CN113413351A

CN113413351A - Fermentation liquor with whitening and anti-aging effects, fermented polypeptide, and preparation method and application thereof

Info

Publication number: CN113413351A
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: 2021-09-21
Anticipated expiration: 2041-07-15
Also published as: CN113413351B

Abstract

The invention relates to a fermentation broth with whitening and anti-aging effects, a fermented polypeptide, a preparation method and application thereof. The rose and sealwort mixed powder is fermented and subjected to ultrasonic treatment by lactobacillus plantarum, and is subjected to low-temperature fermentation and ultrasonic treatment by saccharomyces cerevisiae again, so that the obtained fermentation liquor has a strong antioxidation effect, the polypeptide prepared by enzymolysis of protein in the fermentation liquor has an inhibition effect on tyrosinase, and the preparation processes of the fermentation liquor and the fermented polypeptide are simple and easy to obtain, and no additive or organic reagent is contained, so that the rose and sealwort mixed powder is applied to whitening and anti-aging cosmetics and can improve the healthy microecology of skin.

Description

Fermentation liquor with whitening and anti-aging effects, fermented polypeptide, and preparation method and application thereof

Technical Field

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

Background

The health effect of the probiotics is firstly provided by Russian scientists Mctcnikoff that the probiotics have the effects of prolonging the service life, preventing putrefying bacteria, treating intestinal canal disorder in medicine, resisting tumors and the like, and is widely applied to medical health 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 generate various nutritional active ingredients such as volatile acid, vitamin, folic acid, nicotinic acid, pantothenic acid and the like. On the other hand, the probiotics improve the activity of macrophages, further enhance the immune function of the organism, and promote the growth and development of immune organs by feeding the broiler chickens with the probiotics producing protease. The probiotic preparation has effects of resisting radiation and fatigue through white rat experiment. Yeast is primarily a single cell protein of yeast and is therefore also called single cell protein. The yeast as a fungus culture contains a large amount of effective proteins and beneficial bacteria, inhibits the propagation of pathogenic microorganisms, improves the immunity and disease resistance of organisms, is rich in nutrition and rich in crude protein, B vitamins, minerals, digestive enzymes, growth promotion factors and complete amino acids, and is widely applied to medical health care and industrial production. The yeast can be divided into nutritional yeast and functional yeast, the nutritional yeast is mainly a product prepared by fermenting beer yeast or baker's yeast, performing enzymolysis treatment and spray drying, and the product contains abundant proteins, small peptides, nucleotides, vitamins and the like. The functional yeast is mainly derivatives of some yeast, active dry yeast and the like, and is rich in nutrients such as yeast cell wall polysaccharide, yeast selenium, yeast chromium and the like. The lactobacillus plantarum is one of common probiotics, and plays roles in resisting aging, oxidation, bacteria and the like in daily life.

The ferment is produced by utilizing microbial fermentation plant and fungus sources, contains enzymes, catalytic substances and other multiple biological active substances, is taken as a complex protein participating in metabolism in organisms, widely exists in living cells and plays an important role, so that the ferment product is widely recognized, and various edible ferment products are developed. At present, probiotics are widely applied to traditional Chinese medicine fermentation, wherein the probiotics have multiple effects of improving the content of functional substances of the medicines, improving the efficacy of fermentation products, reducing toxicity and the like. The method has abundant plant resources in China, and the method is applied by extraction and extraction by utilizing a development main mode, so that the process is complicated and toxic and harmful substances are possibly brought into residues. Therefore, the method has important research significance for changing the utilization mode of plant resources and improving the utilization rate of the resources.

Rose has the effects of whitening, improving skin texture and promoting blood circulation and metabolism, and modern researches on polygonatum sibiricum contain polygonatum sibiricum saponin, nicotinic acid, saccharides, quinones, amino acids, trace elements and the like, so that the rose or polygonatum sibiricum has the effects of beautifying, keeping young and resisting aging.

Not only aiming at the rose and polygonatum 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 amount of prebiotics beneficial to human skin absorption through biological fermentation, macromolecules which are difficult to be absorbed by human bodies of the plants are converted into micromolecules, and on the other hand, macromolecular proteins are converted into polypeptides through enzymolysis by the application of an enzymolysis technology, so that the macromolecular proteins can efficiently act on different targets to exert corresponding biological activity. The enzyme serving as a macromolecule is difficult to be directly absorbed and utilized by skin, so that a fermentation liquid and a fermented polypeptide with whitening and anti-aging effects, as well as a preparation method and an application thereof are provided.

