Method for preparing skin-brightening cubilose fermentation filtrate based on precise fermentation technology and application of skin-brightening cubilose fermentation filtrate

Abstract

The invention provides a method for preparing skin-brightening cubilose fermentation filtrate based on a precise fermentation technology and application of the skin-brightening cubilose fermentation filtrate. Dried cubilose and a carbon source solution are sterilized, stood, cooled and mixed into a fermentation culture medium, and then activated bacillus fungicide is inoculated into the fermentation culture medium to obtain a cubilose fermentation culture medium. According to the application, microorganisms are utilized to ferment and metabolize the cubilose, extracellular protease and the like, so that the composition of cubilose substances is changed, the content of functional components in fermentation filtrate is increased, and release of skin color brightening related functional activity is accelerated and optimized. The cubilose filtrate fermented by the specific bacillus obviously reduces the melanin content and the heme content of the skin, improves the brightness of the skin, reduces the redness and the yellowness of the skin, and has the effects of removing black, removing red, removing yellowness, extracting the skin and the like; compared with enzymolysis cubilose peptide fermentation filtrate in the prior art, the accurate cubilose fermentation filtrate is free of bitter taste and peptide taste through sensory evaluation, and a solid technical foundation is laid for developing efficient and easily-absorbed cubilose-source skin-brightening functional products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, in particular to a method for preparing a brightening bird's nest fermentation filtrate based on precise fermentation technology and application. BACKGROUND

[0002] Bird's nest, as a traditional and precious tonic, is favored in the field of beauty and health care due to its rich content of protein, amino acids, minerals and various bioactive components (such as epidermal growth factor EGF analogues, sialic acid, etc.). Among them, the active protein components in bird's nest have been proven to have potential effects such as promoting skin cell repair, delaying aging, brightening skin and lightening spots, and have become an important raw material for high-end skin care products and health foods.

[0003] However, there are two core technical bottlenecks in the application of bird's nest in the prior art: first, the absorption efficiency of active ingredients is low. The natural proteins in bird's nest are mostly macromolecular structures, with a molecular weight usually above 10 kDa. Due to the characteristics of macromolecular structure, the absorption rate through the skin stratum corneum or intestinal mucosa is low, which limits the actual application value of bird's nest. Second, the sensory quality of the processed product is poor. In the process of converting bird's nest macromolecular proteins into small molecular peptides through enzymolysis and other processes, the product is often accompanied by obvious bitterness and characteristic "peptide taste". This unpleasant flavor not only reduces the palatability of the product, but also restricts the market acceptance of bird's nest derivative products (such as oral liquids, mask essences, etc.), which is significantly different from the demand of consumers for high-quality and high-experience products.

[0004] With the increasing demand of consumers for natural functional ingredients, how to break through the above technical bottlenecks and realize the efficient utilization of bird's nest active ingredients and the precise improvement of product sensory quality has become a key problem to be solved in the industry, and is also the core direction of promoting the high-value development of bird's nest resources. SUMMARY

[0005] The present application is carried out in view of the above-mentioned problems, and aims to provide a method for preparing a brightening bird's nest fermentation filtrate based on precise fermentation technology and application.

[0006] In order to achieve the above-mentioned purpose, the present application provides a method for preparing a brightening bird's nest fermentation filtrate and application, which has high absorption efficiency of active ingredients, good sensory quality, and meets the market demand for high-quality bird's nest derivative products.

[0007] The first aspect of the present application provides a method for preparing a brightening bird's nest fermentation filtrate based on precise fermentation technology, comprising the following steps:

[0008] The dried bird's nest is placed in water, sterilized, and cooled after standing; the carbon source solution is sterilized and cooled after standing; the sterilized and cooled bird's nest is mixed with the sterilized and cooled carbon source solution to obtain a fermentation culture medium; the preserved bacillus agent is activated and cultured, and then the activated bacillus agent is inoculated into the fermentation culture medium to obtain a bird's nest fermentation culture medium;

[0009] The bird's nest fermentation culture medium formula comprises 0.1%-20% by weight of a carbon source, 0.1%-10% by weight of a bird's nest, and 0.1%-10% by weight of an activated bacillus agent;

[0010] The bird's nest fermentation culture medium is subjected to fermentation culture based on preset fermentation culture conditions to obtain a culture solution; wherein the preset fermentation culture conditions include a culture temperature of 25-48°C, a shaking speed of 10-250 r / min, and a culture time of 12-96 h;

[0011] The culture solution is centrifuged to obtain a supernatant, and the supernatant is filtered through a filter membrane to obtain a bird's nest fermentation filtrate.

[0012] In any embodiment, the carbon source comprises at least one of sucrose, glucose, and granulated sugar.

[0013] In any embodiment, the carbon source is sucrose, the addition amount of sucrose is 3%-5%, the addition amount of the bird's nest is 2%-7%, and the inoculation amount of the bacillus agent is 1%-5%.

[0014] In any embodiment, the carbon source is glucose, the addition amount of glucose is 3%-5%, the addition amount of the bird's nest is 2%-7%, and the inoculation amount of the bacillus agent is 1%-5%.

[0015] In any embodiment, the carbon source is granulated sugar, the addition amount of granulated sugar is 3%-5%, the addition amount of the bird's nest is 2%-7%, and the inoculation amount of the bacillus agent is 1%-5%.

[0016] In any embodiment, the addition mass ratio of the bird's nest to the inoculation amount of the bacillus agent is 1:10-10:1.

[0017] In any embodiment, the addition mass ratio of the bird's nest to the inoculation amount of the bacillus agent is 1:5-5:1.

[0018] In any embodiment, the culture temperature is 30-40°C, the shaking speed is 50-200 r / min, and the culture time is 24-72 h.

