Preparation method of hydrolyzed collagen product with surface amphipathicity as well as product and application thereof

Abstract

The invention provides a preparation method of a hydrolyzed collagen product with surface amphipathicity as well as the product and application thereof, and the preparation method comprises the following steps: (1) taking hydrolyzed collagen enzymatic hydrolysate as a substrate, then preparing a fermentation culture medium with vegetable oil and sugar, and fermenting by adopting candida bumblebee; (2) after fermentation, adding lysozyme into the fermentation liquor for activating treatment, and then sterilizing, centrifuging and filtering; and (3) carrying out freeze-drying treatment on the filtered fermentation liquor to obtain the hydrolyzed collagen product with surface amphipathicity. The hydrolyzed collagen product prepared by the invention not only has low molecular weight and high permeability to skin, but also has water locking and moistening effects on the skin surface due to the amphiphilic sophorolipid on the surface, so that the moisture retention of the skin is better improved, and the product has excellent product characteristics and has wide market prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrolyzed collagen, and specifically relates to a preparation method of a hydrolyzed collagen product with surface amphiphilicity, and the product and application thereof. Background Art

[0002] Skin is the largest organ in the human body. On the one hand, as people age, their skin loses moisture, loses collagen, and collapses its internal structure. On the other hand, environmental factors such as air pollution, wind and sun exposure also cause dryness, spots, wrinkles, and other problems. With the improvement of living standards, consumers are paying more and more attention to skin care. Therefore, a product is needed that can not only meet the needs of replenishing skin moisture and collagen, but also maintain the effect for a long time.

[0003] Hydrolyzed collagen is a polypeptide mixture made from animal collagen, with a molecular weight ranging from several hundred to tens of thousands. Since hydrolyzed collagen is a hydrolysis product of collagen, it contains all the amino acids of collagen. Collagen contains a large number of polar functional group structures such as hydroxyproline, glycine and aspartic acid, which can bind to the moisture in the skin and form stable hydrogen bonds. At the same time, due to its commonality with the special structure of the skin, collagen replenishes the collagen lost in the skin, maintains the integrity and support of the skin, stimulates the activity of cells in the skin, and maintains the smoothness and elasticity of the skin. However, collagen has the problem of large molecular weight and is not easily absorbed and utilized by the skin. Hydrolyzed collagen has the advantages of good moisturizing properties of collagen and better permeability than collagen.

[0004] CN114516912A discloses a hydrolyzed collagen protein and its preparation method and application. The preparation method of hydrolyzed collagen protein comprises: S1: pre-treating cowhide to obtain pre-treated cowhide; S2: homogenizing and digesting the pre-treated cowhide to obtain a homogenized digestion liquid; wherein the homogenized digestion comprises homogenization treatment and digestion treatment with pepsin; S3: adsorption filtration of the homogenized digestion liquid to obtain an adsorption filtrate; S4: sequentially performing concentration and liquid exchange, ion exchange chromatography, secondary concentration and liquid exchange, filtration and sterilization, and centrifugal purification on at least part of the adsorption filtrate to obtain collagen protein; S5: collecting the waste liquid of steps S1-S4, using the collected waste liquid and / or another part of the adsorption filtrate as a pre-hydrolysis liquid, and performing acid hydrolysis on the pre-hydrolysis liquid to obtain hydrolyzed collagen protein. The invention can simultaneously prepare hydrolyzed collagen protein and collagen protein, and the molecular weight uniformity of the hydrolyzed collagen protein is good, the yield and purity of the collagen protein are high, and the product quality is excellent.

[0005] JP2006152108A discloses a hydrolyzed collagen composition, which is excellent in collagen regeneration and is helpful for beauty and health as a cosmetic, skin preparation and health food. The method comprises the following steps: dissolving, drying and pulverizing a raw material selected from fish scales, fish skin or fish bones with water, and immersing a solution obtained by dissolving one selected from sodium dodecyl sulfate, guanidine hydrochloride or urea in water or immersing the solution in water and irradiating it with ultrasonic wave, and then heating the temperature to 110°C to 150°C to extract the hydrolyzed collagen, filtering with cheesecloth, centrifuging and dehydrating, and preparing the collagen composition by gel filtration process.

[0006] The osmotic absorption of hydrolyzed collagen is related to its molecular weight. Studies have shown that hydrolyzed collagen with a small molecular weight is more easily absorbed and utilized, and its absorption rate gradually decreases as the molecular weight increases. However, hydrolyzed collagen products in the prior art not only have the problem of poor osmotic absorption due to unclear molecular weight range, but also have the inherent defect of insufficient moisture retention. Summary of the invention

[0007] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for preparing a hydrolyzed collagen product with surface amphiphilicity, and its product and application, so as to solve the problems of slow penetration and absorption of hydrolyzed collagen in cosmetics and poor skin moisturizing effect.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for preparing a hydrolyzed collagen product having surface amphiphilicity, characterized in that the preparation method comprises:

[0010] (1) using hydrolyzed collagen hydrolysate as a substrate, then preparing a fermentation medium with vegetable oil and sugar, and fermenting with bumblebee Candida albicans;

[0011] (2) After fermentation, lysozyme is added to the fermentation broth for activation treatment, followed by sterilization, centrifugation, and filtration;

[0012] (3) freeze-drying the filtered fermentation liquid to obtain the hydrolyzed collagen product with surface amphiphilicity.