Disclosure of Invention

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

The invention realizes the 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 primary fermentation products by using saccharomyces cerevisiae.

As a further optimized scheme of the invention, the natural plant raw materials with the whitening and anti-aging effects are a composition of rose and rhizoma polygonati.

Application of the fermentation liquor in preparation of whitening and 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 invention, the extraction method of the protein in the fermentation liquor is an ammonium sulfate precipitation method.

As a further optimization scheme of the invention, the protein in the fermentation liquor is subjected to enzymolysis by using a complex enzyme, and the complex enzyme comprises the following components: pepsin, alkaline protease and trypsin.

Application of the fermented polypeptide in preparing whitening anti-aging cosmetics.

A preparation method of the fermentation liquor or the fermentation polypeptide mainly comprises the following steps:

(1) preparation of culture medium

Mixing the crushed rose and the crushed rhizoma polygonati in a proportion of 1-40: 1-40 mass ratio, and sterilizing;

(2) inoculating fermentation

Inoculating 1-20% of lactobacillus plantarum seed liquid in volume ratio into a fermentation culture medium, placing the lactobacillus plantarum seed liquid into a constant-temperature oscillator at 20-50 ℃ for fermentation for 24-96h to obtain a primary fermentation product, placing the primary fermentation product into an ultrasonic instrument for ultrasonic treatment, inoculating 1-15% of saccharomyces cerevisiae seed liquid into the primary fermentation product after ultrasonic treatment, placing the primary fermentation product into a constant-temperature oscillator at 10-40 ℃ for fermentation for 2-10d, placing the secondary fermentation product into the ultrasonic instrument for ultrasonic treatment again, centrifuging to obtain supernatant fermentation liquid, namely the fermentation liquid, and storing the fermentation liquid for later use;

(3) protein extraction from fermentation broth

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

(4) zymolysis of fermented liquid to prepare fermented polypeptide

Adding to the protein precipitate obtained in step (3) a mixture of 1-20: adding complex enzyme for enzymolysis for 5-30h according to the mass ratio of 1-100 to obtain the fermented polypeptide.

As a further optimization scheme of the invention, in the step (2), both the primary fermentation product and the secondary fermentation product are subjected to ultrasonic treatment at the frequency of 40KHz, wherein the ultrasonic time is 1-20min, and the ultrasonic interval is 3-30 min.

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

The invention has the beneficial effects that:

(1) the invention adopts the composition of natural plant rose and rhizoma polygonati, the rose has the effects of whitening, improving the skin texture and promoting blood circulation and metabolism, and the rhizoma polygonati is discovered by modern researches to contain rhizoma polygonati saponin, nicotinic acid, saccharides, quinones, amino acids, trace elements and the like, has the effects of maintaining beauty, keeping young and resisting aging, and can be applied to the preparation of whitening and antibiotic products through biological fermentation.

(2) According to the invention, saccharomyces cerevisiae and lactobacillus plantarum are used as fermentation strains, and the temperature difference of two probiotics is utilized for fermentation, so that the release of effective components and the biotransformation of secondary metabolites can be indirectly improved by a combined fermentation mode; the probiotics adopted by fermentation are all from daily life, and the generated small molecular substances are easy to be absorbed by human bodies to play a role;

(3) the invention reduces the complicated procedures of the extraction process of the plant source materials and the introduction of organic reagents by adopting a biological fermentation method, reduces the safety risk, and the fermentation broth generated by fermentation has stronger DPPH (dipeptidyl peptidase) removing effect and the fermented polypeptide has stronger tyrosinase inhibiting effect and bacteriostatic effect.

Drawings

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

Detailed Description

The present application will now be described in further detail with reference to the drawings, it should be noted that the following detailed description is given for illustrative purposes only and is not to be construed as limiting the scope of the present application, as those skilled in the art will be able to make numerous insubstantial modifications and adaptations to the present application based on the above 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: pulverizing flos Rosae Rugosae and rhizoma Polygonati with a high speed pulverizer, and mixing at a ratio of 1-40: mixing the raw materials uniformly at a mass ratio of 1-40, adding 1-50g of the mixed raw materials into 100mL of fermentation medium, particularly preferably adding three concentrations of 5g/100mL, 10g/100mL and 15g/100mL of the mixed raw materials to prepare the fermentation medium, and sterilizing the fermentation medium at the temperature of 121 ℃ for 15min for later use;

wherein the fermentation medium comprises the following components: 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 citric acid triamine, 2.0g/L dipotassium hydrogen phosphate, 0.05g/L manganese sulfate and 1.0g/L Tween 80, and the pH value of the culture medium is adjusted to 6.2 +/-0.2;

s2: activating and culturing Lactobacillus plantarum and Saccharomyces cerevisiae, inoculating to MRS and YM culture medium, respectively, culturing at 37 deg.C and 28 deg.C to OD6₀₀Both are 0.8, thus obtaining lactobacillus plantarum seed liquid and saccharomyces cerevisiae seed liquid required by fermentation;

