Method for preparing biological fresh-keeping agent from squid processing waste

Abstract

The invention discloses a method for preparing a biological fresh-keeping agent by using squid processing wastes, which comprises the following steps: drying and crushing the wastes such as squid heads and skins, and performing enzymolysis with papain to obtain a polypeptide mixture; and then grafting with 3-O-ferulic acid quinic acid and 2, 5-dimethoxy cinnamic acid, dialyzing and freeze-drying to obtain the biological fresh-keeping agent. The sleeve-fish processing waste is used as a raw material, the antioxidant and antibacterial properties of the polypeptide are enhanced through a grafting reaction, the cost is low, the process is green, and the sleeve-fish preservative is suitable for coating preservation of aquatic products such as sleeve-fish and shrimps and has the advantages of efficient corrosion prevention and resource recycling.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological preservation, and particularly relates to a method for preparing a biological preservative by using squid processing waste. Background Art

[0002] In today's food industry, aquatic products are deeply loved by consumers due to their rich nutritional value. However, aquatic products have high water content and fragile tissues, and are extremely vulnerable to microbial contamination and oxidation during storage and transportation, resulting in quality degradation, spoilage, and huge economic losses.

[0003] Traditional preservation technologies such as freezing and chemical preservatives can extend the shelf life of aquatic products to a certain extent, but have problems such as high energy consumption, chemical residues, and health risks. Nitrite generates carcinogenic nitrosamines, while freezing causes protein denaturation and juice loss, affecting product quality, and its residues have an adverse impact on human health, which prompts people to actively seek safe and efficient new preservation methods. Biological preservatives have become a research hotspot due to their natural and safe characteristics. In the prior art, active peptides extracted from animals and plants, such as chitosan, protamine, etc., although having antibacterial properties, have defects such as limited sources, high costs, or single functions.

[0004] As an important aquatic resource, a large amount of waste is generated during the processing of squid, such as squid heads, epidermis, fins, and processed minced meat. These wastes are usually directly discarded or used for low-value feed production, not only causing waste of resources, but also causing environmental pollution problems. In fact, squid processing waste contains various potentially valuable components, such as proteins, polypeptides, etc. If it can be effectively utilized to develop a new biological preservative, it can not only solve the problem of aquatic product preservation, but also improve the added value of the squid processing industry and achieve the recycling of resources.

[0005] Therefore, the present invention aims to prepare a new biological preservative by using squid processing waste, fully explore the potential value of squid processing waste, solve the key problems in the preservation of aquatic products, meet the market demand for safe and efficient preservation technologies, and promote the sustainable development of the aquatic product processing industry. Summary of the Invention

[0006] The purpose of the present invention is to provide a biological preservative with excellent antioxidant and antibacterial effects.

[0007] A method for preparing a biological preservative by using squid processing waste, comprising A grafting reaction is carried out between a polypeptide mixture produced by enzymatic hydrolysis of squid processing waste and a carboxylic acid derivative in a phosphate buffer solution.

[0008] Preferably, the mass ratio of the polypeptide mixture to the carboxylic acid derivative is 0.5 - 5:1 - 10.

[0009] Preferably, the carboxylic acid derivative includes 3 - O - feruloylquinic acid and 2,5 - dimethoxycinnamic acid.

[0010] Preferably, the mass - volume ratio of 2,5 - dimethoxycinnamic acid to 3 - O - feruloylquinic acid is 0.6 - 6:0.5 - 5.

[0011] When 2,5 - dimethoxycinnamic acid and 3 - O - feruloylquinic acid act on the polypeptide mixture together, the carboxylic acid derivatives can cooperate with each other, optimize the composition and interaction of the active ingredients in the reaction system, connect with the polypeptide mixture through amide bonds, jointly improve the ability of the biological preservative to capture free radicals, and enhance the overall antioxidant performance; adsorb on the surface of the spoilage bacteria biofilm through hydrophobic interaction, destroy the integrity of the bacterial cell membrane, inhibit the growth and reproduction of microorganisms in aquatic products, and enhance the antibacterial performance.

