A double-enzyme hybrid microsphere for fish oil detection, and a preparation method and application thereof

Abstract

This invention belongs to the field of bionanomaterials and rapid food detection technology, specifically relating to a dual-enzyme hybrid microsphere for fish oil detection, its preparation method, and its application. The dual-enzyme hybrid microsphere comprises a copper-chelated hybrid microsphere framework, and horseradish peroxidase and lipoxygenase co-embedded in the framework, wherein the framework is a copper-phosphate biomineralization network. The microsphere has a porous, perforated mesh structure with a copper loading of 15-22 wt%. After reconstitution, it is added to a fish oil sample diluted with anhydrous ethanol, catalyzing the oxidation of unsaturated fatty acids and producing turbidity differences, which can be detected visually or by measuring OD. 600 / OD 450 This invention provides a quick and easy way to identify adulterated salmon oil. The preparation process is mild and simple, the detection speed is fast, the cost is low, and the operation is convenient, making it suitable for rapid on-site screening of adulterated fish oil.

Description

Technical Field

[0001] This invention belongs to the field of bio-nanomaterials and rapid food detection technology, specifically relating to a dual-enzyme hybrid microsphere for fish oil detection, its preparation method, and its application. Background Technology

[0002] Fish oil, rich in polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), is widely used as a dietary supplement and food ingredient. However, high-value fish oil products such as salmon oil are often adulterated with low-priced vegetable oils or refined fish oil, harming consumer rights and posing challenges to food safety supervision. Currently, the detection of fish oil adulteration mainly relies on high-precision instrumental analysis methods such as gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR). While these techniques are accurate and reliable, the equipment is expensive, the operation is complex, and the time is long, making it difficult to meet the needs of grassroots market supervision and rapid on-site screening. In recent years, enzyme-catalyzed biosensing methods have attracted attention due to their simplicity and low cost. In particular, the strategy of using lipoxygenase (LOX) to catalyze the oxidation of unsaturated fatty acids to generate peroxides, thereby triggering color or turbidity changes, is considered promising for developing portable detection tools. However, free enzymes face problems such as poor stability, easy inactivation, and difficulty in recovery and reuse in practical applications, which seriously limits their promotion in on-site detection.

[0003] To address the issue of enzyme stability, researchers have attempted to immobilize enzymes on various supports. Traditional immobilization methods include adsorption, embedding, and covalent bonding; however, these methods often lead to a significant decrease in enzyme activity, or involve harsh conditions such as organic solvents and high temperatures, which are detrimental to maintaining the enzyme's native conformation. In recent years, self-assembled hybrid materials based on the interaction between metal ions and proteins have become a research hotspot in the field of enzyme immobilization. For example, copper ions (Cu... 2+ Using chelating agents, horseradish peroxidase (HRP) is induced to self-assemble in phosphate buffer to form hybrid materials with nanoflower or microsphere structures, which has been proven to effectively improve the storage stability and reusability of HRP. The preparation of these materials is mild, usually carried out at low or room temperature, and does not require the introduction of additional cross-linking agents, thus being considered a green and efficient immobilization strategy. Building on this, researchers have further explored the possibility of co-immobilization of two enzymes, such as co-embedding glucose oxidase (GOx) and HRP into copper-phosphate hybrid microspheres to construct a cascade catalytic system for glucose detection, achieving good results. Similarly, there are reports of co-immobilizing diamine oxidase (DAO) and HRP into the same microsphere for colorimetric detection of histamine in aquatic products. These works demonstrate that co-embedding two enzymes into hybrid microspheres not only improves enzyme stability but also enhances the efficiency of cascade reactions through spatial proximity effects, expanding the application of hybrid materials in the field of biosensing.