(2) inoculating fermentation

S1: inoculating 1-20% volume ratio of Lactobacillus plantarum seed solution into culture medium containing rhizoma Polygonati and flos Rosae Rugosae, culturing in 20-50 deg.C constant temperature oscillator for 24-96h, optimally fermenting at 37 deg.C, and treating with 40KHz frequency ultrasonic wave at an interval of 1-20 min/3-30 min for ultrasonic treatment, preferably 5min for ultrasonic treatment at an interval of 15 min;

s2: inoculating saccharomyces cerevisiae seed liquid with the volume ratio of 1-15% when the ultrasonic treatment is finished, culturing the saccharomyces cerevisiae seed liquid on a constant temperature oscillator with the temperature of 20-40 ℃ for 2-10d, wherein the optimal fermentation temperature is 15 ℃, the fermentation time is 5d, placing the saccharomyces cerevisiae liquid on 40KHz frequency ultrasonic waves for ultrasonic treatment at the interval of 1-20 min/3-30 min when the saccharomyces cerevisiae fermentation is finished, particularly preferably performing ultrasonic treatment for 5min and 15min, finally placing the fermentation liquid in a centrifuge, centrifuging the fermentation liquid for 5-20min at the speed of 4000 plus 10000rpm/min, particularly preferably centrifuging the fermentation liquid for 5min at the speed of 5000rpm/min, and collecting supernatant fermentation liquid;

(3) preparation of fermentation broth protein

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

(4) zymolysis of fermented liquid to prepare fermented polypeptide

And (3) carrying out enzymolysis on the protein precipitate obtained in the step (3) by using complex enzyme (prepared by mixing pepsin, alkaline protease and trypsin in a mass ratio of 1-10: 1-10: 1-10) to obtain fermented polypeptide, wherein the mass ratio of the protein to the enzyme is 1-50: 1-100, the enzymolysis time is 5-30h, and the alkaline protease is particularly preferred: trypsin: pepsin-1: 1: 1, protein: the complex enzyme is 1: 40, the enzymolysis time is 12 hours.

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 and the crushed rhizoma polygonati in a proportion of 1: 1, adding the mixture into a fermentation medium, and sterilizing the mixture for later use;

(2) inoculating fermentation

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

(3) preparation of fermentation broth protein

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

(4) zymolysis of fermented liquid to prepare fermented polypeptide

Adding to the protein precipitate obtained in step (3) a mixture of 1: adding complex enzyme for enzymolysis for 5h according to the mass ratio of 40 to obtain the fermented polypeptide.

Example 2

(1) preparation of culture medium

Mixing the crushed rose and the crushed rhizoma polygonati in a proportion of 20: 40, adding the mixture into a fermentation medium, and sterilizing the mixture for later use;

(2) inoculating fermentation

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

(3) preparation of fermentation broth protein

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

(4) zymolysis of fermented liquid to prepare fermented polypeptide

Adding to the protein precipitate obtained in step (3) a mixture of 1: adding complex enzyme for enzymolysis for 12h according to the mass ratio of 40 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 and the crushed rhizoma polygonati in a proportion of 40: 20 is added into a fermentation medium in a mass ratio and is used after sterilization;

(2) inoculating fermentation

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

(3) preparation of fermentation broth protein

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

(4) zymolysis of fermented liquid to prepare fermented polypeptide

Adding to the protein precipitate obtained in step (3) a mixture of 1: adding complex enzyme for enzymolysis for 30h according to the mass ratio of 100 to obtain the fermented polypeptide.

Example 4

To verify the beneficial effects of the products of the present invention, the present example provides methods for preparing and verifying fermentation broth or polypeptide, wherein the methods are conventional methods known to those skilled in the art without specific indications, and the materials such as reagents used, without specific indications, are commercially available products.