[0019] In any embodiment, the culture solution is centrifuged under a preset centrifugal condition to obtain a supernatant, and the supernatant is filtered through a 0.22 μm filter membrane to obtain the bird's nest fermentation filtrate; the preset centrifugal condition includes a centrifugal speed of 3000 r / min to 12000 r / min and a centrifugal time of 1 min to 30 min.

[0020] In any embodiment, the centrifugal speed is 8000 r / min to 12000 r / min, and the centrifugal time is 5 min to 15 min.

[0021] In any embodiment, the Bacillus agent corresponds to Bacillus including any one of Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, and Weizmannia coagulans.

[0022] In any embodiment, the bird's nest fermentation filtrate contains soluble protein at 1.95 to 3.14 g / 100 g, polypeptide at 10.70 to 14.86 mg / mL, polysaccharide at 2.13 to 2.81 mg / mL, free amino acid at 2.18 to 2.56 mg / mL, and protein or polypeptide > 500 Da.

[0023] The second aspect of the present application also provides an application of the brightening bird's nest fermentation filtrate prepared based on the precise fermentation technology, and the bird's nest fermentation filtrate prepared by the above method is used in a functional skin care product for brightening skin.

[0024] In any embodiment, the functional skin care product for brightening skin is an oral liquid or a mask essence.

[0025] The present application has the following beneficial effects: the present application inoculates a functional food-grade microorganism into a bird's nest substrate, uses fermentation metabolism and extracellular proteases, changes the material composition of the bird's nest, increases the content of functional ingredients in the fermentation filtrate, and accelerates and optimizes the release of the functional activity related to skin lightening. The bird's nest filtrate fermented by specific Bacillus can significantly reduce the content of skin melanin and hemoglobin, improve skin brightness, reduce skin redness and yellowness, is safe and harmless to the skin, has the effects of removing black, red, and yellow, and improving skin, and can improve skin quality in multiple dimensions; through sensory evaluation, the bird's nest precise fermentation filtrate has no bitter taste and peptide taste, and has better sensory quality, which lays a solid technical foundation for developing efficient and easily absorbed bird's nest source skin lightening functional skin care products or oral beauty products. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 A flowchart of a method for preparing a brightening bird's nest fermentation filtrate based on a precise fermentation technology is provided for the embodiments of the present application. DETAILED DESCRIPTION

[0027] Hereinafter, specific embodiments of the method for preparing a skin-brightening bird's nest fermentation filtrate based on precision fermentation technology and the application thereof disclosed in the present application will be described in detail with appropriate reference to the accompanying drawings. However, there will be cases where unnecessary detailed descriptions are omitted. For example, there will be cases where detailed descriptions of matters known to those skilled in the art, repeated descriptions of substantially identical configurations are omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate understanding by those skilled in the art. In addition, the accompanying drawings and the following description are provided so that those skilled in the art can fully understand the present application, and are not intended to limit the subject matter recited in the claims.

[0028] The ranges disclosed herein are defined by their lower and upper limits. Ranges that are given by selecting a lower limit and an upper limit define the range between the particular lower and upper limits. Ranges defined by these methods can be either inclusive or exclusive of the end values, and can be arbitrarily combined, i.e., any lower limit can be combined with any upper limit to form a range. For example, if a range of 60-120 and a range of 80-110 are listed, it is understood that a range of 60-110 and a range of 80-120 are also contemplated. Furthermore, if a minimum range value of 1 and 2 is listed, and if a maximum range value of 3, 4, and 5 is listed, then the following ranges are all contemplated: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In the present application, unless otherwise stated, a numerical range "a-b" indicates a shorthand way of describing all of the real combinations between "a" and "b", where "a" and "b" are both real numbers. For example, the numerical range "0-5" indicates that all of the real numbers between "0" and "5" have been listed herein, and "0-5" is just a shorthand way of describing these numerical combinations. In addition, when it is stated that a certain parameter is an integer ≥ 2, it is equivalent to disclose that the parameter is, for example, an integer 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0029] If not specifically stated, all embodiments and optional embodiments of the present application can be combined with each other to form new technical solutions.

[0030] If not specifically stated, all technical features and optional technical features of the present application can be combined with each other to form new technical solutions.

[0031] If not specified otherwise, all steps of the present application can be carried out in sequence or randomly, preferably in sequence. For example, the method comprising steps (a) and (b) means that the method can comprise steps (a) and (b) in sequence, or steps (b) and (a) in sequence. For example, the method can further comprise step (c) means that step (c) can be added to the method in any sequence, for example, the method can comprise steps (a), (b) and (c), or steps (a), (c) and (b), or steps (c), (a) and (b), etc.

[0032] If not specified otherwise, the terms "comprising" and "including" as used in the present application are meant to be interpreted open-ended. For example, the terms "comprising" and "including" can mean that other components not listed can also be included or can mean that only the listed components are included.

[0033] If not specified otherwise, the term "or" in the present application is inclusive. For example, the phrase "A or B" means "A, B, or both A and B." More specifically, any of the following satisfy the condition "A or B": A is true (or present) and B is false (or not present); A is false (or not present) and B is true (or present); or both A and B are true (or present).

[0034] Edible bird's nest is rich in protein, amino acids, minerals and various bioactive components (such as epidermal growth factor EGF analogues, sialic acid, etc.), and the active protein components have been proven to have potential effects of promoting skin cell repair, delaying aging, lightening skin and fading spots, etc., but there are still two core technical bottlenecks in the field of beauty and health care.

