A method for preparing a color-changing and freshness-preserving dual-functional film by extracting small molecule antibacterial and antioxidant peptides from crayfish waste

Abstract

The present invention discloses a method for extracting small molecule antibacterial and antioxidant peptides from crayfish waste and preparing a dual-functional color-changing fresh-keeping film. The method uses shrimp heads and shrimp shells as raw materials to extract functional polypeptides therefrom, and then prepares them into small molecule polypeptide microcapsules, and further into a color-changing fresh-keeping film. The antioxidant and antibacterial abilities of the small molecule peptide products prepared by the present invention have been significantly improved; the prepared antioxidant and antibacterial fresh-keeping film can not only improve the fresh-keeping ability, but also detect whether the product deteriorates through color change.

Description

Technical Field

[0001] The present invention belongs to the technical field of comprehensive utilization of shrimp by-products, and particularly relates to a method for preparing a color-changing and freshness-preserving dual-effect film from crayfish waste and the film thereof. Background Art

[0002] Shrimp shells and heads account for 70%-80% of the body mass of crayfish, and contain rich proteins, fats, minerals, as well as active ingredients such as astaxanthin and chitin. The waste of shrimp head shells contains 20%-30% animal protein and 30%-40% inorganic salts. Shrimp shells contain 20%-30% chitin and 4%-8% astaxanthin. If these by-products are directly discarded, it will seriously pollute the environment. How to comprehensively utilize these scraps has important social and economic benefits. In particular, the deep processing of shrimp by-products has great environmental protection value and can increase the added value of products. At present, most of the enzymatic hydrolysis studies on crayfish by-products directly use shrimp shells as the raw materials for enzymatic hydrolysis, with low enzymatic hydrolysis efficiency, low kinetic energy for polypeptide extraction yield, and too low economic benefits. And functional polypeptides are easily inactivated and have poor antibacterial effects.

[0003] Antioxidant peptides are a major category of antioxidant substances. Usually, bioactive peptides with the function of inhibiting the peroxidation of biological macromolecules or scavenging free radicals in the body are called antioxidant peptides. Antioxidant peptides exist in the protein long chain in an inactive state. These oligopeptides can be obtained by hydrolyzing proteins by appropriate methods, and they have functions that the original proteins or constituent amino acids do not have. They are closely related to the metabolic regulation, immunity, endocrine, aging, etc. of the body and play a very important role in life activities.

[0004] At present, there is still a lack of a method for extracting small molecule peptides from shrimp by-products and further processing their raw materials into functional materials. Summary of the Invention

[0005] Aiming at the above technical problems, the purpose of the present invention is to provide a method for preparing a color-changing dual-effect fresh-keeping film from crayfish waste and the film thereof. This method uses shrimp heads and shells as raw materials, extracts the functional polypeptides therein, then prepares them into small molecule polypeptide microcapsules, and further makes a color-changing fresh-keeping film. This color-changing fresh-keeping film can not only improve the freshness-keeping ability, but also detect whether the product has deteriorated by color change.

[0006] The technical solution provided by the present invention is as follows:

[0007] In the first aspect, the present invention provides a method for preparing a color-changing fresh-keeping film from crayfish waste, comprising the following steps:

[0008] (1) Preparing small molecule polypeptide microcapsules:

[0009] (1.1) After cleaning the crayfish by-products, powder A is obtained through freeze-drying and pulverization.

[0010] (1.2) Using powder A as the raw material, crude protein from crayfish by-products is extracted.

[0011] (1.3) The crude protein is dissolved in distilled water, sealed in a bag, subjected to high-pressure treatment, and then freeze-dried to obtain powder B.

[0012] (1.4) Powder B is formulated into a protein hydrolysis solution, and a compound protease is added for hydrolysis. After the hydrolysis is completed, the protease is inactivated, and then centrifuged under freezing conditions. The supernatant is the hydrolyzed protein peptide mixture.

[0013] (1.5) The hydrolyzed protein peptide mixture is filtered, concentrated, centrifuged, and purified by chromatography to obtain small-molecule polypeptides.

[0014] (1.6) Using the small-molecule polypeptides as the core material and sodium alginate as the wall material, the small-molecule polypeptides are microencapsulated by the sharp orifice method and then freeze-dried to obtain powder C.

[0015] (2) Preparation of a color-changing indicator antioxidant and antibacterial packaging film:

[0016] (2.1) 2,6-Dihydroxybenzaldehyde and 4-hydroxyacetophenone are dissolved in an ethyl acetate / methanol mixture, and then trimethylchlorosilane is slowly added at 0 °C. After stirring evenly, ethyl acetate is added to promote precipitation. It is purified by column chromatography and freeze-dried to obtain powder D.