The method specifically comprises the following steps:

1. composition of rose and sealwort by fermenting saccharomyces cerevisiae

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

(2) respectively putting the fermented materials into an ultrasonic instrument for ultrasonic treatment for 5min at intervals of 20min for 4 times after fermentation;

(3) and (3) inoculating 1% volume ratio of saccharomyces cerevisiae seed liquid again when the fermentation liquid is subjected to ultrasonic treatment, then placing the saccharomyces cerevisiae seed liquid on a constant temperature oscillator at 15 ℃ and fermenting for 5d at 200r/min, respectively placing the saccharomyces cerevisiae seed liquid in an ultrasonic instrument after the fermentation is finished, centrifuging the saccharomyces cerevisiae seed liquid in a centrifuge at 5000rpm/min for 15min, and collecting supernatant fermentation liquor in 3 MRS culture media to obtain fermentation liquid.

2. Rose and polygonatum composition fermented by lactobacillus plantarum

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

(2) respectively placing the fermented materials in an ultrasonic instrument for ultrasonic treatment after fermentation is finished, wherein ultrasonic treatment is carried out for 5min at intervals of 20min for 4 times;

(3) and (3) inoculating the lactobacillus plantarum seed liquid with the volume ratio of 1% again when the ultrasonic treatment is finished, then 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, respectively placing the lactobacillus plantarum seed liquid in an ultrasonic instrument after the fermentation is finished, centrifuging the lactobacillus plantarum seed liquid in a centrifuge at the speed of 5000rpm/min for 15min, and collecting the supernatant fermentation liquid in 3 MRS culture media.

3. Rose and polygonatum composition fermented by combining lactobacillus plantarum and saccharomyces cerevisiae

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

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

(4) and when the fermentation is finished, respectively putting the culture medium into an ultrasonic instrument again, performing ultrasonic treatment at the frequency of 20min at intervals of 5min, centrifuging the culture medium at 5000rpm/min for 15min when the ultrasonic treatment is finished, and collecting supernatant fermentation liquid in 3 MRS culture media.

4. Fermentation polypeptide preparation

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

(2) respectively placing the 3 groups of samples processed in the step (1) into a centrifuge to centrifuge for 15min at 5000rpm/min, and sequentially collecting the lower-layer precipitates;

(3) combining 3 sets of pellet samples collected in step (3) at a ratio of 1: 40, sequentially adding complex enzyme (alkaline protease: trypsin: protease: 1: 1) for enzymolysis for 12 hours;

(4) after enzymolysis, putting 3 groups of enzymolysis liquid samples at the temperature of 55 ℃ for passivation for 30min to obtain an enzymolysis product, namely fermented polypeptide for later use;

(5) the fermented polypeptide is prepared into powder for later use through vacuum freeze drying.

In order to verify the efficacy of fermentation broth and fermented polypeptide prepared by fermenting rose and sealwort compositions by using different probiotics, a series of verification experiments are carried out, which are as follows:

DPPH Activity test

DPPH is a stable free radical centered on nitrogen, and its absolute ethanol solution shows maximum absorption at a wavelength of 517 nm. In the presence of a free radical scavenger, DPPH can be combined with or substituted for, reducing the number of free radicals resulting in a decrease in absorbance.

(1) Preparing reagents, precisely weighing DPPH3.0mg, putting the DPPH3.0mg into a 10ml volumetric flask, adding a small amount of absolute ethyl alcohol to dissolve the DPPH3.0mg, fixing the volume to a scale, and shaking up. And (3) taking another 2ml to 100ml volumetric flask, and shaking up to constant volume to obtain a DPPH solution with the concentration of 0.006 mg/ml.

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

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

(4) after reagents are added into the 4 groups of sample tubes, the control tubes and the blank tubes, placing the reaction solution in the dark for reaction for 30 min;

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

(6) Results of DPPH removal experiments

The effect of eliminating DPPH by fermentation broth and fermentation polypeptide prepared by fermentation of fermentation raw materials (Polygonatum sibiricum Red. 1: 1) with different concentrations by saccharomyces cerevisiae, lactobacillus plantarum and two probiotics was compared, and the results are shown in Table 1.

TABLE 1 DPPH inhibitory Effect

Remarking: all samples were diluted with distilled water, and the fermentation broth samples in steps 1, 2, 3 and 4 were diluted 30 times; fermented polypeptide is prepared into 0.1mg/ml stock solution, and then diluted by 30 times to prepare 0.0033mg/ml working solution for experiment.

And (4) experimental conclusion: the effect of the fermentation liquor fermented by the probiotics on removing DPPH is higher than that of unfermented fermentation, and the effect of the combined fermentation of the strains is higher than that of the single strain fermentation.