[0035] The applicant found that the existing prior art has the problem of low absorption of active ingredients of edible bird's nest: the various proteins (such as epidermal growth factor EGF analogues) with potential beauty effects contained in edible bird's nest have a low absorption rate through the stratum corneum of the skin or the intestinal mucosa due to their large molecular weight, which limits the exertion of their lightening and spot-fading potential. The fermentation process can decompose the edible bird's nest into small molecular substances, improve the absorption rate, and improve the value of the edible bird's nest.

[0036] The applicant found that the existing prior art has the problem that the enzymatic bird's nest peptides and the fermentation products of the enzymatic bird's nest peptides usually have obvious bitter taste and peptide taste. Another important purpose of the present application is to solve the above bitter taste problem by means of precise control of the fermentation process, to significantly improve the sensory quality of the edible bird's nest related products, and to meet the market demand for high-quality edible bird's nest derivative products.

[0037] The present application inoculates functional food-grade microorganisms into bird's nest substrate, uses its fermentation metabolism and extracellular protease, etc., to change the material composition of bird's nest, increase the content of functional components in the fermentation filtrate, accelerate and optimize the release of skin lightening related functional activities. Bird's nest, as a natural treasure with both nutritional and medicinal values, has multiple biological activities such as antiviral and immune regulation. The bird's nest filtrate fermented by specific Bacillus is safe and harmless to the skin, and has the effects of removing black, red and yellow spots, and lightening skin. Therefore, developing a skin lightening bird's nest fermentation filtrate based on precise fermentation technology can improve skin quality in multiple dimensions, and has good product sensory quality, which undoubtedly has important practical significance and broad market prospects, and lays a solid technical foundation for developing efficient and easily absorbed skin lightening functional skin care products or oral cosmetic products from bird's nest.

[0038] In one embodiment of the present application, the present application provides a method for preparing a skin lightening bird's nest fermentation filtrate based on precise fermentation technology, comprising the following steps:

[0039] S1, dry bird's nest is placed in water, sterilized, and cooled after standing; the carbon source solution is sterilized and cooled after standing; the sterilized and cooled bird's nest is mixed with the sterilized and cooled carbon source solution to obtain a fermentation medium.

[0040] It should be noted that the dry bird's nest is placed in water of a predetermined volume in the present application, which is set according to the mass of the dry bird's nest. The bird's nest can be crushed or not, soaked or not, stewed or not, and passed through a colloid mill or not.

[0041] S2, the preserved Bacillus inoculum is activated and cultured, and then the activated Bacillus inoculum is inoculated into the fermentation medium to obtain a bird's nest fermentation medium.

[0042] The seed culture medium is used to activate and culture the Bacillus inoculum, and the seed culture medium formula of the Bacillus inoculum at least includes two components of tryptone and sodium chloride. In the seed culture medium formula, the tryptone is 0.1 g / L-50 g / L, for example, 5 g / L, 10 g / L, 20 g / L, 30 g / L or 40 g / L, and the sodium chloride is 0.1 g / L-50 g / L, for example, 5 g / L, 10 g / L, 20 g / L, 30 g / L or 40 g / L.

[0043] It should be noted that the Bacillus inoculum used in the present application corresponds to Bacillus with hydrolysis ability, for example, which can include Bacillus subtilis, Bacillus licheniformis, Weizmannia coagulans and Bacillus megaterium.

[0044] It should be noted that the formula of the bird's nest fermentation medium includes: 0.1% to 20% by weight of a carbon source; 0.1% to 10% by weight of bird's nest; 0.1% to 10% by weight of activated bacillus agent, such as 1%, 2%, 3%, 4%, 5%; and the balance is water.

[0045] The formula of the bird's nest fermentation medium includes 0.1% to 20% by weight of a carbon source, such as 3%, 4%, 5%, 6%, 7%, 8%. The applicant has found that 0.1% to 20% by weight of the carbon source can make the activity of the bacillus strain be within a reasonable range. If the carbon source exceeds 20%, the nutrients are too much, the activity of the bacillus strain is too high, and the ability to produce small molecule peptide fragments is strong, which can produce bitterness.

[0046] The formula of the bird's nest fermentation medium includes 0.1% to 10% by weight of bird's nest, such as 0.5%, 1%, 2%, 4%, and 6%. The formula of the bird's nest fermentation medium includes 0.1% to 10% by weight of activated bacillus agent, such as 1%, 2%, 3%, 4%, 5%.

[0047] It should be noted that in the embodiments of the present application, the carbon source includes at least one of sucrose, glucose, and granulated sugar.

[0048] In any embodiment, the carbon source is sucrose, the addition amount of sucrose is 3% to 5%, the addition amount of bird's nest is 2% to 7%, and the inoculation amount of bacillus agent is 1% to 5%.

[0049] In any embodiment, the carbon source is glucose, the addition amount of glucose is 3% to 5%, the addition amount of bird's nest is 2% to 7%, and the inoculation amount of bacillus agent is 1% to 5%.

[0050] In any embodiment, the carbon source is granulated sugar, the addition amount of granulated sugar is 3% to 5%, the addition amount of bird's nest is 2% to 7%, and the inoculation amount of bacillus agent is 1% to 5%.

[0051] In any embodiment, the mass ratio of the addition amount of bird's nest to the inoculation amount of bacillus agent is 1:10 to 10:1.

[0052] In any embodiment, the mass ratio of the addition amount of bird's nest to the inoculation amount of bacillus agent is 1:5 to 5:1.

[0053] S3, fermenting the bird's nest fermentation medium based on the preset fermentation culture conditions to obtain a culture solution.

[0054] It should be noted that the preset fermentation culture conditions include a culture temperature of 25°C to 48°C, a shaking speed of 10 r / min to 250 r / min, and a culture time of 12 h to 96 h.

