacillus plantarum is used as one of common probiotics, and plays roles in aging resistance, oxidation resistance, bacteria resistance and the like in the daily life.

Enzymes are produced by fermenting plants and fungus sources with microorganisms, and contain enzymes, catalysis and other multiple biological active substances, and the enzymes are taken as complex proteins participating in metabolism in organisms, widely exist in living cells and play important roles, so that enzyme products are widely recognized, and various edible enzyme products are endlessly layered. At present, probiotics are widely applied to traditional Chinese medicine fermentation, wherein the content of medicine effect substances is improved, the effect of a fermented product is improved, toxicity is reduced and the like. I have abundant plant resources, which are applied by extraction and extraction methods in a main development mode, so that the process is complicated and residues of toxic and harmful substances can be brought in. Therefore, the method for changing the utilization mode of plant resources and improving the utilization rate of the resources has important research significance.

The roses have the effects of whitening, improving skin texture and promoting blood circulation and metabolism, and modern researches of the rhizoma polygonati find that the roses contain rhizoma polygonati saponins, nicotinic acid, saccharides, quinones, amino acids, trace elements and the like, have the effects of maintaining beauty and keeping young and resisting aging, and can ferment the roses or the rhizoma polygonati by utilizing microorganisms to generate plant enzymes containing enzymes, catalysis and other multiple biological active substances, so that the roses or the rhizoma polygonati have good whitening and anti-aging effects, but the enzymes serving as macromolecules are difficult to be directly absorbed and utilized by skin at present, and restrict the development of the roses or the rhizoma polygonati in the field of cosmetics.

Not only aiming at the rose and sealwort composition, most of the existing pure plant cosmetic raw materials can fully release and utilize active ingredients and metabolism in plants to generate a large number of prebiotics which are beneficial to human skin absorption, so that macromolecules which are difficult to be absorbed by human bodies of the plants are converted into small molecules, but also the application of enzymolysis technology converts macromolecular proteins into polypeptides through enzymolysis, and further the polypeptides can be effectively acted on different targets to exert corresponding biological activities. The ferment serving as the macromolecule is difficult to be directly absorbed and utilized by skin, so that a fermentation broth and a fermentation polypeptide with whitening and anti-aging effects are provided, and a preparation method and application thereof.

Disclosure of Invention

The application aims to solve the problems and provide a fermentation liquor with whitening and anti-aging effects, a fermentation polypeptide, a preparation method and application thereof.

The application realizes the above purpose through the following technical scheme:

a fermentation liquor with whitening and anti-aging effects is prepared by taking natural plants with whitening and anti-aging effects as raw materials, performing primary fermentation on the raw materials by using lactobacillus plantarum, and performing secondary fermentation on a primary fermentation product by using saccharomyces cerevisiae.

As a further optimized scheme of the application, the natural plant raw material with the whitening and anti-aging effects is a composition of rose and rhizoma polygonati.

An application of the fermentation liquor in preparing whitening anti-aging cosmetics.

A fermented polypeptide with whitening and anti-aging effects is prepared by taking protein in any one of the fermentation liquids as a raw material and performing enzymolysis.

As a further optimization scheme of the application, the extraction method of the protein in the fermentation broth is an ammonium sulfate precipitation method.

As a further optimization scheme of the application, the protein in the fermentation broth is subjected to enzymolysis by utilizing complex enzyme, and the components of the complex enzyme comprise: pepsin, alkaline protease and trypsin.

An application of the fermented polypeptide in preparing whitening and anti-aging cosmetics.

A method for preparing a fermentation broth or a fermentation polypeptide as described above, comprising the steps of:

(1) Preparation of culture medium

Mixing the crushed rose and rhizoma polygonati in a proportion of 1-40:1-40 mass ratio is added into a fermentation culture medium, and the fermentation culture medium is sterilized for later use;

