Preparation method and application of fish maw oligopeptide

Abstract

The invention relates to a preparation method of fish maw oligopeptide, which comprises the following steps: crushing fish maw to obtain fish maw powder, weighing a certain amount of fish maw powder, adding pure water, heating to obtain a fish maw powder solution, adding protease into the fish maw powder solution, carrying out multi-step enzymolysis reaction to obtain enzymatic hydrolysate, adding a composition into the enzymatic hydrolysate, and uniformly stirring to obtain the fish maw oligopeptide. The preparation method comprises the following steps: carrying out enzyme deactivation and filtration treatment on an enzymatic hydrolysate added with the composition to obtain a coarse filtrate, carrying out decoloration and deodorization treatment on the coarse filtrate, filtering to obtain a fine filtrate, and carrying out concentration and drying treatment on the fine filtrate to obtain the fish maw oligopeptide. The fish maw oligopeptide provided by the invention is lower in molecular weight, has better physiological activity, and has the capability of resisting UVR-induced epidermal cell mitochondrial damage.

Description

Technical Field

[0001] The present application relates to the technical field of bioactive peptides, and in particular to a preparation method and application of fish maw oligopeptides. Background Art

[0002] Fish maw, also known as fish glue and fish belly, has been considered an excellent tonic since ancient times and is known as one of the "eight treasures". Modern nutritional research has found that fish maw contains 84.2% protein, mostly high-grade collagen, 0.2% fat, and is rich in calcium, zinc, iron, selenium and other trace elements. It is a high-protein, low-fat healthy food. With the improvement of living standards, the scale of the fish maw market has gradually expanded, and different types of fish maw products have emerged on the market to meet the needs of different groups of people.

[0003] Using bio-enzymatic hydrolysis technology to prepare fish maw into fish maw peptides, which are applied to various products such as liquid beverages, solid beverages, and jelly candies, can better meet the needs of different groups of people and different scenarios and create higher economic value.

[0004] However, in actual production, it was found that fish maw peptides directly made from fish maw using bio-enzymatic hydrolysis technology are prone to precipitation when used in slightly acidic oral beverages, which limits the application of such fish maw peptides.

[0005] In addition, studies have shown that low molecular weight peptides are more stable during gastric and intestinal digestion, short-chain peptides are less likely to be degraded by intestinal epithelial cell peptidases than long-chain peptides, and short-chain peptides are more easily absorbed than long-chain peptides, and small molecular weight peptides are more easily absorbed than large molecular weight peptides. However, the fish maw peptides currently available on the market have a large molecular weight and a long peptide chain, which are easily degraded during the digestion and absorption process and cannot play their due active role.

[0006] Therefore, it is particularly necessary to develop fish maw oligopeptides with smaller molecular weight, better activity and the ability to remain stable in slightly acidic oral beverages. Summary of the invention

[0007] The present application proposes a method for preparing fish maw oligopeptide, comprising the following steps: S1: crushing fish maw to obtain fish maw powder; S2: Weigh a certain amount of fish maw powder, add pure water and heat to obtain a fish maw powder solution, add protease to the fish maw powder solution to perform multi-step enzymolysis reaction to obtain an enzymolysis solution; S3: adding a composition to the enzymatic hydrolyzate and stirring evenly, wherein the composition comprises chicken egg yolk extract, chitosan oligosaccharide and ginger extract; S4: performing enzyme inactivation and filtration treatment on the enzymatic hydrolyzate after adding the composition to obtain a coarse filtrate; S5: decolorizing and deodorizing the coarse filtrate to obtain a refined filtrate after filtering; S6: The refined filtrate is concentrated and dried to obtain fish maw oligopeptides.

[0008] Wherein, in step S2, the weight ratio of the fish maw powder to pure water is 1:(10-20), and the heating temperature is 50-60°C.

[0009] In step S2, alkaline protease is added to the fish maw powder solution heated at 50-60°C and reacted for 0.5-2h, and then flavor protease is added and reacted for 0.5-2h, wherein the amount of alkaline protease added is 1-1.5% of the weight of the fish maw powder, and the amount of flavor protease added is 0.3-0.8% of the weight of the fish maw powder.