In addition, the DPPH removing effect of the fermented polypeptide prepared by fermentation of saccharomyces cerevisiae and lactobacillus plantarum is stronger than that of the unfermented fermented polypeptide, and the DPPH removing effect of the fermented polypeptide is further improved to be higher than that of single strain fermentation by combined fermentation of the two strains.

Inhibition of tyrosinase Activity

Tyrosinase is an oxidase and is the rate-limiting enzyme in the regulation of melanin production. This enzyme is involved in two reactions of melanin synthesis: in the first step, monophenol is hydroxylated into diphenol, and in the second step, o-diphenol is oxidized into o-diquinone. The o-diquinone is reacted in several steps to become melanin. Tyrosinase is found in the melanosomes of skin melanocytes, and therefore, inhibition of tyrosinase activity experiments can be used to assess the efficacy of whitening ingredients.

(1) Reagent preparation

50mM phosphate buffer (pH6.8)

3.42g of potassium dihydrogen phosphate was weighed precisely and made to a volume of 125mL, 0.49g of sodium hydroxide was weighed and made to a volume of 59mL, and the two were mixed and made to a volume of 500mL using water.

1.8mM L-tyrosine: 0.0659g of L-tyrosine was precisely weighed, dissolved and made to 200 mL.

③ 125unit/mL (0.25mg/mL) tyrosinase: 0.25mg of tyrosinase was weighed out and dissolved thoroughly by adding 1mL of 50mM phosphate buffer.

Fourthly, 100mg/mL arbutin stock solution: accurately weighing 200mg of arbutin, adding 2mL of 50mM phosphate buffer solution, preserving at-20 ℃ for later use, and preparing 0.12mg/mL of arbutin: 6mg was weighed out precisely and made to 50 mL.

(2) Referring to a tyrosinase inhibition experiment reaction system shown in Table 2, 50mM phosphate buffer solution, an experiment sample and 1.8mM L-tyrosine are sequentially added into the reaction system, then the reaction system is incubated at 37 ℃ for 5min, after the incubation is finished, 125unit/mL tyrosinase is added, the incubation is carried out at 37 ℃ for 15min, and the mixture is placed at 475nm wavelength to detect the change of absorbance.

Calculating the formula: the inhibition rate (%) [1- (C-D)/(a-B) ] × 100%.

TABLE 2 tyrosinase inhibition experiment reaction System

(3) Results of tyrosinase inhibition experiments

The results of comparison of the tyrosinase inhibition ratios of fermentation broth and fermented polypeptide prepared by the combined fermentation of saccharomyces cerevisiae, lactobacillus plantarum and two probiotics with different concentrations of fermentation raw materials (polygonatum: rose 1: 1) are shown in table 3.

TABLE 3 inhibitory Effect of fermentation broths and fermented Polypeptides on tyrosinase

Remarking: all samples were diluted with distilled water, and the fermentation broth samples in steps 1, 2, 3 and 4 were diluted 30 times; the fermented polypeptide is prepared into lyophilized powder, and the lyophilized powder is prepared into a concentration of 100mg/mL to detect the inhibition effect of the fermented polypeptide on tyrosinase.

And (4) experimental conclusion: the inhibition effect of the fermentation liquor fermented by the single strain and the combined fermentation of the multiple strains on the tyrosinase is enhanced compared with that of the fermentation liquor which is not fermented, the inhibition effect on the tyrosinase is lower than 20%, and the inhibition effect of the positive control on the 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, and the inhibition effect of the polypeptide prepared without fermentation on tyrosinase is only 35.23%. The inhibition effect of the polypeptide prepared by single strain fermentation on tyrosinase is lower than that of the two strains combined fermentation, wherein the inhibition effect of the polypeptide prepared by 15% of the raw material addition amount in the fermentation liquid on tyrosinase is 68.68%.

Experiment for inhibiting bacteria

(1) Activation of bacterial strains

Respectively inoculating Escherichia coli and Staphylococcus aureus to LB culture medium, culturing in 37 deg.C constant temperature incubator for 24 hr, selecting single colony, inoculating to LB liquid culture medium, and culturing at 200r/min to OD6₀₀Diluting the bacterial liquid by 10 times and storing for later use when the bacterial liquid is 0.6;

(2) filtering the culture medium, the fermentation liquid and the polypeptide solution by using a sterile filter head with the diameter of 0.45 mu m;

(3) taking 1mg/mL green-streptomycin mixed liquor 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) into an LB culture medium and uniformly coating, and adding 50 mu L of a sample to be detected and a positive control into the central part of a culture dish;

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

(6) Bacteriostatic effect of fermentation liquor

The results of comparing the inhibitory effect of the fermentation broths on E.coli and S.aureus are shown in Table 4.