(2) Inoculating fermentation

Inoculating lactobacillus plantarum seed liquid with the volume ratio of 1-20% into a fermentation culture medium, fermenting for 24-96 hours by a constant temperature oscillator with the temperature of 20-50 ℃, obtaining a primary fermentation product, then performing ultrasonic treatment in an ultrasonic instrument, inoculating Saccharomyces cerevisiae seed liquid with the volume ratio of 1-15% into the primary fermentation product after ultrasonic treatment, fermenting for 2-10 days by a constant temperature oscillator with the temperature of 10-40 ℃, placing the secondary fermentation product into the ultrasonic instrument again for ultrasonic treatment, and centrifuging to obtain an upper fermentation clear liquid which is the fermentation liquid, and storing the fermentation liquid for later use;

(3) Fermentation broth protein extraction

Adding ammonium sulfate into the fermentation liquor obtained in the step (2) to extract protein in the fermentation clear liquor, and centrifuging to obtain a precipitate, wherein the adding amount of the ammonium sulfate is 10-80g of ammonium sulfate added into each 100ml of fermentation liquor;

(4) Preparation of fermented polypeptide by zymohydrolysis of fermentation liquor

1 to 20% of the protein precipitate obtained in the step (3): and adding a compound enzyme according to a mass ratio of 1-100 for enzymolysis for 5-30 hours to obtain the fermented polypeptide.

As a further optimization scheme of the application, in the step (2), the primary fermentation product and the secondary fermentation product are subjected to ultrasonic treatment at the frequency of 40KHz for 1-20min at intervals of 3-30min.

As a further optimization scheme of the application, the complex enzyme is pepsin, alkaline protease and trypsin with the ratio of 1-10:1-10:1-10 mass ratio.

The application has the beneficial effects that:

(1) The application adopts the natural plant rose and rhizoma polygonati composition, the rose has the effects of whitening skin, improving skin texture and promoting blood circulation and metabolism, and the rhizoma polygonati is found by modern researches to contain rhizoma polygonati saponin, nicotinic acid, sugar, quinone, amino acid, trace elements and the like, has the effects of maintaining beauty and keeping young and resisting aging, and can be applied to preparing whitening and anti-type products through biological fermentation of the two.

(2) The application adopts Saccharomyces cerevisiae and lactobacillus plantarum as fermentation strains, and utilizes the temperature difference of the two probiotics to ferment, and the combined fermentation mode can indirectly improve the release of active ingredients and the bioconversion of secondary metabolites; the probiotics adopted in the fermentation are all derived from daily life, and the produced micromolecular substances are easy to be absorbed by human bodies to play a role;

(3) The application adopts a biological fermentation method to reduce the complicated procedure of the extraction process of plant source materials and the introduction of organic reagents, reduce the safety risk, and the fermentation liquor produced by fermentation has the effect of stronger DPPH clearance and the fermentation polypeptide has the effect of stronger tyrosinase inhibition and bacteriostasis.

Drawings

FIG. 1 is a flow chart of the preparation of fermentation broth and polypeptide provided by the application.

Detailed Description

The present application will be described in further detail with reference to the accompanying drawings, wherein it is to be understood that the following detailed description is for the purpose of further illustrating the application only and is not to be construed as limiting the scope of the application, as various insubstantial modifications and adaptations of the application to those skilled in the art can be made in light of the foregoing disclosure.

A preparation method of fermentation liquor or fermentation polypeptide with whitening and anti-aging effects mainly comprises the following steps:

(1) Preparation of culture medium

S1: crushing the rose and the rhizoma polygonati by using a high-speed crusher and mixing the crushed rose and the rhizoma polygonati at a ratio of 1-40:1-40 mass ratio, adding 1-50g of the mixed raw materials into 100mL of fermentation medium, particularly preferably three concentrations of 5g/100mL, 10g/100mL and 15g/100mL, preparing the fermentation medium, placing the fermentation medium at 121 ℃ and sterilizing for 15min for later use;

wherein, the fermentation medium comprises: 10.0g/L casein peptone, 8.0g/L beef powder, 4.0g/L yeast powder, 20g/L glucose, 0.2g/L magnesium sulfate, 5.0g/L sodium acetate, 2.0g/L triamine citrate, 2.0g/L dipotassium hydrogen phosphate, 0.05g/L manganese sulfate and 1.0g/L tween 80, and the pH of the culture medium is adjusted to 6.2+/-0.2;

s2: activating lactobacillus plantarum and Saccharomyces cerevisiae required by fermentation, inoculating to MRS and YM culture mediums respectively, and culturing at 37deg.C and 28deg.C to OD6 ₀₀ All are 0.8, and lactobacillus plantarum seed solution and saccharomyces cerevisiae seed solution required by fermentation are obtained;