[0010] Wherein, during the enzymatic hydrolysis reaction, the reaction solution is subjected to ultrasonic treatment to obtain an enzymatic hydrolyzate.

[0011] Among them, the weight ratio of chicken egg yolk extract, chitosan oligosaccharide and ginger extract in the composition in step S3 is (2-3): (0.1-0.5): (5-10), and the added amount of the composition is 2-5% of the weight of fish maw powder.

[0012] The temperature of the enzyme inactivation and filtration treatment in step S4 is 85-90° C., the enzyme inactivation time is 20-60 min, and after the enzyme inactivation, filtration treatment is performed using an 800-mesh filter cloth to obtain a coarse filtrate.

[0013] Among them, in step S5, firstly, activated carbon is added to the coarse filtrate, and the added amount of activated carbon is 2-5% of the weight of the fish maw powder. The reaction is carried out at 50-60° C. for 30-90 minutes, and then filtering is performed.

[0014] In step S5, the filtration treatment first uses 800-mesh filter cloth to obtain a filtrate, and the filtrate is further filtered using a filter membrane to obtain a refined filtrate.

[0015] Wherein, the pore size of the filter membrane is 0.22-1um.

[0016] The present application also proposes the use of a fish maw oligopeptide in resisting UVR-induced epidermal cell mitochondrial damage.

[0017] The preparation method of fish maw oligopeptide of the present application uses a multi-step enzymatic hydrolysis method to prepare fish maw oligopeptide, and at the same time adopts a composition synergistic treatment method to remove the easily precipitated part of the fish maw oligopeptide. The composition includes chicken egg yolk extract, chitosan oligosaccharide and ginger extract, which can obtain fish maw oligopeptide with lower molecular weight. The fish maw oligopeptide is more stable, has better physiological activity, and has the ability to resist UVR-induced epidermal cell mitochondrial damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] By reading the detailed description below with reference to the accompanying drawings, the above and other purposes, features and advantages of the exemplary embodiments of the present disclosure will become readily understood. In the accompanying drawings, several embodiments of the present disclosure are shown in an exemplary and non-limiting manner, and the same or corresponding reference numerals represent the same or corresponding parts, wherein: Figure 1 is a state diagram of the solution prepared in an embodiment of the present application after sterilization; Figure 2 This is a state diagram of the solution prepared in a pair of ratios of the present application after being sterilized and stored at 37°C for 7 days; Figure 3 It is a bar graph of ROS content in HaCaT cells induced by UVR in the examples of the present application; Figure 4 is a bar graph of GSH-Px activity in HaCaT cells induced by UVR in the examples of the present application; Figure 5 This is a bar graph of SOD activity in HaCaT cells induced by UVR in the examples of this application; Figure 6 It is a bar graph of mitochondrial membrane potential of HaCaT cells induced by UVR in the examples of the present application; Figure 7 It is a bar graph of ATP production of HaCaT cells induced by UVR in the examples of the present application. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present disclosure to clearly and completely describe the technical solutions in the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present disclosure.

[0020] The present application proposes a method for preparing fish maw oligopeptide, comprising the following steps: S1: crushing fish maw to obtain fish maw powder; This step is a pre-treatment step, in which the fish maw is crushed using a fish maw grinder or other crushing devices to obtain ultra-fine fish maw powder for later use.

[0021] S2: Weigh a certain amount of fish maw powder, add pure water and heat to obtain a fish maw powder solution, add protease to the fish maw powder solution to perform multi-step enzymolysis reaction to obtain an enzymolysis solution; In this step, the amount of pure water added can be determined according to the weight of the fish maw powder, and the weight ratio of the fish maw powder to pure water can be 1: (10-20), which is selected according to the actual mixing situation. After adding the pure water, the temperature is raised to 50-60°C, and protease is added to perform a multi-step enzymatic hydrolysis reaction.

[0022] Add alkaline protease to the fish maw powder solution heated at 50-60°C and react for 0.5-2h, then add flavor protease and continue to react for 0.5-2h, wherein the added amount of alkaline protease is 1-1.5% of the weight of the fish maw powder, and the added amount of flavor protease is 0.3-0.8% of the weight of the fish maw powder.