[0004] However, most existing dual-enzyme hybrid microsphere systems are designed for specific small-molecule substrates such as glucose and histamine, and the enzyme pairs used are usually combinations of oxidases (such as GOx and DAO) and HRP. For the specific scenario of fish oil adulteration detection, the substrate to be catalyzed is long-chain unsaturated fatty acids, whose oxidation process involves free radical chain reactions and the generation of secondary products, which is fundamentally different from the oxidation mechanism of glucose or histamine. Currently, there are no reports of co-immobilizing lipoxygenase and HRP in the same hybrid microsphere for fish oil quality identification. Furthermore, the existing preparation processes for dual-enzyme hybrid microspheres still have some limitations: some methods require long-term low-temperature incubation (e.g., more than 72 hours), or precise control of pH and ionic strength, resulting in a narrow process window; other methods require multiple washings after centrifugation, which can easily lead to enzyme loss. More importantly, the morphology of these microspheres is mostly dense nanoflowers or solid spheres, with limited specific surface area, which is not conducive to substrate diffusion and sufficient contact between the enzyme and substrate, potentially affecting detection sensitivity and response speed. Therefore, developing a dual-enzyme immobilization system with a simpler and milder preparation process, a structure conducive to mass transfer, and the ability to specifically catalyze the oxidation of unsaturated fatty acids in fish oil and generate distinguishable signals is of great significance for promoting the practical application of rapid on-site detection technology for fish oil adulteration.

[0005] In summary, existing technologies for detecting adulteration in fish oil have the following main shortcomings: First, traditional instrumental analysis methods cannot meet the demands for low-cost, rapid, and on-site screening; second, while free enzyme methods show potential, their stability is poor, making long-term preservation and use difficult; third, existing enzyme immobilization technologies, although capable of improving stability, have limited substrate types and lack a dedicated dual-enzyme synergistic system adapted for unsaturated fatty acid oxidation; fourth, the preparation process of existing dual-enzyme hybrid microspheres still has room for optimization, particularly in terms of structural control and ease of operation. These problems necessitate that those skilled in the art explore a new technical solution that balances detection speed, cost, stability, and specificity, thereby filling the technological gap in the field of rapid detection of fish oil adulteration. Summary of the Invention

[0006] This application aims to overcome the shortcomings of existing technologies that lack a dual-enzyme immobilization system that can simultaneously meet the requirements of low cost, rapid detection, simple operation, good enzyme stability, and is specifically adapted to the oxidation of unsaturated fatty acids in fish oil to generate distinguishable signals. Therefore, it provides a dual-enzyme hybrid microsphere for fish oil detection, its preparation method, and its application to overcome the above-mentioned deficiencies.

[0007] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a dual-enzyme hybrid microsphere for fish oil detection. It includes a copper ion-chelated hybrid microsphere framework, and horseradish peroxidase and lipoxygenase co-embedded in the copper ion-chelated hybrid microsphere framework. The hybrid microsphere framework is a copper-phosphate biomineralization network.

[0008] The inventive concept of the dual-enzyme hybrid microspheres protected in this application originated from a reflection on the adaptability of existing enzyme immobilization technologies for the detection of adulteration in fish oil. As mentioned in the background, the material paradigm of dual-enzyme co-embedding in copper-phosphate hybrid microspheres has been validated in scenarios such as glucose detection and histamine detection. The enzyme pairs used are mostly combinations of glucose oxidase and horseradish peroxidase, or diamine oxidase and horseradish peroxidase, targeting substrates such as glucose and histamine, which are water-soluble small molecules. When attempting to transfer this paradigm to the detection of adulteration in salmon oil, the first challenge faced by the art is the change in substrate properties—the core differentiating components that need to be identified in fish oil are long-chain polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These substrates are highly hydrophobic, have large molecular volumes, and their oxidation pathways differ from those of glucose or amines. If the design concept of existing dual-enzyme hybrid microspheres is simply used to replace the enzyme species, those skilled in the art would usually prefer to immobilize lipoxygenase alone or pair it with other universal oxidases, because lipoxygenase itself can catalyze the oxidation of unsaturated fatty acids to generate hydroperoxides, and from the perspective of task decomposition, it seems that the step of "oxidation of unsaturated fatty acids" can be completed.