(2) Inoculating fermentation

S1: inoculating lactobacillus plantarum seed solution with a volume ratio of 1-20% into a culture medium containing rhizoma Polygonati and flos Rosae Rugosae, placing on a constant temperature oscillator with a temperature of 20-50deg.C for culturing for 24-96 hr, optimally fermenting at 37 deg.C, placing in 40KHz ultrasonic wave for ultrasonic treatment at 1-20 min/interval of 3-30min, especially preferably 5min, and interval of 15min when fermentation is completed;

s2: inoculating Saccharomyces cerevisiae seed liquid at a volume ratio of 1-15% at the end of ultrasonic treatment, placing on a constant temperature oscillator at 20-40deg.C for culturing for 2-10d, fermenting at 15 deg.C for 5d, placing on 40KHz ultrasonic wave at 1-20 min/interval for 3-30min for ultrasonic treatment, preferably ultrasonic treatment for 5min, and interval for 15min, and centrifuging the fermentation broth in a centrifuge at 4000-10000rpm/min for 5-20min, preferably 5000rpm/min for 5min, and collecting supernatant;

(3) Preparation of fermentation broth proteins

Treating the fermentation clear liquid obtained in the step (2) by ammonium sulfate, refrigerating overnight in a refrigerator at 4 ℃, centrifuging at 5000rpm/min in a high-speed refrigerated centrifuge for 5min, discarding the supernatant, and collecting bottom sediment, wherein the adding amount of the ammonium sulfate is 10-80g of ammonium sulfate added into every 100ml of fermentation liquor;

Carrying out enzymolysis on the protein precipitate obtained in the step (3) by using complex enzyme (prepared by mixing pepsin, alkaline protease and trypsin according to the mass ratio of 1-10:1-10:1-10), namely obtaining the fermented polypeptide, wherein the mass ratio of protein to enzyme is 1-50 during enzymolysis: 1-100, enzymatic hydrolysis time of 5-30h, alkaline protease being particularly preferred: trypsin: pepsin = 1:1:1, protein: the complex enzyme is 1:40, the enzymolysis time is 12h.

Example 1

The embodiment provides a preparation method of fermentation liquor or fermentation polypeptide with whitening and anti-aging effects, which mainly comprises the following steps:

(1) Preparation of culture medium

Mixing the crushed rose with rhizoma polygonati according to a proportion of 1:1 in mass ratio to fermentation medium, and sterilizing for later use;

(2) Inoculating fermentation

Inoculating lactobacillus plantarum seed liquid with the volume ratio of 1% into a fermentation culture medium, fermenting for 24 hours by a constant-temperature oscillator with the temperature of 20 ℃, obtaining a primary fermentation product, then performing ultrasonic treatment in an ultrasonic instrument, inoculating saccharomyces cerevisiae seed liquid with the volume ratio of 1% into the primary fermentation product after ultrasonic treatment, fermenting for 2 days by a constant-temperature oscillator with the temperature of 10 ℃, placing the secondary fermentation product into the ultrasonic instrument again for ultrasonic treatment, centrifuging to obtain an upper fermentation clear liquid, namely fermentation liquor, and storing for later use;

(3) Preparation of fermentation broth proteins

Adding ammonium sulfate into the fermentation liquor obtained in the step (2) to extract protein in the fermentation clear liquor, and centrifuging to obtain a precipitate, wherein the adding amount of the ammonium sulfate is 10g of ammonium sulfate added into every 100ml of fermentation liquor;

To the protein precipitate obtained in step (3) was added 1:40 mass ratio, and adding compound enzyme for enzymolysis for 5 hours to obtain the fermented polypeptide.

