Preparation of amphiphilic hybrid nanoflower based on polypeptide and application of amphiphilic hybrid nanoflower to degradation of aflatoxin B1 in edible vegetable oil
By designing amphiphilic hybrid nanoflowers based on polypeptides, the problem of expensive equipment when removing aflatoxin B1 in edible vegetable oil in the prior art is solved, and the degradation effect of efficient, stable and without destroying nutrients is achieved.
Patent Information
- Application Number
- CN202510371068.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-27
- Publication Date
- 2025-06-24
AI Technical Summary
In the prior art, when removing aflatoxin B1 in edible vegetable oil, special equipment and high energy consumption are usually required, and may destroy the nutrients in the oil, affect product quality, and pose food safety risks.
An amphiphilic hybrid nanoflower based on a polypeptide was designed to form nanoflower by co-precipitation of the polypeptide with divalent copper and phosphate groups, and modify the amphiphilic polymer on its surface to form amphiphilic hybrid nanoflower with high-performance catalysts.
This method can efficiently degrade aflatoxin B1 in edible vegetable oil, with high degradation activity, stability and reusability, reduce food safety risks, and do not destroy the nutrients in the oil.
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Figure CN120192376A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of polypeptide biomaterials, and particularly to the preparation of an amphiphilic polypeptide - copper phosphate hybrid nanoflower and its application in degrading aflatoxin B1 in edible oil. Background Art
[0002] Mycotoxins are a class of toxic secondary metabolites produced by fungi and are common contaminants in crops and crop processing products. Approximately 60% - 80% of crops worldwide may be contaminated by mycotoxins each year. Aflatoxin B1 (AFB1) is the most potent carcinogen among various known mycotoxins and has been classified as a Group 1 carcinogen by the International Agency for Research on Cancer (IARC). The structure of AFB1 contains a bifuran ring and a coumarin, and its acute toxicity, mutagenicity, and carcinogenicity are closely related to its structure.
[0003] Edible vegetable oil is an important crop processing product and plays an indispensable role in people's daily diet as a source of essential fatty acids and fat - soluble vitamins. However, oil crops such as peanuts, soybeans, rapeseeds, sunflower seeds, and sesame seeds are contaminated by mycotoxin - producing fungi at a rate of approximately 6 - 22% during the entire process of their planting, harvesting, storage, and production. Under suitable environmental conditions, mycotoxins are stimulated to be released, inevitably transferring contaminants to edible oil products. For example, AFB1 is often detected in peanut oil, mainly from contaminated peanuts. These contaminated edible oils enter the human body with food intake, thus having an adverse impact on human health. Therefore, the research on methods for detoxifying aflatoxin in edible oil is of great significance for reducing food contamination and maintaining human health.
[0004] In recent years, methods such as selective adsorption, ultraviolet radiation, alkaline electrolytic water treatment, and photocatalytic degradation have been successfully developed and applied to the detoxification of mycotoxins in edible oil. However, these detoxification methods usually require special equipment and high energy consumption, and may damage nutritional components and affect product quality during the detoxification process of edible oil. Inappropriate treatment may also cause food safety problems. Therefore, the research and development of new aflatoxin hazard control technologies are of great significance for improving the control ability of food biological hazards and food safety risks and ensuring the safety of agricultural products and food in China.
[0005] Polypeptides are a rich source of materials with advantages such as low toxicity, good biocompatibility, adjustable structure, biodegradability, and low production costs. Therefore, constructing catalytic materials for degrading aflatoxins based on polypeptides has great advantages. Catalysts based on polypeptides mainly have the following characteristics: 1) Amino acids not only form the backbone of polypeptides, but also the catalytic functional groups are derived from the side chains of amino acids; 2) Polypeptides mainly drive self-assembly into certain molecular structures through non-covalent interactions (such as hydrogen bonds, hydrophobic or electrostatic interactions, and metal coordination), and the formed hydrophobic domains are conducive to substrate binding and catalytic action; 3) Polypeptides have a smaller molecular weight, and the catalytic activity can be regulated by adjusting the primary and secondary structures of the peptide chain; 4) Polypeptides have high stability and can adapt to complex external environments. In addition, the preparation method of polypeptide-based catalytic materials is simple, and large equipment and high energy consumption are not required during the application in the toxin detoxification process. Therefore, aiming at the advantages of polypeptide materials, designing a polypeptide-based catalytic material for the degradation of aflatoxins has become an urgent technical problem to be solved in this field. Summary of the Invention
[0006] Aiming at the problems existing in the degradation of aflatoxin B1 in edible vegetable oils at the present stage, the present invention proposes the preparation of a polypeptide-based amphiphilic hybrid nanoflower and its application in degrading aflatoxin B1 in edible vegetable oils.
