CN118048423A - Multi-effect bird's nest peptide and preparation method and application thereof - Google Patents
Multi-effect bird's nest peptide and preparation method and application thereof Download PDFInfo
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- CN118048423A CN118048423A CN202410287514.3A CN202410287514A CN118048423A CN 118048423 A CN118048423 A CN 118048423A CN 202410287514 A CN202410287514 A CN 202410287514A CN 118048423 A CN118048423 A CN 118048423A
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- bird
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- nest
- bacillus
- nest peptide
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Classifications
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- A23L33/18—Peptides; Protein hydrolysates
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
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- A—HUMAN NECESSITIES
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- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A61Q19/08—Anti-ageing preparations
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The application relates to the technical field of polypeptide preparation, in particular to a multi-effect bird's nest peptide and a preparation method and application thereof. The bird's nest peptide is prepared by fermenting the bird's nest enzymolysis products by using bacillus subtilis, bacillus cereus, bacillus licheniformis and bacillus amyloliquefaciens, has obvious antioxidation, whitening and moisturizing effects, belongs to pure natural raw materials, and has high use safety; and no chemical reagent is added in the preparation process, the preparation process is simple, the cost is low, the energy conservation and environmental protection are realized, and the preparation method has good application prospect in the fields of foods, health care products, cosmetics and the like.
Description
Technical Field
The application relates to the technical field of polypeptide preparation, in particular to a multi-effect bird's nest peptide and a preparation method and application thereof.
Background
The edible bird's nest contains protein, sialic acid, amino acid, inorganic element, trace element, sialyllactose, epidermal growth factor, etc. The edible bird's nest is considered by the traditional Chinese medicine to have the effects of tonifying qi, resolving phlegm, nourishing kidney, nourishing lung, tonifying spleen, harmonizing stomach, and regulating and tonifying consumptive disease. Modern medical research shows that edible bird's nest has the functions of whitening skin, resisting aging (resisting oxidation), resisting virus, regulating immunity, enhancing intelligence and memory, improving neurodegenerative diseases, etc.
In the prior art, the bird's nest peptide mainly adopts an extraction method, a microbial fermentation method and a hydrolysis method. The extraction method is a method for directly extracting natural polypeptide substances from biological samples, and the content of the polypeptide substances in the samples is required to be rich, but the quantity of the naturally-occurring peptide substances is very limited, and the direct extraction method is difficult to meet the requirement of large-scale production. Hydrolysis methods can be divided into acid hydrolysis, alkali hydrolysis and enzymatic hydrolysis. In contrast, the enzymatic hydrolysis method has strong specificity and high extraction rate, but the process is complicated and the cost is high. Microbial fermentation is a process of hydrolyzing proteins in a raw material using various enzyme catalysts generated during the growth of microorganisms, and then producing hydrolyzed polypeptides. The microbial fermentation method for preparing the polypeptide has the advantages of simple process, high yield efficiency, low cost, mild whole process condition and the like, and is easier to realize industrialized large-scale generation, but the microbial fermentation method has the limitation of low yield. Therefore, it is urgent to find a preparation method of the bird's nest peptide with low cost, high yield and high use safety.
Disclosure of Invention
In order to achieve the purpose, the invention provides a multi-effect bird's nest peptide, a preparation method and application thereof, wherein the bird's nest peptide has good antioxidant, whitening and moisturizing and nourishing skin effects.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides a preparation method of a multi-effect bird's nest peptide, which comprises the following steps:
Cleaning nidus Collocaliae, soaking, stewing, homogenizing, and performing enzymolysis to obtain enzymolysis product;
Inoculating one or more of bacillus subtilis, bacillus cereus, bacillus licheniformis or bacillus amyloliquefaciens into the enzymolysis product, fermenting, culturing and sterilizing to obtain the bird's nest peptide;
The enzymolysis process adopts one or more of alkaline protease, flavourzyme, neutral protease or pancreatin.
