Collagen peptide with anti-aging activity and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine. More particularly, it relates to a collagen peptide and a preparation method and application thereof. The preparation method of the collagen peptide with anti-aging activity comprises the following steps: S1, fish scale pretreatment: washing, crushing, degreasing and removing impurities, and then washing to neutral; S2, decalcification: adding 1-4 times of pure water by weight to the pretreated fish scale, treating in a pulsed electric field for 1-5 min, then adding neutral protease for enzymolysis, and drying to obtain decalcified fish scale; S3, hydrolysis: adding plasma activated water and hydrolysis enzyme to the decalcified fish scale, water bath oscillation for 0.1-1 h, terminating the reaction, ultrafiltration, collecting the filtrate, and obtaining the collagen peptide.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine. More particularly, it relates to a collagen peptide with anti-aging activity and a preparation method and application thereof. BACKGROUND

[0002] Fish scales contain a large amount of inorganic and organic effective components, among which the organic matter accounts for 40% to 55%, mainly composed of a large amount of collagen, scleroprotein, lecithin, fat and various vitamins, etc.; the inorganic matter accounts for 7% to 25%, mainly composed of hydroxyapatite and mineral salts.

[0003] The extraction of fish scale collagen generally includes decalcification and collagen extraction steps. The decalcification method of fish scales mainly adopts acid decalcification, EDTA decalcification and the like. For example, Huang Yu et al. [1] Hydrochloric acid is used to decalcify tilapia fish scales, and the decalcification rate is 99.55% after process optimization. However, acid decalcification is easy to cause hydroxyproline to be dissolved out, affecting the quality of collagen, and if acid method is used for extracting collagen in the later stage, part of the impure protein will be left over; Pati F et al. [2] EDTA is used to decalcify fish scales for 48 hours to achieve the maximum decalcification rate, but the long decalcification time is not conducive to the industrial production of fish scales.

[0004] The collagen in fish scales is usually extracted by acid method, alkali method and enzyme method and the like. For example, Lu Zhao et al. [3] Acetic acid is used to extract ASC of grass carp fish scales, and the test shows that the extraction rate is 15.33% under the condition of 1 mol / L acetic acid, extraction temperature of 28℃ and extraction time of 25h; Zeng Shaokui et al. [4] Citric acid is used to extract tilapia fish scale gelatin, and the optimized conditions are: extraction temperature of 65℃ and extraction time of 3.6h, and the extraction rate is 28.4%; however, acid extraction of fish scale collagen is complete hydrolysis, and tryptophan and other amino acids will be destroyed, and acid decalcification cannot be used, otherwise part of the impure protein will be left over, the subsequent purification is complicated, and the acid corrodes the equipment, which is not conducive to industrial production.

[0005] Plasma activated water (PAW) refers to a kind of activated liquid formed by transferring various high-energy active substances generated by low-temperature plasma discharge above or in water to water. The interaction between plasma and water molecules and a series of chemical reactions will change the physical and chemical properties of water, such as pH value reduction, conductivity increase, oxidation-reduction potential (ORP value) increase and the like, and produce active oxygen (ROS) and active nitrogen (RNS) and other high-energy active components in water. These active substances mainly include long-life substances such as nitrate (NO3 - ), nitrite (NO2 -), hydrogen peroxide (H2O2) and ozone (O3) and short-lived species hydroxyl radical (·OH), singlet oxygen (1O2), superoxide (O2 - ) and peroxynitrite (ONOO - ) and so on [5] . At present, plasma-activated water is mainly used for sterilization, agricultural planting (can promote the growth of some plants) and wound treatment, such as Matthew J Traylor et al. used the interfacial dielectric barrier discharge device to prepare activated distilled water, and when Escherichia coli was cultivated in the activated water for 15 min, the sterilization quantity reached 5 orders of magnitude, but with the increase of storage time, the sterilization effect of activated water was obviously weakened [6] ; KTakaki et al. used PAW to irrigate Chinese cabbage, and the dry weight of Chinese cabbage irrigated with activated water for 10 min and 20 min increased to 3.9 and 6.6 times of the control group, respectively [7] ; Liu Xin et al. found that the active particles of liquid phase plasma (PAW) had stronger transdermal effect than those of gas phase plasma, which could better penetrate into dermal cells and achieve the effect of treating wounds [8] ; Dehui Xu et al. used a portable DBD activated water device to prepare activated tap water to treat mouse skin wounds, and found that the wounds of mice in the activated water treatment group were significantly smaller than those in the control group, and the wounds of mice in the activated water treatment group completely healed in 17 days, while those in the control group needed 22 days [9] .

