Fish collagen peptide with whitening effect and preparation method thereof

Abstract

The invention provides a fish collagen peptide with a whitening effect and a preparation method thereof, and the method comprises the following steps: performing glycosylation modification treatment on fish collagen by using glucose, and then sequentially treating the fish collagen with gastric juice and intestinal juice to obtain the fish collagen peptide with a polypeptide sequence with glycosylation sites on a peptide chain. According to the invention, the fish collagen peptide with a remarkable inhibition effect on tyrosinase is obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of collagen, in particular to a fish collagen peptide with whitening effect and a preparation method thereof. BACKGROUND

[0002] The high activity of tyrosinase can cause excessive production of melanin, so inhibiting the activity of tyrosinase has become a major goal for treating or preventing abnormal skin pigmentation, and the consumer market is increasingly inclined to use natural raw materials as tyrosinase inhibitors. Food-derived bioactive peptides have great potential as new tyrosinase inhibitors due to their high activity, specificity, safety, accessibility and strong targeting. Tyrosinase inhibiting peptides from aquatic sources usually have shorter peptide chains, which are beneficial to skin penetration and intestinal digestion and absorption, and the hydrolysate of fish scales and other processing by-products as potential tyrosinase inhibiting peptide raw materials has attracted widespread attention.

[0003] Therefore, how to obtain a food-derived bioactive peptide capable of effectively inhibiting tyrosinase is a problem that researchers in the field need to solve. SUMMARY

[0004] The purpose of the present application is to provide a fish collagen peptide with whitening effect and a preparation method thereof, and a fish collagen peptide with significant inhibitory effect on tyrosinase is obtained.

[0005] To achieve the above purpose, the present application provides the following technical solutions: A method for preparing a fish collagen peptide with whitening effect, the method comprising the following steps: S1, providing fish scales, removing impurities, decalcifying, and heating to extract collagen; S2, subjecting the collagen obtained in step S1 to ultrafiltration purification, and then vacuum freeze-drying; S3, subjecting the collagen obtained in step S2 to glycosylation modification treatment with glucose; S4, dissolving the material obtained in step S3 in gastric juice and incubating; S5, adjusting the pH of the material obtained in step S4, then adding intestinal juice and culturing; S6, adjusting the pH of the material obtained in step S5, and vacuum freeze-drying; S7, mixing the material obtained in step S6 with tyrosinase, incubating, ultrafiltration centrifuging, collecting the eluate after elution, and drying to obtain the fish collagen peptide with glycosylation sites on the polypeptide sequence of the peptide chain.

[0006] To achieve the above purpose, the present application also provides the following technical solutions: A fish collagen peptide with whitening effect, which is obtained by the above method, wherein the fish collagen peptide has a polypeptide sequence with a glycosylation site on a peptide chain.

[0007] Other suitable fields will become apparent from the description provided in the present disclosure.

[0008] The description in the summary and specific examples are only intended to illustrate and are not intended to limit the scope of the present disclosure.

[0009] Compared with the prior art, the technical solution provided by the present application has the following beneficial effects: 1. The fish collagen peptide with a polypeptide sequence with a glycosylation site on a peptide chain is obtained by using glucose to glycosylate and modify fish-derived collagen, and then sequentially treating it with gastric juice and intestinal juice; 2. The fish collagen peptide with a significant inhibitory effect on tyrosinase is obtained, thereby realizing the whitening effect; 3. The natural tyrosinase inhibitory peptide from aquatic sources provided by the present application has great potential in the field of whitening and skin care due to its high safety, high activity, high absorbability, multifunctionality and good stability; 4. The preparation method provided by the present application can utilize fish scales in a high-value manner, which not only prevents resource waste, but also effectively reduces environmental pollution, and has a very broad development prospect. BRIEF DESCRIPTION OF DRAWINGS

[0010] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other embodiments can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0011] Figure 1 is the DPPH· scavenging capacity of the fish collagen peptide obtained in each embodiment; Figure 2 is the tyrosinase inhibitory activity of the fish collagen peptide obtained in each embodiment; Figure 3 is the melanin content in B16F10 cells of the fish collagen peptide obtained in each embodiment; Figure 4 is the tyrosinase inhibitory activity in B16F10 cells of the fish collagen peptide obtained in each embodiment; Figure 5 is the reduced glutathione content in B16F10 cells of the fish collagen peptide obtained in each embodiment; Figure 6 is the oxidized glutathione content in B16F10 cells of the fish collagen peptide obtained in each embodiment. DETAILED DESCRIPTION

[0012] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0013] Any specific numerical values (including the endpoints of any numerical range) disclosed herein are not to be construed as limiting, but rather as approximations. Any numerical value, however, can contain certain errors associated with measurement of physical quantities, as well as with the rounding off of such values to the required precision. Rounding off errors can result in a slight variation between the values contained in any numerical range disclosed herein and the values of the range intended to be embraced. It is therefore contemplated that any numerical range disclosed herein can in fact be a slightly larger range than that stated, in order to account for the rounding off, as well as to encompass values that are virtually identical to those intended to be embraced within a given numerical range. It is intended, therefore, that any numerical range disclosed herein can actually include values outside its stated limits in the interest of brevity and precision.