[0023] During the multi-step enzymatic hydrolysis reaction, the reaction system can be subjected to ultrasonic treatment, that is, the reaction solution is placed in an ultrasonic device to ensure that the enzymatic hydrolysis reaction is more complete, wherein the ultrasonic power is 200-500w / m 2 .

[0024] S3: adding a composition to the enzymatic hydrolyzate and stirring evenly, wherein the composition comprises chicken egg yolk extract, chitosan oligosaccharide and ginger extract; The chicken egg yolk extract is obtained from chicken egg yolk as raw material after crushing, homogenizing, partially hydrolyzing and drying, and contains ingredients such as lecithin and phosphorylated protein; the chitosan oligosaccharide, also known as chito-oligosaccharide or oligochitosan, is a sugar chain with a polymerization degree of 2-20, has good water solubility, and is the only positively charged cationic alkaline amino oligosaccharide in nature; the ginger extract contains a variety of flavonoids, polysaccharides, and enzyme substances.

[0025] The weight ratio of the chicken egg yolk extract, chitosan oligosaccharide and ginger extract in the composition is (2-3): (0.1-0.5): (5-10), and the added amount of the composition is 2-5% of the weight of the fish maw powder.

[0026] S4: performing enzyme inactivation and filtration treatment on the enzymatic hydrolyzate after adding the composition to obtain a coarse filtrate; The temperature of the enzyme inactivation filtration treatment is 85-90°C, the enzyme inactivation time is 20-60min, and after the enzyme inactivation, filtration treatment is performed using an 800-mesh filter cloth to obtain a coarse filtrate.

[0027] S5: decolorizing and deodorizing the coarse filtrate to obtain a refined filtrate after filtering; First, add activated carbon to the coarse filtrate, the amount of activated carbon added is 2-5% of the weight of the fish maw powder, react at 50-60°C for 30-90 minutes, and filter.

[0028] The filtration treatment first uses 800 mesh filter cloth to obtain a filtrate, and then further uses a filter membrane to obtain a refined filtrate. The pore size of the filter membrane is 0.22-1 um.

[0029] S6: The refined filtrate is concentrated and dried to obtain fish maw oligopeptides.

[0030] In the concentration and drying process, the refined filtrate is first concentrated to 28-36% of solid content using a double-effect concentrator to obtain a concentrated solution, and the concentrated solution is dried using freeze drying to obtain the fish maw oligopeptide of the present application.

[0031] The lecithin in the chicken egg yolk extract in the present application is a lipid substance with a phosphate group head and a fatty acid tail chain, wherein the phosphate group is hydrophilic and the fatty acid tail chain is hydrophobic. When it exists in a solution, the phosphate group and the fatty acid tail chain form tiny inclusions to the outside. At the same time, due to the hydrophobicity of the fatty acid tail chain, it is easier to encapsulate the less hydrophilic impurities in the fish maw peptide; the phosphorylated protein has multiple phosphate groups, and the phosphate groups carry a negative charge. Multiple phosphate groups will form a cavity, which can react with a variety of metal ions in the fish maw polypeptide to remove the metal ions from the solution and prevent the non-enzymatic reaction caused by the metal ions.

[0032] The fish jelly peptides obtained by enzymatic hydrolysis are complex mixtures. When in solution, they exist in various forms such as molecules, ions, and active groups. Among them, the part that exists in the form of ions is more likely to react with other impurities due to electrostatic attraction due to electric charge, thereby deteriorating and precipitating. At the same time, fish jelly peptides contain more carboxyl and amino groups, which tend to ionize hydrogen ions and then exist in the form of anions. The addition of cationic chitosan oligosaccharides can combine with unstable fish jelly peptides and thus remove them.

[0033] The flavonoid compounds in the ginger extract are often connected with functional groups such as phenolic hydroxyl, methoxy, methyl, and isopentenyl in their structures, and short peptide substances can be grafted onto these functional groups to be modified, thereby changing the stability of the short peptides; the ginger extract also contains various enzyme substances, which work together to further change the short peptide substances.