[0007] In order to achieve the above object, the technical solution of the present invention is as follows:
[0008] The present invention provides a polypeptide-based amphiphilic hybrid nanoflower, which is constructed from the polypeptide Ac-ICIHIHIHIHIKI-CONH2, divalent copper, phosphate groups, and an amphiphilic polymer.
[0009] In the peptide sequence, I is isoleucine, C is cysteine, H is histidine, K is lysine, the C-terminus of the peptide chain is amidated, and the N-terminus is amidated.
[0010] The present invention also provides a method for preparing a polypeptide-based amphiphilic hybrid nanoflower. This method uses polypeptides as organic components, divalent copper and phosphate groups as inorganic components, and forms them by co-precipitation crystallization and then modifying amphiphiles on their surfaces.
[0011] Further, the divalent copper is derived from copper chloride, copper sulfate, or copper nitrate;
[0012] Further, the phosphate group is derived from disodium hydrogen phosphate or potassium dihydrogen phosphate.
[0013] Even further, the above preparation method includes the following two steps:
[0014] (1) Preparation of polypeptide-copper phosphate hybrid nanoflowers: The polypeptide and divalent copper are first co-incubated for 24 hours under alkaline conditions (pH 7-10), and then copper phosphate buffer solution is added to form through co-precipitation crystallization.
[0015] (2) Preparation of amphiphilic polypeptide-copper phosphate hybrid nanoflowers: The polypeptide-copper phosphate hybrid nanoflowers are formed by Schiff base reaction with aldehyde-functionalized amphiphilic polymers.
[0016] Furthermore, the mass ratio of copper to peptide in step (1) is 0.8-3.5.
[0017] Furthermore, the concentration of the aldehyde-functionalized amphiphilic polymer in step (2) is 1-64 mg / mL.
[0018] The present invention also provides an application of the amphiphilic hybrid nanoflowers based on polypeptides in the degradation of aflatoxin B1 in edible vegetable oils.
[0019] Furthermore, the above application of degrading aflatoxin in edible vegetable oils includes the following steps:
[0020] (1) Degrading aflatoxin: Take the aflatoxin-contaminated vegetable oil and add the amphiphilic hybrid nanoflowers based on polypeptides for oscillating reaction.
[0021] (2) Repeated application of amphiphilic hybrid nanoflowers: Recover the amphiphilic hybrid nanoflowers in (1) above by centrifugation, and after washing, apply them cyclically.
[0022] As one of the preferred embodiments of the present invention, the edible vegetable oils for application include, but are not limited to, peanut oil, soybean oil, corn oil, and rapeseed oil.
[0023] The present invention provides a design method of an amphiphilic hybrid nanoflower based on polypeptides as a detoxifying agent for aflatoxin B1 in edible vegetable oils,
[0024] including the following steps:
[0025] (1) Analysis of the active domain of the natural detoxifying agent laccase: Laccase belongs to multi-copper oxidase, and its active center contains three copper atoms with different coordination environments, namely T1, T2, and T3; the amino acids in the active center are histidine (H) and cysteine (C), which can coordinate with divalent copper; lysine (K) participates in substrate recognition and catalysis.
[0026] (2) Peptide sequence design: Based on the structural characteristics of natural enzymes and the non-covalent self-assembly characteristics of polypeptides, the peptide sequence Ac-ICIHIHIHIHIKI-CONH2 contains the active amino acids histidine (H), cysteine (C), lysine (K) and the hydrophobic amino acid isoleucine (I) that promotes polypeptide self-assembly. All hydrophilic and hydrophobic amino acids are arranged at intervals. The C-terminus of the peptide chain is amidated and the N-terminus is acetylated to eliminate the unfavorable electrostatic interactions between the charged terminal groups to stabilize the assembled structure.