In some embodiments, the ratio of the red bird's nest to water in the stewing process is 1: (20-60)/g.ml -1, preferably 1: (30-50)/g.mL -1;
in some embodiments, the stewing time is 30min-180min, preferably 90min-120min;
In some embodiments, the homogenizing pressure is 25Mpa-30Mpa, and the homogenizing time is 10min-40min, preferably 20min-30min;
In some embodiments, the alkaline protease, pancreatin, flavourzyme, and neutral protease are purchased from nanin Pang Bo bioengineering, inc;
When the enzyme comprises alkaline protease, pancreatin, flavourzyme and neutral protease, the addition amount of the alkaline protease, pancreatin, flavourzyme and neutral protease is 2000U/g-10000U/g;
When the enzyme comprises alkaline protease and neutral protease, the addition amount ratio of the alkaline protease to the neutral protease is 1:1;
When the enzyme comprises flavourzyme and neutral protease, the addition amount ratio of the flavourzyme to the neutral protease is 1:1;
In some embodiments, the temperature of the enzymatic hydrolysis may be 45 ℃ to 65 ℃, preferably 50 ℃ to 60 ℃ as is conventional in the art;
In some embodiments, the time for the enzymatic hydrolysis may be a time conventional in the art, preferably 120min-300min, more preferably 180min-240min;
in some embodiments, the enzymatic hydrolysis may be performed under agitation conditions, as is conventional in the art, at a rotational speed of 150rpm to 300rpm;
in some embodiments, the step of performing the enzymolysis further comprises centrifuging, and collecting supernatant.
Wherein the centrifugal speed is 5000rpm-12000rpm, preferably 8000rpm-10000rpm, more preferably 10000rpm;
Wherein the centrifugation time is 20min-40min, preferably 25min-40min, more preferably 30min;
wherein the centrifugation temperature is-10 ℃ to 4 ℃, preferably-8 ℃ to 0 ℃, more preferably-4 ℃;
in some embodiments, the fermentation substrate further comprises a carbon source;
Wherein the carbon source is rock sugar and/or glucose;
Wherein the carbon source accounts for 0.1-0.5% of the supernatant of the enzymolysis product of the bird's nest, and is preferably 0.2-0.3%;
in some embodiments, the fermentation substrate further comprises a sterilization operation prior to use, the sterilization process being an autoclaving process;
Wherein, when the fermentation substrate is sterilized by a high-temperature sterilization method, the sterilization temperature is the normal operation temperature in the field, and is preferably 100-125 ℃; more preferably 115 ℃ to 121 ℃;
Wherein, when the fermentation substrate is sterilized by a high-temperature sterilization method, the sterilization time is the conventional operation time in the field, preferably 10min-30min; more preferably 15min-20min;
Wherein, the fermentation substrate is further cooled after high-temperature sterilization, and is normally cooled to room temperature;
In some embodiments, the fermentation bacteria include bacillus subtilis, bacillus licheniformis, bacillus cereus, bacillus amyloliquefaciens;
In some embodiments, the amount of bacillus subtilis, bacillus licheniformis, bacillus cereus, bacillus amyloliquefaciens inoculated per unit volume of the fermentation substrate is 10 5-108 CFU/g;
In some embodiments, the fermentation culture is aerobic culture;
In some embodiments, the fermentation culture is performed on a shaker at a speed of 100rpm to 300rpm, preferably 150rpm to 250rpm
In some embodiments, the fermentation time is 24h-96h, preferably 36h-72h;
In some embodiments, the temperature of the fermentation culture is from 25 ℃ to 48 ℃, preferably from 28 ℃ to 40 ℃, and in some embodiments, the method of sterilization is a high temperature sterilization method;
Wherein the sterilization is performed by the high temperature sterilization method, and the sterilization temperature is 100-125 ℃ which is the temperature conventional in the operation in the field, preferably 115-121 ℃;
Wherein the high temperature sterilization method is adopted for sterilization, and the sterilization time is 10min-40min, preferably 15min-25min which is the conventional time of the operation in the field;
In some embodiments, the centrifugation further comprises cooling, centrifuging, and collecting supernatant;
wherein, the cooling is carried out, and the normal cooling is carried out until the room temperature is reached;
wherein the centrifugal speed is 5000rpm-12000rpm, preferably 8000rpm-10000rpm, more preferably 1000rpm;
Wherein the centrifugation time is 20min-40min, preferably 25min-40min, more preferably 30min;
When the high temperature sterilization method is adopted for the secondary sterilization, the secondary sterilization temperature is 90-110 ℃ which is the temperature conventional in the operation of the field, preferably 95-100 ℃;
Wherein the high-temperature sterilization method is adopted for the secondary sterilization, and the secondary sterilization time is 20min-50min, preferably 25min-35min which are common in the operation in the field;
and freeze-drying the second sterilized operation to obtain the bird's nest peptide.