[0055] In any embodiment, the culture temperature is 30-40°C, the shaking speed is 50-200 r / min, and the culture time is 24-72 h.

[0056] The applicant found that, in the fermentation culture process of the bird's nest fermentation medium, too high culture temperature, too fast shaking speed, no addition of carbon source, too short culture time, and too low shaking speed can significantly affect the activity of the strain, and thus reduce the ability of the strain to hydrolyze bird's nest protein.

[0057] S4, centrifuging the culture solution to obtain supernatant, and filtering the supernatant through a filter membrane to obtain bird's nest fermentation filtrate.

[0058] In any embodiment, the culture solution is centrifuged under preset centrifugation conditions to obtain supernatant, and the supernatant is filtered through a 0.22 pm filter membrane to obtain bird's nest fermentation filtrate; the preset centrifugation conditions include a centrifugation speed of 3000-12000 r / min and a centrifugation time of 1-30 min.

[0059] In any embodiment, the centrifugation speed is 8000-12000 r / min, and the centrifugation time is 5-15 min.

[0060] In any embodiment, the bird's nest fermentation filtrate contains 1.95-3.14 g / 100 g of soluble protein, 10.70-14.86 mg / mL of polypeptide, 2.13-2.81 mg / mL of polysaccharide, 2.18-2.56 mg / mL of free amino acid, and >500 Da of protein or polypeptide.

[0061] In one embodiment of the present application, the present application provides an application of a brightening bird's nest fermentation filtrate prepared based on precise fermentation technology; the bird's nest fermentation filtrate prepared by the above method is used in a brightening functional skincare product.

[0062] In any embodiment, the brightening functional skincare product is an oral liquid or a mask essence.

[0063] The Bacillus provided in the present application has good hydrolysis effect on bird's nest, and can hydrolyze bird's nest protein into soluble protein during fermentation. The protease secreted by the Bacillus provided in the present application can degrade protein with a molecular weight of more than 100 k Da into protein or polypeptide with a molecular weight of 500-10 k Da, and the fermentation filtrate has no bitter taste and no peptide taste, and has good sensory quality.

[0064] It should be noted that, by using the precise fermentation process, the present application produces bird's nest fermentation filtrate with no peptide taste and no bitter taste, significantly improves the sensory quality of bird's nest related products, and meets the market demand for high-quality bird's nest derivative products.

[0065] The human efficacy test results show that the bird's nest fermentation filtrate can significantly reduce the content of skin melanin and hemoglobin, improve skin brightness, and reduce skin redness and yellowness. These experimental results provide strong scientific evidence for its skin lightening efficacy.

[0066] The bird's nest fermentation filtrate is derived from natural bird's nest and Bacillus, and its ingredients are natural and green, which meets the preferences of modern consumers for natural, safe and mild cosmetics. In the future, as the main raw material of food, cosmetics and health food with skin lightening efficacy, the bird's nest fermentation filtrate has broad market prospects.

[0067] Examples

[0068] Hereinafter, the examples of the present application will be described. The examples described below are exemplary and are only for the purpose of explaining the present application and cannot be understood as a limitation of the present application. If the specific technology or condition is not specified in the examples, it is carried out according to the technology or condition described in the literature in the art or according to the product manual. If the reagent or instrument used is not specified by the manufacturer, it is a conventional product that can be obtained by purchase.

[0069] Example 1

[0070] A method for preparing a skin lightening bird's nest fermentation filtrate based on precise fermentation technology, comprising the following steps:

[0071] Seed medium for bacterial agent: 20 g / L of tryptone and 20 g / L of sodium chloride.

[0072] Preparation of bacterial agent: 1 mL of Bacillus licheniformis bacterial agent stored in a glycerol freezing tube in a -80°C freezer was added to 50 mL of seed medium, and activated twice to an OD 600nm greater than 0.6. The seed liquid was centrifuged at 10,000 r / min for 10 min, and the bacterial bodies were washed with sterile water. The washed bacterial bodies were used as inoculated bacterial agent for bird's nest fermentation.

[0073] Fermentation medium: 5% bird's nest and 5% sucrose. After sterilization of the bird's nest and sucrose solution in a high-temperature and high-pressure sterilization pot (121°C, 20 min), they were mixed after cooling to room temperature to obtain a bird's nest fermentation medium.

[0074] Fermentation culture: The fermentation bacterial agent was inoculated into the above fermentation medium at a inoculation amount of 2%, and placed in a 30°C constant temperature shaking fermentation incubator at 100 r / min for fermentation for 48 h. The supernatant was obtained by centrifugation at 10,000 r / min for 10 min, and then filtered through a 0.22 μm filter membrane to obtain a bird's nest fermentation filtrate rich in skin lightening efficacy ingredients.

[0075] Example 2

[0076] Compared with Example 1, the only difference is that the fermentation time is different, specifically, the fermentation time of 48 h is replaced by 24 h, and other condition parameters are the same as Example 1.

[0077] Example 3

[0078] Compared with Example 1, the only difference is that the fermentation time is different, specifically, the fermentation time of 48 h is replaced by 72 h, and other condition parameters are the same as Example 1.

[0079] Example 4

[0080] Compared with Example 1, the only difference is that the inoculation amount of the fermentation agent is different, specifically, the inoculation amount of the fermentation agent of 2% is replaced by 1%, and other condition parameters are the same as Example 1.

[0081] Example 5

[0082] Compared with Example 1, the only difference is that the inoculation amount of the fermentation agent is different, specifically, the inoculation amount of the fermentation agent of 2% is replaced by 5%, and other condition parameters are the same as Example 1.

[0083] Example 6

[0084] Compared with Example 1, the only difference is that the fermentation temperature is different, specifically, the fermentation temperature of 30℃ is replaced by 37℃, and other condition parameters are the same as Example 1.