[0012] More preferably, 3,4 - diaminon - N,N - diethylbenzamide can also be added in the grafting reaction. The mass ratio of 3,4 - diaminon - N,N - diethylbenzamide to the polypeptide mixture is 0.5 - 5:0.5 - 5. The amino group of 3,4 - diaminon - N,N - diethylbenzamide combines with the carboxyl group of the polypeptide to form a stable covalent bond network, reduce the loss of active ingredients, enhance the antioxidant effect; combined with the carboxylic acid derivative to interfere with the structural stability of the bacterial cell membrane, enhance the antibacterial effect, and improve the preservation performance of the biological preservative.

[0013] Preferably, the squid processing waste includes squid heads, epidermis, fins, and processed minced meat.

[0014] Preferably, the enzyme used for enzymatic hydrolysis is papain.

[0015] Preferably, the enzymatic hydrolysis temperature is 50 - 60 °C, and the enzymatic hydrolysis time is 6 - 8 h.

[0016] Preferably, the pH of the grafting reaction is 7.1 - 7.3, and the reaction time is 20 - 25 h.

[0017] A biological preservative is prepared by a method for preparing a biological preservative using squid processing waste.

[0018] Use of the biological preservative in the preservation of aquatic products, where the aquatic products include squid, shrimp, or fish.

[0019] The present invention also provides a preparation method of waste raw materials, including: Preparation of waste raw material: Wash and chop the waste from squid processing, dry it at 45 - 55 °C for 10 - 15 h, crush it and then sieve it through a 95 - 105 mesh sieve to obtain waste powder; Immerse the waste powder in isopropanol, extract it at 35 - 45 °C for 1 - 3 h, and dry it at 45 - 55 °C for 10 - 15 h to obtain the waste raw material.

[0020] Preferably, the waste from squid processing includes squid heads, epidermis, fins, and processed minced meat.

[0021] Preferably, the mass - to - volume ratio of the waste powder to isopropanol is 2 - 20 g:20 - 200 mL.

[0022] The present invention also provides a method for preparing a polypeptide mixture, including: Preparation of polypeptide mixture: Disperse the waste raw material in deionized water, stir evenly, adjust the pH to 6.9 - 7.1, add papain, perform water - bath oscillation at 50 - 60 °C for 6 - 10 h, inactivate the enzyme by boiling water bath for 5 - 15 min, cool to room temperature, adjust the pH to 6.9 - 7.1, centrifuge at 6000 - 10000 rpm for 5 - 15 min, and then lyophilize the supernatant to obtain the polypeptide mixture.

[0023] Preferably, the mass - to - volume ratio of the waste raw material to deionized water is 2 - 20 g:20 - 200 mL.

[0024] Preferably, the mass ratio of papain to the waste raw material is 0.5 - 5:5 - 50.

[0025] The present invention also provides a method for determining the amino acid composition in a polypeptide mixture, including: Determination of amino acid composition in polypeptide mixture: Disperse the polypeptide mixture in deionized water, add a 5.5 - 6.5 mol / L hydrochloric acid solution, hydrolyze it under vacuum sealing at 105 - 115 °C for 20 - 25 h to obtain a hydrolysate, filter it after cooling to room temperature, vacuum - seal and dry the filtrate at 55 - 65 °C, and use an automatic amino acid analyzer to determine the amino acid composition.

[0026] Preferably, the mass - to - volume ratio of the polypeptide mixture to deionized water is 5 - 50 mg:1 - 10 mL.

[0027] Preferably, the mass - to - volume ratio of the polypeptide mixture to the hydrochloric acid solution is 5 - 50 mg:0.5 - 5 mL.

[0028] The present invention also provides a method for preparing a biological preservative, including: Preparation of biological preservative: Disperse the polypeptide mixture in phosphate buffer to obtain a polypeptide mixture dispersion; Dissolve 3-O-feruloylquinic acid and 2,5-dimethoxycinnamic acid in citrate buffer, add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, adjust the pH to 7.1 - 7.3, add the polypeptide mixture dispersion, and react for 20 - 25 h at room temperature with stirring to obtain a reaction solution. Dialyze the reaction solution for 2 - 4 d and then lyophilize to obtain the biological preservative.