TABLE 4 bacteriostatic effect of fermentation broth

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

And (4) experimental conclusion: the bacteriostatic effect of the fermentation liquor obtained by fermenting a single strain is poorer than that of the combined fermentation of two strains. The effect of the fermentation liquor for inhibiting staphylococcus aureus is stronger than that of escherichia coli, and the effect of the lactobacillus plantarum in inhibiting bacteria is stronger than that of saccharomyces cerevisiae.

(7) Bacteriostatic effect of fermented polypeptide

The collected polypeptide powder was formulated to 0.1mg/mL to study bacteriostatic effects, and the results are shown in Table 5, comparing the inhibitory effects of the fermented polypeptides on Escherichia coli and Staphylococcus aureus.

TABLE 5 bacteriostatic Effect of fermented Polypeptides

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

And (4) experimental conclusion: the polypeptide prepared by fermenting lactobacillus plantarum has stronger antibacterial activity than that of saccharomyces cerevisiae, the antibacterial activity of the polypeptide can be improved by the combined fermentation of the two strains, and the effect of the polypeptide on inhibiting staphylococcus aureus is stronger than that of escherichia coli.

As used in the specification and claims, certain terms are used to refer to particular components or methods. As one skilled in the art will appreciate, different regions may refer to a component by different names. The present specification and claims do not intend to distinguish between components that differ in name but not in name. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. "substantially" means within an acceptable error range, and a person skilled in the art can solve the technical problem within a certain error range to substantially achieve the technical effect. The description which follows is a preferred embodiment of the present application, but is made for the purpose of illustrating the general principles of the application and not for the purpose of limiting the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.

It is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a good 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 good or system. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other like elements in a commodity or system that includes the element.

While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. The fermentation liquor with the whitening and anti-aging effects is characterized in that natural plants with the whitening and anti-aging effects are used as raw materials, lactobacillus plantarum is used for carrying out primary fermentation on the raw materials, and then saccharomyces cerevisiae is used for carrying out secondary fermentation on primary fermentation products to prepare the fermentation liquor.

2. The fermentation broth with whitening and anti-aging effects as claimed in claim 1, wherein the natural plant materials with whitening and anti-aging effects are a composition of rose and polygonatum.

3. Use of the fermentation broth according to any one of claims 1-2 in the preparation of whitening anti-aging cosmetics.

4. A fermented polypeptide with whitening and anti-aging effects, which is characterized in that the fermented polypeptide is prepared by taking protein in the fermentation liquid of any one of claims 1-2 as a raw material and performing enzymolysis.

5. The fermented polypeptide with whitening and anti-aging effects as claimed in claim 4, wherein the extraction method of the protein in the fermentation liquid is ammonium sulfate precipitation.

6. The fermented polypeptide with whitening and anti-aging effects as claimed in claim 4, wherein the enzymolysis is performed on the protein in the fermentation liquid by using a complex enzyme, and the components of the complex enzyme comprise: pepsin, alkaline protease and trypsin.

7. Use of the fermented polypeptide according to any one of claims 4-6 for preparing whitening anti-aging cosmetics.

8. A method for producing the fermentation broth or polypeptide according to claim 1 or 4, comprising essentially the steps of:

(1) preparation of culture medium

(2) inoculating fermentation

(3) protein extraction from fermentation broth

(4) zymolysis of fermented liquid to prepare fermented polypeptide

9. The method for preparing the fermentation broth or the fermented polypeptide with whitening and anti-aging effects according to claim 8, wherein the fermentation broth or the fermented polypeptide with whitening and anti-aging effects is prepared from the following raw materials: in the step (2), both the primary fermentation product and the secondary fermentation product are subjected to ultrasonic treatment at the frequency of 40KHz, wherein the ultrasonic time is 1-20min, and the ultrasonic interval is 3-30 min.

10. The method for preparing the fermentation broth or the fermented polypeptide with whitening and anti-aging effects according to claim 8, wherein the fermentation broth or the fermented polypeptide with whitening and anti-aging effects is prepared from the following raw materials: the compound enzyme is pepsin, alkaline protease and trypsin which are mixed according to the weight ratio of 1-10: 1-10: 1-10 by mass ratio.