Preparation method of low-molecular-weight takifugu rubripes fishbone collagen peptide
By treating the red-fin oriental fish bones under low temperature conditions, removing impurities and preparing them by enzymatic methods, the problems of fish bone waste and environmental pollution are solved, and the effect of efficient extraction of low molecular weight collagen peptides is achieved.
Patent Information
- Application Number
- CN202411649728.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-19
- Publication Date
- 2025-03-11
AI Technical Summary
The prior art is difficult to effectively utilize the red-fin oriental squirrel bones, resulting in waste of by-products and polluting the environment.
The bones of red-fin oriental scorpion were naturally thawed and impurities were removed under low temperature conditions. Then, the impurities, minerals and fat were removed using NaOH, EDTA-2Na and isopropanol solutions, and finally low molecular weight collagen peptides were prepared by enzymatic method.
It has achieved efficient extraction of low molecular weight collagen peptides from redfin oriental fish bones, reducing production costs, improving resource utilization, and reducing environmental pollution.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of aquatic animal by-product processing, and particularly relates to a method for preparing low-molecular weight redfin puffer fish bone collagen peptide. Background Art
[0002] The redfin pufferfish (Takifugu rubripes) is large in size, has a short growth cycle, and has great breeding potential. According to the 2024 China Fisheries Statistical Yearbook, the average breeding output of pufferfish in China in the past three years was about 29,000 tons, showing an increasing trend year by year. The processing process will produce a large number of by-products such as fish bones and fish skin, which are often made into low-value feed or landfilled and discarded, polluting the environment and wasting resources. The bones of redfin pufferfish are low-cost, widely available, and rich in collagen, making them a high-quality source of aquatic collagen.
[0003] Collagen is mainly distributed in load-bearing tissues such as skin, bones, and tendons, accounting for about 30% of the total protein. After collagen is hydrolyzed, collagen peptides with small molecular weight, biological activity, and can be almost completely absorbed by the human body can be obtained. Depending on the hydrolysis conditions, the molecular weight of the hydrolyzate obtained is generally 100-10000Da, and the peptides containing 2-6 amino acids and a molecular weight of less than 1000Da are called collagen oligopeptides, which are mostly absorbed and utilized by the human body in this form. Collagen peptides from aquatic organisms such as fish have the characteristics of low allergenicity and low antigenicity compared to collagen peptides from terrestrial organisms such as chickens, cattle, and sheep. According to statistics, the market value of collagen peptides can reach US$22.6 billion in 2027, and it will increase year by year at a growth rate of 5.4%, and the market prospects are broad. Red fin pufferfish bones are a major by-product in the processing process, accounting for about 20% of the individual. The raw materials are cheap and easy to obtain, and the collagen content is high, which has a high development and utilization value. Summary of the invention
[0004] The purpose of the present invention is to provide a method for preparing low molecular weight redfin puffer fish bone collagen peptide.
[0005] A method for preparing low molecular weight redfin puffer fish bone collagen peptide is carried out according to the following steps:
[0006] (1) Under low temperature conditions, the bones of redfin pufferfish were naturally thawed at room temperature or in running water, the bones were rinsed with distilled water, cut into pieces, added with NaOH solution and stirred for 30-40 hours, and the solution was changed 2-4 times in the middle to remove foreign proteins;
[0007] (2) Add EDTA-2Na solution and stir for 64-80 hours, changing the solution 2-4 times to remove minerals;
[0008] (3) Add isopropanol aqueous solution and stir for 20-28 hours, changing the solution 1-3 times to remove fat, and then rinse with distilled water;
[0009] (4) drying the redfin pufferfish bones processed in step (3) in an oven at 40-60° C., crushing them using a universal grinder, and passing them through a 30-50 mesh sieve;
[0010] (5) Add water to the redfin puffer fish bones treated in step (4), adjust the pH to 8-9, add composite protease, place in a 40-50° C. water bath for enzymatic hydrolysis for 4-6 hours, then inactivate in a boiling water bath for 5-15 minutes, centrifuge at 6000-10000 g for 10-20 minutes, take the supernatant, and freeze-dry to obtain collagen peptide powder.
[0011] The low temperature condition is 2-8°C.
[0012] The concentration of the NaOH solution is 0.05-0.15 mol / L, and the amount added is 15-25 times the mass of the redfin pufferfish bone.