[0006] However, there is no research on the use of low-temperature plasma-activated water to assist enzymatic hydrolysis to extract anti-aging active collagen from fish scales.

[0007] [1] Huang Y, Wang Y, Nguyen VD, et al. Optimization of fish scale decalcification process by response surface methodology[J]. Fujian Fisheries, 2012, 34(2): 127-133.

[0008] [2] PATI F, ADHIKARI B, DHARA S. Isolation and characterization of fish scale collagen of higher thermal stability[J]. Bioresource technology, 2010, 101(10): 3737-3742.

[0009] [3] Lu Z, Li LX, Zhang YF, et al. Study on extraction process parameters of acid-soluble collagen from grass carp scale[J]. Food Science and Technology, 2013, 38(5): 250-254.

[0010] [4]Zeng S, Liu K, Wu Y, et al. Optimization of extraction process and physicochemical properties of decalcified tilapia scale gelatin [J]. South China Fisheries Science, 2013, 9(2): 38-44.

[0011] [5]Yury Gorbanev, Deborah O’Connell, Victor Chechik. Non-Thermal Plasma in Contact with Water: The Origin of Species. Chemistry-A European Journal, 2016, 22(10): 3496-3505.

[0012] [6]Matthew J. Traylor, Matthew J. Pavlovich, Sharmin Karim, et al. Long-term antibacterial efficacy of air plasma-activated water. Journal of Physics D: Applied Physics, 2011, 44(47): 472001.

[0013] [7]K. Takaki, J. Takahata, S. Watanabe, et al. Improvements in plant growth rate using underwater discharge. Journal of Physics: Conference Series, 2013, 418(1): 012140.

[0014] [8]Liu X. Transdermal effect of active components of gas and liquid plasma [Master's thesis]. Wuhan: Huazhong University of Science and Technology, 2019.

[0015] [9]Dehui Xu, Shuai Wang, Bing Li, et al. Effects of Plasma-Activated Water on Skin Wound Healing in Mice. Microorganisms, 2020, 8(7): 1091. SUMMARY

[0016] The technical problem to be solved by the present application is to overcome the defects of low extraction rate of collagen peptide, long time and low quality of collagen peptide in the existing fish scale collagen extraction process, and to provide a new process.

[0017] The application aims to provide a preparation method of collagen peptide with anti-aging activity, comprising the following steps:

[0018] S1, fish scale pretreatment: washing, crushing, defatting and removing impurities, and then washing to neutral;

[0019] S2, decalcification: adding 1-4 times of pure water to the pretreated fish scale, treating in a pulsed electric field, then adding neutral protease for enzymolysis, and drying to obtain decalcified fish scale;

[0020] S3, hydrolysis: adding plasma activated water and hydrolysis enzyme to the decalcified fish scale, water bath oscillation for 0.1-1h, terminating the reaction, ultrafiltration, collecting the filtrate, and obtaining collagen peptide.

[0021] Further, in the step S1, the defatting and removing impurities comprises the following steps: adding reagent to the fish scale according to the solid-liquid ratio of 1:10-15, oscillating and treating at 30-40℃ and 100-300rpm for 15-40min.

[0022] Further, in the step S1, the reagent is 1-3% NaHCO3+0.1-0.5% Tween-80.