[0085] Example 7

[0086] Compared with Example 1, the only difference is that the shaking speed during the fermentation culture is different, specifically, the shaking speed of 100 r / min is replaced by 180 r / min, and other condition parameters are the same as Example 1.

[0087] Example 8

[0088] Compared with Example 1, the only difference is that the fermentation strain is different, specifically, the fermentation strain of Bacillus licheniformis is replaced by Bacillus megaterium, and other condition parameters are the same as Example 1.

[0089] Comparative Example 1

[0090] Compared with Example 1, the only difference is that no fermentation agent is added, specifically, the inoculation amount of the fermentation agent of 2% is replaced by 0%, and other condition parameters are the same as Example 1.

[0091] Comparative Example 2

[0092] Compared with Example 1, the only difference is that the fermentation agent is different, specifically, Bacillus is replaced by Lactobacillus plantarum, and other condition parameters are the same as Example 1.

[0093] Comparative Example 3

[0094] Comparative Example 3 is the same as Example 1 except that the fermentation inoculum is different, specifically Bacillus is replaced by Lactobacillus helveticus, and other conditions are the same as Example 1.

[0095] Comparative Example 4

[0096] Comparative Example 4 is the same as Example 1 except that the fermentation temperature is different, specifically 30℃ is replaced by 50℃, and other conditions are the same as Example 1.

[0097] Comparative Example 5

[0098] Comparative Example 5 is the same as Example 1 except that the shaking speed during fermentation is different, specifically 100 r / min is replaced by 300 r / min, and other conditions are the same as Example 1.

[0099] Comparative Example 6

[0100] Comparative Example 6 is the same as Example 1 except that the fermentation medium is different, specifically no sucrose is added to the fermentation medium, and only 5% bird’s nest is contained, and other conditions are the same as Example 1.

[0101] Comparative Example 7

[0102] Comparative Example 7 is the same as Example 1 except that the fermentation time is different, specifically 72 h is replaced by 10 h, and other conditions are the same as Example 1.

[0103] Comparative Example 8

[0104] Comparative Example 8 is the same as Example 1 except that the fermentation inoculum amount is different, specifically 2% is replaced by 0.4%, and other conditions are the same as Example 1.

[0105] Comparative Example 9

[0106] Comparative Example 9 is the same as Example 5 except that the fermentation medium is different, specifically the 5% bird’s nest content in the fermentation medium is reduced to 0.4%, and other conditions are the same as Example 5.

[0107] Comparative Example 10

[0108] Comparative Example 10 is the same as Example 1 except that the fermentation time is different, specifically 72 h is replaced by 100 h, and other conditions are the same as Example 1.

[0109] Comparative Example 11

[0110] Comparative Example 11 is the same as Example 1 except that the fermentation medium is different, specifically the 5% sucrose content in the fermentation medium is increased to 25%, and other conditions are the same as Example 1.

[0111] Comparative Example 12

[0112] Compared with Example 1, the only difference is that the fermentation medium is different, specifically, 5% bird's nest in the fermentation medium is replaced by 5% enzymatic bird's nest peptide (average molecular weight 3000-10000 Da), and other condition parameters are the same as those in Example 1.

[0113] In order to further understand the technical effects of the present application, the filtrates obtained in Examples 1-8 and Comparative Examples 1-12 are tested.

[0114] Effect Experiment Example 1. Determination of soluble protein content

[0115] The soluble protein content of the samples in Examples 1-8 and Comparative Examples 1-11 is determined by GB5009.5-2016 Determination of Protein in Foods, i.e. Kjeldahl method, and the results are shown in Table 1.

[0116] It should be noted that the soluble protein content of Examples 1-8 is significantly higher than that of Comparative Example 1, indicating that the precise fermentation process of Bacillus can fully hydrolyze the proteins in bird's nest. The soluble protein content of Comparative Examples 2 and 3 is almost zero, indicating that Lactobacillus plantarum and Lactobacillus helveticus do not have the ability to hydrolyze bird's nest proteins.

[0117] The soluble protein content of Comparative Examples 4-5 is almost zero, indicating that too high fermentation temperature and too fast fermentation speed can significantly affect the activity of the strain, thereby reducing its ability to hydrolyze bird's nest proteins.

[0118] The soluble protein content of Comparative Examples 6-8 is significantly lower than that of Example 1, indicating that the strain cannot fully hydrolyze bird's nest when no carbon source is added, the fermentation time is too short, and the ratio of bird's nest addition amount to Bacillus inoculum amount is greater than 10:1. The soluble protein content of Comparative Example 9 is significantly lower than that of Example 1, which is because the bird's nest content in the substrate is very low, and the protein content is also very low after being fully hydrolyzed.

[0119] The soluble protein content of Comparative Examples 10 and 11 has no significant difference with that of Example 1, indicating that bird's nest has been fully hydrolyzed at 48 h of fermentation, and the activity of the strain is higher under high carbon source conditions, and the proteins can also be fully hydrolyzed.

[0120] Because the enzymatic bird's nest peptide in Comparative Example 12 is itself soluble protein, it is not compared in Table 1.

[0121] Table 1. Soluble protein content of examples and comparative examples

[0122]

[0123] Effect Experiment Example 2. Determination of polypeptide content

[0124] Take examples 1-8 and comparative examples 1-12 samples, respectively, add equal volume of 35% TCA solution, vortex oscillation 30s fully mixed. The mixture was placed at room temperature for 10 min, so that the protein was fully precipitated. Then, 12000r / min under the condition of centrifugation for 15 min, take supernatant, using the biuret method, determination of polypeptide content, as shown in table 2.