[0014] The terminology used in the present disclosure is for the purpose of describing particular example embodiments only and is not intended to be limiting. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms "comprises," "comprising," "includes," "including," and "has," "having," and the like are inclusive and therefore specify the presence of stated features, integers, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Although the open-ended term "comprising," when used in the description and the claims, is intended to be construed as a non-limiting term, in certain aspects, the term "comprising" is alternatively construed to be more limiting, e.g., as "consisting of or "consisting essentially of. Thus, any given embodiment of a recited composition, material, component, element, feature, integer, operation, and / or process step that is recited as "comprising" one or more components, elements, steps, etc. also can consist of or consist essentially of such recited components, elements, steps, etc. In the case of "consisting of," the alternative embodiment excludes any additional components, elements, steps, etc. not specified in the claim. In the case of "consisting essentially of," any additional components, elements, steps, etc. that do not materially affect the basic and novel characteristics of the claimed embodiment can be included in the alternative embodiment.

[0015] Any method steps, processes and operations described in the present disclosure should not be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless expressly specified as such. It is also to be understood that additional or alternative steps can be employed, unless otherwise stated.

[0016] In the present application, except for the explicitly stated content, any matter or item not mentioned is directly applicable to those known in the art without any change. Moreover, any embodiment described in the present disclosure can be freely combined with one or more other embodiments described in the present disclosure, and the technical solutions or technical ideas formed thereby are considered as part of the original disclosure or original description of the present application, and should not be considered as new content that has not been disclosed or anticipated by the present disclosure, unless the combination is considered to be obviously unreasonable by those skilled in the art.

[0017] Unless otherwise stated, the terms used herein have the same meaning as generally understood by those skilled in the art. If the term is defined herein and its definition is different from the general understanding in the art, the definition herein shall prevail.

[0018] Unless otherwise stated, as referred to herein, refers to wt%.

[0019] First aspect The present application provides a method for preparing fish collagen peptide, comprising the following steps: S1, providing fish scales, removing impurities, decalcifying, heating to extract collagen; S2, the collagen obtained in step S1 is subjected to ultrafiltration purification, and then vacuum freeze-dried; S3, the collagen obtained in step S2 is subjected to glycosylation modification treatment by using glucose; S4, the material obtained in step S3 is dissolved in gastric juice and incubated; S5, the pH of the material obtained in step S4 is adjusted, then enterogastric juice is added and cultured; S6, the pH of the material obtained in step S5 is adjusted, and vacuum freeze-dried; S7, the material obtained in step S6 is mixed with tyrosinase, incubated, subjected to ultrafiltration centrifugation, and then the eluate is collected after elution, dried, to obtain the fish collagen peptide with glycosylation site on the polypeptide sequence of the peptide chain.

[0020] It should be noted that the fish scale protein glue after glycosylation reaction is used as raw material, and it is found through in vitro digestion simulation experiment that the glycosylation reaction can improve the antioxidant property of the digestion product; the tyrosinase is incubated with the fish collagen peptide hydrolysate solution of the best concentration through bioaffinity ultrafiltration, ultrafiltration centrifugation is carried out after the incubation is completed, the cut-off material of the centrifugation is eluted, and the eluate is collected and dried to obtain the polypeptide; the polypeptide sequence with a glycosylation site on the peptide chain is selected through liquid chromatography-mass spectrometry high-throughput identification, so that the purpose of inhibiting the tyrosinase is realized; the bioaffinity ultrafiltration is combined with the mass spectrometry technology to further screen the fish collagen peptides capable of inhibiting the tyrosinase, the amount of the enzyme and the sample in the separation and purification steps and the screening process is reduced, and the preparation efficiency is improved; and the bioaffinity ultrafiltration is especially suitable for rapidly screening the candidate peptide segments with specific biological activity from a complex peptide library due to the advantages of high specificity, simple operation and low cost. Therefore, the fish collagen peptide with whitening effect obtained through glycosylation and subsequent simulation of gastrointestinal digestion and bioaffinity ultrafiltration using fish processing by-products as raw material can improve the utilization rate of aquatic resources and provide a new active ingredient for natural whitening products, and has important economic value and application prospect. The present application is based on the above background, and provides a fish collagen peptide with high whitening activity and excellent physicochemical properties and an efficient preparation method thereof.

[0021] It should be noted that from the micro mechanism, the glycosylation modification of glucose can change the conformation of the collagen peptide through the steric hindrance effect, expose or form amino acid sequences (such as histidine, cysteine residues) that can specifically bind to the active center of tyrosinase (containing copper ion binding site); and the subsequent enzymatic hydrolysis of gastric juice and intestinal juice can accurately cut the peptide chain, release the glycosylated peptide segments with smaller molecular weight and better water solubility, and the sugar chains and the peptide segments can synergistically bind to the substrate binding site of tyrosinase or chelate the copper ions in the active center, block the reaction path of the enzyme catalyzed melanin synthesis, and finally form a functional peptide segment with tyrosinase inhibition activity.

[0022] It should be noted that the innovation of the present application lies in that the collagen is first glycosylated and modified with glucose, and then treated with gastric juice and intestinal juice in sequence; specifically, the glycosylation site (Lys / Arg residue modified by glycosylation reaction) on the peptide chain of the fish collagen peptide obtained after the above operation treatment forms a synergistic structure with the polypeptide sequence, and the hydroxyl group of the sugar chain chelates the Cu 2+, the hydrophobic region of the peptide segment is combined with the substrate channel of the enzyme through van der Waals force, and the hydration layer of the sugar chain hinders the diffusion of the product, forming a double inhibition mechanism of "competitive binding + spatial shielding", which can significantly enhance the inhibition effect on tyrosinase, that is, the fish collagen peptide provided by the present application can limit the inhibition activity of tyrosinase, thereby realizing the effect of whitening.

[0023] In some embodiments of the present application, in the step S1, the fish scales are from any one of grass carp, carp and tilapia.