[0023] In the above steps, NaHCO3 can remove most of the lipids through saponification reaction, and emulsify the residual lipid droplets with Tween-80, enhancing the flushing effect; secondly, NaHCO3 can remove impurities through isoelectric point precipitation and salt solubilization effect of Na + , and its pH is much lower than that of strong alkali (such as NaOH), which will not destroy the triple helix structure of collagen; it can also inhibit the activity of endogenous proteases (such as metalloproteinase) in weak alkaline environment fish scales, preventing collagen degradation. Although the defatting rate is slightly lower (about 90%) compared with NaOH, the risk of collagen loss is greatly reduced.

[0024] Further, in the step S2, the specific parameters of the pulsed electric field treatment are: electric field strength of 8-12kV / cm, pulse frequency of 20-50Hz, and treatment time of 1-5min. The effect of pulsed electric field (PEF) on enzyme activity has a dual nature (may activate or inhibit), and its effect depends on the electric field parameters, the type of enzyme and the treatment environment. High-intensity electric field (>20kV / cm) may cause the enzyme molecules α-helix / β-fold to unwind, and the active center to deform (such as pepsin activity decreased by 40% at 25kV / cm). High pulse frequency (200Hz) will cause overheating inactivation.

[0025] Compared with acid decalcification, neutral protease decalcification has no defects such as residual impurities and tryptophan destruction, but also has the disadvantage of long time (2-4 h). High-voltage pulsed electric field (PEF) can cause irreversible electroporation of fish scale cell membrane and collagen fiber network, forming microporous channels, promoting the dissolution of Ca 2+ and PO4 3- , and collagen (non-conductive) remains stable in PEF, while hydroxyapatite (ionic crystal) dissociates preferentially. The inventors found that PEF pretreatment not only significantly improves the enzyme decalcification rate (>95%), but also shortens the decalcification time by more than 80% (compared with single enzyme method), while the yield of collagen peptide is increased by about 20%.

[0026] Further, in the step S2, the neutral protease is Subtilisin Carlsberg, the enzymolysis pH is 7.0, the enzymolysis temperature is 40-45℃, the enzyme addition amount is 0.1-1% w / w, and the enzymolysis time is 30-50 min.

[0027] In the enzyme decalcification process, the selection of enzyme needs to meet the dual requirements of high-efficiency decalcification and protection of collagen integrity. Calcium in fish scales mainly exists in the form of hydroxyapatite, which is closely combined with collagen fiber network. Enzymatic decalcification does not directly dissolve calcium salt, but releases calcium crystals by degrading collagen matrix, which is divided into two steps: ① Enzymatic degradation of collagen fiber: destroying the organic matrix wrapping hydroxyapatite; ② Calcium ion diffusion: free calcium ions are removed through the solution. Subtilisin can degrade the hydrophobic peptide bond of the non-helical region of collagen, release the wrapped hydroxyapatite, has low cost and good temperature resistance. The PEF synergistic enzyme decalcification process can efficiently remove calcium in fish scales under mild conditions, with less hydroxyproline dissolution, and can maximize the retention of the functional properties of collagen protein, and the prepared collagen protein has high hydroxyproline retention rate and high activity.

[0028] Further, in the step S3, the ratio of decalcified fish scales to plasma-activated water is 1:8-15 (w / v).

[0029] In this paper, plasma-activated water can be prepared by dielectric barrier discharge (DBD) or radio frequency (RF) plasma generator, and dielectric barrier discharge (DBD) device is preferred, and its working parameters are shown in Table 1:

[0030] Table 1: Working parameters of dielectric barrier discharge (DBD) device

[0031]

[0032]

[0033] The directional oxidative enhanced plasma activated water (PAW) is prepared by optimizing the parameters of the device, and the directional oxidative enhanced plasma activated water contains high concentration of active oxygen, in which ·OH attacks the peptide bond (such as Gly-Pro) in collagen protein which is not tryptophan; H2O2 and O3 oxidize the collagen fiber network, promote loosening; NO· regulates the redox balance, inhibits the degradation of tryptophan, and the indole ring of tryptophan is protected due to steric hindrance and reducing environment.