[0013] The method provided by the present invention ferments the hydrolyzed collagen hydrolysate by bumblebee Candida, thereby improving the conversion rate and small molecule level of the hydrolyzed collagen and producing surface amphiphilic sophorolipids. The hydrolyzed collagen product prepared by the present invention not only has a low molecular weight and high permeability to the skin, but also has a water-locking and moisturizing effect on the skin surface due to the amphiphilic sophorolipids on the surface, thereby better improving the moisture retention of the skin, and the product has excellent product characteristics and has broad market prospects.

[0014] Preferably, the method for preparing the hydrolyzed collagenase solution in step (1) comprises:

[0015] (a) soaking the minced fish skin in alkali solution and then washing and removing the alkali;

[0016] (b) mixing the cleaned fish skin with water, soaking and stirring at 90-100° C. (for example, 91° C., 93° C., 95° C., 97° C. or 99° C., etc.) for 50-100 min (for example, 60 min, 70 min, 80 min, 90 min or 100 min, etc.);

[0017] (c) enzymolyzing the fish skin after the immersion treatment to obtain a hydrolyzed collagen enzymolysis solution.

[0018] In the preparation method of the hydrolyzed collagen enzymatic hydrolyzate provided by the present invention, some impurity proteins in the fish skin can be removed by alkali washing and high-temperature treatment of the fish skin, thereby improving the purity of the hydrolyzed collagen; by using enzymatic hydrolysis technology, the conversion rate of collagen hydrolysis is improved, and deep hydrolysis is performed to achieve a minimized molecular weight level, thereby ensuring that the hydrolyzed collagen quickly penetrates and is absorbed on human skin, thereby improving the moisturizing effect of the skin.

[0019] Preferably, the concentration of the alkali solution in step (a) is 0.3-1%, for example, it can be 0.4%, 0.5%, 0.6%, 0.7%, 0.8% or 0.9%.

[0020] Preferably, the material-liquid ratio of the fish skin to the alkali solution in step (a) is 1:(20-30) g / mL, for example, it can be 1:21 g / mL, 1:23 g / mL, 1:25 g / mL, 1:27 g / mL or 1:29 g / mL, etc.

[0021] Preferably, the soaking time in step (a) is 5-10 hours, for example, 6 hours, 7 hours, 8 hours, 9 hours or 10 hours.

[0022] Preferably, the liquid-to-material ratio of the fish skin to water in step (b) is 1:(4-8) g / mL, for example, it can be 1:4.5 g / mL, 1:5 g / mL, 1:5.5 g / mL, 1:6 g / mL, 1:6.5 g / mL, 1:7 g / mL or 1:7.5 g / mL, etc.

[0023] Preferably, the enzyme used for the enzymatic treatment in step (c) includes any one of alkaline protease, papain, bromelain, pepsin, and a complex protease, or a combination of at least two thereof.

[0024] Preferably, the enzyme is a complex protease and an alkaline protease.

[0025] Preferably, the enzymatic hydrolysis step is: firstly using a composite protease for enzymatic hydrolysis, and then trying to use an alkaline protease for enzymatic hydrolysis.

[0026] Preferably, the dosage of the composite protease is 0.2-0.5% of the mass of the fish skin, for example, it can be 0.25%, 0.28%, 0.3%, 0.35%, 0.38%, 0.4%, 0.45% or 0.48%, etc.

[0027] Preferably, the amount of the alkaline protease is 1-3% of the mass of the fish skin, for example, it can be 1.1%, 1.3%, 1.5%, 1.7%, 2%, 2.2%, 2.5%, 2.7% or 2.9%, etc.

[0028] Preferably, the enzymatic hydrolysis in step (d) is followed by filtration.

[0029] Preferably, the pore size of the filtering membrane is 0.1-0.5 μm, for example, it can be 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm or 0.5 μm.

[0030] Preferably, the sugar in step (1) comprises glucose.

[0031] Preferably, the vegetable oil in step (1) comprises rapeseed oil.

[0032] Preferably, the fermentation medium in step (1) further comprises a nitrogen source and / or an inorganic salt.

[0033] Preferably, the nitrogen source comprises malt powder and / or yeast powder.

[0034] Preferably, the inorganic salt comprises magnesium sulfate.

[0035] Preferably, in terms of mass percentage, the fermentation medium contains 0.5-1.5% vegetable oil, 0.5-1.5% sugar, 0.1-1% nitrogen source, 0.01-0.1% inorganic salt, and the remainder is enzymolysis solution.

[0036] Preferably, the inoculation amount of the seed solution of bumblebee Candida albicans in step (1) is 1-3% (v / v), for example, it can be 1.1% (v / v), 1.3% (v / v), 1.5% (v / v), 1.7% (v / v), 2% (v / v), 2.2% (v / v), 2.5% (v / v), 2.7% (v / v) or 2.9% (v / v), etc.