[0009] However, during the initial exploration, this application found that when relying solely on a single-enzyme system of lipoxygenase to catalyze the oxidation of unsaturated fatty acids in fish oil, the reaction product is mainly hydroperoxide. Its aggregation degree in the ethanol-fish oil mixture is limited, making it difficult to form a turbidity signal that can be stably captured by the naked eye or an enzyme-linked immunosorbent assay (ELISA) reader. This results in insufficient difference in response between pure and adulterated oils to support differentiation. The problem lies in the fact that if the hydroperoxide produced by lipoxygenase catalysis does not undergo subsequent secondary transformation, the system lacks the driving force to further cross-link and aggregate the secondary products of lipid oxidation. This secondary transformation is precisely the typical catalytic function of horseradish peroxidase. That is, using hydroperoxide as a substrate to drive phenolic or mediator reactions, indirectly promoting the extension and polymerization of lipid free radical chains. This leads to the enzyme pair selection logic of this application: co-embedding lipoxygenase and horseradish peroxidase in the same hybrid microsphere is not a simple continuation of the existing "oxidase + horseradish peroxidase" paradigm, but a targeted match for the specific cascade pathway of unsaturated fatty acid oxidation. Lipoxygenase is responsible for initiating the oxygenation reaction of EPA / DHA to generate hydroperoxides, while horseradish peroxidase takes over the hydroperoxides generated in situ to drive subsequent conversions, promoting lipid oxidation products to an aggregated state that can produce turbidity. The catalytic steps of the two enzymes are connected in the temporal and spatial domains, forming a complete cascade for fish oil substrates.

[0010] After the enzyme pair is determined, another key aspect of the design is how to co-immobilize the two enzymes on the same carrier and achieve a cascade efficiency superior to that of a physical mixing system. This application selects a copper-chelated hybrid microsphere framework as the immobilization platform, specifically a copper-phosphate biomineralization network. This choice is not a direct application of existing hybrid microsphere paradigms, but rather an adaptation based on the specific needs of fish oil detection. The construction process of the copper-phosphate biomineralization network is essentially a process of self-assembly of copper ions with enzyme molecule surface groups (such as carboxyl and amino groups) and phosphate ions. The enzyme molecule itself, as an organic template for mineralization nucleation, is "frozen" within the inorganic network, forming a porous, perforated mesh structure.

[0011] The significance of this structure in this case is reflected in three aspects: First, the porous and hollow shape gives the microspheres a large internal surface area and good pore connectivity, which is conducive to the diffusion of hydrophobic unsaturated fatty acids in the ethanol-fish oil mixture into the interior of the microspheres and their contact with the embedded enzymes during subsequent detection. This is particularly crucial for fish oil substrates because, compared to water-soluble substrates such as glucose and histamine, the mass transfer of unsaturated fatty acids in conventional aqueous or weakly polar media is inherently limited. If the microspheres have a dense, solid structure, the substrate may have difficulty reaching the embedded enzyme molecules. Secondly, the simultaneous embedding of the two enzymes in the copper-phosphate network allows lipoxygenase and horseradish peroxidase to be spatially adjacent. The hydroperoxides generated in situ by lipoxygenase can be catalyzed without diffusing to distant horseradish peroxidase sites, shortening the mass transfer path of the cascade intermediates and reducing the loss of hydroperoxides during the transfer to the second-order reaction. Thirdly, the coordination between copper ions and the enzyme protein restricts the conformational freedom of the enzyme molecules to a certain extent, allowing them to maintain their active conformation after leaving the natural aqueous environment. At the same time, the mineralization network forms a physical encapsulation of the enzyme molecules, which can slow down the leakage and inactivation of the enzymes during storage and use. Of the three points mentioned above, the first two (pore mass transfer and spatial proximity of the two enzymes) work together to improve cascade efficiency, while the third point serves to improve stability. All three rely on the same copper-phosphate biomineralization network and do not involve any additional modification steps.

[0012] In terms of the preparation process, this application adopts a stepwise strategy of first pre-chelating copper ions with horseradish peroxidase at low temperature, and then adding lipoxygenase for co-assembly at room temperature. This stepwise approach is not arbitrary. If both enzymes are simultaneously exposed to copper ions and phosphate, the lipoxygenase may participate in the mineralization process differently from horseradish peroxidase due to its own characteristics, which may lead to uneven distribution of the two enzymes in the final network or a low encapsulation rate of one enzyme. However, by first forming a pre-chelated complex between copper ions and horseradish peroxidase at low temperature, it is equivalent to providing an enzyme template that has been "attached" with copper for subsequent mineralization. Then, the lipoxygenase is added for co-assembly at room temperature, which allows the two enzymes to participate in the network construction more evenly. The relative activities of the two enzymes in the final microspheres are closer to the feed ratio.