[0024] In some embodiments of the present application, in the step S1, the fish scales of grass carp are preferably used for extracting collagen; specifically, grass carp is selected because of its dual adaptation of practicality and functionality. As a bulk freshwater fish, the fish scales (processing by-products) are widely available and low in cost, and the Gly-Pro-Hyp sequence in the rich type I collagen accounts for a high proportion, and the thermal stability and water solubility are adapted to glycosylation modification and gastrointestinal enzymolysis process, which can efficiently generate tyrosinase inhibitor peptides with glycosylation sites, and the raw material economy and active peptide preparation efficiency are considered.

[0025] In some embodiments of the present application, in the step S1, the fish scales are soaked in 0.3-0.8 mol / L hydrochloric acid for decalcification. It should be noted that 0.3-0.8 mol / L hydrochloric acid is selected for decalcification because this concentration can effectively dissolve hydroxyapatite (Ca + (Ca 10 (PO4)6(OH)2) in the fish scales to achieve decalcification, and can avoid excessive concentration (>0.8 mol / L) to cause the amide bond of the collagen peptide chain to break and the amino acid residues to degrade, or excessive dilution (<0.3 mol / L) to cause incomplete decalcification and residual calcium salt to interfere with subsequent glycosylation modification and enzymolysis reaction. This range can maximize the structural integrity and active sites of collagen while efficiently decalcifying.

[0026] In some embodiments of the present application, in the step S1, 0.5 mol / L hydrochloric acid is used for decalcification.

[0027] In some embodiments of the present application, in the step S1, the solid-liquid ratio of the fish scales to hydrochloric acid is 1:(20-30), and more preferably the solid-liquid ratio is 1:25. It should be noted that the solid-liquid ratio of 1:(20-30) is selected because this ratio can ensure that the hydrochloric acid fully contacts the fish scales and dissolves the calcium salt, avoid excessive concentration of the hydrochloric acid in the local area to damage the structure of the collagen, or excessive concentration of the hydrochloric acid to increase the amount of the hydrochloric acid, increase the burden of the subsequent neutralization step, and reduce the yield of the active peptide. This range can balance the reagent cost and the retention rate of collagen while efficiently decalcifying.

[0028] In some embodiments of the present application, the decalcification time in step S1 is 0.5-2 h, and more preferably 1 h. It should be noted that the decalcification time of 0.5-2 h is selected because this time can allow hydrochloric acid to fully penetrate the fish scales to dissolve calcium salt, avoid incomplete decalcification caused by too short time (<0.5 h), and residual calcium salt affecting subsequent reactions, or excessive hydrolysis of collagen caused by too long time (>2 h), damage to glycosylation sites, and this range can ensure the decalcification effect while maximizing the structural integrity and active sites of collagen.

[0029] In some embodiments of the present application, after decalcification in step S1, the collagen is extracted by heating at 75-90°C for 1-3 h, and preferably at 80°C for 2 h. It should be noted that the collagen is extracted by heating at 75-90°C for 1-3 h because this temperature range can allow the triple helix structure of collagen in fish scales to be moderately unwound into soluble collagen, and the time is matched with the dissolution rate; too low temperature (<75°C) or too short time will result in low extraction rate, and too high temperature (>90°C) or too long time will cause the collagen to be denatured, the peptide chain to be broken, and the glycosylation sites and active sequences to be damaged, and this range can ensure the extraction efficiency while preserving the structural and functional integrity of collagen.

[0030] In some embodiments of the present application, the ultrafiltration purification in step S2 is performed using a membrane with a molecular weight cut-off of 10-30 kDa. It should be noted that the molecular weight cut-off of 10-30 kDa is selected because this range can accurately retain collagen peptides with tyrosinase inhibitory activity (mainly in this interval), while filtering out small molecular impurities (<10 kDa) and large molecular undegraded collagen (>30 kDa); if the cut-off is too low (<10 kDa), the target active peptide will be lost, and if the cut-off is too high (>30 kDa), large molecular impurities will be left, which will interfere with the subsequent glycosylation efficiency, and this range can balance the purification accuracy and recovery rate of active ingredients. In some embodiments of the present application, the ultrafiltration purification in step S2 is performed using a membrane made of any one of polyether sulfone (PES), polypropylene (PP), and polyvinylidene fluoride (PVDF). It should be noted that the membrane made of polyether sulfone (PES), polypropylene (PP), and polyvinylidene fluoride (PVDF) is selected because these materials have strong chemical stability, suitable acid and alkali resistance for the collagen peptide solution environment, moderate hydrophilicity that does not easily adsorb the target peptide, and good mechanical strength and pore size stability; if other materials (such as cellulose acetate) are used, the membrane may be degraded due to poor solvent resistance, or the recovery rate of active peptides may be reduced due to strong adsorption, or the purification accuracy may be affected due to the change in pore size, while the above-mentioned materials can ensure the separation efficiency while prolonging the service life of the membrane and maintaining the stability of the purification process.

[0031] In some embodiments of the present application, in step S2, any one of hollow fiber membrane, spiral wound membrane, and flat sheet membrane is used for ultrafiltration purification. It should be noted that hollow fiber membrane, spiral wound membrane, and flat sheet membrane are selected because these forms of membranes have high specific surface area and good mass transfer efficiency, adapt to the viscosity characteristics of the collagen peptide solution, and can achieve efficient rejection and permeation; hollow fiber membrane has high flux and simple operation, spiral wound membrane has high packing density and is suitable for large-scale, flat sheet membrane is easy to clean and maintain, and can meet the needs of different processing scales; if other forms (such as tubular membrane) are selected, the efficiency may be low due to small specific surface area, or the cleaning difficulty may be increased due to complex structure, which affects the continuity and cost control of purification. The above-mentioned membrane forms can balance efficiency, operability and economy.