[0037] Preferably, the fermentation temperature in step (1) is 20-35°C (for example, 23°C, 25°C, 28°C, 30°C, 32°C or 34°C, etc.), and the fermentation time is 45-60h (for example, 46h, 48h, 50h, 52h, 55h, 57h or 59h, etc.).

[0038] Preferably, the fermentation is carried out under stirring, and the stirring speed is 200-400 rpm, for example, it can be 220 rpm, 250 rpm, 280 rpm, 300 rpm, 320 rpm, 350 rpm or 380 rpm.

[0039] Preferably, the sterilization temperature in step (2) is 75-85°C (for example, 76°C, 78°C, 80°C, 82°C or 84°C, etc.), and the time is 20-40min (for example, 22min, 25min, 28min, 30min, 32min, 35min or 38min, etc.).

[0040] Preferably, the centrifugal speed in step (2) is 5000-7000 rpm, for example, it can be 5200 rpm, 5500 rpm, 5800 rpm, 6000 rpm, 6200 rpm, 6500 rpm or 6800 rpm.

[0041] Preferably, the filtration treatment in step (2) is graded membrane filtration.

[0042] Preferably, the step of graded membrane treatment comprises:

[0043] The centrifuged liquid is filtered through a 9000-11000Da membrane and a 900-1100Da membrane in sequence to obtain the filtrate, and the filtrate is filtered through a 150-250Da membrane to obtain the retentate, which is the fermented liquid after filtration treatment.

[0044] Preferably, the freeze-drying process in step (3) is carried out in a freeze dryer.

[0045] Any specific point value within the above numerical range can be selected, and will not be described in detail here.

[0046] In a second aspect, the present invention provides a hydrolyzed collagen product with surface amphiphilicity, wherein the hydrolyzed collagen product with surface amphiphilicity is prepared by the method described in the first aspect.

[0047] In a third aspect, the present invention provides a use of the hydrolyzed collagen product with surface amphiphilicity as described in the second aspect in the preparation of cosmetics.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] (1) In the preparation method of the present invention, the incompletely hydrolyzed collagen is secondary-molecularized by fermentation technology, so that the molecular weight of the hydrolyzed collagen is minimized and the concentration is higher; at the same time, a surface amphiphilic substance, a sophorolipid, is produced. From the properties of the sophorolipid, it can be seen that it is beneficial to promote the compatibility of hydrolyzed collagen with the skin, improve the penetration and absorption effect of hydrolyzed collagen, and effectively promote the improvement of the skin moisturizing effect. In addition, as an amphiphilic substance, sophorolipid also plays a role in locking water and moisturizing on the skin surface, which doubles the moisturizing effect of this product.

[0050] (2) The present invention uses a specific cold enzymatic hydrolysis technology in the preparation process of the hydrolyzed collagen hydrolyzate to improve the conversion rate of collagen hydrolysis and achieve deep hydrolysis to achieve a minimized molecular weight level, thereby ensuring that the hydrolyzed collagen quickly penetrates and is absorbed into human skin, thereby improving the moisturizing effect of the skin.

[0051] (3) The present invention uses multiple fine filtration technologies during the preparation process to ensure that the hydrolyzed collagen has high purity, low conductivity, and controllable molecular weight distribution level, which is concentrated in the range of 200-1000, reaching the oligopeptide level, thereby improving the permeability and absorbability of the hydrolyzed collagen on the skin, ensuring rapid skin absorption, and improving the moisturizing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is a molecular weight test chart of the hydrolyzed collagen product prepared in Example 1;

[0053] Figure 2 This is a molecular weight test chart of the hydrolyzed collagen product prepared in Example 1. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0055] The Candida bombicola described in the present invention was purchased from Bioboway Biotechnology Co., Ltd., with the deposit number being ATCC 22214;

[0056] The Saccharomyces cerevisiae was purchased from Bio-Bowei Biotechnology Co., Ltd. with the number CICC 32359;

[0057] The compound protease was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd.

[0058] The alkaline protease was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd.

[0059] The rest of the raw materials used can be used as long as they are purchased from regular dealers.

[0060] Example 1

[0061] This embodiment provides a hydrolyzed collagen product with surface amphiphilicity, and the preparation method thereof comprises:

[0062] (1) Pretreatment of fish skin: put frozen fish skin into a container, thaw it with room temperature pure water, wash the thawed fish skin, select fish bones, fish meat and other debris, and then dry it; put it into a meat grinder perforated plate and grind it for later use;

[0063] (2) Protein removal: minced fish skin was stirred and soaked in 0.6% NaOH solution for 6 h, with a solid-liquid ratio of 1:24. After the soaking, the fish skin was repeatedly washed with clean water to remove alkali, and the washed fish skin was dried and set aside;

[0064] (3) High temperature treatment of fish skin: fish skin and pure water were mixed in a material-liquid ratio of 1:5, set at 95°C for 1 h, and then cooled to room temperature;