[0013] In terms of technical effectiveness, when the aforementioned dual-enzyme hybrid microspheres were applied to the detection of adulteration in salmon oil, a distinguishable difference in turbidity was observed between pure salmon oil and samples adulterated with vegetable oil or low-priced fish oil after the reaction. This difference can be traced back to the aforementioned cascade mechanism: pure salmon oil has a higher EPA / DHA content, resulting in a relatively higher total amount of hydroperoxides catalyzed by lipoxygenase, leading to more aggregated products formed after subsequent catalysis by horseradish peroxidase, resulting in a stronger turbidity response; in adulterated oil, the proportion of unsaturated fatty acids decreases, leading to a corresponding reduction in the amount of cascade products generated, and a weaker turbidity response. This turbidity difference can be initially judged visually or measured using an ELISA reader using OD values. 600 OD 450 Quantitative reading at the same wavelength is possible, and the detection process only requires mixing the reconstituted microspheres with the oil sample diluted with ethanol, without the need for chromatographic or nuclear magnetic resonance equipment.

[0014] Compared to the physical mixture of free horseradish peroxidase and lipoxygenase, the dual-enzyme hybrid microspheres of this application exhibit a more significant turbidity response in the same oil sample due to the shortened mass transfer pathway of the cascade intermediates and improved enzyme stability. Compared to single-enzyme (lipoxygenase only or horseradish peroxidase only) hybrid microspheres, only the dual-enzyme co-embryosystem can fully cover the chain of "unsaturated fatty acid oxygenation—hydroperoxide subsequent conversion—product aggregation and turbidity formation," in which single-enzyme systems cannot form a stable and discernible turbidity signal. These effects qualitatively demonstrate the difference between the overall scheme of "specific dual-enzyme pairing + copper-phosphate co-embryostructure" and similar existing dual-enzyme hybrid microspheres (such as glucose oxidase + horseradish peroxidase, diamine oxidase + horseradish peroxidase). The enzyme pairs and substrate combinations in those schemes do not target the unsaturated fatty acid oxidation cascade, and therefore cannot be directly transferred to the application scenario of this application to obtain the same turbidity discrimination effect.

[0015] In summary, the synergy of enzyme pair selection, cascade mechanism matching, carrier structure adaptation, and preparation process of the dual-enzyme hybrid microspheres in this application constitutes the core of its inventive concept relative to the prior art, rather than a conventional replacement of the existing dual-enzyme hybrid microsphere paradigm.

[0016] Preferably, the mass loading of copper in the hybrid microspheres is 15~22 wt%.

[0017] Preferably, the hybrid microspheres have a porous, hollowed-out mesh-like microsphere structure.

[0018] Preferably, the mass ratio of horseradish peroxidase to lipoxidase in the hybrid microspheres is 1:0.5 to 1:2.

[0019] Secondly, the present invention also provides a method for preparing the aforementioned dual-enzyme hybrid microspheres, comprising the following steps: (1) Dissolve horseradish peroxidase in phosphate buffer to form HRP solution; (2) Add copper salt solution to the HRP solution and react at a temperature below 10°C to pre-chelate HRP with copper ions to form HRP-Cu complex solution; (3) Dissolve lipoxygenase in phosphate buffer to form LOX solution; (4) Add LOX solution to the HRP-Cu complex solution and mix. React at room temperature to allow HRP, LOX and copper ions to self-assemble into hybrid microspheres in the presence of phosphate. (5) Separate and purify to remove free copper ions and unreacted enzymes, and collect the solid phase to obtain the dual-enzyme hybrid microspheres.

[0020] Preferably, the copper salt in step (2) is selected from copper sulfate, copper chloride or copper nitrate.

[0021] Preferably, the final concentration of copper ions in the reaction system in step (2) is 2~5 mM.

[0022] Preferably, the reaction time in step (2) is 8~16 h; the reaction time in step (4) is 12~36 h.

[0023] Preferably, the separation and purification in step (5) is centrifugal separation, with a centrifugation speed of 8000~12000 rpm and a centrifugation time of 5~15 min; the solid phase collection also includes a freeze-drying step.