[0029] Preferably, the mass-to-volume ratio of the polypeptide mixture to the phosphate buffer is 0.5 - 5 g:50 - 500 mL.

[0030] Preferably, the mass-to-volume ratio of 3-O-feruloylquinic acid to the citrate buffer is 2 - 20 g:20 - 200 mL.

[0031] Preferably, the mass-to-volume ratio of 2,5-dimethoxycinnamic acid to 3-O-feruloylquinic acid is 0.6 - 6:0.5 - 5.

[0032] Preferably, the mass-to-volume ratio of N-hydroxysuccinimide to the citrate buffer is 0.6 - 6 g:50 - 500 mL.

[0033] Preferably, the mass-to-volume ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the citrate buffer is 1 - 10 g:50 - 500 mL.

[0034] Preferably, the mass of the polypeptide mixture dispersion is measured by the mass of the polypeptide mixture therein, and the mass ratio of the polypeptide mixture to 3-O-feruloylquinic acid is 0.5 - 5:0.5 - 5.

[0035] Preferably, the molecular weight of the dialysis bag used for dialysis is 3500 - 5000 Da, and the dialysis buffer used for dialysis is deionized water.

[0036] The present invention also provides a method for preparing a biological preservative, including: Preparation of biological preservative: Disperse the polypeptide mixture in phosphate buffer to obtain a polypeptide mixture dispersion; Dissolve 3-O-feruloylquinic acid and 2,5-dimethoxycinnamic acid in citrate buffer, add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, adjust the pH to 7.3 - 7.4, add the polypeptide mixture dispersion, and react for 20 - 25 h at room temperature with stirring to obtain a reaction solution. Add 3,4-diamino-N,N-diethylbenzamide and stir for 20 - 25 h to obtain a mixed solution. Dialyze the mixed solution for 2 - 4 d and then lyophilize to obtain the biological preservative.

[0037] Preferably, the mass-volume ratio of the polypeptide mixture to the phosphate buffer solution is 0.5 - 5 g : 50 - 500 mL.

[0038] Preferably, the mass-volume ratio of 3-O-feruloylquinic acid to the citrate buffer solution is 2 - 20 g : 20 - 200 mL.

[0039] Preferably, the mass-volume ratio of 2,5-dimethoxycinnamic acid to 3-O-feruloylquinic acid is 0.6 - 6 : 0.5 - 5.

[0040] Preferably, the mass-volume ratio of N-hydroxysuccinimide to the citrate buffer solution is 0.6 - 6 g : 50 - 500 mL.

[0041] Preferably, the mass-volume ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the citrate buffer solution is 1 - 10 g : 50 - 500 mL.

[0042] Preferably, the mass of the polypeptide mixture dispersion is measured by the mass of the polypeptide mixture therein, and the mass ratio of the polypeptide mixture to 3-O-feruloylquinic acid is 0.5 - 5 : 0.5 - 5.

[0043] Preferably, the mass of the polypeptide mixture dispersion is measured by the mass of the polypeptide mixture therein, and the mass ratio of 3,4-diamino-N,N-diethylbenzamide to the polypeptide mixture is 0.5 - 5 : 0.5 - 5.

[0044] Preferably, the molecular weight of the dialysis bag used for dialysis is 3500 - 5000 Da, and the dialysis buffer solution used for dialysis is deionized water.

[0045] Since the present invention uses papain to enzymatically hydrolyze squid processing waste to obtain a polypeptide mixture and carry out a grafting reaction with 3-O-feruloylquinic acid and 2,5-dimethoxycinnamic acid, it has the following beneficial effects: excellent antioxidant effect and excellent antibacterial effect on aquatic products. Therefore, the present invention is a biological preservative with excellent preservation performance. Description of the Drawings

[0046] Figure 1 It is a schematic diagram of the infrared absorption spectrum of the biological preservative.

[0047] Figure 2 It is a schematic diagram of the results of the biological safety test of the biological preservative.