[0065] (4) Enzymolysis: Using SCN fixed-cut cold enzymolysis technology (first by compound protease enzymolysis, enzyme addition amount is 0.3% of fish skin, temperature 55°C, 3h, pH 7.0±0.5; then by alkaline protease enzymolysis, enzyme addition amount is 2% of fish skin, temperature 55°C, 6h, pH 9.0±0.5), the macromolecular collagen is hydrolyzed into small molecular peptides to form an enzymolysis solution of a polypeptide mixture; after the enzymolysis is completed, it is filtered through a 0.2 μm filter membrane to obtain a clear and transparent enzymolysis solution;

[0066] (5) Seed liquid culture: Candida bombicola ATCC 22214 preserved in a glycerol tube was thawed at room temperature, and the bacterial liquid was streaked onto YM solid medium (1% glucose, 0.3% malt powder, 0.3% yeast powder, 0.5% tryptone, 1.5% agar, pH adjusted to 6.0-6.4), and cultured at 30°C for 72 h. A single colony was picked and inoculated into a seed medium (1% glucose, 0.3% malt powder, 0.3% yeast powder, 0.5% tryptone), and cultured at 30°C and 200 r / min for 48 h. The cultured strain was centrifuged to remove the medium, and the culture was repeatedly rinsed with sterile water three times. Finally, the clean Candida bombicola was added to sterile water for suspension and set aside.

[0067] (6) Fermentation: Using the enzymatic hydrolyzate as a substrate, adding 1% rapeseed oil, 1% glucose, 0.3% malt powder, 0.3% yeast powder, and 0.03% MgSO4 to prepare a fermentation medium, inoculating the seed liquid at an inoculum rate of 1.7%, adjusting the stirring speed to 300 r / min and the fermentation temperature to 25° C. through the operating system, and adjusting the temperature to 30° C. after fermenting for 48 h;

[0068] (7) Lysozyme is then added to break the cell wall and release intracellular active substances;

[0069] (8) sterilizing the mixture at 80°C for 30 min, then centrifuging at 6000 rpm to remove the bacteria and collect the centrifuge liquid;

[0070] (9) Passing the centrifuged liquid through 10000Da and 1000Da filter membranes to obtain the filtrate, and then filtering through a 200Da membrane to obtain the retentate; and then freeze-drying the retentate using a freeze dryer to obtain the hydrolyzed collagen product with surface amphiphilicity.

[0071] Example 2

[0072] This embodiment provides a hydrolyzed collagen product with surface amphiphilicity, and the preparation method thereof comprises:

[0073] (1) Pretreatment of fish skin: put frozen fish skin into a container, thaw it with room temperature pure water, wash the thawed fish skin, select fish bones, fish meat and other debris, and then dry it; put it into a meat grinder perforated plate and grind it for later use;

[0074] (2) Protein removal: minced fish skin was stirred and soaked in 0.3% NaOH solution for 8 h, with a solid-liquid ratio of 1:20. After the soaking, the fish skin was repeatedly washed with clean water to remove alkali, and the washed fish skin was dried and set aside;

[0075] (3) High temperature treatment of fish skin: fish skin and pure water were mixed in a material-liquid ratio of 1:4, set at 98°C for 50 min, and then cooled to room temperature;

[0076] (4) Enzymolysis: Using SCN fixed-cut cold enzymolysis technology (first by compound protease enzymolysis, the enzyme addition amount is 0.2% of the fish skin, temperature 55°C, 3h, pH 7.0±0.5; then by alkaline protease enzymolysis, the enzyme addition amount is 2% of the fish skin, temperature 55°C, 6h, pH 9.0±0.5), the macromolecular collagen is hydrolyzed into small molecular peptides to form an enzymolysis solution of a polypeptide mixture; after the enzymolysis is completed, it is filtered through a 0.4 μm filter membrane to obtain a clear and transparent enzymolysis solution;

[0077] (5) Seed liquid culture refers to Example 1;

[0078] (6) Fermentation: using the enzymatic hydrolysate as a substrate, adding 1% glucose, 1% rapeseed oil, 0.3% malt powder, 0.3% yeast powder, and 0.03% MgSO4 to prepare a fermentation medium, inoculating the seed liquid at an inoculum rate of 2%, adjusting the stirring speed to 200 r / min through the operating system, adjusting the fermentation temperature to 28° C., and adjusting the temperature to 32° C. after fermenting for 45 h;

[0079] (7) Lysozyme is then added to break the cell wall and release intracellular active substances;

[0080] (8) sterilizing the mixture at 75°C for 40 min, then centrifuging at 7000 rpm to remove the bacteria and collect the centrifuge liquid;

[0081] (9) Passing the centrifuged liquid through 9000Da and 1100Da filter membranes to obtain the filtrate, and then filtering through a 250Da membrane to obtain the retentate; and then freeze-drying the retentate using a freeze dryer to obtain the hydrolyzed collagen product with surface amphiphilicity.