[0024] Thirdly, the present invention also provides the application of the aforementioned dual-enzyme hybrid microspheres in the detection of unsaturated fatty acid oxidation in fish oil.

[0025] Preferably, the fish oil is salmon oil, and the application is for identifying adulteration of salmon oil, including the following steps: (a) The dual-enzyme hybrid microspheres were redissolved in water and dispersed by ultrasonication to form a detection solution; (b) Dilute the fish oil sample to be tested with anhydrous ethanol to obtain a diluted oil solution; (c) Add the test solution to the oil sample dilution solution to allow the dual-enzyme hybrid microspheres to catalyze the oxidation of unsaturated fatty acids in the oil sample; (d) By measuring the absorbance values ​​OD at wavelengths of 600 nm and / or 450 nm. 600 and / or OD 450 Fish oil quality can be judged based on differences in turbidity.

[0026] Therefore, the beneficial effects of this application are mainly reflected in the following aspects: (1) The detection speed is fast and the operation is simple. The microspheres are simply reconstituted and mixed with the fish oil sample diluted with anhydrous ethanol. The turbidity difference can be read by the naked eye or enzyme-linked immunosorbent assay (ELISA) in a short time. No large and precise instruments such as chromatography or mass spectrometry are required, which significantly reduces the detection threshold and time cost. (2) The synergistic effect of the two enzymes is significant. Horseradish peroxidase and lipoxygenase are co-embedded in the same copper-phosphate biomineralization network. After lipoxygenase catalyzes the formation of hydroperoxide from unsaturated fatty acids, horseradish peroxidase can take over the intermediate in situ to drive subsequent transformations, shortening the mass transfer path of the intermediate in the cascade reaction, and greatly improving the turbidity response compared with the free enzyme physical mixture system or the single enzyme system. (3) The porous mesh structure of the microspheres facilitates the diffusion of hydrophobic fish oil substrates into the interior, improving the contact efficiency between the enzyme and the substrate. At the same time, the copper-phosphate mineralization network forms a physical encapsulation and coordination stabilization effect on the enzyme molecules, which significantly improves the stability of the two enzymes during storage and use. (4) The preparation process is mild and simple, requiring only low-temperature pre-chelation, room-temperature co-assembly and centrifugal drying. The raw materials are all commercially available conventional reagents, which are easy to mass-produce and promote. (5) The detection cost is low, the amount of microspheres used in a single test is only microliters, and high-throughput parallel detection can be achieved using a 96-well plate, which is suitable for grassroots market supervision and on-site rapid screening scenarios. Attached Figure Description

[0027] Figure 1 This is a scanning electron microscope image of the hybrid microspheres prepared in Embodiment 1 of this patent.

[0028] Figure 2 This is an X-ray photoelectron spectroscopy elemental diagram of the hybrid microspheres prepared in Embodiment 1 of this patent.

[0029] Figure 3 This is a scanning electron microscope elemental mapping image of the hybrid microspheres prepared in Embodiment 1 of this patent.

[0030] Figure 4 This is a turbidity bar chart of salmon oil (Y) and adulterated salmon oil (YS) prepared by hybrid microspheres in Embodiment 2 of this patent. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0032] Example 1: Preparation of HRP@LOX HM hybrid microspheres This embodiment provides a method for preparing HRP@LOX HM hybrid microspheres, the specific steps of which are as follows: (1) Dissolve 10 mg of horseradish peroxidase (HRP) in 1 mL of PBS to prepare a 10 mg / mL HRP solution, and pipette until completely dissolved.

[0033] (2) Weigh 191.8 mg of anhydrous copper sulfate, dissolve it in 10 mL of ultrapure water, and prepare a 120 mM CuSO4 solution.

[0034] (3) Take 30 μL of CuSO4 solution and mix it with HRP solution, and react at 4 °C for 12 h to obtain HRP-CuSO4 mixture.

[0035] (4) Dissolve 10 mg of lipoxygenase (LOX) in 1 mL of PBS to prepare a 10 mg / mL LOX solution, and pipette until completely dissolved.

[0036] (5) Mix the LOX solution with the HRP-CuSO4 mixture until homogeneous.

[0037] (6) The mixture was reacted at room temperature for 24 h.