[0034] Further, in the step S3, the enzyme is acid protease, the enzyme substrate ratio in the enzymolysis process is 1-2% (w / w), and the enzymolysis temperature is 35-55 DEG C.

[0035] In the selection of hydrolytic enzymes, alkaline protease, trypsin, collagenase, flavor protease, bromelain, pepsin, acid protease and papain can all hydrolyze fish scale collagen to obtain collagen peptides with high antioxidant activity, but through screening, it is found that acid protease can not only improve the extraction rate of collagen peptides in cooperation with PAW, but also can significantly shorten the hydrolysis time, and the prepared collagen peptides have the characteristics of high hydroxyproline retention rate, high purity and activity.

[0036] Further, in the step S3, the ultrafiltration is performed by using an ultrafiltration membrane with a molecular weight cut-off of 3 kDa.

[0037] Another object of the present application is to provide collagen peptides prepared by the preparation method. The collagen peptides prepared by the present application have a high hydroxyproline retention rate (> 95%), and hydroxyproline as an indirect indicator of the functional activity of collagen peptides, the peptide segment containing hydroxyproline has the following clear biological activities:

[0038] Promoting cell adhesion (through integrin receptors);

[0039] Antioxidation (scavenging hydroxyl radicals);

[0040] Skin repair (stimulating fibroblast proliferation);

[0041] The content of hydroxyproline is usually positively correlated with these activities (such as collagen peptides with Hyp>9% promoting cell migration by 2 times), and the collagen peptides prepared by the present application are suitable for medical-grade cosmetics and drugs.

[0042] Another object of the present application is to provide the use of the collagen peptides prepared by the preparation method in the preparation of anti-aging cosmetics or drugs.

[0043] The present application has the following beneficial effects:

[0044] (1) The fish scale decalcification process is innovated, PEF cooperates with enzyme to remove calcium in fish scale under mild conditions, with high decalcification rate, short time, less hydroxyproline dissolution, and the prepared collagen protein has the characteristics of high hydroxyproline retention rate and high activity.

[0045] (2) The application innovatively uses PAW to cooperate with enzyme to significantly improve the extraction rate of collagen peptide, and can significantly shorten the hydrolysis time, and the peptide yield reaches 82.66%.

[0046] (3) The prepared collagen peptide has high activity, and the process is mild, which is suitable as a medical grade product. DETAILED DESCRIPTION

[0047] The application can be further described by the following examples, but the scope of the application is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the application without departing from the spirit and scope of the application.

[0048] I. Comparison of decalcification effects of different processes

[0049] Enzyme + PEF decalcification: S1, fish scale pretreatment: 100g of fresh fish scale is washed with clean water, dried and crushed, 1% NaHCO3+0.5% Tween-80 is added to the fish scale according to the solid-liquid ratio of 1:10, and the fish scale is treated at 40℃ and 200rpm for 30min for degreasing and impurity removal, and washed with water until neutral; S2, decalcification: 3 times the weight of pure water is added to the pretreated fish scale, and the fish scale is treated in a pulse electric field with an electric field intensity of 8kV / cm and a pulse frequency of 40Hz for 3min, then 0.5%w / w of enzyme is added for enzyme hydrolysis for 40min, the enzyme hydrolysis pH is 7.0, the enzyme hydrolysis temperature is 40℃, and the enzyme is extracted by suction filtration, and the filtrate is collected.

[0050] Acid decalcification: refer to steps S1 and S2, the difference is that 0.3mol / L of HCl solution is used for decalcification in step S2, the acid immersion time is 1.5h, and the solid-liquid ratio is 1:30(g:mL).