Example 2

(1) Preparation of culture medium

Mixing the crushed rose with rhizoma polygonati according to a proportion of 20:40 mass ratio is added into a fermentation medium, and the fermentation medium is sterilized for later use;

(2) Inoculating fermentation

Inoculating lactobacillus plantarum seed liquid with the volume ratio of 10% into a fermentation culture medium, placing the lactobacillus plantarum seed liquid into a constant-temperature oscillator at 37 ℃ for fermentation for 48 hours, placing the fermentation product into an ultrasonic instrument for ultrasonic treatment after obtaining a primary fermentation product, inoculating saccharomyces cerevisiae seed liquid with the volume ratio of 8% into the primary fermentation product after ultrasonic treatment, placing the fermentation product into a constant-temperature oscillator at 15 ℃ for fermentation for 5 days, placing the secondary fermentation product into the ultrasonic instrument for ultrasonic treatment again, and centrifuging to obtain an upper fermentation clear liquid which is the fermentation liquid, and storing the fermentation clear liquid for later use;

(3) Preparation of fermentation broth proteins

Adding ammonium sulfate into the fermentation liquor obtained in the step (2) to extract protein in the fermentation clear liquor, and centrifuging to obtain a precipitate, wherein the adding amount of the ammonium sulfate is 50g of ammonium sulfate added into each 100ml of fermentation liquor;

To the protein precipitate obtained in step (3) was added 1:40 mass ratio, and adding compound enzyme for enzymolysis for 12 hours to obtain the fermented polypeptide.

Example 3

The embodiment provides a preparation method of fermentation liquor or fermentation polypeptide, which mainly comprises the following steps:

(1) Preparation of culture medium

Mixing the crushed rose with rhizoma polygonati according to a proportion of 40:20 mass ratio is added into a fermentation medium, and the fermentation medium is sterilized for later use;

(2) Inoculating fermentation

Inoculating lactobacillus plantarum seed liquid with the volume ratio of 20% into a fermentation culture medium, placing the lactobacillus plantarum seed liquid into a constant-temperature oscillator with the temperature of 50 ℃ for fermentation for 96 hours, placing the fermentation product into an ultrasonic instrument for ultrasonic treatment after obtaining a primary fermentation product, inoculating saccharomyces cerevisiae seed liquid with the volume ratio of 15% into the primary fermentation product after ultrasonic treatment, placing the fermentation product into a constant-temperature oscillator with the temperature of 40 ℃ for fermentation for 10 days, placing the secondary fermentation product into the ultrasonic instrument for ultrasonic treatment again, and centrifuging to obtain upper fermentation clear liquid which is fermentation liquid, and storing the fermentation liquid for later use;

(3) Preparation of fermentation broth proteins

Adding ammonium sulfate into the fermentation liquor obtained in the step (2) to extract protein in the fermentation clear liquor, and centrifuging to obtain a precipitate, wherein the adding amount of the ammonium sulfate is 80g of ammonium sulfate added into every 100ml of fermentation liquor;

To the protein precipitate obtained in step (3) was added 1: and (3) adding a compound enzyme according to the mass ratio of 100 for enzymolysis for 30 hours to obtain the fermented polypeptide.

Example 4

In order to verify the beneficial effects of the product of the present application, the present embodiment provides a method for preparing and verifying a fermentation broth or a fermentation polypeptide, wherein the methods are conventional methods known to those skilled in the art unless otherwise specified, and the materials such as reagents are commercially available products unless otherwise specified.

The method specifically comprises the following steps:

1. fermenting flos Rosae Rugosae and rhizoma Polygonati composition with Saccharomyces cerevisiae

(1) Mixing the crushed rose with rhizoma polygonati according to a proportion of 1:1 mass ratio, adding into 3 MRS culture mediums respectively at 5%, 10% and 15% mass ratio, cooling to room temperature after sterilization, sequentially inoculating 1% of Saccharomyces cerevisiae seed solution at volume ratio, placing on 15 ℃ constant temperature oscillator, and fermenting for 5d at 200 r/min;

(2) Respectively placing the fermented materials into an ultrasonic instrument for ultrasonic treatment after fermentation is finished, and performing ultrasonic treatment for 5min at intervals of 20min for 4 times;

(3) Inoculating and inoculating 1% of Saccharomyces cerevisiae seed liquid by volume ratio again after ultrasonic treatment of the fermentation liquid is finished, placing the fermentation liquid on a constant temperature oscillator at 15 ℃ and fermenting for 5d at 200r/min, placing the fermentation liquid in an ultrasonic instrument for ultrasonic treatment after the fermentation is finished, placing the fermentation liquid in a centrifugal machine for centrifugal treatment at 5000rpm/min for 15min, and collecting upper fermentation clear liquid in 3 MRS culture mediums to obtain the fermentation liquid.