[0125] It can be seen that the polypeptide content of examples 1-8 samples is significantly higher than that of comparative example 1, which shows that the precise fermentation process of bacillus can improve the polypeptide content in the sample. The polypeptide content of comparative examples 2-7 is significantly lower than that of examples 1-8, which shows that lactobacillus plantarum and lactobacillus helveticus cannot hydrolyze bird's nest protein to produce polypeptide, and high fermentation temperature, high fermentation speed, no carbon source, short fermentation time and replacing bird's nest with bird's nest peptide can significantly affect the activity of the strain, thereby reducing the polypeptide content in the filtrate.

[0126] The polypeptide content of comparative example 8 is significantly lower than that of example 1, which shows that when the ratio of bird's nest addition amount to bacillus inoculum amount is greater than 10:1, the strain cannot fully hydrolyze bird's nest protein to polypeptide.

[0127] The polypeptide content of comparative example 9 is significantly lower than that of example 1, which is due to the small amount of bird's nest in the fermentation substrate, and only a small amount of polypeptide is generated after the protein is fully hydrolyzed.

[0128] The polypeptide content of comparative examples 10 and 11 has no significant difference with that of example 1, which shows that when the fermentation time is 48h, the bird's nest has been fully hydrolyzed to polypeptide, and the strain can also generate polypeptide under high carbon source. The polypeptide content of comparative example 12 has no significant difference with that of example 1.

[0129] Table 2 polypeptide content of examples and comparative examples

[0130]

[0131]

[0132] Effect example 3. Polysaccharide content determination

[0133] The polysaccharide content of examples 1-8 and comparative examples 1-12 samples was determined by phenol-sulfuric acid colorimetry, and the results are shown in table 3. The polysaccharide content of examples 1-8 is 2.13mg / mL-2.78mg / mL, which is significantly higher than that of comparative example 1, which shows that the bacillus fermentation process improves the polysaccharide content in the filtrate. The polysaccharide content of comparative examples 2, 3, 4, 5, 6, 7 and 12 is significantly lower than that of examples 1-8, which shows that lactobacillus plantarum and lactobacillus helveticus cannot utilize bird's nest medium to generate polysaccharide, and high fermentation temperature, high fermentation speed, no carbon source, short fermentation time and replacing bird's nest with bird's nest peptide can significantly affect the activity of the strain, thereby reducing the polysaccharide content in the filtrate.

[0134] The polysaccharide content of Comparative Example 8 was significantly lower than that of Example 1, indicating that when the ratio of the amount of bird's nest added to the amount of Bacillus inoculum was greater than 10:1, the strain could not fully utilize the bird's nest, thereby reducing the polysaccharide content in the filtrate. The polysaccharide content of Comparative Example 9 was significantly lower than that of Example 1, which was due to the fact that the bird's nest content in the substrate was very small, and the strain could only generate a small amount of polysaccharide even if it fully utilized the bird's nest.

[0135] The polysaccharide contents of Comparative Examples 10 and 11 were not significantly different from that of Example 1, indicating that when the fermentation time was 48 h, the nutrients in the bird's nest had been fully utilized, and extending the fermentation time or increasing the carbon source content in the culture medium would not increase the polysaccharide content in the filtrate.

[0136] Table 3 Polysaccharide content of examples and comparative examples

[0137]

[0138] Effect Experiment Example 4. Determination of free amino acid content

[0139] The samples of Examples 1-8 and Comparative Examples 1-12 were mixed with an equal volume of sulfosalicylic acid solution, centrifuged at 12000 r / min for 10 min, and the supernatant was passed through a 0.22 μm water filter membrane. The free amino acid content in different samples was determined using an amino acid automatic analyzer, and the results are shown in Table 4.

[0140] The protease secreted by Bacillus during fermentation can efficiently degrade large molecular weight proteins (such as collagen and glycoprotein) in bird's nest, hydrolyzing them into small molecular weight polypeptides and amino acids. During the continuous action of the fermentation system, the protein molecules gradually undergo enzymatic hydrolysis, converting into free amino acid form, which promotes the upward trend of the amino acid content. Compared with the unfermented bird's nest of Comparative Example 1, the free amino acid content of Examples 1-8 increased significantly. Comparative Examples 2-7 were significantly lower than Examples 1-8, indicating that Lactobacillus plantarum and Lactobacillus helveticus could not utilize the bird's nest medium to generate free amino acids, and high fermentation temperature, high fermentation speed, no addition of carbon source, and short fermentation time would significantly affect the activity of the inoculum, thereby reducing the free amino acid content in the filtrate.

[0141] The free amino acid content of Comparative Example 8 was significantly lower than that of Example 1, indicating that when the ratio of the amount of bird's nest added to the amount of Bacillus inoculum was greater than 10:1, the strain could not fully hydrolyze the bird's nest. The free amino acid content of Comparative Example 9 was significantly lower than that of Example 1, which was due to the fact that the bird's nest content in the substrate was very small, and only a small amount of free amino acid was generated after the protein was fully hydrolyzed.

[0142] The polysaccharide content of Comparative Examples 10 and 11 has no significant difference with the sample of Example 1, which indicates that the nutritional ingredients in bird's nest have been fully utilized when the fermentation time is 48 h, and extending the fermentation time or increasing the carbon source content in the culture medium will not increase the free amino acid content in the filtrate.

[0143] The free amino acid content of Comparative Example 12 is significantly less than Example 1, which indicates that replacing bird's nest with bird's nest peptide will reduce the free amino acid content in the fermentation filtrate.