[0017] (2.2) Powder D is dissolved in a mixture of ethanol and acetone, vinylphenol is added, the pH is adjusted to acidic, and it is stirred and mixed evenly. Pyran flavone is obtained by column chromatography separation.

[0018] (2.3) Powder C is added to the chitosan solution and stirred evenly, and then a methanol solution of pyran flavone is added. After homogenization, the film is obtained.

[0019] Furthermore, in the step (1.2), the extraction method is as follows: Powder A is sieved and then dissolved in absolute ethanol, heated and shaken, and then centrifuged. The precipitate is washed with absolute ethanol, then added to a NaOH solution, and then heated, shaken, centrifuged, and freeze-dried to obtain.

[0020] Furthermore, in the step (1.3), the high-pressure treatment method is as follows: The sealed bag containing the crude protein dispersion is immersed in the pressurizing medium, and the pressure is increased, maintained, and then decreased to obtain.

[0021] Still further, in the step (1.3), the pressure during pressure maintenance is 500 Mpa, the pressure maintenance time is 5 min, and both the pressure increase and decrease processes are completed within 1 min.

[0022] Further, in the step (1.4), the compound protease includes pepsin, alkaline protease, and trypsin.

[0023] Further, the steps of the step (1.5) are as follows: adjust the hydrolyzed protein peptide mixture to neutral, filter, and then use a pressure filtration system to concentrate and retain with a 300 molecular weight nanofiltration membrane; centrifuge the concentrated solution, take the supernatant and filter it through micropores, then use a superdex 30pg dextran gel column, and use phosphate buffer as the eluent for protein chromatography purification. The flow rate of the eluent is 1.0 mL / min, collect the components of each separation peak at a wavelength of 280 nm, and then concentrate by rotary evaporation, and further lyophilize the concentrated solution to obtain.

[0024] Further, in the step (2.1), the dosage ratio of 2,6-dihydroxybenzaldehyde, 4-hydroxyacetophenone, and trimethylchlorosilane is 1 mol:1 mol:20 mL; use C18 silica gel and a methanol-acidified aqueous solution with a concentration of 20-40% for column chromatography purification.

[0025] Further, in the step (2.2), use C18 silica gel and a methanol-acidified aqueous solution with a concentration of 10-20% for column chromatography separation.

[0026] Further, in the step (2.3), the addition amount of powder C is 1 wt% of the chitosan solution; the mass ratio of powder C to pyrano-flavone is 10:1.

[0027] In the second aspect, the present invention provides a color-changing and freshness-preserving dual-effect film prepared by using the method described in the first aspect. Anthocyanin can change color with the change of pH value, but anthocyanin is unstable and easily decomposes under light and high temperature. Pyrano-flavone is a derivative of anthocyanin and also has the characteristic of changing color according to the pH value, but has stronger stability. Using chitosan as a film-forming matrix, adding the antioxidant antibacterial peptide prepared above and the synthesized pyrano-flavone color indicator to prepare a color-changing fresh-keeping film.

[0028] The beneficial effects of the present invention are as follows:

[0029] 1. The present invention adopts a new process of first performing high-pressure treatment on the crude protein of crayfish by-products and then treating it with a mixed enzyme. Compared with the control group (without high-pressure treatment, directly hydrolyzed with alkaline protease), the combination of prior high pressure and mixed enzyme hydrolysis adopted this time significantly increases the content of small-molecule peptides. The proportion of peptides with a molecular weight below 500 Da reaches 80%, and the content of small-molecule peptides with a molecular weight of 30 Da - 50 Da increases by more than 70% compared with the control group, and the antioxidant ability is significantly enhanced. When the shrimp shell antioxidant small-molecule peptides prepared by the process of the present invention are formulated at a concentration of 10 mg / mL, in terms of antioxidant performance, the ABTS free radical scavenging rate reaches 95%, the DPPH free radical reaches 92%, and the hydroxyl free radical (·OH) scavenging rate reaches 75%, while for the control group (without high-pressure treatment, directly hydrolyzed with alkaline protease), they are 65%, 68%, and 33% respectively; in terms of antibacterial performance, when the polypeptide solution prepared by the new process has a concentration of 10 mg / mL, the inhibition zone diameter of the Escherichia coli culture dish reaches 8 mm, while for the control group at 10 mg / mL, the inhibition zone of the Escherichia coli culture dish is not significant, and when the concentration reaches 30 mg / mL, the inhibition zone diameter is 7.8 mm. The data comparison shows that the antioxidant and antibacterial abilities of the small-molecule peptide products from crayfish by-products have been significantly improved by the ultra-high pressure treatment combined with the composite enzyme hydrolysis process.