2. Fermented rose and rhizoma polygonati composition adopting lactobacillus plantarum

(1) Mixing the crushed rose with rhizoma polygonati according to a proportion of 1:1 mass ratio, adding the mixture into 3 MRS culture mediums at the mass ratio of 5%, 10% and 15%, sterilizing the culture mediums at 121 ℃ for 15min, after the sterilization is finished and the culture mediums are cooled to room temperature, sequentially inoculating lactobacillus plantarum seed liquid at the volume ratio of 1%, and then placing the lactobacillus plantarum seed liquid on a constant-temperature oscillator at 37 ℃ and fermenting the lactobacillus plantarum seed liquid at 200r/min for 48h;

(2) Respectively placing the fermentation products into an ultrasonic instrument for ultrasonic treatment after fermentation is finished, wherein the interval between ultrasonic treatment and ultrasonic treatment is 20min, and the ultrasonic treatment is carried out for 4 times;

(3) Inoculating lactobacillus plantarum seed liquid with the volume ratio of 1% again after the ultrasonic treatment is finished, placing the lactobacillus plantarum seed liquid on a constant-temperature oscillator with the temperature of 37 ℃ and fermenting for 48 hours at the speed of 200r/min, placing the fermentation finished products in an ultrasonic instrument for ultrasonic treatment respectively, placing the ultrasonic treatment in a centrifuge for centrifugation at the speed of 5000rpm/min for 15 minutes respectively, and collecting upper-layer fermentation clear liquid in 3 MRS culture mediums.

3. Rose and rhizoma polygonati composition prepared by adopting lactobacillus plantarum and saccharomyces cerevisiae to jointly ferment

(1) Mixing the crushed rose with rhizoma polygonati according to a proportion of 1:1 mass ratio, adding the mixture into 3 MRS culture mediums at the mass ratio of 5%, 10% and 15%, sterilizing the culture mediums at 121 ℃ for 15min, after the sterilization is finished and the culture mediums are cooled to room temperature, sequentially inoculating lactobacillus plantarum seed liquid at the volume ratio of 1%, and fermenting the lactobacillus plantarum seed liquid at the temperature of 37 ℃ for 48h at 200 r/min;

(3) Inoculating 1% of wine yeast seed liquid by volume ratio in sequence when the ultrasonic treatment is finished, placing on a constant-temperature oscillator at 37 ℃ and fermenting for 5d at 200 r/min;

(4) And after fermentation is finished, respectively placing the fermentation products into an ultrasonic instrument again, carrying out ultrasonic treatment at ultrasonic 5min intervals of 20min, centrifuging the fermentation products for 15min at 5000rpm/min at the end of ultrasonic treatment, and collecting upper fermentation clear liquid in 3 MRS culture mediums.

4. Fermented polypeptide production

(1) Taking 10mL of the samples collected in the steps 1, 2 and 3 into a container respectively, sequentially adding 5g of ammonium sulfate powder, and placing the mixture in a refrigerator at 4 ℃ for refrigeration overnight;

(2) Respectively placing the 3 groups of samples treated in the step (1) into a centrifugal machine to be centrifuged at 5000rpm/min for 15min, and sequentially collecting the lower-layer sediment;

(3) 3 groups of sediment samples collected in step (3) were mixed with 1:40, sequentially adding compound enzyme (alkaline protease: trypsin: protease=1:1:1) for enzymolysis for 12h;

(4) Passivating 3 groups of enzymolysis liquid samples at 55 ℃ for 30min after enzymolysis is finished to obtain an enzymolysis product, namely fermented polypeptide;

(5) The fermented polypeptide is prepared into powder by vacuum freeze drying for standby.

In order to verify the efficacy of fermentation broth and fermented polypeptide prepared by fermenting rose and rhizoma Polygonati composition with different probiotics, a series of verification experiments are made as follows:

DPPH Activity test

DPPH is a nitrogen-centered stable radical whose absolute ethanol solution exhibits a maximum absorption at a wavelength of 517 nm. In the presence of a radical scavenger, DPPH may be combined or replaced to reduce the number of radicals resulting in a decrease in absorbance.