[0013] The concentration of the EDTA-2Na solution is 0.3-0.7 mol / L, and the added amount is 3-7 times the mass of the redfin puffer fish bone.
[0014] The volume concentration of isopropanol in the isopropanol aqueous solution is 10-20%, and the added amount is 5-15 times the mass of the redfin puffer fish bone.
[0015] The amount of water added in step (5) is 5-15 times the mass of the redfin puffer fish bone.
[0016] The composite protease is a mixed enzyme of alkaline protease and sugar palm plant protease, and the added amount is 6000-10000 U / g.
[0017] The composite protease is a mixed enzyme of Neutrase enzyme and Alcalase enzyme, and the added amount is 6000-10000 U / g.
[0018] Beneficial effects of the present invention: The present invention selects red fin puffer fish bones as raw materials for the production of collagen peptides, the raw materials are cheap and easy to obtain, the red fin puffer fish bones are processing byproducts, the cost is low, and the crude protein and collagen content are high. The extraction method selects a relatively mild enzymatic preparation method, which has little effect on the collagen peptides, wherein alkaline protease and sugar palm plant protease are used in combination, and Neutrase enzyme and Alcalase enzyme are used in combination to synergistically enhance the effect. DETAILED DESCRIPTION
[0019] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.
[0020] The red fin puffer fish bones used in the following examples were provided by Tangshan Haidu Aquatic Food Co., Ltd., Tangshan City, Hebei Province, and transported to the laboratory in a low-temperature environment. Sodium hydroxide, disodium ethylenediaminetetraacetic acid, isopropanol, formaldehyde solution, etc. were purchased from Fuchen (Tianjin) Chemical Reagent Co., Ltd.; alkaline protease, Neutrase enzyme and Alcalase enzyme were purchased from Beijing Solebaugh Technology Co., Ltd., and the sugar palm plant protease was prepared according to the method disclosed in patent CN 1066468A; all the reagents required in the experiment were analytical grade unless otherwise specified.
[0021] Example 1
[0022] A method for preparing low molecular weight redfin puffer fish bone collagen peptide is carried out according to the following steps:
[0023] (1) Under low temperature conditions of 4°C, the bones of redfin pufferfish were naturally thawed at room temperature or in running water, the bones were rinsed with distilled water, cut into pieces, and a 0.1 mol / L NaOH solution was added and stirred for 35 h, with the solution changed 3 times in the middle to remove foreign proteins; the amount of the NaOH solution added was 20 times the mass of the redfin pufferfish bones;
[0024] (2) adding 0.5 mol / L EDTA-2Na solution and stirring for 72 h, changing the solution three times in the middle to remove minerals; the amount of EDTA-2Na solution added is 5 times the mass of the redfin pufferfish bone;
[0025] (3) adding an isopropanol aqueous solution with a volume concentration of 15%, stirring for 24 hours, changing the solution twice in the middle to remove fat, and then rinsing with distilled water; the amount of the isopropanol aqueous solution added is 10 times the mass of the redfin puffer fish bone;
[0026] (4) drying the redfin pufferfish bones treated in step (3) in an oven at 50° C., crushing them using a universal grinder, and passing them through a 40-mesh sieve;
[0027] (5) adding 10 times the weight of water to the redfin puffer fish bones treated in step (4), adjusting the pH to 8.8, adding a composite protease, placing the bones in a 45°C water bath for enzymolysis for 5 hours, then inactivating the bones in a boiling water bath for 10 minutes, centrifuging at 8000g for 15 minutes, taking the supernatant, and freeze-drying to obtain collagen peptide powder; the composite protease is a mixed enzyme of alkaline protease and galangal plant protease, the alkaline protease added in an amount of 4000U / g, and the galangal plant protease added in an amount of 4000U / g.