The invention also provides a multi-effect bird's nest peptide.
The invention also provides application of the multi-effect bird's nest peptide in the fields of food, health care products and cosmetics.
The invention also provides application of the multi-effect bird's nest peptide in preparing foods, health products and cosmetics with antioxidant effect, whitening effect and moisturizing effect.
The invention also provides application of the multi-effect bird's nest peptide directly as a product, as an ingredient or as a base substance.
Compared with the prior art, the invention has the following beneficial effects:
The application adopts bacillus subtilis or bacillus cereus or bacillus licheniformis or bacillus amyloliquefaciens to ferment the enzymolysis product of the bird's nest, and the prepared bird's nest peptide has obvious antioxidation, whitening, moisturizing and anti-aging effects. The preparation process does not add any chemical components, has high use safety, simple and easy operation of the preparation process and low preparation cost, realizes energy conservation and environmental protection, and has wide market application prospect in the aspects of food, health care products, cosmetics and the like.
Drawings
In order to more clearly illustrate the embodiments of the application or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 Multi-effect bird's nest peptide ABTS radical clearance of examples and comparative examples
FIG. 2 Multi-effect bird's nest peptide DPPH radical scavenging Rate of examples and comparative examples
FIG. 3 inhibition of tyrosinase activity by the pleiotropic bird's nest peptides of the examples and comparative examples
FIG. 4 moisturizing effect of the multiple-effect bird's nest peptides of the examples and comparative examples
FIG. 5 survival of the pleiotropic bird's nest peptides of the examples and comparative examples to HaCat cells
Detailed Description
The following detailed description of embodiments of the invention, provided in the accompanying drawings, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention.
Example 1
A preparation method of a multi-effect bird's nest peptide comprises the following steps:
Weighing 10g of edible bird's nest raw material, adding water for cleaning and soaking for 120min, adding distilled water according to the feed liquid ratio of 1:30 g.mL -1, stewing for 2h. Homogenizing the stewed edible nidus Collocaliae under 28MPa for 30min, adding 6000U/g neutral protease, reacting for 4 hr, and maintaining enzymolysis temperature at 55deg.C. Centrifuging the enzymolysis solution at 10000rpm for 30min to obtain supernatant, adding 5g glucose into the supernatant, mixing well, and maintaining at 121deg.C for 15min to completely inactivate enzyme. Cooling the supernatant to normal temperature after enzyme deactivation to obtain a fermentation substrate;
Adding bacillus subtilis into the fermentation substrate, wherein the number of bacillus subtilis added into the fermentation substrate per unit volume is 10 7 CFU/g, carrying out aerobic fermentation for 48 hours at the temperature of 32 ℃, carrying out fermentation culture on a shaking table, sterilizing for 20 minutes at the temperature of 115 ℃ after the fermentation is finished, cooling to room temperature after the sterilization is finished, centrifuging for 30 minutes at the temperature of 10000rpm to obtain supernatant, then carrying out secondary sterilization, carrying out secondary sterilization for 40 minutes at the temperature of 100 ℃, and finally freeze-drying to obtain the bird's nest peptide.