(1) The reagent is prepared, DPPH3.0mg is precisely weighed and placed in a 10ml volumetric flask, a small amount of absolute ethyl alcohol is added to dissolve the reagent, the volume is fixed to the scale, and the solution is uniformly shaken. And (3) taking another 2ml to 100ml volumetric flask, and shaking uniformly to obtain DPPH solution with the concentration of 0.006 mg/ml.

(2) Diluting the samples to be tested collected in the steps 1, 2, 3 and 4 into different multiples;

(3) Adding 3.0mL of LDPPH solution and 0.5mL of 2.1, 2.2, 2.3 and 2.4 sample solutions to be detected into the 4 groups of sample tubes, and adding 3.0mL of absolute ethyl alcohol and 0.5mL of sample solutions to be detected into the control tube; the blank was added with 3.0mL of LDPPH solution and 0.5mL of distilled water;

(4) After the reagent is added into the 4 groups of sample tubes, the control tube and the blank tube, the reaction solution is placed in a dark condition for reaction for 30min;

(5) After the reaction is finished, 3.0mL of absolute ethyl alcohol and 0.5mL of distilled water are used for zeroing, and the zeroes are respectively placed under the condition of 517nm wavelength to detect the change of the empty absorbance of each reaction, and 3 parallel reaction holes are arranged to calculate the change of the average value of the empty absorbance.

(6) DPPH clearing test results

The effect of fermentation broths prepared by combined fermentation of Saccharomyces cerevisiae, lactobacillus plantarum and two probiotics and the effect of fermentation polypeptides on DPPH removal were compared with fermentation broths prepared by fermenting different concentrations of fermentation raw materials (Polygonatum sibiricum: roseum = 1:1), and the results are shown in Table 1.

TABLE 1 DPPH inhibitory Effect

Remarks: all samples were diluted with distilled water, and the broth samples in steps 1, 2, 3 and 4 were diluted 30-fold; the fermentation polypeptide was formulated as a stock solution of 0.1mg/ml, and then diluted 30-fold to a working solution of 0.0033mg/ml for the experiment.

Conclusion of experiment: the fermentation liquor after probiotic fermentation has higher DPPH removing effect than unfermented fermentation and the combined fermentation of the strains is higher than the single strain fermentation removing effect.

In addition, the effect of the fermentation polypeptide prepared by fermenting Saccharomyces cerevisiae and lactobacillus plantarum on eliminating DPPH is stronger than that of unfermented fermentation polypeptide, and the combined fermentation of the two strains further improves the effect of the fermentation polypeptide on eliminating DPPH, which is higher than that of single strain fermentation.

Experiment for inhibiting tyrosinase activity

Tyrosinase is an oxidase and is the rate-limiting enzyme that regulates melanin production. This enzyme is involved in two reactions of melanin synthesis: the first step is to oxidize monophenols to diphenols and the second step is to oxidize ortho-diphenols to ortho-diphenoquinones. The o-biquinone turns into melanin after several steps of reaction. Tyrosinase can be found in melanosomes of skin melanocytes, and thus, inhibition of tyrosinase activity assays can be used to assess the efficacy of whitening ingredients.

(1) Reagent preparation

(1) 50mM phosphate buffer (pH 6.8)

3.42g of monopotassium phosphate is precisely weighed to a volume of 125mL, 0.49g of sodium hydroxide is weighed to a volume of 59mL, and the two are mixed and water is used to a volume of 500mL.

(2) 1.8mM L-tyrosine: 0.0659 gL-tyrosine was precisely weighed to dissolve and to a volume of 200mL.

(3) 125unit/mL (0.25 mg/mL) tyrosinase: 0.25mg of tyrosinase was precisely weighed and 1mL of 50mM phosphate buffer was added to make it fully dissolved.

(4) 100mg/mL arbutin stock: 200mg of arbutin is precisely weighed, added with 2mL of 50mM phosphate buffer solution, and placed at the temperature of minus 20 ℃ for standby, and 0.12mg/mL of arbutin is prepared: 6mg was weighed precisely and its volume was fixed to 50mL.