[0030] 2. The present invention also prepares an antioxidant and antibacterial preservative film based on small-molecule peptides. The thickness of the finished film is about 80 μm, the moisture content is 10.5%, the swelling index is 12.8%, the transparency is 77%, the thermal stability is good below 200 °C, and the water vapor permeability (WVP) is 2.5×10 -10 g.m -1 .s -1 .Pa -1 , the color difference values a* and b* are about 10 and 20, and the fresh film presents a light yellow color. Through the 0 °C cold storage preservation experiment of shrimp, compared with the control group which has deteriorated after 4 days, using the prepared preservative film for packaging can extend the shelf life to 8 days. And at the initial stage, the packaging film is light yellow. When the pH value of the shrimp exceeds 7 on the 5th day, the packaging film presents a light purple color, and at the end of the shelf life, it presents a dark purple color, which can intuitively judge the freshness of the shrimp products. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a chitosan film containing soluble small-molecule peptide microcapsules and pyrano flavones;

[0032] Figure 2 is a change diagram of the film color changing from light yellow (left) to purple (right) with the change of pH value. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention will be further described below in conjunction with specific embodiments, and the content of the present invention is not limited thereto at all.

[0034] Example 1

[0035] I. Preparation of small molecule polypeptide microcapsules:

[0036] (1) The by-products (shrimp heads and shrimp shells) remaining after removing the shrimp meat from crayfish are rinsed clean in clear water to ensure that there is no sediment and dirt after cleaning, and then placed in a freeze dryer. The vacuum degree is controlled at 20 Pa, and after freeze-drying for 48 h, it is taken out and pulverized by a pulverizer.

[0037] (2) The freeze-dried crayfish by-product powder is sieved through a 60-mesh sieve and dissolved in absolute ethanol with a solid-liquid ratio of 1:10. It is shaken in a water bath at 45 °C for 20 min, centrifuged (3000 r / min, 15 min), and the precipitate is washed 3 times with absolute ethanol. Then, 0.1 mol / L NaOH (1:10, w / v) solution is added, and it is shaken in a water bath at 45 °C for 1 h, centrifuged (8000 r / min, 10 min). The supernatant is adjusted to pH 4.0 with 1 mol / L HCl, allowed to stand for 12 h, and then centrifuged (8000 r / min, 10 min). The precipitate is freeze-dried to obtain crude crayfish by-product protein.

[0038] (3) The obtained crude protein powder is dissolved in an appropriate amount of distilled water. The amount of distilled water should not be too much. It is packed in two layers of polyethylene plastic bags, evacuated and sealed to ensure complete sealing without leakage, and placed in the high-pressure chamber of a high-pressure device, immersed in a pressure medium (tap water), and subjected to high-pressure treatment. It is required that the equipment completes the pressure increase and decrease within 1 min, and the pressure holding time is 5 min under the condition of 500 Mpa. After the high-pressure treatment is completed, it is freeze-dried again.

[0039] (4) The above-mentioned high-pressure-treated crude protein powder is prepared into a protein hydrolysis solution according to a solid-liquid ratio of 1:15 (volume ratio). Subsequently, a compound protease (pepsin, alkaline protease, trypsin, with enzyme activities of 500 U / mg, 250 U / mg, and 200 U / mg respectively, mixed in a mass ratio of 1:2:1) is added. The usage amount of the compound enzyme is 3 wt% of the protein solution. The pH is adjusted to 6.0, the enzyme hydrolysis temperature is stabilized at 45 °C, and the reaction time is 3 h. After the hydrolysis is completed, it is heated to 100 °C and kept for 1 min to inactivate the protease. After cooling to room temperature, it is frozen and centrifuged (4 °C, 8000 r / min, 10 min). The supernatant is the hydrolyzed protein peptide mixture.

[0040] (5) Adjust the pH of the obtained enzymatically hydrolyzed polypeptide mixture to 7.0, filter it through a 200-mesh filter paper, place the obtained filtrate in a pressure filtration system and concentrate it by retention with a 300-molecular weight nanofiltration membrane. Centrifuge the concentrated solution at 1000 r / min, take the supernatant and filter it through a 0.45-μm microporous filter head, and purify it by protein chromatography on a Superdex 30 pg Sephadex gel column. Use phosphate buffer as the eluent, with an eluent flow rate of 1.0 mL / min, and collect the components of each separated peak at a wavelength of 280 nm. The collection time periods are 15 - 20 min and 42 min - 55 min respectively. Then concentrate by rotary evaporation, and further lyophilize the concentrated solution to obtain antioxidant and antibacterial polypeptides.