[0028] Example 2
[0029] A method for preparing low molecular weight redfin puffer fish bone collagen peptide is carried out according to the following steps:
[0030] (1) Under low temperature conditions of 6°C, the bones of redfin pufferfish were naturally thawed at room temperature or in running water, the bones were rinsed with distilled water, cut into pieces, and a 0.08 mol / L NaOH solution was added and stirred for 38 h, with the solution changed 3 times in the middle to remove foreign proteins; the amount of the NaOH solution added was 18 times the mass of the redfin pufferfish bones;
[0031] (2) adding 0.6 mol / L EDTA-2Na solution and stirring for 66 h, changing the solution twice in the middle to remove minerals; the amount of EDTA-2Na solution added is 6 times the mass of the redfin pufferfish bone;
[0032] (3) adding an isopropanol aqueous solution with a volume concentration of 18%, stirring for 21 hours, changing the solution three times in the middle to remove fat, and then rinsing with distilled water; the amount of the isopropanol aqueous solution added is 12 times the mass of the redfin puffer fish bone;
[0033] (4) drying the redfin pufferfish bones treated in step (3) in an oven at 55° C., crushing them using a universal grinder, and passing them through a 50-mesh sieve;
[0034] (5) adding 12 times the weight of water to the redfin puffer fish bones treated in step (4), adjusting the pH to 8.5, adding a composite protease, placing the bones in a 50° C. water bath for enzymolysis for 6 h, then inactivating the bones in a boiling water bath for 10 min, centrifuging at 8000 g for 15 min, taking the supernatant, and freeze-drying the mixture to obtain collagen peptide powder; the composite protease is a mixed enzyme of Neutrase enzyme and Alcalase enzyme, the amount of Neutrase enzyme added is 4000 U / g, and the amount of Alcalase enzyme added is 6000 U / g.
[0035] Comparative Example 1
[0036] A method for preparing low molecular weight redfin puffer fish bone collagen peptide is carried out according to the following steps:
[0037] (1) Under low temperature conditions of 4°C, the bones of redfin pufferfish were naturally thawed at room temperature or in running water, the bones were rinsed with distilled water, cut into pieces, and a 0.1 mol / L NaOH solution was added and stirred for 35 h, with the solution changed 3 times in the middle to remove foreign proteins; the amount of the NaOH solution added was 20 times the mass of the redfin pufferfish bones;
[0038] (2) adding 0.5 mol / L EDTA-2Na solution and stirring for 72 h, changing the solution three times in the middle to remove minerals; the amount of EDTA-2Na solution added is 5 times the mass of the redfin pufferfish bone;
[0039] (3) adding an isopropanol aqueous solution with a volume concentration of 15%, stirring for 24 hours, changing the solution twice in the middle to remove fat, and then rinsing with distilled water; the amount of the isopropanol aqueous solution added is 10 times the mass of the redfin puffer fish bone;
[0040] (4) drying the redfin pufferfish bones treated in step (3) in an oven at 50° C., crushing them using a universal grinder, and passing them through a 40-mesh sieve;
[0041] (5) adding 10 times the weight of water to the redfin puffer fish bones treated in step (4), adjusting the pH to 8.8, adding alkaline protease, placing in a 45° C. water bath for enzymatic hydrolysis for 5 h, then inactivating in a boiling water bath for 10 min, centrifuging at 8000 g for 15 min, taking the supernatant, and freeze-drying to obtain collagen peptide powder; the amount of alkaline protease added is 8000 U / g.
[0042] Comparative Example 2
[0043] A method for preparing low molecular weight redfin puffer fish bone collagen peptide is carried out according to the following steps:
[0044] (1) Under low temperature conditions of 4°C, the bones of redfin pufferfish were naturally thawed at room temperature or in running water, the bones were rinsed with distilled water, cut into pieces, and a 0.1 mol / L NaOH solution was added and stirred for 35 h, with the solution changed 3 times in the middle to remove foreign proteins; the amount of the NaOH solution added was 20 times the mass of the redfin pufferfish bones;
[0045] (2) adding 0.5 mol / L EDTA-2Na solution and stirring for 72 h, changing the solution three times in the middle to remove minerals; the amount of EDTA-2Na solution added is 5 times the mass of the redfin pufferfish bone;
[0046] (3) adding an isopropanol aqueous solution with a volume concentration of 15%, stirring for 24 hours, changing the solution twice in the middle to remove fat, and then rinsing with distilled water; the amount of the isopropanol aqueous solution added is 10 times the mass of the redfin puffer fish bone;
[0047] (4) drying the redfin pufferfish bones treated in step (3) in an oven at 50° C., crushing them using a universal grinder, and passing them through a 40-mesh sieve;
[0048] (5) Add 10 times the weight of water to the redfin puffer fish bones treated in step (4), adjust the pH to 8.8, add sugar palm plant protease, place in a 45°C water bath for enzymolysis for 5 hours, then inactivate in a boiling water bath for 10 minutes, centrifuge at 8000g for 15 minutes, take the supernatant, and freeze-dry to obtain collagen peptide powder; the added amount of sugar palm plant protease is 8000U / g.