Example 2
The difference compared with example 1 is only that the enzyme type is different, specifically 6000U/g neutral protease is changed into 6000U/g alkaline protease, and other condition parameters are the same as in example 1.
Example 3
The difference compared with example 1 is only that the enzyme type is different, specifically 6000U/g neutral protease is changed into 6000U/g flavor protease, and other condition parameters are the same as in example 1.
Example 4
The difference compared with example 1 is only that the enzyme type is different, specifically 6000U/g neutral protease is changed into 6000U/g pancreatin, and other condition parameters are the same as in example 1.
Example 5
The difference compared with example 1 is only that the enzyme type is different, specifically 6000U/g neutral protease is changed into 6000U/g mixed alkaline protease and neutral protease, 1:1, and other condition parameters are the same as in example 1.
Example 6
The difference from example 1 is that the enzyme type is different, specifically 6000U/g neutral protease is changed to 6000U/g mixed flavor protease and neutral protease, 1:1, and other condition parameters are the same as in example 1.
Example 7
The difference compared with example 1 is only that the amount of bacillus subtilis to be inoculated is different, specifically, the quantity of bacillus subtilis is 10 7 CFU/g, the quantity of bacillus subtilis is 10 5 CFU/g, and other condition parameters are the same as those of example 1.
Example 8
The difference compared with example 1 is only that the amount of bacillus subtilis to be inoculated is different, specifically, the quantity of bacillus subtilis is 10 7 CFU/g, the quantity of bacillus subtilis is 10 6 CFU/g, and other condition parameters are the same as those of example 1.
Example 9
The difference compared with example 1 is only that the amount of bacillus subtilis to be inoculated is different, specifically, the quantity of bacillus subtilis is 10 7 CFU/g, the quantity of bacillus subtilis is 10 8 CFU/g, and other condition parameters are the same as those of example 1.
Example 10
The difference from example 1 is only that the fermentation time is different, specifically, 48h is changed to 36h, and other conditions are the same as in example 1.
Example 11
The difference from example 1 is only that the fermentation time is different, specifically 48h is replaced by 72h, and other conditions are the same as in example 1.
Example 12
The difference compared with example 1 is that the fermentation bacteria are different, and the bacillus subtilis is replaced by bacillus licheniformis, and other condition parameters are the same as those of example 1.
Example 13
The difference compared with example 1 is that the fermentation tubes are different, and the bacillus subtilis is replaced by bacillus cereus, and other condition parameters are the same as those of example 1.
Example 14
The difference compared with example 1 is that the fermentation bacteria are different, and the bacillus subtilis is specifically replaced by bacillus amyloliquefaciens, and other condition parameters are the same as those of example 1.
Comparative example 1
The difference from example 1 is that no enzymatic treatment was performed, and other conditions and parameters were the same as in example 1. The method specifically comprises the following steps:
Weighing 10g of edible bird's nest raw material, adding water for cleaning and soaking for 120min, adding distilled water according to the feed liquid ratio of 1:30 g.mL -1, and stewing for 2h. And homogenizing the stewed edible bird's nest for 30min under the condition of 28 MPa. Centrifuging the sample at 10000rpm for 30min to obtain supernatant, adding 5g glucose into the supernatant, mixing well, maintaining at 121deg.C for 15min, and cooling the supernatant to room temperature to obtain fermentation substrate;
Adding bacillus subtilis into the fermentation substrate, wherein the number of bacillus subtilis added into the fermentation substrate per unit volume is 10 7 CFU/g, carrying out aerobic fermentation for 48 hours at 37 ℃, carrying out fermentation culture on a shaking table, sterilizing for 20 minutes at 115 ℃ after the fermentation is finished, cooling to room temperature after the sterilization is finished, centrifuging for 30 minutes at 10000rpm to obtain supernatant, then carrying out secondary sterilization treatment, carrying out secondary sterilization for 40 minutes at 100 ℃, and finally freeze-drying to obtain the bird's nest peptide.