[0027] (3) Design of amphiphilic hybrid nanoflowers based on polypeptides: In order to further improve the stability of polypeptides, polypeptides are used as organic components, and divalent copper and phosphate groups are used as inorganic components to induce the formation of nanoflowers through coprecipitation crystallization. In order to improve the degradation effect of edible oil, amphiphilic polymers are covalently modified on the hybrid nanostructured surface to form amphiphilic hybrid nanoflowers, which promotes the dispersibility, stability and detoxification effect in edible oil.
[0028] Compared with the prior art, the present invention has the following beneficial effects:
[0029] The polypeptide-based amphiphilic hybrid nanoflower of the present invention is a high-performance catalyst formed by using polypeptide as the main functional material and by means of hybrid nanoflower technology and amphiphilic modification. The entire preparation method is simple, stable and inexpensive.
[0030] The prepared peptide-based amphiphilic hybrid nanoflowers can be used for the degradation of aflatoxin in edible vegetable oils. They have high degradation activity, stability and reusability, and are beneficial to promoting the improvement of the control capabilities of food biological hazards and food safety risks. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 This is a scanning electron micrograph of the peptide-based amphiphilic hybrid nanoflower;
[0032] Figure 2 is the contact angle diagram of the peptide-based amphiphilic hybrid nanoflower;
[0033] Figure 3 This is the degradation rate diagram of aflatoxin B1 in peanut oil by peptide-based amphiphilic hybrid nanoflowers;
[0034] Figure 4 The first-order reaction kinetic diagram of the degradation of aflatoxin B1 by amphiphilic hybrid nanoflowers based on peptides;
[0035] Figure 5 The reusability diagram of peptide-based amphiphilic hybrid nanoflowers;
[0036] Figure 6 This is a scanning electron micrograph of the peptide-based amphiphilic hybrid nanoflower after recycling;
[0037] Figure 7 It is a detection graph of fatty acids in edible oil after degradation of aflatoxin in peanut oil by polypeptide-based amphiphilic hybrid nanoflowers.
[0038] Figure 8 It is a graph of acid value (AV), peroxide value (POV) and iodine value (IV) of edible oil after degradation of aflatoxin in peanut oil by polypeptide-based amphiphilic hybrid nanoflowers. Detailed implementation manners
[0039] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments. Unless otherwise specified, the technical means used in the specific implementation manners of the present invention are all methods well known to those skilled in the art.
[0040] For those not specified in the embodiments in terms of specific technologies or conditions, they are all conventional methods or carried out according to the technologies or conditions described in the literature in this field, or according to the product specifications. For those reagents and instruments not specified in terms of the manufacturer, they are all conventional products that can be obtained through regular channels.
[0041] The present invention provides a method for structural analysis of polypeptide-based amphiphilic hybrid nanoflowers, including: scanning electron microscopy, contact angle analysis, etc.
[0042] The present invention provides a method for evaluating the activity of polypeptide-based amphiphilic hybrid nanoflowers: detecting the degradation efficiency of aflatoxin B1 in edible oil catalyzed by the amphiphilic hybrid nanoflowers by high performance liquid chromatography.
[0043] Example 1: Preparation and characterization of polypeptide-based amphiphilic hybrid nanoflowers
[0044] The freeze-dried peptide was taken out of the refrigerator in advance to restore it to room temperature. 2 mg of peptide powder was first dissolved in a small amount of acetonitrile, and then gradually added dropwise to a PB buffer solution (20 mM, pH 8) containing CuCl2. After mixing evenly, it was incubated at 25 °C for 24 h in the dark; the incubated polypeptide was diluted with phosphate buffer solution, and then copper sulfate solution (25 mM) was added, and it was incubated at 4 °C for 24 h; after incubation, it was centrifuged at 5000 g and 4 °C for 5 minutes to collect the precipitate to obtain hybrid nanoflowers (EmNF); the hybrid nanoflowers were added to an aldehyde-functionalized polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer (Polyether F127) phosphate buffer solution with a concentration of 16 mg / mL, and reacted at room temperature for 2 h, then sodium cyanoborohydride was added to reduce the Schiff base to amine, and after continuing the reaction for 15 h, it was centrifuged at 5000 g and 4 °C for 5 minutes to collect the precipitate to obtain amphiphilic hybrid nanoflowers (EmNF-P).