[0048] Figure 3 It is for the ABTS of the biological preservative + Schematic diagram of the results of the free radical scavenging test.

[0049] Figure 4Schematic diagram of the test results of volatile basic nitrogen of biological preservatives used in aquatic products. Specific implementation mode

[0050] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0051] The concepts involved in the present application will be described below with reference to the accompanying drawings first. It should be noted here that the following descriptions of each concept are only for making the content of the present application easier to understand, and do not represent the limitation of the protection scope of the present application; at the same time, without conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the drawings and in combination with the embodiments.

[0052] Example 1: Preparation of waste raw materials: Wash and chop the waste from squid processing, dry it at 50 °C for 12 h, crush it and pass through a 100-mesh sieve to obtain waste powder; Immerse the waste powder in isopropanol, extract it at 40 °C for 2 h, and dry it at 50 °C for 12 h to obtain waste raw materials. The waste from squid processing includes squid heads, skins, fins, and processed minced meat; The mass-volume ratio of waste powder to isopropanol is 10 g: 100 mL.

[0053] Preparation of polypeptide mixture: Disperse the waste raw materials in deionized water, stir evenly, adjust the pH to 7.0, add papain, and after shaking in a water bath at 55 °C for 8 h, inactivate the enzyme in a boiling water bath for 10 min, cool to room temperature, adjust the pH to 7.0, centrifuge at 8000 rpm for 10 min, and then freeze-dry the supernatant to obtain a polypeptide mixture. The mass-volume ratio of waste raw materials to deionized water is 10 g: 100 mL, and the mass ratio of papain to waste raw materials is 1:10.

[0054] Determination of amino acid composition in the polypeptide mixture: The polypeptide mixture was dispersed in deionized water, and a hydrochloric acid solution with a concentration of 5.5 - 6.5 mol / L was added. It was hydrolyzed under vacuum sealing at 105 - 115 °C for 20 - 25 h to obtain a hydrolyzate. After cooling the temperature to room temperature, it was filtered, and the filtrate was dried under vacuum sealing at 55 - 65 °C. The amino acid composition was determined using an automatic amino acid analyzer, and polyamino acids including lysine, arginine, and histidine were detected, indicating that there are multiple amino reaction sites in the polypeptide mixture. The mass - volume ratio of the polypeptide mixture to deionized water is 10 mg:2 mL, and the mass - volume ratio of the polypeptide mixture to the hydrochloric acid solution is 10 mg:1 mL.

[0055] Preparation of the biological preservative: The polypeptide mixture was dispersed in a phosphate buffer solution to obtain a polypeptide mixture dispersion; 3 - O - feruloylquinic acid and 2,5 - dimethoxycinnamic acid were dissolved in a citrate buffer solution, N - hydroxysuccinimide and 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide were added, the pH was adjusted to 7.2, and the polypeptide mixture dispersion was added. The reaction was carried out at room temperature with stirring for 24 h to obtain a reaction solution. The reaction solution was dialyzed for 3 d and freeze - dried to obtain the biological preservative. The mass - volume ratio of the polypeptide mixture to the phosphate buffer solution is 1 g:100 mL, the mass - volume ratio of 3 - O - feruloylquinic acid to the citrate buffer solution is 1 g:100 mL, the mass - volume ratio of 2,5 - dimethoxycinnamic acid to 3 - O - feruloylquinic acid is 1.2:1, the mass - volume ratio of N - hydroxysuccinimide to the citrate buffer solution is 1.2 g:100 mL, and the mass - volume ratio of 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide to the citrate buffer solution is 2 g:100 mL; the mass of the polypeptide mixture dispersion is measured by the mass of the polypeptide mixture therein, and the mass ratio of the polypeptide mixture to 3 - O - feruloylquinic acid is 1:1; the molecular weight of the dialysis bag used for dialysis is 4000 Da, and the dialysis buffer solution used for dialysis is deionized water.

[0056] Example 2: Compared with Example 1, the only difference in this example lies in the preparation of the biological preservative.