[0049] Comparative Example 3
[0050] A method for preparing low molecular weight redfin puffer fish bone collagen peptide is carried out according to the following steps:
[0051] (1) Under low temperature conditions of 6°C, the bones of redfin pufferfish were naturally thawed at room temperature or in running water, the bones were rinsed with distilled water, cut into pieces, and a 0.08 mol / L NaOH solution was added and stirred for 38 h, with the solution changed 3 times in the middle to remove foreign proteins; the amount of the NaOH solution added was 18 times the mass of the redfin pufferfish bones;
[0052] (2) adding 0.6 mol / L EDTA-2Na solution and stirring for 66 h, changing the solution twice in the middle to remove minerals; the amount of EDTA-2Na solution added is 6 times the mass of the redfin pufferfish bone;
[0053] (3) adding an isopropanol aqueous solution with a volume concentration of 18%, stirring for 21 hours, changing the solution three times in the middle to remove fat, and then rinsing with distilled water; the amount of the isopropanol aqueous solution added is 12 times the mass of the redfin puffer fish bone;
[0054] (4) drying the redfin pufferfish bones treated in step (3) in an oven at 55° C., crushing them using a universal grinder, and passing them through a 50-mesh sieve;
[0055] (5) Add 12 times the weight of water to the redfin puffer fish bones treated in step (4), adjust the pH to 8.5, add Neutrase enzyme, place in a 50° C. water bath for enzymolysis for 6 hours, then inactivate in a boiling water bath for 10 minutes, centrifuge at 8000 g for 15 minutes, take the supernatant, and freeze-dry to obtain collagen peptide powder; the amount of Neutrase enzyme added is 10,000 U / g.
[0056] Comparative Example 4
[0057] A method for preparing low molecular weight redfin puffer fish bone collagen peptide is carried out according to the following steps:
[0058] (1) Under low temperature conditions of 6°C, the bones of redfin pufferfish were naturally thawed at room temperature or in running water, the bones were rinsed with distilled water, cut into pieces, and a 0.08 mol / L NaOH solution was added and stirred for 38 h, with the solution changed 3 times in the middle to remove foreign proteins; the amount of the NaOH solution added was 18 times the mass of the redfin pufferfish bones;
[0059] (2) adding 0.6 mol / L EDTA-2Na solution and stirring for 66 h, changing the solution twice in the middle to remove minerals; the amount of EDTA-2Na solution added is 6 times the mass of the redfin pufferfish bone;
[0060] (3) adding an isopropanol aqueous solution with a volume concentration of 18%, stirring for 21 hours, changing the solution three times in the middle to remove fat, and then rinsing with distilled water; the amount of the isopropanol aqueous solution added is 12 times the mass of the redfin puffer fish bone;
[0061] (4) drying the redfin pufferfish bones treated in step (3) in an oven at 55° C., crushing them using a universal grinder, and passing them through a 50-mesh sieve;
[0062] (5) Add 12 times the weight of water to the redfin puffer fish bones treated in step (4), adjust the pH to 8.5, add Alcalase enzyme, place in a 50° C. water bath for enzymolysis for 6 hours, then inactivate in a boiling water bath for 10 minutes, centrifuge at 8000 g for 15 minutes, take the supernatant, and freeze-dry to obtain collagen peptide powder; the amount of Alcalase enzyme added is 10,000 U / g.
[0063] Experimental Example 1:
[0064] Determination of hydrolysis degree: Use formaldehyde titration to determine the free amino acid nitrogen content in the hydrolyzate. Mix the hydrolyzate and distilled water, use NaOH to titrate the pH to 8.2, add 37% formaldehyde solution, mix, titrate the pH to 9.2, and record the titrated NaOH volume as V1. Take the same volume of distilled water as a blank control, and record the titrated NaOH volume as V2. Calculate the free amino acid nitrogen content using the formula:
[0065]
[0066] In the formula: C: concentration of NaOH standard titration solution (mol / L); 0.014: gram equivalent of N; 100: unit conversion factor; V: volume of the sample to be absorbed (mL).