Comparative example 2
Weighing 10g of edible bird's nest raw material, adding water for cleaning and soaking for 120min, adding distilled water according to the feed liquid ratio of 1:30 g.mL -1, and stewing for 2h. And homogenizing the stewed edible bird's nest for 30min under the condition of 28 MPa. The pH of the system was stabilized in the range of 7.0 using 0.01% sodium carbonate, and 6000U/g neutral protease was added for reaction for 4 hours, wherein the enzymolysis temperature was maintained at 55 ℃. Centrifuging the sample at 10000rpm for 30min to obtain supernatant, adding 5g glucose into the supernatant, mixing well, and maintaining at 121deg.C for 15min to completely inactivate enzyme. And after enzyme deactivation, cooling the supernatant to normal temperature, and finally, freeze-drying to obtain the bird's nest peptide.
Comparative example 3
The difference compared with example 1 is only that the amount of bacillus subtilis to be inoculated is different, specifically, the quantity of bacillus subtilis is 10 7 CFU/g, the quantity of bacillus subtilis is 10 4 CFU/g, and other condition parameters are the same as those of example 1.
Comparative example 4
The difference from example 1 is only that the fermentation time is different, specifically 48h is changed to 30h, and other conditions are the same as in example 1.
Comparative example 5
Compared with example 1, the difference is only that the fermentation strains are different, and the bacillus subtilis is replaced by equal amount of saccharomyces cerevisiae, and other condition parameters are the same as those of example 1.
Comparative example 6
The difference compared with example 1 is that the fermentation species are different, and the bacillus subtilis is replaced by lactobacillus reuteri in equal amount, and other condition parameters are the same as those of example 1.
Comparative example 7
The difference compared with example 1 is that the fermentation species are different, specifically, bacillus subtilis is replaced by Lactobacillus plantarum with the same amount, and other condition parameters are the same as those of example 1.
Comparative example 8
The difference compared with example 1 is that the fermentation species are different, specifically, bacillus subtilis is replaced by equal amount of Lactobacillus helveticus, and other condition parameters are the same as those of example 1.
Comparative example 9
The difference compared with example 1 is that the fermentation species are different, specifically, bacillus subtilis is replaced by Lactobacillus casei of the same amount, and other conditions and parameters are the same as those in example 1.
Comparative example 10
Compared with example 1, the difference is only that the fermentation strains are different, and the bacillus subtilis is specifically replaced by lactobacillus rhamnosus with the same quantity, and other condition parameters are the same as those of example 1.
Effect example 1:
K 2S208 is used for directly generating stable cation free radical ABTS+ with ABTS, and the antioxidant substance reacts with ABT S + to fade the reaction system. 7.0 mmol/L2, 2' -diazabis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt aqueous solution and 2.45mmol/L potassium persulfate are mixed according to the ratio of 1:1 (v/v), and react for 12-16h at room temperature in a dark place, thus obtaining the ABTS + solution. Then diluted with 1.0mmol/L PBS buffer to give a absorbance at 734nm of 0.70.+ -. 0.02. Taking 0.4mL of water-soluble samples (the samples prepared in examples 1-14 and comparative examples 1-10 are respectively and uniformly mixed with tertiary water, and respectively prepared as 1 mg/mL), adding 4.0mL of diluted ABTS + solution, measuring absorbance A1 at 734nm after light-shielding reaction for 6min, measuring absorbance A2 by using distilled water instead of ABTS+ solution as a blank, and measuring absorbance A0 by using distilled water instead of the sample as a control group.
The results are shown in FIG. 1 (** P < 0.01 in FIG. 1, very significant statistical difference compared to example 1, very significant increase; ## P < 0.01, very significant statistical difference compared to example 1, very significant decrease; ns indicates no statistical difference compared to example 1).