[0034] The chicken egg yolk extract in the present application can change the solution environment, stabilize the metal ions in the fish glue peptide solution and further remove impurities, and work together with chitosan oligosaccharides to remove the unstable part of the fish glue peptide. The ginger extract changes some properties of the fish glue peptide and affects the stability of the fish glue peptide. The chicken egg yolk extract, chitosan oligosaccharides and ginger extract work together to make the fish glue peptide more stable. At the same time, since the unstable part of the fish glue peptide is removed, its activity is further improved.

[0035] It should be noted that the chicken egg yolk extract and chitosan oligosaccharide in the present application are commercially available, and the ginger extract is obtained by crushing and homogenizing ginger, adding pectinase and cellulase in a ratio of 1:1 to 1% of the weight of ginger, and then filtering for 2-4 hours.

[0036] It needs to be further explained that peptide substances are derived from the decomposition of proteins and have a wide range of sources. Those with functional activity are called active polypeptides, and those with a molecular weight of less than 1000Da are called oligoactive peptides. Oligoactive peptides are generally composed of 2-6 amino acids and have the characteristics of fast transport, low energy consumption, and not easy to saturate. They can reach cells faster and better and bind to active sites within cells, thus having better functional activity.

[0037] The fish maw oligopeptide prepared by the method of the present application is prepared by a multi-step enzymatic hydrolysis method, and a composition synergistic treatment method is used to remove the easily precipitated part of the fish maw oligopeptide. The composition includes chicken egg yolk extract, chitosan oligosaccharide and ginger extract, which can obtain fish maw oligopeptide with lower molecular weight. The fish maw oligopeptide is more stable, has better physiological activity, and has the ability to resist UVR-induced epidermal cell mitochondrial damage, and can meet different needs.

[0038] The present application also proposes an application of a fish maw oligopeptide in resisting UVR-induced mitochondrial damage of epidermal cells. The fish maw oligopeptide is prepared by the preparation method of the fish maw oligopeptide in the present application.

[0039] Specific examples and comparative examples The method for preparing fish maw oligopeptide in Example 1 comprises the following steps: S1: using a fish maw grinder to grind fish maw to obtain fish maw powder, wherein the fish maw uses white jade fish maw (basa fish maw) produced in Vietnam; S2: Weigh a certain amount of fish maw powder, add pure water and heat it, the weight ratio of fish maw powder to pure water can be 1:15, to obtain a fish maw powder solution, add alkaline protease to the fish maw powder solution heated at 50-60°C and react for 1 hour, then add flavor protease and continue to react for 1 hour, wherein the amount of alkaline protease added is 1% of the weight of the fish maw powder, and the amount of flavor protease added is 0.3% of the weight of the fish maw powder; perform ultrasonic treatment on the reaction system, and the ultrasonic power is 300w / m 2 .

[0040] S3: Add a composition to the enzymatic hydrolyzate and stir evenly, wherein the composition includes chicken egg yolk extract, chitosan oligosaccharide and ginger extract, wherein the weight ratio of the chicken egg yolk extract, chitosan oligosaccharide and ginger extract in the composition is 2:0.1:5, and the added amount of the composition is 3% of the weight of the fish maw powder.

[0041] S4: the enzymatic solution after adding the composition is subjected to enzyme inactivation and filtration treatment, the temperature of the enzyme inactivation and filtration treatment is 85-90°C, the enzyme inactivation time is 20-60min, and after the enzyme inactivation, it is subjected to filtration treatment using 800 mesh filter cloth to obtain a coarse filtrate.

[0042] S5: Add activated carbon to the enzymatic hydrolyzate, the amount of activated carbon added is 3% of the weight of the fish maw powder, react at 50-60°C for 60 minutes, and filter. The filtration process first uses an 800-mesh filter cloth to obtain a filtrate, and further filters the filtrate with a filter membrane to obtain a refined filtrate. The pore size of the filter membrane is 0.5um.

[0043] S6: Use a double-effect concentrator to concentrate the refined filtrate to a solid content of 28-36% to obtain a concentrated solution, and use freeze drying to dry the concentrated solution to obtain the fish maw oligopeptide of this embodiment.

[0044] The steps and parameters for preparing fish maw oligopeptides in Examples 2-3 and Comparative Examples 1-9 are consistent with those in Example 1, wherein the contents of alkaline protease, flavor protease, chicken egg yolk extract, chitosan oligosaccharide and ginger extract are different, as shown in Table 1.