[0082] Example 3

[0083] This embodiment provides a hydrolyzed collagen product with surface amphiphilicity, and the preparation method thereof comprises:

[0084] (1) Pretreatment of fish skin: put frozen fish skin into a container, thaw it with room temperature pure water, wash the thawed fish skin, select fish bones, fish meat and other debris, and then dry it; put it into a meat grinder perforated plate and grind it for later use;

[0085] (2) Protein removal: minced fish skin was stirred and soaked in 0.8% NaOH solution for 5 h, with a solid-liquid ratio of 1:30. After the soaking, the fish skin was repeatedly washed with clean water to remove alkali, and the washed fish skin was dried and set aside;

[0086] (3) High temperature treatment of fish skin: fish skin and pure water were mixed in a material-liquid ratio of 1:8, set at 90°C for 80 min, and then cooled to room temperature;

[0087] (4) Enzymolysis: The SCN fixed-cut cold enzymolysis technology (firstly by compound enzyme proteolysis, the enzyme addition amount is 0.4% of the fish skin, temperature 55°C, 3h, pH 7.0±0.5; then by alkaline protease enzymolysis, the enzyme addition amount is 1% of the fish skin, temperature 55°C, 6h, pH 9.0±0.5) is used to hydrolyze the macromolecular collagen into small molecular peptides to form an enzymolysis solution of a polypeptide mixture; after the enzymolysis is completed, the solution is filtered through a 0.2 μm filter membrane to obtain a clear and transparent enzymolysis solution;

[0088] (5) Seed liquid culture refers to Example 1;

[0089] (6) Fermentation: using the enzymatic hydrolysate as a substrate, adding 1% glucose, 1% rapeseed oil, 0.3% malt powder, 0.3% yeast powder, and 0.03% MgSO4 to prepare a fermentation medium, inoculating the seed liquid at an inoculum rate of 1.5%, adjusting the stirring speed to 400 r / min and the fermentation temperature to 23° C. through the operating system, and adjusting the temperature to 28° C. after fermenting for 60 h;

[0090] (7) Lysozyme is then added to break the cell wall and release intracellular active substances;

[0091] (8) sterilizing the mixture at 85°C for 20 min, then centrifuging at 5000 rpm to remove the bacteria and collect the centrifuge liquid;

[0092] (9) Passing the centrifuged liquid through 11000Da and 900Da filter membranes to obtain the filtrate, and then filtering through a 150Da membrane to obtain the retentate; and then freeze-drying the retentate using a freeze dryer to obtain the hydrolyzed collagen product with surface amphiphilicity.

[0093] Example 4

[0094] This embodiment provides a hydrolyzed collagen product with surface amphiphilicity. The preparation method thereof is different from that of embodiment 1 only in that step (2) comprises: the minced fish skin is soaked in clean water with stirring for 6 hours. After the soaking, the cleaned fish skin is dried and set aside. The remaining steps and process parameters are the same as those of embodiment 1.

[0095] Example 5

[0096] This embodiment provides a hydrolyzed collagen product with surface amphiphilicity. The preparation method thereof is different from that of embodiment 1 only in that step (3) is not included, and the remaining steps and process parameters are consistent with those of embodiment 1.

[0097] Example 6

[0098] This embodiment provides a hydrolyzed collagen product with surface amphiphilicity. The preparation method thereof is different from that of Example 1 only in that the enzyme used in step (4) is a composite protease and the amount of the enzyme is kept unchanged. The remaining steps and process parameters are consistent with those of Example 1.

[0099] Example 7

[0100] This embodiment provides a hydrolyzed collagen product with surface amphiphilicity. The preparation method thereof is different from that of Example 1 only in that the enzyme used in step (4) is alkaline protease, and the amount of the enzyme is kept unchanged. The remaining steps and process parameters are consistent with those of Example 1.

[0101] Example 8

[0102] This embodiment provides a hydrolyzed collagen product with surface amphiphilicity. The preparation method thereof is different from that of embodiment 1 only in that step (9) comprises: filtering the centrifuged liquid through a 10000Da filter membrane to obtain a filtrate, and then filtering the retentate through a 200Da filter membrane to obtain a retentate; and then freeze-drying the retentate using a freeze dryer to obtain the hydrolyzed collagen product with surface amphiphilicity.

[0103] The remaining steps and process parameters are consistent with those in Example 1.

[0104] Example 9

[0105] This embodiment provides a hydrolyzed collagen product with surface amphiphilicity. The preparation method thereof is different from that of embodiment 1 only in that step (9) comprises: filtering the centrifuged liquid through a 1000Da filter membrane to obtain a filtrate, and then filtering the filtrate through a 200Da filter membrane to obtain a retentate; and then freeze-drying the retentate using a freeze dryer to obtain the hydrolyzed collagen product with surface amphiphilicity.

[0106] The remaining steps and process parameters are consistent with those in Example 1.

[0107] Example 10

[0108] This embodiment provides a hydrolyzed collagen product with surface amphiphilicity. The preparation method thereof is different from that of embodiment 1 only in that step (9) comprises: filtering the centrifuged liquid through a 200Da membrane to obtain a retentate; and then freeze-drying the retentate using a freeze dryer to obtain the hydrolyzed collagen product with surface amphiphilicity.

[0109] The remaining steps and process parameters are consistent with those in Example 1.