[0144] Table 4 Free amino acid content of examples and comparative examples

[0145]

[0146]

[0147] Effect Example 5 Determination of molecular weight distribution

[0148] The samples of Examples 1-8 and Comparative Examples 1-12 were taken to determine the relative molecular weight distribution, and the results are shown in Table 5. Compared with Comparative Example 1, the proportion of soluble proteins with large molecular weight (>100 k Da) in the samples of Examples 1-8 decreased significantly, and most of them were converted into proteins or polypeptides with molecular weight of 3 k Da-10 k Da and 500 Da-3 k Da. Comparative Examples 2-5 and Comparative Example 8 still contain more than 90% of soluble proteins with large molecular weight (>100 k Da), which indicates that most of the large molecular bird's nest has not been decomposed. Comparative Examples 6 and 7 contain more than 60% of soluble proteins with large molecular weight (>100 k Da), which has a significant difference with the samples of Examples 1-8, which indicates that insufficient carbon source and too short fermentation time will affect the activity of the strain, making it unable to hydrolyze large molecular proteins. Comparative Examples 9-12 contain small molecular peptides less than 500 Da, which indicates that the ratio of bird's nest addition to Bacillus inoculation is less than 1:10, the Bacillus fermentation time is too long, the carbon source content in the culture medium is too high, and the fermentation of bird's nest peptide will generate small molecular peptides less than 500 Da.

[0149] And according to Examples 1 and 8 in Effect Examples 1-5, the soluble protein content of Example 8 is similar to that of Example 1, the polypeptide content, polysaccharide content, and free amino acid content of Example 8 are all higher than those of Example 1, the large molecular weight (>100 k Da) of Example 8 is lower than that of Example 1, and the appropriate molecular weight (molecular weight of 3 k Da-10 k Da and 500 Da-3 k Da) of Example 8 is higher than that of Example 1, that is, compared with Bacillus licheniformis, the culture solution obtained by using Bacillus megaterium for fermentation culture has higher content of effective ingredients.

[0150] Because the exoprotease secreted by B. megaterium is few in kind and low in activity, it will not produce a large amount of hetero-protease to "attack" and degrade the target protein during fermentation. B. licheniformis itself secretes a large amount of strong exoprotease, which is easy to degrade the target protein just expressed, resulting in a decrease in yield. In summary, the exoprotease secreted by B. megaterium is far lower than that of B. licheniformis in both kind and activity, so the risk of degradation of the target protein by the host is greatly reduced, and the yield and stability are significantly improved.

[0151] Table 5 Molecular weight distribution of examples and comparative examples

[0152]

[0153]

[0154] Example 6. Sensory evaluation

[0155] Select 20 sensory evaluation personnel (require healthy, visual, olfactory, gustatory acuity) to establish a sensory evaluation team, and use the method of sensory analysis to evaluate the smell, taste and acceptance of the samples of examples 1-8 and comparative examples 1-12. Select a clean and tidy sensory room for sensory analysis, and the evaluators score the intensity of each attribute of the samples according to the score of table 6.

[0156] Table 6 Sensory evaluation score standard

[0157]

[0158] The results of sensory evaluation are shown in table 7. The scores of smell, taste and acceptance of comparative examples 10-12 are significantly lower than those of examples 1-8 and comparative examples 1-8. Because the ratio of the amount of bird's nest added to the amount of bacillus inoculum is less than 1:10, the fermentation time of bacillus is too long, the carbon source content of the culture medium is too high, and the fermentation of bird's nest peptide by bacillus produces small molecular peptides with a molecular weight less than 500 Da, the samples have a peptide taste and a bitter taste. This result shows that the sensory quality of the bird's nest precisely fermented by bacillus is significantly better than that of the bird's nest filtrate over-fermented by bacillus and the bird's nest peptide filtrate fermented by bacillus.

[0159] It should be noted that the sensory evaluation score of example 8 is slightly higher than that of example 1. Because the metabolic products of B. licheniformis are complex, they may bring additional and unexpected flavors. The fermentation process of B. megaterium is more gentle, and its exoprotease activity is lower, which avoids the excessive decomposition of proteins and the production of complex secondary metabolites. That is, the flavor substances produced by B. megaterium are more pure, and it is not easy to produce unpleasant odors, and it is easier to retain the original flavor of bird's nest. In summary, compared with B. licheniformis, the flavor of the product fermented by B. megaterium is easier to control and accept.

[0160] Table 7 Comparison of sensory evaluation scores of examples and comparative examples

[0161]

[0162]

[0163] Effect Example 7. Determination of skin melanin content, hematin content, brightness L*, redness a*, yellowness b*

[0164] The samples of Example 1 and Comparative Examples 1, 10, 11 and 12 were prepared into bird's nest fermentation filtrate dressings for human efficacy testing.

[0165] Female subjects aged 18-50 years old, with healthy skin in the test area, without obvious redness, skin lesions, scars, pigmentation, inflammation, etc., were selected for the test, without serious systemic diseases, skin diseases (such as active acne, eczema, psoriasis, severe allergy history), immune deficiency, etc., with 15 subjects in each sample group.

[0166] After cleaning every night, the subjects took out the product, unfolded the film cloth, and pasted it on the flexor of the left / right forearm. After about 10 minutes, it was taken off and gently massaged to be absorbed.