[0038] (7) After the reaction is complete, the product is centrifuged at 10,000 rpm for 10 min to remove free copper ions and unreacted HRP and LOX.

[0039] (8) The purified HRP@LOX HM hybrid microsphere solution was freeze-dried for 48 h (-50 °C, vacuum degree <20 Pa) to obtain light blue HRP@LOX HM hybrid microsphere solid, which was stored at 4 °C for later use.

[0040] The mass fraction of copper in the HRP@LOXHM-1 microspheres prepared in this example was determined to be 18.1 wt% by ICP-OES. The morphology was characterized by scanning electron microscopy (SEM), as shown below. Figure 1 As shown, the microspheres exhibit a porous, hollowed-out mesh structure with rounded outlines and a particle size distribution between 5 and 15 μm, demonstrating good dispersibility. X-ray photoelectron spectroscopy (XPS) was used to analyze the elemental composition of the microsphere surface; the complete elemental composition and total atomic content are shown below. Figure 2 As shown. Further analysis is performed using SEM element mapping (...). Figure 3The distribution of each element on the surface of the microspheres was observed. The results showed that the microspheres prepared in this embodiment contained elements such as C, N, O, S, Cu, and Fe. Cu mainly existed in the form of Cu(II), while Fe was a trace element inherent in HRP. The detection of Fe confirmed the successful intercalation of HRP. C, N, O, Cu, and Fe were uniformly distributed within the microsphere region, with no obvious local enrichment observed. This indicates that the two enzymes and copper ions were relatively uniformly distributed throughout the microsphere framework, further confirming the successful self-assembly of the dual-enzyme hybrid microspheres.

[0041] Example 2: Preparation of HRP@LOX HM hybrid microspheres This embodiment provides a method for preparing HRP@LOX HM hybrid microspheres, the specific steps of which are as follows: (1) Dissolve 10 mg HRP in 1 mL PBS (0.01 mol / L, pH 7.4) to obtain a 10 mg / mL HRP solution.

[0042] (2) Weigh 134.5 mg of copper chloride dihydrate (CuCl2·2H2O, analytical grade, Sinopharm Group) and dissolve it in 10 mL of ultrapure water to prepare an 80 mM CuCl2 solution.

[0043] (3) Take 25 μL of the CuCl2 solution and add it to the HRP solution (so that the final concentration of copper ions in the reaction system is about 2mM), mix well, and let it stand at 4 ℃ for 8 h.

[0044] (4) Take another 5 mg LOX and dissolve it in 0.5 mL PBS to obtain a 10 mg / mL LOX solution.

[0045] (5) Add LOX solution to HRP-Cu complex solution, mix well, and let stand at room temperature for 12 h.

[0046] (6) After the reaction was completed, the supernatant was discarded by centrifuging at 8000 rpm for 15 min. The precipitate was washed twice with ultrapure water and freeze-dried for 48 h to obtain light blue hybrid microsphere solids, which were stored at 4 °C for later use.

[0047] Example 3: Preparation of HRP@LOX HM hybrid microspheres This embodiment provides a method for preparing HRP@LOX HM hybrid microspheres, the specific steps of which are as follows: (1) Dissolve 10 mg HRP in 1 mL PBS (0.01 mol / L, pH 7.4) to obtain a 10 mg / mL HRP solution.

[0048] (2) Weigh 241.6 mg of copper nitrate trihydrate (Cu(NO3)2·3H2O, analytical grade, Sinopharm Group) and dissolve it in 10 mL of ultrapure water to prepare a 100 mM Cu(NO3)2 solution.

[0049] (3) Take 50 μL of the Cu(NO3)2 solution and add it to the HRP solution (so that the final concentration of copper ions in the reaction system is about 5mM), mix well, and let it stand at 4 ℃ for 16 h.

[0050] (4) Take another 20 mg LOX and dissolve it in 2 mL PBS to obtain a 10 mg / mL LOX solution.

[0051] (5) Add LOX solution to HRP-Cu complex solution, mix well, and let stand at room temperature for 36 h.

[0052] (6) After the reaction was completed, the supernatant was discarded after centrifugation at 12000 rpm for 5 min. The precipitate was washed twice with ultrapure water and freeze-dried for 48 h to obtain light blue hybrid microsphere solids, which were stored at 4 °C for later use.