[0041] (6) Use the prepared small molecule peptide as the core material and sodium alginate as the wall material, and microencapsulate the antioxidant peptide from crayfish shrimp shells by the sharp orifice method (the mass ratio of core material to wall material is 0.25∶1, the mass fraction of sodium alginate is 2.6%, the embedding temperature is 49 °C, and the mass fraction of CaCl2 is 2.48%). The embedding rate reaches 90%, then vacuum freeze-dry it, and place the dry powder in a sealed PE bag and store it at 4 °C.

[0042] II. Preparation of a color-changing indicator antioxidant and antibacterial packaging film (as Figure 1 shown)

[0043] (1) Dissolve 1 mol of 2,6-dihydroxybenzaldehyde and 1 mol of 4-hydroxyacetophenone in an ethyl acetate / methanol mixed solution (volume ratio 2:1), then slowly add 20 mL of trimethylchlorosilane at 0 °C, stir for 1 hour, add 20 mL of ethyl acetate to promote the formation of a precipitate, and purify the precipitate by column chromatography using C18 silica gel with a methanol-acidified aqueous solution with a concentration of 20 - 40% and lyophilize it.

[0044] (2) Dissolve the above product in an ethanol-acetone solution (volume ratio 2:1), with a prepared concentration of 1 mg / mL, add vinylphenol with the same mass as the dissolved product, adjust the pH to 3.5, stir at 37 °C for 2 hours, and then separate to obtain pyran flavone by column chromatography using C18 silica gel and a methanol-acidified aqueous solution with a concentration of 10 - 20%.

[0045] (3) Prepare a 2% chitosan solution (w / w) at room temperature, add the antioxidant polypeptide microcapsules prepared in step one to the chitosan solution, with a dosage of 1 wt% of the chitosan solution, stir at high speed for 12 hours (12000 r / min), then add the prepared pyran flavone (dissolved in methanol, 1 mg / mL) solution, with a pyran flavone dosage of 0.1 wt% of the chitosan solution, homogenize for 1 hour, pour it into a petri dish, and then dry it in an oven at 45 °C for 5 hours to obtain a color-changing indicator antioxidant and antibacterial packaging film.

[0046] Performance testing:

[0047] 1. Performance testing of small molecule polypeptide products:

[0048] For the polypeptide small molecule product prepared in Example 1, the content of small molecule peptides has increased significantly. The proportion of peptides with a molecular weight below 500 Da reaches 80%. The content of small molecule peptides with a molecular weight of 30 Da - 50 Da has increased by more than 70% compared with the control group (without high-pressure treatment, hydrolyzed by a single alkaline protease), and the antioxidant capacity has been significantly enhanced.

[0049] For the polypeptide small molecule product prepared in Example 1, when the concentration is 10 mg / mL, in terms of antioxidant performance, the ABTS radical scavenging rate reaches 95%, the DPPH radical scavenging rate reaches 92%, and the hydroxyl radical (·OH) scavenging rate reaches 75%. For the control group (without high-pressure treatment, directly hydrolyzed by alkaline protease), the corresponding values are 65%, 68%, and 33% respectively; in terms of antibacterial performance, when the concentration of the polypeptide solution in Example 1 is 10 mg / mL, the diameter of the inhibition zone on the Escherichia coli culture dish reaches 8 mm. For the control group, when the concentration is 10 mg / mL, the inhibition zone on the Escherichia coli culture dish is not significant. When the concentration reaches 30 mg / mL, the diameter of the inhibition zone is 7.8 mm. The data comparison shows that the antioxidant and antibacterial abilities of the small molecule peptide product from crayfish by-products have been significantly improved by the combination of ultra-high pressure treatment and complex enzyme hydrolysis process.

[0050] 2. Testing of food wrap:

[0051] The packaging film prepared in Example 1 was used to package shrimps for testing, and the results are as follows: In the 0°C refrigerated fresh-keeping experiment, compared with the control group (commercially available PE food wrap) which became spoiled after 4 days, using the food wrap of Example 1 can extend the shelf life to 8 days. And at the initial stage, the packaging film is light yellow. When the pH value of the shrimp exceeds 7 on the 5th day, the packaging film turns light purple until dark purple, enabling the intuitive judgment of the freshness of the shrimp product.