[0032] In some embodiments of the present application, in step S2, a transmembrane pressure of 0.1-0.5 MPa is used for ultrafiltration purification. It should be noted that the transmembrane pressure of 0.1-0.5 MPa is selected because this range can drive the collagen peptide solution to efficiently permeate the membrane pores, while avoiding the problems of insufficient flux and low purification efficiency caused by too low pressure (<0.1 MPa), or the problems of intensified concentration polarization on the membrane surface, pore blockage, and even damage to the membrane structure caused by too high pressure (>0.5 MPa), and the problem of inactivation of active peptides caused by too large shear force; this range can maintain stable membrane performance while ensuring separation rate, and can balance purification efficiency and activity retention of target components.

[0033] In some embodiments of the present application, in step S2, an operating temperature of 4-10°C is used for ultrafiltration purification. It should be noted that the operating temperature of 4-10°C is selected because this temperature range can inhibit the growth of microorganisms and the activity of proteases, reduce the degradation of target peptides, and reduce the viscosity of the solution to improve the flux; if the temperature is too low (<4°C), the membrane pores will shrink and the flux will drop sharply, and if the temperature is too high (>10°C), the enzymatic reaction will be accelerated and the active structure will be damaged, and the membrane pollution will be aggravated and the service life will be shortened; this range can maximize the retention of the structural integrity and tyrosinase inhibitory activity of collagen peptides while ensuring the purification efficiency.

[0034] In some embodiments of the present application, in step S2, a pH of 5.5-7.5 is used for ultrafiltration purification. It should be noted that the pH range of 5.5-7.5 is selected because this range is close to the isoelectric point of collagen peptides, which can reduce the membrane adsorption caused by the charge effect of collagen peptides, and avoid the problems of denaturation of peptide chains, destruction of glycosylation sites, and degradation of membrane materials caused by extreme pH (<5.5 or >7.5); this range can maintain the stability of peptide segments, reduce membrane pollution, and ensure the efficiency of ultrafiltration and the activity of target components.

[0035] In some embodiments of the present invention, in step S2, ultrafiltration purification is performed using a stirring speed of 100-300 rpm. It should be noted that a stirring speed of 100-300 rpm is chosen because this range allows for moderate turbulence to reduce concentration polarization at the membrane surface, increasing flux, while avoiding insufficient mass transfer and low purification efficiency due to excessively low speed (<100 rpm), or excessively high speed (>300 rpm) generating strong shear forces that damage peptide chain structures and exacerbate membrane wear. This range balances mass transfer efficiency and protection of active ingredients, maintaining ultrafiltration stability.

[0036] In some embodiments of the present invention, in step S2, 10~50 L / m 2 Ultrafiltration purification was performed using a membrane flux of 10–50 L / m³. It should be noted that this value is appropriate. 2 A membrane flux of ·h is chosen within this range to balance mass transfer efficiency and membrane fouling rate, ensuring effective permeation and retention of collagen peptide solution while avoiding excessive flux (>50 L / m). 2 • h) leads to increased membrane pore blockage, severe concentration polarization, or excessively low concentration (<10 L / m) 2 •h) This results in low processing efficiency and excessive time consumption; this range can maintain stable membrane performance while balancing purification efficiency and operational economy.

[0037] In some embodiments of the present invention, in step S2, ultrafiltration purification is performed with a feed concentration of 10-20 g / L. It should be noted that a feed concentration of 10-20 g / L is chosen because this range allows for a balance between the viscosity and diffusion properties of the collagen peptide solution: too low a concentration (<10 g / L) results in high flux but low recovery efficiency of the target component, and increases subsequent concentration costs; too high a concentration (>20 g / L) significantly increases solution viscosity, exacerbates concentration polarization and gel layer formation on the membrane surface, leading to a sharp drop in flux or even membrane pore blockage, while also increasing transmembrane pressure requirements and energy consumption. This range ensures purification efficiency while avoiding excessive membrane fouling and increased operating costs.

[0038] In some embodiments of the present application, in step S2, the temperature during vacuum freeze-drying is -60 to -80 DEG C, and the vacuum degree is 10 to 40 Pa. It should be noted that the temperature of -60 to -80 DEG C and the vacuum degree of 10 to 40 Pa are selected because this range can quickly freeze the material into a stable solid state, avoiding the destruction of the molecular structure of collagen peptide caused by large ice crystals, and the vacuum environment can make the ice sublimate directly, thereby retaining the active ingredients and nutrients to the greatest extent; if the temperature is too high (> -60 DEG C), the ice crystals grow, the peptide chains aggregate and denature, and if the vacuum degree is too low (< 10 Pa), the sublimation rate is too slow and the energy consumption increases dramatically, and if the vacuum degree is too high (> 40 Pa), the peptide segments may be oxidized or agglomerated due to residual moisture, and this range can ensure the drying efficiency while maintaining the activity, solubility and dispersibility of the product.

[0039] In some embodiments of the present application, in step S3, the temperature during glycosylation modification is 45 to 55 DEG C, preferably 50 DEG C. It should be noted that the temperature of 45 to 55 DEG C is selected because this temperature range can effectively activate the activity of glycosyltransferase, promote the covalent combination of sugar chains and peptide segments, and avoid the thermal denaturation of peptide chains or sugar chains, the destruction of active sites caused by high temperature (> 55 DEG C), or the slow reaction rate and low modification efficiency caused by low temperature (< 45 DEG C); this range can ensure the degree of glycosylation and the stability of the product while reducing side reactions (such as peptide degradation and non-specific crosslinking), and the enzyme activity and reaction specificity are optimally balanced at 50 DEG C, and deviating from this temperature may reduce the uniformity of the modified product or lose the biological activity.