[0057] Preparation of biological preservative: The polypeptide mixture is dispersed in phosphate buffer to obtain a polypeptide mixture dispersion; 3-O-feruloylquinic acid and 2,5-dimethoxycinnamic acid are dissolved in citrate buffer, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are added, the pH is adjusted to 7.2, the polypeptide mixture dispersion is added, and the reaction is carried out at room temperature with stirring for 24 h to obtain a reaction solution. The reaction solution is dialyzed for 3 d and freeze-dried to obtain the biological preservative. The mass-volume ratio of the polypeptide mixture to the phosphate buffer is 1 g:100 mL, the mass-volume ratio of 3-O-feruloylquinic acid to the citrate buffer is 1 g:100 mL, the mass-volume ratio of 2,5-dimethoxycinnamic acid to 3-O-feruloylquinic acid is 2:1, the mass-volume ratio of N-hydroxysuccinimide to the citrate buffer is 1.2 g:100 mL, and the mass-volume ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the citrate buffer is 2 g:100 mL; the mass of the polypeptide mixture dispersion is measured by the mass of the polypeptide mixture therein, and the mass ratio of the polypeptide mixture to 3-O-feruloylquinic acid is 1:1; the molecular weight of the dialysis bag used for dialysis is 4000 Da, and the dialysis buffer used for dialysis is deionized water.

[0058] Example 3: This example is different from Example 1 only in the preparation of the biological preservative.

[0059] Preparation of biological preservative: Disperse the polypeptide mixture in phosphate buffer to obtain a polypeptide mixture dispersion; Dissolve 3-O-feruloylquinic acid and 2,5-dimethoxycinnamic acid in citrate buffer, add N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide, adjust the pH to 7.2, add the polypeptide mixture dispersion, react for 24 h at room temperature with stirring to obtain a reaction solution, add 3,4-diamino-N,N-diethylbenzamide, stir for 24 h to obtain a mixed solution, dialyze the mixed solution for 3 d, and freeze-dry to obtain the biological preservative. The mass-volume ratio of the polypeptide mixture to the phosphate buffer is 1 g:100 mL, the mass-volume ratio of 3-O-feruloylquinic acid to the citrate buffer is 1 g:100 mL, the mass-volume ratio of 2,5-dimethoxycinnamic acid to 3-O-feruloylquinic acid is 1.2:1, the mass-volume ratio of N-hydroxysuccinimide to the citrate buffer is 1.2 g:100 mL, and the mass-volume ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the citrate buffer is 2 g:100 mL; The mass of the polypeptide mixture dispersion is measured by the mass of the polypeptide mixture therein, the mass ratio of the polypeptide mixture to 3-O-feruloylquinic acid is 1:1, and the mass ratio of 3,4-diamino-N,N-diethylbenzamide to the polypeptide mixture is 1:1; The molecular weight of the dialysis bag used for dialysis is 4000 Da, and the dialysis buffer used for dialysis is deionized water.

[0060] Example 4: This example is different from Example 1 only in the preparation of the biological preservative.

[0061] Preparation of biological preservative: The polypeptide mixture was dispersed in phosphate buffer to obtain a polypeptide mixture dispersion; 3-O-feruloylquinic acid and 2,5-dimethoxycinnamic acid were dissolved in citrate buffer, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, the pH was adjusted to 7.2, the polypeptide mixture dispersion was added, and the reaction was carried out at room temperature with stirring for 24 h to obtain a reaction solution. 3,4-Diamino-N,N-diethylbenzamide was added and stirred for 24 h to obtain a mixed solution. The mixed solution was dialyzed for 3 d and freeze-dried to obtain the biological preservative. The mass-volume ratio of the polypeptide mixture to the phosphate buffer was 1 g:100 mL, the mass-volume ratio of 3-O-feruloylquinic acid to the citrate buffer was 1 g:100 mL, the mass-volume ratio of 2,5-dimethoxycinnamic acid to 3-O-feruloylquinic acid was 1.2:1, the mass-volume ratio of N-hydroxysuccinimide to the citrate buffer was 1.2 g:100 mL, and the mass-volume ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the citrate buffer was 2 g:100 mL; the mass of the polypeptide mixture dispersion was measured by the mass of the polypeptide mixture therein, the mass ratio of the polypeptide mixture to 3-O-feruloylquinic acid was 1:1, and the mass ratio of 3,4-diamino-N,N-diethylbenzamide to the polypeptide mixture was 2:1; the molecular weight of the dialysis bag used for dialysis was 4000 Da, and the dialysis buffer used for dialysis was deionized water.