(2) Referring to the tyrosinase inhibition assay reaction system shown in Table 2, 50mM phosphate buffer, assay sample and 1.8mM L-tyrosine were added in sequence to the reaction system, and then incubated at 37℃for 5min, 125unit/mL tyrosinase was added after the incubation was completed, and incubated at 37℃for 15min, and the absorbance change was detected at 475nm wavelength.

The calculation formula is as follows: inhibition ratio (%) = [1- (C-D)/(a-B) ]x100%.

TABLE 2 tyrosinase inhibition assay reaction system

(3) Tyrosinase inhibition assay results

The tyrosinase inhibition rates of fermentation broths and fermentation polypeptides prepared by combined fermentation of Saccharomyces cerevisiae, lactobacillus plantarum and two probiotics were compared with fermentation broths prepared from different concentrations of fermentation raw materials (Polygonatum sibiricum: roseum=1:1), and the results are shown in Table 3.

TABLE 3 inhibition of tyrosinase by fermentation broths and fermentation polypeptides

Remarks: all samples were diluted with distilled water, and the broth samples in steps 1, 2, 3 and 4 were diluted 30-fold; the fermented polypeptide is prepared into freeze-dried powder, and the freeze-dried powder is prepared into 100mg/mL concentration to detect the inhibition of the fermented polypeptide on tyrosinase.

Conclusion of experiment: the inhibition effect of the fermentation liquor of single strain fermentation and multi-strain combined fermentation on tyrosinase is enhanced compared with that of the fermentation liquor of non-fermentation, the inhibition effect on tyrosinase is lower than 20%, and the inhibition effect of positive control on tyrosinase is far higher than that of the fermentation liquor.

In addition, the prepared polypeptide powder is prepared into 100mg/mL by using 50mM phosphate buffer solution, and the inhibition effect of the unfermented polypeptide on tyrosinase is only 35.23%. The inhibition effect of the polypeptide prepared by fermenting the single strain on the tyrosinase is lower than that of the polypeptide prepared by fermenting the two strains, wherein the inhibition effect of the polypeptide prepared by fermenting the single strain on the tyrosinase reaches 68.68 percent, and the raw material addition amount of the fermentation liquor is 15 percent.

Bacteriostasis experiment

(1) Strain activation

Inoculating Escherichia coli and Staphylococcus aureus into LB medium respectively, culturing in 37 deg.C constant temperature incubator for 24 hr, inoculating single colony into LB liquid medium, culturing at 200r/min to OD6 ₀₀ Diluting the bacterial liquid for 10 times to be stored for standby after the bacterial liquid is 0.6;

(2) Filtering the culture medium, fermentation broth and polypeptide solution with 0.45 μm sterile filter head;

(3) Taking 1mg/mL of the green-streptomycin mixed solution as a positive control, and taking a culture medium as a negative control;

(4) Inoculating 100 mu L of the diluted bacterial liquid in the step (1) to an LB culture medium, uniformly coating, and adding 50 mu L of a sample to be detected and a positive control in the central part of a culture dish;

(5) And (3) placing the culture medium in the step (4) at 37 ℃ for culturing for 24 hours, and detecting the diameter of the inhibition zone by using a vernier caliper.

(6) Antibacterial effect of fermentation broth

The inhibitory effect of the fermentation broth on E.coli and Staphylococcus aureus was compared and the results are shown in Table 4.

TABLE 4 bacteriostatic effects of fermentation broths

Remarks: all samples were diluted with distilled water and the broth samples in steps 1, 2, 3 and 4 were diluted 30-fold.

Conclusion of experiment: the bacteria inhibition effect of the fermentation liquor obtained by fermenting the single strain is poorer than that of the combined fermentation effect of the two strains. The fermentation liquor has stronger effect of inhibiting staphylococcus aureus than escherichia coli, and the bacteriostasis effect of lactobacillus plantarum is stronger than that of saccharomyces cerevisiae.

(7) Antibacterial effect of fermented polypeptide

The collected polypeptide powder was formulated into 0.1mg/mL to investigate the bacteriostatic effect, and the inhibitory effects of the fermented polypeptide on E.coli and Staphylococcus aureus were compared, and the results are shown in Table 5.