Conclusion: after the ABTS + radical is scavenged, its absorbance at 734nm is reduced, based on which the scavenging ability of a substance to ABTS + radicals can be determined to characterize the antioxidant activity of the substance. Under the condition of the same concentration, the product prepared by the embodiment of the application has good antioxidation effect, and when the bird's nest is treated by adopting the mixed enzyme of alkaline protease and neutral protease and the mixed enzyme of flavourzyme and neutral protease, the antioxidation effect of the product prepared by fermenting bacillus subtilis, bacillus cereus, bacillus licheniformis and bacillus amyloliquefaciens is more ideal. The edible bird's nest is only subjected to enzymolysis treatment, only fermentation, other bacteria are adopted, fermentation time and unit volume of the bacteria are out of range, and the prepared product ABTS + has poor free radical scavenging capability and is not beneficial to extracting effective components with antioxidation efficacy from the edible bird's nest.
Effect example 2:
DPPH is a stable nitrogen-centered radical. Its stability is mainly due to the steric hindrance of its conjugated structure and the three benzene rings, which makes unpaired electrons on the intermediate nitrogen atom difficult to pair. When an antioxidant is added, H+ is released from the self-reduction and combined with DPPH, so that the characteristic color of DPPH disappears. Therefore, the DPPH-scavenging ability of an antioxidant can be determined by the change in absorbance. Respectively taking 2mL of sample solutions with different concentrations, adding 2mL of DPPH solution, uniformly mixing, standing at room temperature for 30min, and centrifuging at 5000r/min for 10min. The supernatant was taken and absorbance was measured at 517 nm. Preparing a liquid to be tested: samples prepared in examples 1 to 14 and comparative examples 1 to 10 were mixed with tertiary water to prepare 1mg/mL.
The DPPH radical scavenging rate was calculated according to the formula:
Wherein, A0: absorbance value of 2mL absolute ethanol+2 mL DPPH radical solution; a1: absorbance value of 2mL sample solution+2 mL DPPH radical solution; a2: absorbance value of 2mL sample solution+2 mL absolute ethanol.
The results are shown in FIG. 2 (** P < 0.01 in FIG. 2, very significant statistical difference compared to example 1, very significant increase; ## P < 0.01, very significant statistical difference compared to example 1, very significant decrease; ns indicates no statistical difference compared to example 1).
Conclusion: under the condition of the same concentration, the product prepared by the embodiment of the application has good antioxidation effect, and the DPPH free radical scavenging capability of the product is poor after the bird's nest is subjected to enzymolysis treatment, fermentation, other bacteria and fermentation time and the unit volume of the bacteria are out of range, so that the effective antioxidation component in the bird's nest is not easy to extract.
Effect example 3:
Tyrosinase is a copper-containing glycoprotein located in the melanosome and has an active structure in which two copper ions are surrounded by three histidine residues, respectively. The interaction of two copper ions of the active site with molecular oxygen forms a highly reactive chemical intermediate, which directly participates in the hydroxylation of monophenol to diphenol and the oxidation of diphenol to dopaquinone of melanin substrate tyrosine, is the rate limiting enzyme in the melanin production process. Therefore, inhibition of tyrosinase activity is most important, and many whitening active substances take inhibition of tyrosinase activity as evaluation indexes.
Samples prepared in examples 1-14 and comparative examples 1-10 were reconstituted with phosphate buffer (0.05 mol/L, pH 6.8) to 10mg/mL of sample solution and the in vitro mushroom tyrosinase inhibition rates of the different enzymatic hydrolysis products were determined as follows.
The absorbance at 475nm was measured using a microplate reader in a water bath at 28℃for 10 min. The mushroom tyrosinase inhibition rate was calculated as follows:
The results are shown in FIG. 3 (** P < 0.01 in FIG. 3, very significant statistical difference compared to example 1, very significant increase; ## P < 0.01, very significant statistical difference compared to example 1, very significant decrease; ns indicates no statistical difference compared to example 1).