[0110] Embodiment 11

[0111] This embodiment provides a hydrolyzed collagen product with surface amphiphilicity. The preparation method thereof is different from that of embodiment 1 only in that step (9) comprises: filtering the centrifuged liquid through a 1000Da membrane to obtain a retentate; and then freeze-drying the retentate using a freeze dryer to obtain the hydrolyzed collagen product with surface amphiphilicity.

[0112] The remaining steps and process parameters are consistent with those in Example 1.

[0113] Comparative Example 1

[0114] This comparative example provides a hydrolyzed collagen product, and its preparation method comprises:

[0115] (1) Pretreatment of fish skin: put frozen fish skin into a container, thaw it with room temperature pure water, wash the thawed fish skin, select fish bones, fish meat and other debris, and then dry it; put it into a meat grinder perforated plate and grind it for later use;

[0116] (2) Protein removal: minced fish skin was stirred and soaked in 0.6% NaOH solution for 6 h, with a solid-liquid ratio of 1:24. After the soaking, the fish skin was repeatedly washed with clean water to remove alkali, and the washed fish skin was dried and set aside;

[0117] (3) High temperature treatment of fish skin: fish skin and pure water were mixed in a material-liquid ratio of 1:5, set at 95°C for 1 h, and then cooled to room temperature;

[0118] (4) Enzymatic hydrolysis: The SCN fixed-cut cold enzymatic hydrolysis technology (the enzymes used are a composite protease and alkaline protease in a mass ratio of 1:1) is used to hydrolyze the macromolecular collagen into small molecular peptides to form an enzymatic hydrolyzate of a polypeptide mixture; after the enzymatic hydrolysis is completed, the solution is filtered through a 0.2 μm filter membrane to obtain a clear and transparent enzymatic hydrolyzate, the enzyme is inactivated, the supernatant is separated by centrifugation, and the hydrolyzed collagen product is obtained after concentration.

[0119] Comparative Example 2

[0120] This comparative example provides a hydrolyzed collagen product with surface amphiphilicity. The preparation method thereof is different from that of Example 1 only in that the bacteria used in step (5) is Saccharomyces cerevisiae, and the remaining steps and process parameters are consistent with those of Example 1.

[0121] Comparative Example 3

[0122] This comparative example provides a hydrolyzed collagen product with surface amphiphilicity. The preparation method thereof is different from that of Example 1 only in that rapeseed oil is not included in the fermentation medium in step (6), and the reduced amount is supplemented by enzymatic hydrolysis liquid. The remaining steps and process parameters are consistent with those of Example 1.

[0123] Comparative Example 4

[0124] This comparative example provides a hydrolyzed collagen product with surface amphiphilicity. The preparation method thereof is different from that of Example 1 only in that step (7) is not included, and the remaining steps and process parameters are consistent with those of Example 1.

[0125] Test Example 1 Molecular Weight Determination

[0126] The molecular weight of the products of Example 1 and Comparative Example 1 was tested by gel permeation chromatography. The results are shown in Figure 1 and 2 .

[0127] Depend on Figure 1 It can be seen that the molecular weight distribution of the hydrolyzed collagen product prepared by the method of the present invention is more uniform and controllable, concentrated in the range of 200-1000, reaching the oligopeptide level, improving the permeability and absorbability of hydrolyzed collagen on the skin, ensuring rapid skin absorption, and improving the moisturizing effect.

[0128] Depend on Figure 2 It is known that when the fermentation treatment is not performed, the molecular weight of the hydrolyzed collagen product is large, and the product will have the problem of being difficult to be absorbed and utilized by the skin.

[0129] Test Example 2 Determination of Sophorolipid Yield

[0130] Test method: 500 μl of the fermentation broth of Examples 1-3 and Comparative Examples 2-4 was drawn into a test tube, 1 ml of ethanol was added, the mixture was placed in a vortex shaker and vigorously shaken for 1 min, and centrifuged at 25°C 8000 r / min for 10 min to obtain a supernatant. The residual sugar content in the supernatant was determined by the DNS method, and the total sugar content of the supernatant was determined by the sulfuric acid-anthrone method, i.e., the amount of sophorolipids = (total sugar content - residual sugar content) × 1.91.

[0131] Drawing of the DNS glucose standard curve: Prepare a glucose solution with a concentration of 1000 mg / L, take a 2 ml test tube and add 0, 50, 100, 200, 300, 400 and 500 μl of the solution, then add deionized water in turn until the liquid in the test tube is 500 μl. Then add 500 μl of DNS reagent to each test tube and mix well, and boil in a water bath for 10 minutes. Quickly cool to room temperature with ice water, measure the absorbance at 540 nm, and draw a standard curve with glucose concentration as the horizontal axis and absorbance as the vertical axis.

[0132] Drawing of the standard curve of glucose by sulfuric acid-anthrone method: Prepare a glucose solution with a concentration of 1000 mg / L, take a 2 ml test tube and add 0, 50, 100, 200 and 250 μl of the solution respectively, and then add deionized water in turn until the liquid in the test tube is 250 μl. Then add 1 ml of sulfuric acid-anthrone reagent to each test tube, put it in ice water to cool quickly, mix it, and boil it in water for 10 minutes. Cool it quickly to room temperature with ice water, measure the absorbance value at 625 nm, and draw the standard curve with glucose concentration as the horizontal axis and absorbance value as the vertical axis.