[0051] Single enzyme method: refer to steps S1 and S2, the difference is that no pulse electric field treatment is added in step S2, and the enzyme hydrolysis time is extended to 6h.

[0052] PEF method: refer to steps S1 and S2, the difference is that no subtilisin is added in step S2.

[0053] Decalcification rate determination: The total calcium content in the raw fish scales was determined by EDTA titration method GB5009.92-2016, and the calcium content in the decalcification solution was determined by EDTA complexometric titration method according to Hu Aijun et al. (Hu Aijun, Song Feiyin, Zheng Jie, et al. Process condition optimization for removing calcium from sliver carp scales by citric acid [J]. Food Research and Development, 2017, 38(16): 77-81). The decalcification rate was calculated by the following formula (1), and the specific operation was as follows:

[0054]

[0055] In the formula: C1- concentration of calcium ions in the decalcification solution, mg / mL;

[0056] V1- volume of the decalcification solution, mL;

[0057] f- dilution factor;

[0058] m- total calcium content in the fish scales, mg.

[0059] Hydroxyproline dissolution amount determination: The content of hydroxyproline in the filtrate was determined by chloramine T-p-dimethylaminobenzaldehyde method (national standard GB / T 9695.23-2008): 2 mL of the decalcification solution was taken and 1 mL of chloramine T solution (1.41 g / L) was added, and then it was oxidized at room temperature for 10 min, and then 1 mL of Ehrlich reagent (10% w / v, dissolved in perchloric acid: propanol = 1:4) was added, and then it was placed in a water bath at 60°C for 20 min, and then the absorbance at 560 nm was measured after cooling (with ultrapure water as blank), and then a series of concentrations were prepared with L-hydroxyproline standard, and then the hydroxyproline dissolution amount of different decalcification processes was calculated according to the standard curve, and the results are shown in Table 2.

[0060] Table 2. Comparison of decalcification rates and hydroxyproline dissolution amounts (per 100 g of fish scales) of different decalcification processes

[0061] Decalcification process Decalcification rate (%) Hydroxyproline leaching (pg) Acid method 93.3％ 5162.3 Single enzyme method 50.7％ 2243.1 PEF method 78.6％ 1615.8 Enzyme + PEF method 98.5％ 357.5

[0062] As shown in Table 2, after decalcification with 0.3 mol / L HCl solution for 1.5 h, the decalcification rate was 93.3%, and the dissolution amount of hydroxyproline was the largest, reaching 5162.3 μg; compared with the acid method, although the dissolution amount of hydroxyproline was significantly reduced in the single enzyme method and the PEF method, the protection effect on collagen was better, but the decalcification rates were all lower than 80%, especially after decalcification for 6 h by the enzyme method, the decalcification rate was only 50.7%, which could not meet the requirements of subsequent collagen extraction; and the decalcification rate could be increased to more than 95% by the combined decalcification of the enzyme method and the PEF method, and the dissolution amount of hydroxyproline was the least, not only the decalcification time was significantly shortened, but also the collagen retention rate was the highest, and the combination of the two achieved the best combined effect.

[0063] Application example

[0064] After the rohu fish scales were washed clean, the moisture content was determined according to the constant weight drying method at 105°C (GB / T 5009.3-2003); the ash content was determined according to the high temperature ignition method (GB / T 5009.4-2003); the crude protein content was determined according to the micro-Kjeldahl method (GB / T 5009.5-2003); and the fat content was determined according to the Soxhlet extraction method (GB / T 5009.6-2003), and the results are shown in Table 3 below.

[0065] Table 3, Main component composition of rohu fish scales (w / w%)

[0066] Moisture Ash Crude protein Fat Other ingredients 12.69 42.25 44.36 0.62 0.7

[0067] The above rohu fish scales were used to prepare collagen peptides, and the specific implementation was as follows:

[0068] Example 1

[0069] S1, Fish scale pretreatment: Fresh rohu fish scales were washed with clean water, dried in the air, and then crushed. 1% NaHCO3+0.5% Tween-80 was added to the fish scales according to the solid-liquid ratio of 1:10, and the mixture was subjected to defatting and impurity removal by oscillation treatment at 40°C and 200 rpm for 30 min. Then, the fish scales were washed with water until neutral.