[0045] Table 1: Comparison of the parameters of each component in Examples 2-3 and Comparative Examples 1-9 and Example 1.

[0046]

[0047] Comparative Example 10: The parameters of each component in this comparative example are consistent with those in Example 1, except that, during the enzymatic hydrolysis reaction in step S2, flavor protease is added first and then alkaline protease is added.

[0048] Comparative Example 11: The parameters of each component in this comparative example are consistent with those in Example 1, except that, during the enzymatic hydrolysis reaction in step S2, no ultrasonic treatment is performed.

[0049] Control group: Commercially available fish glue peptide was used as the control.

[0050] Specific test examples Test Example 1 The fish glue peptides prepared in Examples 1-3, Comparative Examples 1-11 and the control group were respectively prepared into solutions, the pH value of the solution was about 4.0, and the specific components were: 20g of fish glue peptide, 6g of concentrated lemon juice, 0.6g of anhydrous citric acid, 8g of maltitol, 0.1g of erythritol, and 65.3g of pure water.

[0051] The prepared solutions were divided into 30ml PP bottles, placed in a sterilizer, sterilized at 105℃ for 25 minutes, and the precipitation of the solutions was observed after cooling to room temperature. The cooled solutions were then stored in a constant temperature incubator at 37℃ and 55℃, and samples were taken at 1, 3, 5, and 7 days to observe the precipitation of each solution and evaluate its stability.

[0052] See Figure 1 and Figure 2 , Figure 1 This is a state diagram of the solution prepared in Example 1 after sterilization. The solution is clear and transparent without precipitation. Figure 2 This is a state diagram of the solution prepared in Comparative Example 3 after being sterilized and stored at 37°C for 7 days, with a large amount of precipitation in the solution.

[0053] It should be noted that the precipitate formed in the solution is a suspended floc, which is not easy to settle to the bottom of the solution and cannot be removed by conventional filtration processes. When there is a precipitate inside the solution, it greatly affects the consumer experience.

[0054] See Table 2 for the precipitation conditions of each solution in Examples 1-3, Comparative Examples 1-11 and the control group.

[0055] Table 2: Precipitation of solutions of Examples 1-3, Comparative Examples 1-11 and the control group at different stages.

[0056]

[0057] Note: “ / ” indicates no precipitation and the solution is clear and transparent; “*” indicates a small amount of precipitation in the solution; “**” indicates obvious precipitation in the solution; and “***” indicates a large amount of precipitation in the solution.

[0058] According to Table 1, Examples 1-3 and Comparative Examples 8-11 can all obtain stable fish glue peptides, indicating that the synergistic treatment of the composition of the present application can obtain stable fish glue peptides, and has a wide range of adaptability. Comparative Example 1 shows that the fish glue peptides obtained by direct enzymatic hydrolysis without the synergistic treatment of the composition are unstable; Comparative Examples 2-7 show that the synergistic treatment of the composition with only one component or one less component cannot obtain stable fish glue peptides, which may be due to the synergistic effect of the three components in the combination on the fish glue peptide, and stable fish glue peptides can only be obtained under multiple effects.

[0059] Test Example 2 The molecular weight of the fish glue peptides in each embodiment and comparative example was detected according to the molecular weight detection method in Appendix A of GB 31645-2018.

[0060] Specifically, the determination is carried out by high-performance size exclusion chromatography. That is, porous fillers are used as stationary phases, sample components are separated according to the difference in molecular volume, and the detection is carried out under the condition of the ultraviolet absorption wavelength of 220nm of the peptide bond. The chromatograms and data of the standard and sample are processed using the dedicated data processing software for relative molecular mass distribution determination (i.e., GPC software), and the relative molecular mass size and distribution range of the collagen peptide are calculated according to the relative molecular mass calibration curve equation. The specific molecular weight test results are shown in Table 3.

[0061] Table 3 Molecular weight distribution (%) and average molecular weight of samples in each embodiment, comparative example and control group.