[0040] In some embodiments of the present application, in step S3, the time for glycosylation modification is 6 to 10 h, preferably 8 h. It should be noted that the time of 6 to 10 h is selected because this range can ensure that the glycosylation reaction proceeds sufficiently, and the covalent combination of peptide segments and sugar chains reaches an ideal degree, while avoiding incomplete modification, low glycosylation rate of the product caused by too short reaction time (< 6 h), or excessive glycosylation, peptide chain aggregation and precipitation, and even intensified side reactions (such as oxidation and degradation) caused by too long reaction time (> 10 h); this range can balance the modification efficiency and the stability of the product, and the degree of glycosylation and the uniformity of the product are best at 8 h, and deviating from this time may reduce the activity of the target product or increase the difficulty of purification.

[0041] In some embodiments of the present application, in step S3, the mass ratio of glucose to collagen obtained in step S2 is (1 to 2) : (1 to 2), preferably 1:1. It should be noted that the above-mentioned mass ratio of glucose to collagen is the optimal choice after considering the reaction efficiency, product uniformity, activity retention and process economy, and the ratio of 1:1 can achieve the best balance in all aspects, ensuring the effectiveness and practicability of glycosylation modification.

[0042] In some embodiments of the present application, in step S4, the mass ratio of the material obtained in step S3 to the gastric juice is 1:7. It should be noted that the mass ratio of 1:7 of the material to the gastric juice is the optimal choice for comprehensively simulating the physiological digestive environment of the human body, ensuring the authenticity and controllability of the experimental data, objectively reflecting the digestion characteristics of the glycosylated collagen peptide, avoiding the deviation of the results caused by the imbalance of the proportion, and providing reliable basis for the function verification and application of the product.

[0043] In some embodiments of the present application, in step S4, the material obtained in step S3 is incubated in the gastric juice for 50-100 min, preferably 60 min. It should be noted that the gastric juice incubation time of 50-100 min is the optimal choice for balancing the experimental efficiency and data reliability on the basis of simulating the physiological digestion process of the human body, which can truly reflect the in-gastric digestion characteristics of the product and accurately evaluate the protection effect of glycosylation modification. Deviating from this range may lead to distorted results or lose the practical application reference value.

[0044] In some embodiments of the present application, in step S4, the material obtained in step S3 is incubated in the gastric juice at a temperature of 36-38℃, preferably 37℃. It should be noted that the incubation temperature of 36-38℃ is the optimal choice for balancing the physiological authenticity and enzyme activity stability, and its core value lies in simulating the normal digestive environment of the human body to ensure that the digestion experiment results of the glycosylated collagen peptide can directly map the in-vivo process. Deviating from this range may lead to unreliable data due to abnormal enzyme activity or distorted reaction conditions, thereby affecting the judgment of the product function.

[0045] In some embodiments of the present application, in step S5, the pH of the material obtained in step S4 is adjusted to 6.5. It should be noted that adjusting the pH to 6.5 is a key step to realize the physiological transition from gastric digestion to intestinal environment, and its core value lies in maintaining the stability of the material composition, matching the intestinal physiological conditions, and providing a compatible basis for subsequent experiments. Deviating from this value may cause the experimental environment to be out of line with the in-vivo physiological state, which may damage the material structure or interfere with the enzyme activity, and ultimately affect the authenticity and application reference value of the research results.

[0046] In some embodiments of the present application, in step S5, the mass ratio of the material obtained in step S4 to the intestinal juice is 1:7. It should be noted that the mass ratio of 1:7 of the material to the intestinal juice is the optimal choice for balancing the physiological authenticity, enzyme digestion efficiency and experimental reliability, and its core value lies in simulating the normal digestion ratio in the intestine to ensure that the intestinal enzyme digestion experiment results of the glycosylated collagen peptide can truly reflect the in-vivo process. Deviating from this ratio may lead to abnormal enzyme digestion, thereby affecting the accurate evaluation of the digestion stability and function retention of the product, and reducing the application value of the experimental data.

[0047] In some embodiments of the present application, in step S5, the material is incubated at 36-38℃ for 50-100min after the addition of the intestinal juice, preferably at 37℃ for 60min. It should be noted that 36-38℃ and 50-100min of intestinal juice treatment are key parameters for simulating the physiological environment of the intestine and ensuring enzyme activity and enzymolysis degree. Deviation will lead to data distortion.

[0048] In some embodiments of the present application, in step S6, the pH of the material obtained in step S5 is adjusted to 3.0. It should be noted that the selection of pH 3.0 is a balance of enzyme inactivation efficiency, target component stability, and process adaptability: it can effectively terminate the hydrolysis of protease, maximize the retention of material function, and provide a suitable initial environment for subsequent steps. Deviation from this range may lead to incomplete enzyme inactivation, component destruction, or increased process complexity, ultimately affecting product quality.

[0049] In some embodiments of the present application, in step S6, the temperature is -60 to -80℃ and the vacuum degree is 10-40Pa during vacuum freeze-drying. It should be noted that the selection of -60 to -80℃ and 10-40Pa is because this range can quickly freeze the material into a stable solid state, avoiding the destruction of the molecular structure of collagen peptides caused by large ice crystals. At the same time, the vacuum environment can make the ice sublimate directly, maximizing the retention of active ingredients and nutrients. If the temperature is too high (>-60℃), it will lead to ice crystal growth and peptide chain aggregation denaturation. If the vacuum degree is too low (<10Pa), the sublimation rate will be too slow and the energy consumption will increase dramatically. If it is too high (>40Pa), it may cause peptide segment oxidation or clumping due to residual moisture. This range can maintain the activity, solubility, and dispersibility of the product while ensuring drying efficiency.