[0070] S2, Decalcification: The pretreated fish scales were added with 3 times the weight of pure water, and then subjected to treatment in a pulsed electric field with an electric field intensity of 8 kV / cm and a pulse frequency of 40 Hz for 3 min. Then, 0.5% w / w of enzyme was added for enzymolysis at a pH of 7.0 and a temperature of 40°C for 40 min. The filtrate was removed by suction filtration, and the decalcified fish scales were dried.

[0071] S3, PAW preparation: The parameters of the dielectric barrier discharge low-temperature plasma device (DBD-100A / B type low-temperature plasma device: Shanghai Wanmochun Biological Engineering Co., Ltd.) were adjusted as follows: the output voltage was 10 kV, the frequency was 500 Hz, 80% O2+20% N2 was used as the working gas, and the gas flow rate was 2 L / min. Sterile deionized water was placed in the hopper of the device, and the dielectric barrier discharge plasma device electrode plate was placed on the horizontal surface with a distance of 5 mm from the liquid surface. The power was turned on, and the discharge was carried out for 3 min to obtain low-temperature plasma activated water.

[0072] S4, PAW was added to the decalcified fish scales at a ratio of 1:10 (w / v), and acid protease was added at an enzyme substrate ratio of 1% (w / w). The reaction was terminated after water bath oscillation at 40°C for 0.5 h. The filtrate was collected by ultrafiltration using an ultrafiltration membrane with a molecular weight cut-off of 3 kDa to obtain collagen peptides.

[0073] Example 2

[0074] S1, fish scale pretreatment: fresh tilapia fish scales were washed with clean water, dried and crushed, 1.5% NaHCO3+0.5% Tween-80 was added to the fish scales according to the solid-liquid ratio of 1:12, and the defatting and impurity removal were carried out under the condition of 40°C and 200 rpm for 15 min, and then washed with water until neutral;

[0075] S2, decalcification: 4 times the weight of pure water was added to the pretreated fish scales, and the fish scales were treated in a pulsed electric field with an electric field intensity of 10 kV / cm and a pulse frequency of 50 Hz for 3 min, then trypsin was added for enzymolysis for 30 min, the enzymolysis pH was 7.0, the enzymolysis temperature was 40°C, the enzyme addition amount was 1% w / w, and the filtrate was removed by suction filtration, and then dried to obtain decalcified fish scales;

[0076] S3, PAW preparation: the parameters of DBD were adjusted as follows: output voltage was 8 kV, frequency was 800 Hz, 80% O2+20% N2 was used as working gas, and gas flow rate was 1 L / min; sterile deionized water was placed in the hopper of the device, and the medium barrier discharge plasma device electrode plate was placed on the horizontal plane, the distance between the electrode plate and the liquid surface was 5 mm, the power was turned on, and the discharge was carried out for 5 min to obtain low-temperature plasma activated water;

[0077] S4, PAW was added to the decalcified fish scales at a ratio of 1:8 (w / v), and acid protease was added at an enzyme substrate ratio of 1.5% (w / w), and the reaction was terminated after water bath shaking at 40°C for 0.5 h, then ultrafiltration was carried out using an ultrafiltration membrane with a molecular weight cut-off of 3 kDa, the filtrate was collected, and collagen peptide was obtained.