[0045] Characterize the morphology and contact angle of amphiphilic hybrid nanoflowers (EmNF-P), as shown in Figure 1 and 2 . Figure 1 Figure Figure 1 is the scanning electron microscopy image of EmNF-P. It can be clearly seen from the figure that EmNF-P forms a flower-like structure. Figure 2 Figure Figure 2 is the contact angle image. It can be seen from the figure that the contact angle of amphiphilic nanoflowers is larger than that of hybrid nanoflowers, indicating the successful modification of Polyether F127.
[0046] Example 2: Degradation of Aflatoxin B1 in Edible Oil by Peptide-Based Amphiphilic Hybrid Nanoflowers
[0047] Dilute the amphiphilic hybrid nanoflowers with ultrapure water, and then add them to peanut oil containing aflatoxin B1 at a volume ratio of 1:1 for the degradation reaction. Sampling is carried out every 0.5 h to detect the degradation effect of aflatoxin in peanut oil.
[0048] The effect of amphiphilic hybrid nanoflowers on the degradation of aflatoxin B1 in edible oil is shown in Figure 3 and Figure 4 As shown. As the degradation reaction time progresses, the degradation rate of aflatoxin B1 gradually increases, reaching a degradation rate of 73% within 1 h and 98% at 3 h. According to pseudo-first-order kinetics, the rate constant (k) of EmNF-P for the degradation of AFB1 in peanut oil is 1.06 h -1 ( Figure 4 ).
[0049] Example 3: Reusability of Peptide-Based Amphiphilic Hybrid Nanoflowers for Degrading Aflatoxin B1 in Edible Oil
[0050] After one round of the reaction of amphiphilic hybrid nanoflowers degrading aflatoxin, centrifuge at 5000 g for 5 minutes at room temperature, collect the precipitate, wash it, dissolve it with ultrapure water and then add it to edible oil containing aflatoxin for the degradation reaction, and repeat this cycle.
[0051] As shown in Figure 5 , after 5 cycles, the degradation rate of EmNF-P for AFB1 remains at 51.9%, indicating that EmNF-P has good reusability ( Figure 5 ). The scanning electron microscopy (SEM) image shows that after the degradation reaction, EmNF-P still maintains a flower-like structure, indicating high stability ( Figure 6 ).
[0052] Example 4: Effect of EmNF-P on the Quality of Edible Oil after Degradation Reaction
[0053] Measure the quality of edible oil after the degradation reaction, as shown in Figure 7and 8 As shown, after the degradation reaction, it did not cause significant effects on the physicochemical indexes of edible oil, including fatty acid (FA), acid value (AV), peroxide value (POV) and iodine value (IV), indicating that the polypeptide-based amphiphilic hybrid nanoflowers have good application prospects.
Claims
1. An amphiphilic polypeptide-copper phosphate hybrid nanoflower, characterized in that: It is formed by inducing self-assembly through co-precipitation method, with polypeptide as organic component and copper phosphate as inorganic component.
2. According to claim 1, an amphiphilic polypeptide-copper phosphate hybrid nanoflower, characterized in that: The metallic copper in copper phosphate comes from copper chloride, copper sulfate, and copper nitrate, and the phosphate group comes from disodium hydrogen phosphate or potassium dihydrogen phosphate.
3. According to claim 1, an amphiphilic polypeptide-copper phosphate hybrid nanoflower, characterized in that: Peptides It is a self-assembling polypeptide capable of orderly arrangement, specifically Ac-ICIHIHIHIHIKI-CONH2.
4. The preparation method according to claims 1-3, characterized in that: The method comprises the following two preparation steps: (1) Preparation of polypeptide-copper phosphate hybrid nanoflowers: The polypeptide and divalent copper were first incubated under alkaline conditions (pH 7-10) for 24 h, and then copper phosphate buffer solution was added to form crystals through coprecipitation. (2) Preparation of amphiphilic polypeptide-copper phosphate hybrid nanoflowers: Peptide-copper phosphate hybrid nanoflowers are formed by reacting with aldehyde-modified amphiphilic polymers via Schiff base.
5. The preparation method according to claim 5, characterized in that: The aldehyde-modified amphiphilic polymer is a polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer Polyether F127 containing an aldehyde group at the end.
6. Use of the amphiphilic polypeptide-copper phosphate hybrid nanoflowers according to claim 1 in degrading aflatoxin B1 in edible oil.
7. The use according to claim 6, characterized in that: The edible vegetable oil may be one of peanut oil, soybean oil, corn oil and rapeseed oil.