[0050] In some embodiments of the present application, in step S7, the mass ratio of the material obtained in step S6 to tyrosinase is 10:1. It should be noted that the ratio of 10:1 is a balance point of substrate conversion efficiency, cost control, and product quality: it can ensure that the enzymatic reaction proceeds sufficiently, avoid enzyme waste and the risk of side reactions, and adapt to the time and cost requirements of the process. Deviation from this range may lead to incomplete reaction, increased cost, or decreased product quality, ultimately affecting the implementation effect of the invention.

[0051] In some embodiments of the present application, in step S7, after the material obtained in step S6 is mixed with tyrosinase, it is incubated at 37℃ for 1h.

[0052] In some embodiments of the present application, in step S7, a membrane with a molecular weight cut-off of 10-30kDa is used for ultrafiltration centrifugation.

[0053] In some embodiments of the present application, in step S7, any one of polyether sulfone (PES), cellulose or regenerated cellulose is used for ultrafiltration centrifugation.

[0054] In some embodiments of the present application, in step S7, a tubular centrifugal ultrafiltration tube or a conical centrifugal ultrafiltration tube is used for ultrafiltration centrifugation.

[0055] In some embodiments of the present application, in step S7, the ultrafiltration centrifugation is performed for 10-60 min, preferably 20 min, at a speed of 4000 rpm.

[0056] In some embodiments of the present application, in step S7, the ultrafiltration centrifugation is performed at a temperature of 4-25℃.

[0057] In some embodiments of the present application, in step S7, acetonitrile is used to elute the retentate obtained by ultrafiltration centrifugation.

[0058] In some embodiments of the present application, in step S7, the fish collagen peptide obtained has a molecular weight of less than 3000 Da and a polypeptide sequence with a glycosylation site on the peptide chain.

[0059] Second aspect The present application provides a fish collagen peptide obtained by the above method, wherein the fish collagen peptide has a polypeptide sequence with a glycosylation site on the peptide chain.

[0060] In some embodiments of the present application, the fish collagen peptide has a molecular weight of less than 3000 Da.

[0061] Example 1 The present embodiment provides a fish collagen peptide with whitening effect, and each raw material includes the following components in weight parts: grass carp protein glue 10 parts, glucose 10 parts, gastric juice 80 parts, intestinal juice 80 parts, tyrosinase 1.0 parts, and deionized water 10 parts; the preparation method of the fish collagen peptide includes the following steps: (1) The fish scales are pretreated to remove impurities, soaked in 0.5 mol / L hydrochloric acid to remove calcium, the solid-liquid ratio is 1:25, the soaking time is 1 h, and then the fish scales are heated at 80℃ for 2 h to extract collagen, which is vacuum freeze-dried for the first time and then used; (2) The glucose glycosylation fish protein glue is regulated, the glycosylation temperature is 50℃, the time is 8 h, and the mass ratio of fish protein glue to sugar is 1:1; (3) The glycosylation product is dissolved in gastric juice and incubated at 37°C for 60 min, then the pH of the gastric juice is adjusted to 6.5, the intestinal juice is added to the gastric juice, and incubated at 37°C for 60 min, after incubation, the pH of the digestion solution is adjusted to 3.0, the fish collagen peptide is released by simulating digestion, and is used after the second vacuum freeze-drying; (4) In bioaffinity ultrafiltration, the tyrosinase is incubated with the fish collagen peptide hydrolysate solution (5 mg / mL) at 37°C for 1 h, after incubation, ultrafiltration centrifugation (4000 rpm, 20 min) is performed, the retentate after centrifugation is eluted with acetonitrile, and the eluate is collected and dried to obtain the polypeptide, which is subjected to liquid chromatography-mass spectrometry high-throughput identification to select polypeptide sequences with a molecular weight less than 3000 Da and at the same time meet the glycosylation site on the peptide chain, and the fish collagen protein capable of inhibiting tyrosinase is obtained.

[0062] The fish scales are from grass carp; The ultrafiltration purification is performed using a membrane with a molecular weight cut-off of 20 kDa; The ultrafiltration purification is performed using a membrane made of polyether sulfone (PES) material; The ultrafiltration purification is performed using a membrane in the form of a hollow fiber membrane; The ultrafiltration purification is performed using a transmembrane pressure of 0.3 MPa; The ultrafiltration purification is performed using an operating temperature of 6°C; The ultrafiltration purification is performed at a pH of 6; The ultrafiltration purification is performed using a stirring speed of 200 rpm; The ultrafiltration purification is performed using a membrane flux of 30 L / m 2 The ultrafiltration purification is performed using a membrane flux of 30 L / m The ultrafiltration purification is performed using a feed liquid concentration of 15 g / L; In the first vacuum freeze-drying, the temperature is -70°C, and the vacuum degree is 25 Pa; The mass ratio of the material obtained in step S3 to the gastric juice is 1:7; The mass ratio of the material obtained in step S4 to the intestinal juice is 1:7; In the second vacuum freeze-drying, the temperature is -70°C, and the vacuum degree is 25 Pa; In bioaffinity ultrafiltration, the mass ratio of tyrosinase to fish collagen peptide hydrolysate solution is 1:10; The ultrafiltration centrifugation is performed using a membrane with a molecular weight cut-off of 20 kDa; The ultrafiltration centrifugation is performed using a membrane made of polyether sulfone (PES) material; The ultrafiltration centrifugation is performed using a tubular centrifugal ultrafiltration tube; The temperature of the ultrafiltration centrifugation is 25°C; The retentate after completing the ultrafiltration centrifugation is eluted with acetonitrile; The molecular weight of the fish collagen peptide obtained is 3000 Da, and the polypeptide sequence on the peptide chain has a glycosylation site.