[0078] Example Three

[0079] S1, fish scale pretreatment: fresh tilapia fish scales were washed with clean water, dried and crushed, 1% NaHCO3+0.5% Tween-80 was added to the fish scales according to the solid-liquid ratio of 1:15, and the defatting and impurity removal were carried out under the condition of 40°C and 200 rpm for 40 min, and then washed with water until neutral;

[0080] S2, decalcification: 3 times the weight of pure water was added to the pretreated fish scales, and the fish scales were treated in a pulsed electric field with an electric field intensity of 12 kV / cm and a pulse frequency of 20 Hz for 5 min, then trypsin was added for enzymolysis for 40 min, the enzymolysis pH was 7.0, the enzymolysis temperature was 40°C, the enzyme addition amount was 0.5% w / w, the filtrate was removed by suction filtration, and then dried to obtain decalcified fish scales;

[0081] S3, PAW preparation: the parameters of DBD were adjusted as follows: output voltage was 12 kV, frequency was 600 Hz, working gas was 80% O2+20% N2, and gas flow rate was 2 L / min; sterile deionized water was placed in the hopper of the equipment, and the medium resistance discharge plasma equipment electrode plate was placed on the horizontal surface, the distance between the electrode plate and the liquid surface was 5 mm, the power was turned on, and the discharge was carried out for 3 min to obtain low-temperature plasma activated water;

[0082] S4, PAW was added to the de-calcified fish scales at a ratio of 1:15 (w / v), and acid protease was added at an enzyme substrate ratio of 2% (w / w), and the reaction was terminated after water bath shaking at 40°C for 0.5 h, and ultrafiltration was carried out using an ultrafiltration membrane with a molecular weight cut-off of 3 kDa, and the filtrate was collected to obtain collagen peptides.

[0083] Comparative Example 1, which is different from Example 1 in that PAW is not added in step S4.

[0084] Comparative Example 2, which is different from Example 1 in that acid protease is not added in step S4.

[0085] Comparative Example 3, which is different from Example 1 in that the pulse electric field treatment is not added in step S2.

[0086] Comparative Example 4, which is different from Example 1 in that the subtilisin treatment is not added in step S2.

[0087] Comparative Example 5, which uses the enzymatic extraction method in Zhu Yuhui ① , and the enzymatic parameters are 25°C, pepsin to substrate ratio of 3%, enzymatic time of 2 h, and pH of 3.0.

[0088] ①: Zhu Yuhui, Comparison of Two Extraction Processes of Collagen from Grass Carp Scales [J], Journal of Mountain Agriculture and Biology, 2013, 32(3): 254-257.

[0089] (1) Collagen peptide yield determination: trichloroacetic acid (TCA) can dissolve small molecule proteins or peptide segments, and react with large molecule proteins to form white precipitate. 5 mL of filtrate from Examples 1-3 and Comparative Examples 1-5 was added with 15% (w / v) TCA solution at a volume ratio of 1:1, mixed on a vortex mixer, and then placed for 10 min. After centrifugation at 5000 r / min for 20 min, 1 mL of supernatant was added with 4 mL of biuret reagent, and then placed at room temperature for 30 min. The absorbance was measured at 540 nm, and the corresponding concentration was obtained by comparing with the standard curve. The polypeptide content was calculated according to formula (2), and the results are shown in Table 4:

[0090]

[0091] C- concentration of polypeptide in the sample solution, mg / mL;

[0092] V- volume of the sample solution, mL;

[0093] F- dilution factor;

[0094] M- total mass of the sample, mg.

[0095] (II) DPPH scavenging rate determination: the filtrates of Examples 1-3 and Comparative Examples 1-5 were lyophilized, and then prepared into a sample solution at a concentration of 10 μg / mL. 0.1 mL of the sample solution was added to 2.9 mL of a DPPH ethanol solution (0.1 mM), and then mixed and shaken. The mixture was placed in a water bath at room temperature for 30 min in the dark, and then the absorbance (A517) was measured. VC was used as a positive control, and each group was measured in triplicate. The average value was taken, and the results are shown in Table 5. 样品

[0096]

[0097] (III) ORAC (oxygen radical absorbance capacity) determination: 50 μL of the sample solution was taken, and then 150 μL of fluorescein (40 nM) was added. The mixture was incubated at 37°C for 10 min, and then 25 μL of AAPH (153 mM) was added. The fluorescence decay curve was detected (for 90 min). Trolox was used as a standard curve, and the results were expressed as μmol TE / g. The results are shown in Table 5.