The results show that under the condition of the same concentration, the product prepared by the embodiment of the application has good whitening effect, the bird's nest is only subjected to enzymolysis treatment, only fermentation, other bacteria are adopted, the fermentation time and the unit volume of the bacteria are out of range, and the whitening effect of the product is poor, so that the extraction of the components with moisturizing effect in the bird's nest is not facilitated.
Effect example 4:
Hyaluronic acid is a natural moisturizing factor, and participates in skin moisturizing, and is an important component of extracellular matrix. Its content in human cells decreases with age. Human cells actively synthesize hyaluronic acid to ensure cell humidity, and an important example is HaCat cells that produce hyaluronic acid on the inner surface of cell membranes under the catalysis of hyaluronidase. Thus, the determination of the hyaluronic acid content in a cell may reflect the water holding capacity of the cell to some extent, thereby evaluating the moisture holding capacity of the intervening cells.
HaCat cell hyaluronic acid content was determined using a commercially available hyaluronic acid ELISA kit (EU 2556). First, haCat cells in the logarithmic growth phase were suspended at 5X 10 5 cells/mL, inoculated on a 24-well plate, inoculated with 1.5mL each, and cultured in an incubator at 37℃in 5% CO 2 for 24 hours to allow the cells to adhere completely. The medium was then removed, washed 2 times with PBS buffer, and the sample solutions prepared from the complete medium (samples prepared in examples 1-14 and comparative examples 1-0 were mixed with the complete medium, respectively, to 1 mg/mL) were added and incubated in a 5% CO 2 incubator at 37℃for 24 hours. After the incubation, the adherent cells were digested with trypsin to prepare a cell suspension. Subsequently, the cells were lysed by adding a cell lysate and hyaluronic acid was extracted from the cells using the method described in the kit. Finally, the absorbance value is measured at 450nm, and the hyaluronic acid content is calculated according to the calibration curve. The correction curve for the experiment was y= (0.1311+0.0879 x) (-0.5586),R2 =0.9995, where y is the absorbance value and x is the hyaluronic acid content (ng/mL).
Results FIG. 4 (** P < 0.01 in FIG. 4, very significant statistical difference compared to example 1, very significant increase; ## P < 0.01, very significant statistical difference compared to example 1, very significant decrease; ns indicates no statistical difference compared to example 1).
The results show that under the condition of the same concentration, the product prepared by the embodiment of the application has good moisturizing effect, the bird's nest is only subjected to enzymolysis treatment, only fermented, other bacteria are adopted, the fermentation time and the unit volume of the bacteria are out of range, and the moisturizing capability of the prepared product is poor, so that the components with moisturizing effect in the bird's nest are not beneficial to extraction.
Effect example 5:
the products prepared in examples 1-14 and comparative examples 1-10, respectively, were formulated as complete media into 1000. Mu.g/mL test groups for testing. HaCat cells in logarithmic growth phase were suspended at 5X 10 6/mL, inoculated on 96-well plates, each well was inoculated with 1001L, and cultured in an incubator at 37℃in 5% CO 2 for 24 hours to allow the cells to adhere completely. The medium was removed and washed 2 times with PBS buffer. Adding 100 mu L of the prepared filtered and sterilized experimental group to-be-tested liquid into each hole of the experimental group, and making 6 compound holes for each to-be-tested liquid; the control group was also set to contain cells and the blank group was set to be cell-free, and incubated at 37℃in a 5% CO 2 incubator for 24 hours. After the incubation, 1L of CCK-8 was added to each well and incubated at 37℃for 2h. Finally, the absorbance was measured at 450nm and used to calculate the cell viability. Cell viability was calculated using the following formula:
wherein, sample group: sample + cells;
Control group: culture medium + cells;
blank group: a culture medium.
The results are shown in FIG. 5 (** P < 0.01 in FIG. 5, very significant statistical difference compared to example 1, very significant increase; ## P < 0.01, very significant statistical difference compared to example 1, very significant decrease; ns indicates no statistical difference compared to example 1).