[0063] Example 2 The preparation of the fish collagen peptide with whitening effect in this example is basically the same as that in Example 1, except that the raw materials of Example 2 include grass carp fish protein glue 10 parts, glucose 40 parts, gastric juice 80 parts, intestinal juice 80 parts, tyrosinase 1.0 parts, and deionized water 10 parts by weight. The molecular weight of the fish collagen peptide obtained is 3000 Da, and the polypeptide sequence on the peptide chain has a glycosylation site.

[0064] Example 3 The preparation of the fish collagen peptide with whitening effect in this example is the same as that in Example 1, except that the raw materials of Example 3 include grass carp fish protein glue 10 parts, glucose 1 part, gastric juice 80 parts, intestinal juice 80 parts, tyrosinase 1.0 parts, and deionized water 10 parts by weight. The molecular weight of the fish collagen peptide obtained is 3000 Da, and the polypeptide sequence on the peptide chain has a glycosylation site.

[0065] Example 4 The raw materials of the fish collagen peptide with whitening effect in this example are the same as those in Example 1 by weight, except that the preparation steps of this example further include: regulating the glycosylation of fish protein glue with glucose, the glycosylation temperature is 50 ℃, the time is 14 h, and the mass ratio of fish protein glue to glucose is 1:1. The molecular weight of the fish collagen peptide obtained is 3000 Da, and the polypeptide sequence on the peptide chain has a glycosylation site.

[0066] Example 5 The raw materials of the fish collagen peptide with whitening effect in this example are the same as those in Example 1 by weight, except that the preparation steps of this example further include: regulating the glycosylation of fish protein glue with glucose, the glycosylation temperature is 50 ℃, the time is 0 h, and the mass ratio of fish protein glue to glucose is 1:1. The molecular weight of the fish collagen peptide obtained is 3000 Da, and the polypeptide sequence on the peptide chain has a glycosylation site.

[0067] It should be noted that the glycosylation treatment time in this example is 0 h, which is to set a control group without glycosylation modification, so as to compare with other examples with glycosylation modification.

[0068] Test Example The fish collagen peptides obtained in the above examples are evaluated for activity according to the following method, and the results are shown in the following chart.

[0069] DPPH· scavenging capacity 600 μL 1 mg / mL of sample mixed with 600 μL 0.1 mM of DPPH (dissolved in methanol) was reacted for 20 min in the dark, and the absorbance was measured at 517 nm. 600 μL DPPH· was reacted with an equal volume of distilled water as a control group, and 600 μL sample was reacted with an equal volume of methanol as a control group. The calculation formula is as follows: DPPH clearance capacity (%) = [1 - (absorbance of sample group - absorbance of control group) / absorbance of blank group] x 100%.

[0070] Tyrosinase inhibitory activity For each reaction system, 0.75 mL of sodium phosphate buffer (0.1 mol / L, pH 6.8), 0.3 mL of L-DOPA (2 mmol / L), 0.1 mL of inhibitor solution, and 1.8 mL of ultrapure water were added, followed by the addition of 50 μL of tyrosinase solution (0.2 mg / L); the measurement was performed at 475 nm at 30°C. The calculation formula is as follows: tyrosinase inhibitory activity (%) = (1 - absorbance of sample group / absorbance of blank group) x 100%.

[0071] Melanin content, tyrosinase, and antioxidant enzyme activity B16F10 cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C with 5% CO2. Fish collagen peptides were added to the cells for further culture. After incubation, the cells were lysed with lysis buffer containing 1% volume of protease inhibitor (200 μL). Centrifugation was performed at 4000 rpm for 10 min, and the precipitate and supernatant were used to determine antioxidant enzyme activity, melanin content, and tyrosinase activity, respectively. NaOH solution (250 μL, 1 M, containing 10% DMSO) was added to the precipitate, which was then incubated at 85°C for 2 h. The precipitate reaction solution (100 μL) was measured in a 96-well plate at 405 nm; at the same time, the supernatant (20 μL) and L-dopa (180 μL, 1 mg / mL) were incubated in the dark at 37°C for 2 h, and the measurement was performed at 475 nm to obtain the melanin content and tyrosinase activity. The intracellular reduced glutathione (GSH) and oxidized glutathione (GSSG) were determined using a kit.

[0072] From Figure 1 , 2 It can be seen that the DPPH clearance capacity and tyrosinase inhibitory activity of fish collagen peptides obtained in Example 1 are stronger than those of the other examples, indicating that appropriate glucose concentration and glycosylation time can improve the in vitro activity of fish collagen peptides. From Figure 3 , 4It can be seen that the melanin content in B16F10 cells containing fish collagen peptides obtained in Example 1 was lower than that in the other examples, and the tyrosinase inhibitory activity in B16F10 cells containing fish collagen peptides obtained in Example 1 was higher than that in the other examples. This indicates that appropriate glucose concentration and glycosylation time can enhance the tyrosinase inhibitory activity of fish collagen peptides, thereby inhibiting melanin deposition in cells. Figure 5 , 6 It can be seen that the content of reduced glutathione in B16F10 cells containing fish collagen peptides obtained in Example 1 is higher than that in other examples, and the content of oxidized glutathione in B16F10 cells containing fish collagen peptides obtained in Example 1 is lower than that in other examples, indicating that appropriate glucose concentration and glycosylation time can improve the antioxidant activity of fish collagen peptides in cells.