[0098] (IV) Results

[0099] Table 4: Comparison of the yields of collagen peptides prepared by different processes

[0100]

[0101]

[0102] As shown in Table 4, there were significant differences in the yields of collagen peptides prepared by different processes. The yields of collagen peptides prepared by the processes of Examples 1-3 were the highest, and the highest yield was 36.67%. The content of crude protein in the rohu fish scales was 44.36%, and thus the extraction rate of collagen peptides prepared by the processes of Examples 1-3 was 82.66% (the mass of the extracted collagen peptides / the mass of the peptides in the raw material). The yields of collagen peptides prepared by Comparative Examples 1 and 2 were significantly lower than that of Example 1. The yields of collagen peptides prepared by Comparative Examples 3 and 4 were low because the decalcification processes were changed, and the decalcification was not thorough enough due to the use of a single enzyme or a single PEF, which affected the dissolution of collagen peptides.

[0103] Table 5: DPPH scavenging rates and ORAC values of collagen peptides prepared by different processes

[0104] Group DPPH scavenging rate ORAC value / pmol TE / g Example 1 97.5％ 965 Example 2 95.2％ 924 Example 3 95.9％ 893 Comparative Example 1 65.0％ 594 Comparative Example 2 78.4％ 653 Comparative Example 3 58.2％ 506 Comparative Example 4 80.6％ 701 Comparative Example 5 85.3％ 727 ​

[0105] From Table 5 above, it can be seen that the collagen peptide of the present application has the highest antioxidant activity, and the DPPH clearance rate is more than 95%, and the ORAC value can reach 965 μmol TE / g.

[0106] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A method for preparing collagen peptides with anti-aging activity, characterized in that, Includes the following steps: S1. Fish scale pretreatment: Wash fresh fish scales with water, crush them, degrease and remove impurities, and then wash them until neutral. S2, Decalcification: Add 1 to 4 times the weight of pure water to the pretreated fish scales, place them in a pulsed electric field for treatment, then add neutral protease for enzymatic hydrolysis, and dry to obtain decalcified fish scales. S3. Hydrolysis: Add plasma-activated water and hydrolytic enzyme to decalcified fish scales, shake in a water bath for 0.1-1 h to terminate the reaction, ultrafilter, collect the filtrate, and obtain collagen peptides. In step S2, the specific parameters of the pulsed electric field treatment are: electric field strength of 8~12kV / cm, pulse frequency of 20~50Hz, and treatment time of 1~5min; the neutral protease is subtilisin, the enzymatic hydrolysis pH is 7.0, the enzymatic hydrolysis temperature is 40~45℃, the enzyme addition amount is 0.1~1%w / w, and the enzymatic hydrolysis time is 30~50min; In step S3, the enzyme is an acidic protease, the enzyme-to-water ratio during enzymatic hydrolysis is 1-2% (w / w), and the hydrolysis temperature is 35-55℃; the ratio of decalcified fish scales to plasma-activated water is 1:8-15 (w / v). The plasma-activated water was prepared using a dielectric barrier discharge low-temperature plasma device with the following parameters: output voltage of 8~12kV, frequency of 500Hz~800Hz, discharge time of 3~5min; gas composition: 80%O2+20%N2, gas flow rate of 1~2L / min.

2. The preparation method according to claim 1, characterized in that, In step S1, degreasing and impurity removal includes the following steps: adding reagent to fish scales at a material-to-liquid ratio of 1:10~15, and shaking the mixture at 30~40℃ and 100~300rpm for 15~40min.

3. The preparation method according to claim 2, characterized in that, In step S1, the reagent is 1~3% NaHCO3 + 0.1~0.5% Tween-80.

4. The preparation method according to claim 1, characterized in that, In step S3, the ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa.

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