[0167] The test adopts the method of combination of before and after comparison and parallel blank control. On the 0th day and the 14th day of the test, the skin melanin content, blood red content, and the brightness L*, redness a*, and yellowness b* indexes of the left and right forearms of the subjects were measured by using the German Courage & Khazaka skin tester. Three data of each index of each subject were collected, and the test results were compared by statistical test method to determine whether there was a statistical difference. The human test results are shown in Table 8. The yellow-removing effect of Example 8 is significantly better than that of Example 1. During the fermentation of Bacillus megaterium, vitamin B12 is produced, which can promote skin metabolism to some extent, indirectly help the skin restore healthy luster by promoting cell renewal, improving blood circulation, and correcting skin problems caused by deficiency (such as dark yellow and pigmentation), while Bacillus licheniformis lacks the ability to produce B12. The melanin-removing, red-removing, lightening, and yellow-removing effects of Comparative Examples 1, 10, 11, and 12 are significantly weaker than those of Example 1. The method for preparing the brightening bird's nest fermentation filtrate adopted in Example 1 can effectively retain the key melanin-removing, lightening, red-removing, and yellow-removing active functional fragments, significantly reduce the content of skin melanin and hemoglobin, significantly improve the brightness of the skin, and significantly reduce the redness and yellowness of the skin. Comparative Example 1 is unfermented bird's nest filtrate, 95% of the protein molecules have a molecular weight greater than 100 kDa, and cannot better play the functions of melanin-removing, red-removing, lightening, and yellow-removing. The excessive fermentation of bird's nest filtrate and enzymatic bird's nest peptides contain more peptides with a molecular weight less than 500 Da, and excessive hydrolysis destroys the key melanin-removing, lightening, red-removing, and yellow-removing active functional fragments, resulting in a decrease in activity. Table 8 Statistical analysis results of skin melanin content, blood red content, brightness L*, redness a*, and yellowness b*

[0168]

[0169] Note: ① The data in the table are mean ± standard error.

[0170] ② The data in the table are obtained by randomly testing the left / right forearm of the subject.

[0171] ③ Statistical method: T test method was used for analysis, and the test level a = 0.05.

[0172] ④ Significance marking method: compared with Example 1, “n.s” indicates no statistical difference, P≥0.05; P<0.05 indicates significant difference (“*”

[0173] indicates 0.01≤P<0.05; “**” indicates 0.001≤P<0.01;

[0174] “***” indicates P<0.001).

[0175] ⑤ Sample size: N = 45.

[0176] Note that the present application is not limited to the above-described embodiments. The above-described embodiments are merely examples, and embodiments having substantially the same configuration, function, and effect as the technical idea of the present application are included in the technical scope of the present application. Furthermore, other modes constructed by combining a part of the configurations of the embodiments in a manner that a person skilled in the art can think of within the scope of the present application are also included in the scope of the present application.

Claims

1. A method for preparing a brightening bird's nest fermentation filtrate based on precision fermentation technology, characterized in that, The method comprises the following steps: The dried bird's nest is placed in water, sterilized, and cooled; the carbon source solution is sterilized and cooled; the sterilized and cooled bird's nest and the sterilized and cooled carbon source solution are mixed to obtain a fermentation culture medium; the preserved bacillus agent is activated and cultured, and the activated bacillus agent is inoculated into the fermentation culture medium to obtain a bird's nest fermentation culture medium; The bird's nest fermentation culture medium formula comprises 0.1%-20% by weight of a carbon source, 0.1%-10% by weight of a bird's nest, and 0.1%-10% by weight of an activated bacillus agent; The bird's nest fermentation culture medium is subjected to fermentation culture based on preset fermentation culture conditions to obtain a culture solution; the preset fermentation culture conditions include a culture temperature of 25-48°C, an oscillation speed of 10-250 r / min, and a culture time of 12-96 h; The culture solution is centrifuged to obtain a supernatant, and the supernatant is filtered through a filter membrane to obtain a bird's nest fermentation filtrate.

2. The method of claim 1, wherein the method of preparing the skin-brightening cubilose fermentation filtrate is characterized by, The carbon source comprises at least one of sucrose, glucose, and granulated sugar.

3. The method of claim 1, wherein the method of preparing the skin-brightening cubilose fermentation filtrate is characterized by, When the carbon source is sucrose, the addition amount of the sucrose is 3%-5%, the addition amount of the bird's nest is 2%-7%, and the inoculation amount of the bacillus agent is 1%-5%.

4. The method of claim 1-3 for preparing the skin lightening cubilose fermentation filtrate, characterized in that, The addition mass ratio of the bird's nest to the inoculation amount of the bacillus agent is 1:10-10:

1.

5. The method of claim 4, wherein the method of preparing the skin-brightening cubilose fermentation filtrate is characterized by, The addition mass ratio of the bird's nest to the inoculation amount of the bacillus agent is 1:5-5:

1.

6. The method of claim 1, wherein the method of preparing the skin-brightening cubilose fermentation filtrate is characterized by, The culture temperature is 30-40°C, the oscillation speed is 50-200 r / min, and the culture time is 24-72 h.

7. The method of claim 1, wherein the method of preparing the skin-brightening cubilose fermentation filtrate is characterized by, The culture solution is centrifuged under preset centrifugal conditions to obtain a supernatant, and the supernatant is filtered through a 0.22-μm filter membrane to obtain a bird's nest fermentation filtrate; the preset centrifugal conditions include a centrifugal speed of 3,000-12,000 r / min and a centrifugal time of 1-30 min.

8. The method for preparing skin-brightening bird's nest fermentation filtrate according to claim 7, characterized in that, The centrifugal speed is 8,000-12,000 r / min, and the centrifugal time is 5-15 min.

9. The method of claim 1, 3 or 5, wherein the method of preparing the skin- lightening cubilose fermentation filtrate is characterized by, The bacillus agent corresponds to any one of bacillus subtilis, bacillus licheniformis, weissella coagulans, and bacillus megaterium.

10. The use of a brightening bird's nest fermentation filtrate prepared based on a precision fermentation technology, characterized in that, The bird's nest fermentation filtrate obtained by the preparation method of any one of claims 1-9 is used in a skin-lightening functional skincare product; the skin-lightening functional skincare product includes but is not limited to oral liquid and mask essence.

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