TABLE 5 bacteriostatic effects of fermented polypeptides

Remarks: the fermented polypeptide is prepared into freeze-dried powder, and the freeze-dried powder is prepared into 100mg/mL concentration to detect the inhibition effect of the fermented polypeptide on escherichia coli and staphylococcus aureus.

Conclusion of experiment: the antibacterial activity of the polypeptide prepared by lactobacillus plantarum fermentation is stronger than that of saccharomyces cerevisiae, and the combined fermentation of the two strains can improve the antibacterial activity of the polypeptide, so that the effect of the polypeptide in inhibiting staphylococcus aureus is stronger than that of escherichia coli.

Certain terms are used throughout the description and claims to refer to particular components or methods. It will be appreciated by those of ordinary skill in the art that different regions may be referred to by different terms as a single component. The description and claims do not take the difference in name as a way of distinguishing components. As used throughout the specification and claims, the word "comprise" is an open-ended term, and thus should be interpreted to mean "include, but not limited to. By "substantially" is meant that within an acceptable error range, a person skilled in the art is able to solve the technical problem within a certain error range, substantially achieving the technical effect. The description hereinafter sets forth a preferred embodiment for practicing the application, but is not intended to limit the scope of the application, as the description is given for the purpose of illustrating the general principles of the application. The scope of the application is defined by the appended claims.

It should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a product or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such product or system. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a commodity or system comprising such elements.

While the foregoing description illustrates and describes several preferred embodiments of the application, it is to be understood that the application is not limited to the forms disclosed herein, but is not to be construed as limited to other embodiments, and is capable of use in various other combinations, modifications and environments and is capable of changes or modifications within the spirit of the application described herein, either as a result of the foregoing teachings or as a result of the knowledge or skill of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the application are intended to be within the scope of the appended claims.

Claims

1. The fermentation liquor with the whitening and anti-aging effects is characterized in that the fermentation liquor is prepared by taking a combination of rose and rhizoma polygonati as raw materials, adding the crushed raw materials into a fermentation medium, fermenting the raw materials for the first time by using lactobacillus plantarum seed liquor with the volume ratio of 1-20%, and fermenting the primary fermentation product for the second time by using saccharomyces cerevisiae seed liquor with the volume ratio of 1-15%;

the mass ratio of the rose to the rhizoma polygonati is 1:1.

2. Use of the fermentation broth according to claim 1 for preparing a whitening anti-aging cosmetic.

3. A fermented polypeptide with whitening and anti-aging effects is characterized in that the fermented polypeptide is prepared by taking protein in the fermentation broth as a raw material through enzymolysis.

4. A fermented polypeptide having whitening and anti-aging effects according to claim 3, wherein the protein in the fermentation broth is extracted by ammonium sulfate precipitation.

5. A fermented polypeptide having whitening and anti-aging effects according to claim 3, wherein the protein in the fermentation broth is enzymatically hydrolyzed by a complex enzyme comprising: pepsin, alkaline protease and trypsin.

6. Use of a fermented polypeptide according to any one of claims 3 to 5 for the preparation of a cosmetic product with whitening and anti-ageing properties.

7. A process for the preparation of a fermentation broth or a fermented polypeptide according to claim 1 or 3, comprising essentially the steps of:

preparation of culture medium

Adding the crushed rose and rhizoma polygonati into a fermentation medium according to the mass ratio of 1:1, and sterilizing for later use;

inoculating fermentation

fermentation broth protein extraction

preparation of fermented polypeptide by zymohydrolysis of fermentation liquor

8. The method for preparing the fermentation broth or the fermentation polypeptide with the whitening and anti-aging effects according to claim 7, wherein the method comprises the following steps of: in the step (2), the primary fermentation product and the secondary fermentation product are treated by ultrasonic waves with the frequency of 40KHz for 1-20min at intervals of 3-30min.

9. The method for preparing the fermentation broth or the fermentation polypeptide with the whitening and anti-aging effects according to claim 7, wherein the method comprises the following steps of: the complex enzyme is pepsin, alkaline protease and trypsin with a weight ratio of 1-10:1-10:1-10 mass ratio.