[0062]

[0063] It can be seen from the average molecular weight that the average molecular weights of the fish glue peptides obtained in Examples 1-3 are 853.19Da, 792.37Da, and 831.40Da, respectively, which are significantly smaller than the fish glue peptides obtained in the comparative examples, and less than 1000Da, and can be called fish glue oligopeptides. The molecular weight of the commercially available fish glue peptide (control group) is 2004.81Da, which is relatively large, and the part >10000 is significantly higher than the fish glue oligopeptide provided by the present invention, while the part <500 is significantly lower than the fish glue peptide provided by the present invention, which may be the reason why the functional activity of the commercially available fish glue peptide is significantly worse than that of the fish glue oligopeptide provided by the present invention.

[0064] From the molecular weight distribution, it can be seen that in Example 1-3, the >10000 and 10000-5000 parts are significantly reduced compared to Comparative Example 1-11, while the 500-189 part is significantly increased, indicating that after the synergistic treatment of the composition, the original large molecular weight part of the fish maw peptide is basically removed, and the part with a molecular weight below 500 is increased. According to existing research reports, peptides with a molecular weight less than 500 are mostly 2-5 peptides, which have better activity stability than peptides with larger molecular weight and longer peptide chains. This is also the reason why the fish maw oligopeptides obtained by the present invention have better stability and activity.

[0065] Test Example 3 In this test example, cells were first cultured and treated.

[0066] Human immortalized keratinocytes (HaCaT cells) were routinely passaged and cultured in DMEM medium containing 10% FBS and 1% streptomycin solution and grown in a 37°C incubator containing 5% carbon dioxide.

[0067] HaCaT cells were seeded in 24-well plates (1 × 10 6 After culturing in the incubator for 24 h, the supernatant was discarded and 1 mL of culture medium containing the same concentration of fish glue peptides from different examples and comparative examples was added to each well. After culturing for 2 h, UVR (UVA+UVB) irradiation was performed at a parameter of 300 mJ / cm 2 , lasting for 30 minutes, and set up a blank group and a model group. The blank group was not irradiated, and the model group was irradiated.

[0068] This test example further tests the intracellular ROS content.

[0069] After the experimental treatment, the cells were washed three times with ice-cold phosphate-buffered saline (PBS) to remove the residual and extracellular stain, and the fluorescence intensity of each well was measured by a fluorescence microplate reader at 485 nm as the excitation wavelength and 535 nm as the emission wavelength.

[0070] ROS is a natural byproduct of normal oxygen metabolism in cells, which plays an important role in cell signaling and intracellular balance. Studies have found that under the stimulation of UVR, the level of ROS in cells increases significantly, resulting in excessive ROS, which can cause oxidative stress and cause serious damage to cell structures. Mitochondria are the energy metabolism center in cells, and the unique mtDNA in mitochondria is not protected by histones, making mitochondria more susceptible to damage from excessive ROS than other structures in cells, resulting in mitochondrial dysfunction. Figure 3 The effects of the treatments in the examples and comparative examples on the ROS content in HaCaT cells induced by UVR are shown in Figure 2. Figure 3 As shown, the experimental groups treated with fish maw peptides had lower ROS levels than the model group, indicating that fish maw peptides have the function of reducing ROS produced by cells after UVR induction, and Examples 1-3 are significantly lower than Comparative Examples 8-10. Existing studies have shown that the amount of enzyme preparations used and the order of use of enzyme preparations profoundly affect the structure of the obtained polypeptides, and thus affect the activity of the polypeptides. This may be the reason why the fish maw oligopeptides provided by the present invention have better functional activity.

[0071] This test example detects the activity of intracellular GSH-Px and SOD.

[0072] Intracellular GSH-Px and SOD activity detection Kits were used to detect the activities of GSH-Px and SOD in HaCaT cells.

[0073] GSH-Px and SOD are both intracellular peroxidases that can remove excess ROS in cells and are important for maintaining redox homeostasis in cells. However, reduced activity of GSH-Px and SOD will result in the inability to remove excess ROS in cells in a timely manner, further exacerbating mitochondrial structural damage and mitochondrial functional impairment. Figure 4 and Figure 5 As shown, the fish maw oligopeptides provided by the present invention can significantly increase the activity of GSH-Px and SOD, and can further prevent the granule structure from being damaged. Moreover, Examples 1-3 can increase the activity of GSH-Px and SOD more than Comparative Examples 5-7, which indicates that using any one component of the composition alone has no significant effect.