[0062] Comparative Example 1: This comparative example is different from Example 1 only in that 3-O-feruloylquinic acid was not used in the preparation of the biological preservative.

[0063] Comparative Example 2: This comparative example is different from Example 1 only in that 2,5-dimethoxycinnamic acid was not used in the preparation of the biological preservative.

[0064] Comparative Example 3: This comparative example is different from Example 1 only in that neither 3-O-feruloylquinic acid nor 2,5-dimethoxycinnamic acid was used in the preparation of the biological preservative.

[0065] Test Example 1: Microstructural characterization of the biological preservative.

[0066] Test sample: The biological preservative prepared in Example 1.

[0067] Test method: The biological preservative was dried at 80 °C for 5 h, mixed and ground with dry potassium bromide powder, pressed into a thin slice, and the infrared absorption of the hyaluronic acid composite hydrogel dressing was detected in the range of 400 - 4000 cm -1 .

[0068] The infrared absorption spectrum of the biological preservative prepared by the present invention is as Figure 1As shown, it can be seen from the figure that there is a relatively broad characteristic absorption peak of O-H near 3400 cm -1 and a characteristic absorption peak of C-H near 2850 cm -1 and a characteristic absorption peak of C=O near 1750 cm -1 and there is an absorption peak of the aromatic ring skeleton near 1600 cm -1 and a characteristic absorption peak of C-N near 1550 cm -1 and a characteristic absorption peak of C-O appears near 1150 cm -1 , indicating that the biological preservative has been successfully obtained.

[0069] Test Example 2: Biological safety test of the biological preservative.

[0070] Test samples: Biological preservatives prepared in each example and comparative example.

[0071] Test method: Mouse RAW264.7 macrophages were selected and cultured in an incubator under the conditions of 37 °C and 5% CO2. When the cells were in the logarithmic growth phase, trypsin was used to digest the cells to make them detached, and they were inoculated into a 96-well culture plate. 100 μL of cell suspension was added to each well, and after sedimentation, it was placed in the incubator overnight; the old culture medium was discarded, and after gently washing with PBS buffer, the experimental groups were respectively added with 200 μL of a culture medium containing 200 μg / mL of the biological preservative, and at the same time, a blank control group was set up. The blank control group was added with a culture medium without the biological preservative, and 3 replicates were set in each group. The culture plate was placed in the cell culture incubator and continued to be cultured for 24 h; 10 μL of 10% CCK-8 solution was added to each well, and after incubating for 2 h, the absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate was calculated.

[0072] The cell survival rate was calculated as follows:

[0073] In the formula: A 实 is the absorbance of the experimental group, and A 空 is the absorbance of the blank control group.

[0074] The results of the biological safety test of the biological preservative prepared by the present invention are as Figure 2 shown. The biological preservatives prepared in all examples and comparative examples have no obvious toxicity to mouse RAW264.7 macrophages at a concentration of 200 μg / mL, and at the same time can promote a certain amount of cell growth and improve cell viability, indicating that the biological preservative prepared by the present invention has good biological safety.

[0075] Test Example 3: ABTS + free radical scavenging test.

[0076] Test samples: The biological preservatives prepared in each example and comparative example.

[0077] Test method: Mix 7 mol / L ABTS solution with 2.45 mol / L potassium persulfate, react in the dark for 12 h, and dilute with phosphate buffer solution until the absorbance at 734 nm is 0.700 to obtain ABTS + radical solution. Mix 0.1 mL of the biological preservative with 0.9 mL of ABTS + radical solution, react in the dark for 5 min at room temperature, and measure the absorbance at 734 nm with a UV spectrophotometer to obtain ABTS + radical scavenging rate.