[0133] The results are shown in Table 1.

[0134] Table 1

[0135]

[0136]

[0137] As can be seen from Table 1, the surface of the hydrolyzed collagen product prepared by the method of the present invention has a high level of amphiphilic sophorolipids, which can help promote the compatibility of hydrolyzed collagen with the skin, improve the penetration and absorption effect of hydrolyzed collagen, and effectively promote the moisturizing effect of the skin.

[0138] It can be seen from Comparative Example 2 that when the fermentation bacteria is not the Candida bumblebee of the present invention, the product cannot produce sophorolipids;

[0139] It can be seen from Comparative Example 3 that when there is no vegetable oil in the fermentation medium, sophorolipids cannot be produced;

[0140] It can be seen from Comparative Example 4 that when the fermentation product is not subjected to lysozyme cell wall breaking treatment, the release amount of sophorolipids is poor.

[0141] Test Example 3: Penetration Test

[0142] Select mice that have been fed for one week to prepare ex vivo mouse skin, and remove the hair on the back with a 6% sodium sulfide solution. 2 The mouse skin was removed from the subcutaneous fat and fixed between the supply pool and the receiving pool with the surface layer facing upward. The modified Franz diffusion cell was used, and the physiological saline containing 0.5% sodium azide was used as the receiving fluid. The transdermal absorption test was carried out at 32°C and 600 r / min. The transdermal area was 1.77 cm 2 , the volume of the receiving solution is 12mL. The products of Examples 1-11 and Comparative Examples 1-4 were prepared into a solution with a mass concentration of 10% with the receiving solution, 1mL of the prepared solution was added to the supply pool, and 1mL of the receiving solution was taken after 2h, 4h, 6h, 8h, 10h and 24h of transdermal permeation and supplemented with fresh receiving solution. The content of hydrolyzed collagen in the receiving solution was determined. The cumulative permeation amount per unit area was calculated according to the formula.

[0143]

[0144] Where Qn is the cumulative permeation per unit area of ​​the sample at time t, μg / cm 2 ; A is the penetration area, 1.77 cm 2 ρ n is the mass concentration measured at time t; ρ i is the mass concentration measured before time t; V is the total volume of the receiving solution, 12 ml; V0 is the sampling volume, 1 ml.

[0145] The results are shown in Table 2.

[0146] Table 2

[0147] 2h 4h 6h 8h 10h 24h Example 1 1126 1235 1462 1796 2016 3155 Example 2 1033 1095 1124 1350 1723 1922 Example 3 1065 1101 1197 1263 1655 1874 Example 4 965 1022 1086 1155 1304 1388 Example 5 857 904 1055 1142 1198 1301 Example 6 899 966 1041 1046 1229 1233 Example 7 941 992 1069 1089 1122 1185 Example 8 691 701 722 803 814 834 Example 9 895 925 1008 1073 1084 1099 Example 10 675 721 782 807 822 834 Embodiment 11 859 876 963 1034 1123 1167 Comparative Example 1 652 695 705 711 755 785 Comparative Example 2 689 715 732 757 761 799 Comparative Example 3 537 561 602 622 648 651 Comparative Example 4 584 592 624 633 657 695

[0148] Test Example 4 Moisture Retention Test

[0149] The moisturizing properties of the samples were tested according to the "Evaluation of In Vitro Moisturizing Efficacy of Cosmetics (Weighing Method)". A negative control group and a positive control group were set up under constant environmental conditions. The positive control group was 10% glycerol, and the negative control group used deionized water.

[0150] Experimental steps:

[0151] 1. Place color-changing silica gel on the bottom of the dryer, stick 3M breathable tape (3M) on the glass plate and put it into the dryer one day in advance, and weigh its weight M0;

[0152] 2. According to the size of the tape, weigh the samples of Examples 1-11, Comparative Examples 1-4 and the reference product at 2 mg ± 0.1 mg / cm2, spread them evenly on the tape, and weigh their weight M1;

[0153] 3. Place the glass plate coated with the object to be tested into the desiccator and accurately weigh its mass Mt after 2h, 4h, and 6h;

[0154] Moisture retention rate (P) formula: (Mt-M0) / (M1-M0)×100%.

[0155] The results are shown in Table 3.

[0156] Table 3

[0157]

[0158]

[0159] It can be seen from the data in Tables 2 and 3 that the hydrolyzed collagen product prepared by the present invention has better permeability, so that the active ingredients can be better absorbed by the skin and its moisturizing effect is more significant.

[0160] It can be seen from Examples 4 and 5 that when the fish skin is not subjected to alkali washing or high temperature treatment, the purity of the hydrolyzed collagen is low, thereby affecting the permeability and moisturizing effect of the final product;

[0161] It can be seen from Examples 6-7 that when the enzyme used for enzymatic hydrolysis is not the enzyme combination used in the present invention, the molecular weight of the hydrolyzed collagen obtained is higher, thereby affecting its permeability;

[0162] It can be seen from Examples 8-11 that only by using the specific multiple fine filtration technology of the present invention can the product purity be higher and the molecular weight distribution be more uniform, thereby making the product's permeability and absorption on the skin better and improving the moisturizing effect.