[0074] In this test example, mitochondrial membrane potential was further detected.

[0075] Add JC-1 working solution to the cells treated in each embodiment and comparative example, incubate for 30 minutes (37°C), and centrifuge for 4 minutes. Wash the collected cells with PBS and analyze the results with a fluorescence spectrophotometer. Set the excitation wavelength to 515nm and the emission wavelength to 529nm to measure the fluorescence intensity of JC-1 monomer. Set the excitation wavelength to 585nm and the emission wavelength to 590nm to measure the fluorescence intensity of JC-1 polymer. Evaluate the change in mitochondrial membrane potential by calculating the ratio of the fluorescence intensity of JC1 monomer to polymer.

[0076] It should be noted that mitochondria are organelles composed of two layers of membranes, inner and outer, and most of the physiological reactions in mitochondria occur on the inner membrane of mitochondria. Therefore, maintaining the stability of the membrane structure is of great significance to maintaining the normal function of mitochondria. The different ion concentrations inside and outside the intact mitochondrial membrane lead to different potential differences inside and outside the membrane. When the mitochondrial membrane structure is destroyed, the free flow of ions on both sides of the membrane increases and the potential difference decreases. Therefore, evaluating the potential difference on both sides of the mitochondrial membrane can more objectively evaluate the integrity of the mitochondrial membrane structure, thereby evaluating the integrity of mitochondrial function.

[0077] Figure 6 The effects of various examples and comparative examples on the mitochondrial membrane potential of HaCaT cells induced by UVR are shown in FIG. Figure 6 As shown, the fish glue peptide provided by the present invention can significantly increase the mitochondrial membrane potential of HaCaT cells after UVR induction, indicating that the fish glue peptide provided by the present invention can maintain the integrity of the mitochondrial membrane structure of HaCaT cells after UVR induction. And Examples 1-3 are significantly higher than Comparative Examples 2-4, which indicates that the lack of any one of the components in the composition will lead to a decrease in the effect or failure to achieve the effect, which is related to the synergistic effect of the three components in the composition.

[0078] In this test example, the amount of ATP generated in the cells was further detected.

[0079] After being treated in each embodiment and comparative example, Ripa lysis buffer was added for lysis, and the cell lysate was collected in a 1.5 mL centrifuge tube, centrifuged at 12000 g and 4 ° C for 5 min, and the supernatant was taken. 100 μL of ATP detection working solution was added to a black 96-well plate, and the plate was placed at room temperature for 3-5 min. 20 μL of sample or standard was added to the detection well, mixed quickly, and the RLU value was determined by a multifunctional microplate reader. The ATP content of each group was calculated according to the concentration-RLU value curve.

[0080] According to the instructions of the BCA protein concentration determination kit, the absorbance of each group of samples at 562 nm was detected on a multifunctional microplate reader, and the protein concentration of the sample to be tested was calculated according to the concentration-absorbance curve.

[0081] ATP content is expressed as follows: ATP content (nmol / mg protein) = ATP content of experimental group / protein content of experimental group It should be noted that mitochondria are the energy metabolism center in cells, which specifically generate ATP and transport it to other organelles to meet the energy needs of normal physiological activities. Therefore, evaluating the amount of ATP generated in cells can intuitively evaluate the functional integrity of mitochondria.

[0082] like Figure 7 As shown, the fish maw oligopeptide provided in the present application can significantly increase the ATP production of HaCaT cells after UVR induction, indicating that the fish maw oligopeptide provided in the present application can maintain the functional integrity of the mitochondria of HaCaT cells after UVR induction. And Examples 1-3 are significantly higher than Comparative Example 11. Ultrasonic treatment will produce cavitation, fragmentation and other effects in the solution, and these effects have an impact on the enzymatic hydrolysis process, the structure of proteins and polypeptides, which is the reason why the fish maw oligopeptide provided in the embodiments of the present invention is more active than Comparative Example 11.

[0083] According to the above test results of this test example, the fish maw oligopeptide provided by the present application can reduce the ROS content in HaCaT cells after UVR induction, increase the activity of GSH-Px and SOD in the cells, maintain the membrane potential of mitochondria in the cells, and increase the ATP production in the cells, indicating that the fish maw oligopeptide provided by the present invention has the function of resisting mitochondrial damage in HaCaT cells after UVR induction.