[0078] ABTS + The calculation of the ABTS

[0079] In the formula: R is the ABTS + radical scavenging rate, As is the absorbance of the sample-added solution, Ac is the absorbance of the sample-free solution.

[0080] The results of the ABTS + radical scavenging test of the biological preservative prepared by the present invention are as Figure 3 shown. In Example 1, the molecular weight distribution of the polypeptide mixture is uniform, more active groups are exposed, and the grafting efficiency with carboxylic acid derivatives is higher, thus enhancing the radical scavenging ability; compared with Example 1, in Example 2, the dosage of 2,5-dimethoxycinnamic acid is increased, which affects the composition and interaction of active ingredients in the reaction system, promotes intramolecular electron transfer, and enhances the antioxidant activity; compared with Examples 1-2, in Examples 3-4, the addition of 3,4-diamino-N,N-diethylbenzamide provides more electron donors for the biological preservative, enhancing its ability to capture ABTS + radicals, thus increasing the radical scavenging rate; in Comparative Example 1, 3-O-feruloylquinic acid was not used in the preparation of the biological preservative, and in Comparative Example 2, 2,5-dimethoxycinnamic acid was not used in the preparation of the biological preservative, making the biological preservative unable to effectively provide electrons or hydrogen atoms to stabilize radicals, resulting in a decrease in its antioxidant ability and a significantly lower radical scavenging rate than in the examples; Comparative Example 3 lacks both of these carboxylic acid derivatives, and the antioxidant ability is significantly decreased, and the radical scavenging rate is the lowest, indicating that 3-O-feruloylquinic acid and 2,5-dimethoxycinnamic acid play a key role in the antioxidant process of the biological preservative, and their presence helps to improve the antioxidant performance of the biological preservative.

[0081] Test Example 4: Determination of Volatile Basic Nitrogen in Aquatic Products.

[0082] Test Samples: The biological preservatives prepared in each example and comparative example.

[0083] Test Method: Fresh squid meat was respectively coated with the biological preservative and a control group without treatment, and stored at 4°C. On the 7th day of storage, 10 g of the sample was taken and minced, 100 mL of deionized water was added for homogenization, and the filtrate was obtained after filtration. 5 mL of the filtrate was taken. The semi-micro Kjeldahl method was used, 5 mL of 1% magnesium oxide solution was added for distillation, the volatile ammonia was absorbed with 2% boric acid solution, and titrated with 0.01 mol / L hydrochloric acid to obtain the value of volatile basic nitrogen.

[0084] The calculation of volatile basic nitrogen is as follows:

[0085] In the formula: TVB-N is the volatile basic nitrogen, V 1 is the total volume of the sample filtrate, V 2 is the volume of 0.01 N hydrochloric acid solution consumed during the titration of the sample solution, V 3 is the volume of hydrochloric acid solution consumed by the blank, V 4 is the volume of the sample solution taken during the determination, N is the equivalent concentration of the hydrochloric acid solution, W is the weight of the sample.