[0067] The total nitrogen content in the raw materials was determined using the Kjeldahl method in GB 5009.5-2016. The degree of hydrolysis was calculated using the following formula:
[0068]
[0069] The collagen peptides prepared in Examples 1-2 and Comparative Examples 1-4 were respectively taken, and each material was measured three times to obtain the average value. SPSS 24.0 software was used for statistical analysis, and the quantitative data results were used as follows: (mean ± standard deviation) was expressed, and the Kolmogorov-Smirnov test was used for data normality test. For data that met the normal distribution, the mean difference between the two groups was compared using the t test, and the difference was considered statistically significant when P < 0.05. The test results are shown in Table 1-2:
[0070] Table 1
[0071]
[0072] Note: * represents P < 0.05 compared with the group in Example 1.
[0073] Table 2
[0074]
[0075] Note: * represents P < 0.05 compared with the group in Example 2.
[0076] Experimental Example 2:
[0077] The molecular weight distribution of the prepared collagen peptides was determined by high-performance size exclusion chromatography with reference to GB 31645-2018 National Food Safety Standard Collagen Peptides, and the proportion of peptides <3 kDa (%) was determined. The results are shown in Table 3-4:
[0078] Table 3
[0079]
[0080] Note: * represents P < 0.05 compared with the group in Example 1.
[0081] Table 4
[0082]
[0083] Note: * represents P < 0.05 compared with the group in Example 2.
[0084] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.
Claims
1. A method for preparing low molecular weight redfin puffer fish bone collagen peptide, characterized in that: Follow these steps: (1) Under low temperature conditions, the bones of redfin pufferfish were naturally thawed at room temperature or in running water, the bones were rinsed with distilled water, cut into pieces, and NaOH solution was added and stirred for 30-40 hours, and the solution was changed 2-4 times in the middle to remove foreign proteins; (2) Add EDTA-2Na solution and stir for 64-80 hours, changing the solution 2-4 times to remove minerals; (3) Add isopropanol aqueous solution and stir for 20-28 hours, changing the solution 1-3 times to remove fat, and then rinse with distilled water; (4) drying the redfin pufferfish bones processed in step (3) in an oven at 40-60° C., crushing them using a universal grinder, and passing them through a 30-50 mesh sieve; (5) Add water to the redfin puffer fish bones treated in step (4), adjust the pH to 8-9, add composite protease, place in a 40-50°C water bath for enzymatic hydrolysis for 4-6 hours, then inactivate in a boiling water bath for 5-15 minutes, centrifuge at 6000-10000g for 10-20 minutes, take the supernatant, and freeze-dry to obtain collagen peptide powder.
2. The method for preparing the low molecular weight red fin puffer fish bone collagen peptide according to claim 1, characterized in that: The low temperature condition is 2-8°C.
3. The method for preparing low molecular weight red fin puffer fish bone collagen peptide according to claim 1, characterized in that: The concentration of the NaOH solution is 0.05-0.15 mol / L, and the amount added is 15-25 times the mass of the redfin pufferfish bone.
4. The method for preparing low molecular weight red fin puffer fish bone collagen peptide according to claim 1, characterized in that: The concentration of the EDTA-2Na solution is 0.3-0.7 mol / L, and the added amount is 3-7 times the mass of the redfin puffer fish bone.
5. The method for preparing low molecular weight red fin puffer fish bone collagen peptide according to claim 1, characterized in that: The volume concentration of isopropanol in the isopropanol aqueous solution is 10-20%, and the added amount is 5-15 times the mass of the redfin pufferfish bone.
6. The method for preparing low molecular weight red fin puffer fish bone collagen peptide according to claim 1, characterized in that: The amount of water added in step (5) is 5-15 times the mass of the redfin puffer fish bone.
7. The method for preparing low molecular weight red fin puffer fish bone collagen peptide according to claim 1, characterized in that: The composite protease is a mixed enzyme of alkaline protease and sugar palm plant protease, and the added amount is 6000-10000 U / g.
8. The method for preparing low molecular weight red fin puffer fish bone collagen peptide according to claim 1, characterized in that: The composite protease is a mixed enzyme of Neutrase enzyme and Alcalase enzyme, and the added amount is 6000-10000 U / g.
Citation Information
Patent Citations
Gomuti palm plant protease
CN1066468A