The results show that under the same concentration, the product prepared by the embodiment can promote the growth of HaCat cells, improve the survival rate of the cells, and has the anti-aging effect, while the product prepared by the comparative embodiment does not promote the survival of the HaCat cells, and is not beneficial to extracting the components with the cell proliferation effect from the bird's nest.
Based on the above description, one skilled in the art will appreciate that the present invention may be embodied in different specific forms without changing its technical spirit or essential characteristics. It should be understood, therefore, that the above-described embodiments are not limiting, but are exemplary in all respects. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (10)
1. The preparation method of the multi-effect bird's nest peptide is characterized by comprising the following steps of:
Cleaning nidus Collocaliae, soaking, stewing, homogenizing, and performing enzymolysis to obtain enzymolysis product;
Inoculating one or more of bacillus subtilis, bacillus cereus, bacillus licheniformis or bacillus amyloliquefaciens into the enzymolysis product, fermenting, culturing and sterilizing to obtain the multi-effect bird's nest peptide;
The enzymolysis process adopts one or more of alkaline protease, flavourzyme, neutral protease or pancreatin, wherein the addition amounts of the alkaline protease, the flavourzyme, the neutral protease and the pancreatin are respectively 2000U/g-10000U/g.
2. The method for preparing the multi-effect bird's nest peptide according to claim 1, wherein the ratio of bird's nest to water in the stewing process is 1: (20-60)/g.mL -1;
the enzymolysis temperature is 45-60 ℃ and the enzymolysis time is 120-300 min;
The enzymolysis is carried out under the condition of stirring, and the rotating speed of the stirring is 150rpm-300rpm.
3. The method of producing the multi-effect bird's nest peptide according to any one of claims 1 to 2, wherein the fermentation substrate further includes a carbon source;
The fermentation substrate also comprises a sterilization operation before use, and the sterilization method is a high-temperature sterilization method;
The inoculation amount of the bacillus grass, bacillus cereus, bacillus licheniformis and bacillus amyloliquefaciens is 10 5-108 CFU/g;
the fermentation is carried out on a shaking table, and the rotating speed of the shaking table is 100rpm-300rpm;
the fermentation culture time is 24-96 hours, and the temperature is 25-45 ℃;
the enzyme deactivation method is a high-temperature sterilization method;
after the sterilization step, the method further comprises cooling, centrifuging and collecting supernatant.
4. A method of producing a multi-effect bird's nest peptide according to claim 3, wherein the carbon source comprises rock sugar and/or glucose;
the carbon source accounts for 1-5% of the supernatant of the bird's nest enzymolysis product by mass percent;
The inoculation amount of the bacillus grass, bacillus cereus, bacillus licheniformis and bacillus amyloliquefaciens is 10 6-108 CFU/g;
The fermentation culture is carried out on a shaking table, and the rotating speed of the shaking table is 150rpm-250rpm;
The fermentation culture time is 36-72 h, and the temperature is 28-40 ℃;
The centrifugal operation further comprises a secondary sterilization operation, and high-temperature sterilization is adopted;
and further performing freeze drying after the secondary sterilization operation to obtain the bird's nest peptide.
5. A multi-effect bird's nest peptide, characterized in that it is produced by the production method of the multi-effect bird's nest peptide as claimed in any one of claims 1 to 4.
6. The application of the multi-effect bird's nest peptide in the fields of food, health care products and cosmetics.
7. The application of the multi-effect bird's nest peptide in preparing food with antioxidant effect, whitening effect and moisturizing effect.
8. The application of the multi-effect bird's nest peptide in preparing a health care product with antioxidant effect, whitening effect and moisturizing effect.
9. The use of the multi-effect bird's nest peptide according to claim 5 in the preparation of cosmetics having antioxidant effect, whitening effect and moisturizing effect.
10. The use of the multi-effect bird's nest peptide of claim 5 directly as a product, as an ingredient or as a base substance.
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