[0086] The test results of the biological preservative prepared by the present invention for the volatile basic nitrogen in aquatic products are as Figure 4As shown, in Example 1, the polypeptide mixture in the biological preservative synergistically acts with components such as 3-O-feruloylquinic acid and 2,5-dimethoxycinnamic acid, which can effectively inhibit the growth and reproduction of microorganisms in aquatic products, reduce the production of alkaline nitrogen-containing substances such as ammonia and amines generated by protein decomposition, thereby resulting in a lower volatile basic nitrogen value; compared with Example 1, Example 2 increased the dosage of 2,5-dimethoxycinnamic acid. This change optimized the proportion of each component in the biological preservative, enhanced the destruction effect on the microbial cell membrane or interfered with the metabolic pathway of microorganisms, further inhibited the growth of microorganisms, reduced the degree of protein decomposition, and the growth rate of the volatile basic nitrogen value further slowed down compared with Example 1; in Examples 3-4, 3,4-diamino-N,N-diethylbenzamide was added. 3,4-diamino-N,N-diethylbenzamide produced a synergistic effect with other components in the biological preservative. On the one hand, it enhanced the inhibitory effect on microorganisms, and on the other hand, it affected the enzyme activity of microorganisms, making it difficult for them to decompose the protein in aquatic products, thus significantly reducing the growth rate of the volatile basic nitrogen value and effectively delaying the spoilage process of aquatic products. In Comparative Example 1, 3-O-feruloylquinic acid was not used in the preparation of the biological preservative, and in Comparative Example 2, 2,5-dimethoxycinnamic acid was not used in the preparation of the biological preservative. This made the antibacterial system of the biological preservative incomplete and unable to fully exert the inhibitory effect on microorganisms. Microorganisms could multiply in large numbers and decompose proteins, resulting in a rapid increase in the volatile basic nitrogen value, and its growth rate was significantly higher than that of the examples; in Comparative Example 3, both of these carboxylic acid derivatives were lacking, and the biological preservative almost lost its inhibitory ability on the growth of microorganisms. Microorganisms multiplied in large numbers, proteins were decomposed in large quantities, and the volatile basic nitrogen value increased sharply to reach the highest value. This indicates that 3-O-feruloylquinic acid and 2,5-dimethoxycinnamic acid play an indispensable and crucial role in the biological preservative in inhibiting the growth of microorganisms and delaying the spoilage of aquatic products, and their presence is an important factor in ensuring the antibacterial performance of the biological preservative.

[0087] The above-described embodiments and / or implementation manners are only used to illustrate the preferred embodiments and / or implementation manners for realizing the technology of the present invention, and do not impose any formal restrictions on the implementation manners of the technology of the present invention. Any person skilled in the art, without departing from the scope of the technical means disclosed in the content of the present invention, may make some modifications or changes to other equivalent embodiments, but it should still be regarded as the same technology or embodiment as the present invention in essence.

[0088] In this text, specific examples are used to elaborate on the principles and implementation manners of the present application. The description of the above embodiments is only used to help understand the method and its core idea of the present application. The above is only the preferred implementation manner of the present application. It should be noted that due to the limited nature of literal expression and objectively infinite specific structures, for those of ordinary skill in the art, without departing from the principles of the present application, several improvements, embellishments or changes can be made, or the above technical features can be combined in an appropriate manner; these improvements, embellishments, changes or combinations, or directly applying the inventive concept and technical solution to other occasions without improvement, shall all be regarded as the protection scope of the present application.

Claims

1. A method for preparing a biological preservative using squid processing waste, comprising: The peptide mixture produced by enzymatic hydrolysis of squid processing waste was grafted with carboxylic acid derivatives in phosphate buffer and then dialyzed and freeze-dried.

2. The method for preparing a biological preservative using squid processing waste according to claim 1, characterized in that: The mass ratio of the polypeptide mixture to the carboxylic acid derivative is 0.5-5:1-10.

3. The method for preparing a biological preservative using squid processing waste according to claim 1, characterized in that: The carboxylic acid derivatives include 3-O-feruloquinic acid and 2,5-dimethoxycinnamic acid.

4. The method for preparing a biological preservative using squid processing waste according to claim 3, characterized in that: The mass volume ratio of the 2,5-dimethoxycinnamic acid to 3-O-feruloquinic acid is 0.6-6:0.5-5.

5. The method for preparing a biological preservative using squid processing waste according to claim 1, characterized in that: The squid processing waste includes squid heads, skins, fins and processed minced meat.

6. The method for preparing a biological preservative using squid processing waste according to claim 1, characterized in that: The enzyme used in the enzymolysis is papain.

7. The method for preparing a biological preservative using squid processing waste according to claim 1, characterized in that: The enzymolysis temperature is 50-60° C., and the enzymolysis time is 6-8 hours.

8. The method for preparing a biological preservative using squid processing waste according to claim 1, characterized in that: The pH of the grafting reaction is 7.1-7.3, and the reaction time is 20-25 hours.

9. A biological preservative prepared by the method for preparing a biological preservative using squid processing waste as described in claims 1-8.

10. Use of the biological preservative according to claim 9 in preserving aquatic products, wherein the aquatic products include squid, shrimp or fish.

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