[0073] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims. Furthermore, specific examples have been used in the specification to illustrate the principles and implementation methods of the present invention. The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention, and the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for preparing fish collagen peptides with whitening effects, characterized in that, The method includes the following steps: S1 provides fish scales, removes impurities, decalcifies, and heats to extract collagen; S2, the collagen obtained in step S1 is purified by ultrafiltration and then freeze-dried under vacuum; S3, Glycosylation modification of the collagen obtained in step S2 using glucose; S4, Dissolve the material obtained in step S3 in gastric juice and incubate; S5, adjust the pH of the material obtained in step S4, then add intestinal fluid and incubate; S6, Adjust the pH of the material obtained in step S5, and freeze-dry it under vacuum; S7. The material obtained in step S6 is mixed with tyrosinase, incubated, ultrafiltered and centrifuged, the eluent is collected after elution and dried to obtain the fish collagen peptide with a polypeptide sequence having glycosylation sites on the peptide chain.

2. The method according to claim 1, characterized in that, It has at least one of the following characteristics: In step S1, the fish scales are from any one of grass carp, common carp, or tilapia; In step S1, the fish scales are soaked in 0.3~0.8 mol / L hydrochloric acid to remove calcium, preferably 0.5 mol / L hydrochloric acid, preferably the ratio of fish scales to hydrochloric acid is 1:(20~30), more preferably the ratio is 1:25, preferably the decalcification time is 0.5~2h, more preferably the decalcification time is 1h; In step S1, after decalcification, the collagen is extracted by heating at 75-90°C for 1-3 hours, preferably at 80°C for 2 hours.

3. The method according to claim 1, characterized in that, It has at least one of the following characteristics: In step S2, ultrafiltration purification is performed using a membrane molecular weight cutoff of 10~30kDa. In step S2, ultrafiltration purification is performed using a membrane made of any one of the following materials: polyethersulfone (PES), polypropylene (PP), or polyvinylidene fluoride (PVDF). In step S2, ultrafiltration purification is performed using any one of hollow fiber membranes, spiral wound membranes, or flat sheet membranes. In step S2, ultrafiltration purification is performed using a transmembrane pressure of 0.1~0.5MPa. In step S2, ultrafiltration purification is performed at an operating temperature of 4~10℃. In step S2, ultrafiltration purification is performed using a pH of 5.5 to 7.

5. In step S2, ultrafiltration purification is performed using a stirring speed of 100~300 rpm; In step S2, 10~50 L / m 2 Ultrafiltration purification was performed using a membrane flux of ·h. In step S2, ultrafiltration purification is performed with a feed concentration of 10-20 g / L. In step S2, during vacuum freeze drying, the temperature is -60 to -80°C and the vacuum degree is 10 to 40 Pa.

4. The method according to claim 1, characterized in that, It has at least one of the following characteristics: In step S3, the temperature during glycosylation modification is 45~55℃, preferably 50℃; In step S3, the duration of the glycosylation modification treatment is 6-10 hours, preferably 8 hours. In step S3, the mass ratio of glucose to collagen obtained in step S2 is (1~2):(1~2), preferably 1:

1.

5. The method according to claim 1, characterized in that, It has at least one of the following characteristics: In step S4, the mass ratio of the material obtained in step S3 to the gastric juice is 1:

7. In step S4, the material obtained in step S3 is incubated in the gastric fluid for 50-100 minutes, preferably 60 minutes. In step S4, the material obtained in step S3 is incubated in the gastric fluid at a temperature of 36~38℃, preferably 37℃.

6. The method according to claim 1, characterized in that, It has at least one of the following characteristics: In step S5, the pH of the material obtained in step S4 is adjusted to 6.5; In step S5, the mass ratio of the material obtained in step S4 to the intestinal fluid is 1:

7. In step S5, after adding intestinal fluid, the mixture is cultured at 36-38°C for 50-100 min, preferably at 37°C for 60 min.

7. The method according to claim 1, characterized in that, In step S6, the pH of the material obtained in step S5 is adjusted to 3.0; And / or, in step S6, during vacuum freeze drying, the temperature is -60 to -80°C and the vacuum degree is 10 to 40 Pa.

8. The method according to claim 1, characterized in that, It has at least one of the following characteristics: In step S7, the mass ratio of the material obtained in step S6 to tyrosinase is 10:

1. In step S7, the material obtained in step S6 is mixed with tyrosinase and then incubated at 37°C for 1 hour. In step S7, an ultrafiltration centrifugation is performed using a membrane with a molecular weight cutoff of 3-5 kDa. In step S7, ultrafiltration centrifugation is performed using any one of polyethersulfone (PES), cellulose, or regenerated cellulose. In step S7, ultrafiltration centrifugation is performed using a tubular ultrafiltration tube or a conical ultrafiltration tube. In step S7, the ultrafiltration centrifugation time is 10~60min, preferably 20min, and the rotation speed is 4000rpm; In step S7, the temperature of ultrafiltration centrifugation is 4~25℃; In step S7, acetonitrile is used to elute the residue after ultrafiltration centrifugation. In step S7, the obtained fish collagen peptide has a molecular weight of less than 3000 Da and a polypeptide sequence with glycosylation sites on the peptide chain.

9. A fish collagen peptide with whitening effects, characterized in that, The fish collagen peptide is obtained by the method described in any one of claims 1 to 8, wherein the peptide chain of the fish collagen peptide has a polypeptide sequence with glycosylation sites.

10. The fish collagen peptide according to claim 9, characterized in that, The molecular weight of the fish collagen peptide is less than 3000 Da.

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