[0084] The preparation method of fish maw oligopeptide of the present application uses a multi-step enzymatic hydrolysis method to prepare fish maw oligopeptide, and at the same time adopts a composition synergistic treatment method to remove the easily precipitated part of the fish maw oligopeptide. The composition includes chicken egg yolk extract, chitosan oligosaccharide and ginger extract, which can obtain fish maw oligopeptide with lower molecular weight. The fish maw oligopeptide is more stable, has better physiological activity, and has the ability to resist UVR-induced epidermal cell mitochondrial damage.

[0085] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0086] Although the preferred embodiments of the present application have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present application.

[0087] Finally, it should be noted that, in this application, relational terms such as first and second, etc., are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article including the elements.

[0088] Specific examples are used in this application to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core idea of ​​this application. At the same time, for those skilled in the art, according to the idea of ​​this application, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as a limitation on this application.

Claims

1. A method for preparing fish maw oligopeptide, characterized in that: The following steps are involved: S1: crushing fish maw to obtain fish maw powder; S2: Weigh a certain amount of fish maw powder, add pure water and heat to obtain a fish maw powder solution, add protease to the fish maw powder solution to perform multi-step enzymolysis reaction to obtain an enzymolysis solution; S3: adding a composition to the enzymatic hydrolyzate and stirring evenly, wherein the composition comprises chicken egg yolk extract, chitosan oligosaccharide and ginger extract; S4: performing enzyme inactivation and filtration treatment on the enzymatic hydrolyzate after adding the composition to obtain a coarse filtrate; S5: decolorizing and deodorizing the coarse filtrate to obtain a refined filtrate after filtering; S6: The refined filtrate is concentrated and dried to obtain fish maw oligopeptides.

2. The method for preparing fish maw oligopeptide according to claim 1, characterized in that: In step S2, the weight ratio of the fish maw powder to pure water is 1:(10-20), and the heating temperature is 50-60°C.

3. The method for preparing fish maw oligopeptide according to claim 2, characterized in that: In step S2, alkaline protease is added to the fish maw powder solution heated at 50-60°C and reacted for 0.5-2h, and then flavor protease is added and reacted for 0.5-2h, wherein the amount of alkaline protease added is 1-1.5% of the weight of the fish maw powder, and the amount of flavor protease added is 0.3-0.8% of the weight of the fish maw powder.

4. The method for preparing fish maw oligopeptide according to claim 3, characterized in that: During the enzymatic hydrolysis reaction, the reaction solution is subjected to ultrasonic treatment to obtain an enzymatic hydrolyzate.

5. The method for preparing fish maw oligopeptide according to claim 1, characterized in that: The weight ratio of the chicken egg yolk extract, chitosan oligosaccharide and ginger extract in the composition in step S3 is (2-3): (0.1-0.5): (5-10), and the added amount of the composition is 2-5% of the weight of the fish maw powder.

6. The method for preparing fish maw oligopeptide according to claim 1, characterized in that: The temperature of the enzyme inactivation and filtration treatment in step S4 is 85-90° C., the enzyme inactivation time is 20-60 min, and after the enzyme inactivation, filtration treatment is performed using an 800-mesh filter cloth to obtain a coarse filtrate.

7. The method for preparing fish maw oligopeptide according to claim 1, characterized in that: In step S5, firstly, activated carbon is added to the coarse filtrate, the amount of activated carbon added is 2-5% of the weight of the fish maw powder, the reaction is carried out at 50-60° C. for 30-90 minutes, and then filtering is performed.

8. The method for preparing fish maw oligopeptide according to claim 7, characterized in that: In step S5, the filtration treatment first uses 800-mesh filter cloth to obtain a filtrate, and the filtrate is further filtered through a filter membrane to obtain a refined filtrate.

9. The method for preparing fish maw oligopeptide according to claim 8, characterized in that: The pore size of the filter membrane is 0.22-1um.

10. Use of a fish maw oligopeptide in resisting UVR-induced epidermal cell mitochondrial damage, wherein the fish maw oligopeptide is prepared by any method of claims 1-9.

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