[0052] The above is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the technical scope disclosed by the present invention shall be included within the scope of protection of the invention.

Claims

1. A method for extracting small molecule antibacterial and antioxidant peptides from crayfish waste and preparing a color-changing and freshness-preserving dual-effect film, which is characterized in that, It includes the following steps: (1) Prepare small molecule polypeptide microcapsules: (1.1) After cleaning the crayfish by-products, they are freeze-dried, crushed to obtain powder A; (1.2) Using powder A as the raw material, crude protein from crayfish by-products is extracted; (1.3) The crude protein is dissolved in distilled water, sealed in a bag, subjected to high-pressure treatment, and then freeze-dried to obtain powder B; (1.4) Powder B is formulated into a protein hydrolysis solution, and a compound protease is added for hydrolysis; after hydrolysis, the protease is inactivated, and after centrifugation at low temperature, the supernatant is taken as the hydrolyzed protein peptide mixture; (1.5) The hydrolyzed protein peptide mixture is filtered, concentrated, centrifuged, and purified by chromatography to obtain small molecule polypeptides; (1.6) Using small molecule polypeptides as the core material and sodium alginate as the wall material, the small molecule polypeptides are microencapsulated by the sharp orifice method, and then freeze-dried to obtain powder C; (2) Prepare a color-changing indicator antioxidant and antibacterial packaging film: (2.1) 2,6-dihydroxybenzaldehyde and 4-hydroxyacetophenone are dissolved in an ethyl acetate / methanol mixture, and then trimethylchlorosilane is slowly added at 0 °C, stirred evenly, and ethyl acetate is added to promote precipitation. It is purified by column chromatography and freeze-dried to obtain powder D; (2.2) Powder D is dissolved in a mixture of ethanol and acetone, vinylphenol is added, the pH is adjusted to acidic, stirred evenly, and pyran flavone is obtained by column chromatography separation; (2.3) Powder C is added to the chitosan solution and stirred evenly, and then a methanol solution of pyran flavone is added, and the film is prepared after homogenization.

2. The method according to claim 1, wherein: In the step (1.2), the extraction method is as follows: After sieving powder A, it is dissolved in absolute ethanol, heated and shaken, and then centrifuged; the precipitate is washed with absolute ethanol, then added to the NaOH solution, and then heated, shaken, centrifuged, and freeze-dried to obtain.

3. The method according to claim 1, wherein: In the step (1.3), the high-pressure treatment method is as follows: The sealed bag containing the crude protein dispersion is immersed in the pressurizing medium, and the pressure is increased, maintained, and then decreased to obtain.

4. The method according to claim 3, wherein: In the step (1.3), the pressure for maintaining pressure is 500 Mpa, the time for maintaining pressure is 5 min, and both the pressure increasing and decreasing processes are completed within 1 min.

5. The method according to claim 1, wherein: In the step (1.4), the compound protease includes pepsin, alkaline protease, and trypsin.

6. The method according to claim 1, wherein: The steps of the step (1.5) are as follows: The hydrolyzed protein peptide mixture is adjusted to neutral, filtered, and then concentrated by using a pressure filtration system with a 300 molecular weight nanofiltration membrane; the concentrated solution is centrifuged, the supernatant is taken and filtered through micropores, and then a superdex 30 pg dextran gel column is used, and phosphate buffer solution is used as the eluent for protein chromatography purification. The eluent flow rate is 1.0 mL / min, and each separated peak component is collected at a wavelength of 280 nm, and then concentrated by rotary evaporation, and the concentrated solution is further freeze-dried to obtain.

7. The method according to claim 1, wherein: In the step (2.1), the dosage ratio of 2,6-dihydroxybenzaldehyde, 4-hydroxyacetophenone, and trimethylchlorosilane is 1 mol:1 mol:20 mL; column chromatography purification is carried out using C18 silica gel and a methanol acidified aqueous solution with a concentration of 20-40%.

8. The method according to claim 1, wherein: In the step (2.2), column chromatography separation is carried out using C18 silica gel and a methanol acidified aqueous solution with a concentration of 10-20%.

9. The method according to claim 1, wherein: In the step (2.3), the addition amount of powder C is 1 wt% of the chitosan solution; the mass ratio of powder C to pyrano flavone is 10:

1.

10. A color-changing and freshness-preserving dual-effect film prepared from crayfish waste, which is characterized in that: Prepared by the method according to any one of claims 1-9.

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