[0163] It can be seen from Comparative Example 1 that when the hydrolyzed collagen is not fermented, the amphiphilic sophorolipids cannot be produced on its surface, and thus its compatibility with the skin is poor, which affects its skin penetration effect.

[0164] It can be seen from Comparative Example 2 that when the fermentation bacteria is not the bumblebee Candida albicans of the present invention, the product cannot produce sophorolipids, which will also affect the penetration effect and moisturizing effect of the product;

[0165] It can be seen from Comparative Example 3 that when there is no vegetable oil in the fermentation medium, the content of sophorolipids produced is low, and thus the penetration effect of the product is also poor;

[0166] It can be seen from Comparative Example 4 that when the fermentation product is not subjected to lysozyme wall-breaking treatment, the release amount of active substances is poor, which will also affect the effect of the final product.

[0167] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the preparation method of a hydrolyzed collagen product with surface amphiphilicity, its product and application, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0168] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0169] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing a hydrolyzed collagen product with surface amphiphilicity, characterized in that: The preparation method comprises: (1) using hydrolyzed collagen hydrolysate as a substrate, then preparing a fermentation medium with vegetable oil and sugar, and fermenting with bumblebee Candida albicans; (2) After fermentation, lysozyme is added to the fermentation broth for activation treatment, followed by sterilization, centrifugation, and filtration; (3) freeze-drying the filtered fermentation liquid to obtain the hydrolyzed collagen product with surface amphiphilicity.

2. The preparation method according to claim 1, characterized in that: The method for preparing the collagen hydrolyzate in step (1) comprises: (a) soaking the minced fish skin in alkali solution and then washing and removing the alkali; (b) mixing the cleaned fish skin with water, and soaking and stirring at 90-100° C. for 50-100 min; (c) enzymolyzing the fish skin after the immersion treatment to obtain a hydrolyzed collagen enzymolysis solution.

3. The preparation method according to claim 2, characterized in that: The concentration of the alkali solution in step (a) is 0.3-1%; Preferably, the material-liquid ratio of the fish skin to the alkali solution in step (a) is 1:(20-30) g / mL; Preferably, the soaking time in step (a) is 5-10 hours.

4. The preparation method according to claim 2 or 3, characterized in that: The material-liquid ratio of the fish skin to water in step (b) is 1:(4-8) g / mL.

5. The preparation method according to any one of claims 2 to 4, characterized in that: The enzyme used in the enzymatic treatment in step (c) includes any one of alkaline protease, papain, bromelain, pepsin, and a composite protease, or a combination of at least two thereof; Preferably, the enzyme is a composite protease and an alkaline protease; Preferably, the enzymatic hydrolysis step is: firstly using a composite protease for enzymatic hydrolysis, and then trying alkaline protease for enzymatic hydrolysis; Preferably, the amount of the composite protease is 0.2-0.5% of the fish skin mass; Preferably, the amount of alkaline protease used is 1-3% of the weight of the fish skin; Preferably, the enzymatic hydrolysis in step (c) is followed by filtration; Preferably, the pore size of the filtering membrane is 0.1-0.5 μm.

6. The preparation method according to any one of claims 1 to 5, characterized in that The sugar in step (1) includes glucose; Preferably, the vegetable oil in step (1) comprises rapeseed oil; Preferably, the fermentation medium in step (1) further comprises a nitrogen source and / or an inorganic salt; Preferably, the nitrogen source comprises malt powder and / or yeast powder; Preferably, the inorganic salt comprises magnesium sulfate.

7. The preparation method according to any one of claims 1 to 6, characterized in that The inoculation amount of the seed solution of the bumblebee-grown Candida in step (1) is 1-3% (v / v); Preferably, the fermentation temperature in step (1) is 20-35°C and the fermentation time is 45-60h; Preferably, the fermentation is carried out under stirring, and the stirring speed is 200-400 rpm.

8. The preparation method according to any one of claims 1 to 7, characterized in that The sterilization temperature in step (2) is 75-85°C and the time is 20-40 minutes; Preferably, the centrifugal speed in step (2) is 5000-7000 rpm; Preferably, the filtration treatment in step (2) is graded membrane filtration; Preferably, the step of graded membrane treatment comprises: The centrifuged liquid is filtered through a 9000-11000Da membrane and a 900-1100Da membrane in sequence to obtain the filtrate, and the filtrate is filtered through a 150-250Da membrane to obtain the retentate, which is the fermented liquid after the filtration treatment; Preferably, the freeze-drying process in step (3) is carried out in a freeze dryer.

9. A hydrolyzed collagen product with surface amphiphilicity, characterized in that: The hydrolyzed collagen product with surface amphiphilicity is prepared by the method described in any one of claims 1 to 8.

10. Use of the hydrolyzed collagen product with surface amphiphilicity as claimed in claim 9 in the preparation of cosmetics.

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