[0053] Application Example: Application of dual-enzyme hybrid microspheres in the detection of adulteration in salmon oil This embodiment provides a method for detecting adulteration in salmon oil using HRP@LOX HM hybrid microspheres. The specific steps are as follows: (1) The HRP@LOX HM hybrid microspheres prepared in Example 1 were reconstituted using 2 mL of ultrapure water and sonicated for 5 min to assist dissolution.

[0054] (2) Weigh 130 mg of salmon oil as the normal salmon oil group (Y), and weigh 110 mg of salmon oil and 20 mg of edible oil as the adulterated salmon oil group (YS). The amount of edible oil added is about 19%. Add anhydrous ethanol to make up to 1 mL.

[0055] (3) Take 100 μL of the test solution of Y and YS groups, add 900 μL of anhydrous ethanol to dilute 10 times, and mix well.

[0056] (4) Take 100 μL of the diluted test solution into a 96-well plate, and set up 3 parallels for each group.

[0057] (5) Add 10 μL of HRP@LOX HM hybrid microsphere solution to the well to be tested.

[0058] (6) Measure the OD value at wavelengths of 600 nm and 450 nm.

[0059] Measurement results: such as Figure 4The HRP@LOX HM hybrid microspheres shown have an effective ability to identify adulterated salmon oil. The OD of group Y... 600 =0.6947, OD 450 =0.8007; OD of group YS 600 =0.9066, OD 450 =1.0035, indicating that adulterated salmon oil produces more turbid substances. This example demonstrates that HRP@LOX HM hybrid microspheres can be used for the detection of adulteration in salmon oil.

[0060] Industrial applicability This invention provides a horseradish peroxidase-lipoxygenase (HRP@LOX HM) hybrid microsphere and its preparation method, as well as its application in detecting adulteration in salmon oil. It possesses complete and practical value, specifically reflected in the following aspects: (1) Raw materials are readily available and the preparation process is simple: The horseradish peroxidase, lipoxygenase, copper sulfate, PBS buffer and anhydrous ethanol required for preparation are all commercially available conventional biochemical reagents with wide procurement channels and low cost; The preparation process only includes low-temperature pre-chelation, room temperature self-assembly, centrifugal purification and freeze drying. No high temperature, high pressure and special closed reaction equipment are required. Ordinary biochemical laboratories and small food testing companies can set up production lines to prepare hybrid microspheres in batches and stably.

[0061] (2) Stable materials and high detection throughput: The hybrid microsphere-copper ion biomineralization structure of this invention stably chelates and embeds HRP and LOX dual enzymes, greatly improving the enzyme's environmental tolerance and facilitating storage and use. The single usage volume is only 10 μL, which is small; using a 96-well plate as the detection carrier, dozens of samples can be detected simultaneously in parallel at one time, and the detection efficiency is much higher than that of traditional chromatography and mass spectrometry methods, which can effectively shorten the detection cycle.

[0062] (3) Simple operation and suitable for on-site sampling: The detection uses only anhydrous ethanol as the dispersion medium, without complicated organic extraction, derivatization and other pretreatment steps; the detection is carried out based on the general 96-well plate, the equipment is widely available, the operation threshold is low, and the operators do not need to have professional chromatographic or mass spectrometry instrument operation qualifications. After the sample is added to the microsphere dispersion, a white turbid liquid is generated in a short time, without the need for large-scale precision analytical instruments, and it can be directly used for rapid on-site sampling.

Claims

1. A dual-enzyme hybrid microsphere for fish oil detection, characterized in that, It includes a copper ion-chelated hybrid microsphere framework, and horseradish peroxidase and lipoxygenase co-embedded in the copper ion-chelated hybrid microsphere framework. The hybrid microsphere framework is a copper-phosphate biomineralization network.

2. The dual-enzyme hybrid microspheres according to claim 1, characterized in that, The mass loading of copper in the hybrid microspheres is 15~22 wt%.

3. The dual-enzyme hybrid microspheres according to claim 1, characterized in that, The hybrid microspheres have a porous, hollowed-out mesh-like microsphere structure.

4. The dual-enzyme hybrid microspheres according to claim 1, characterized in that, The mass ratio of horseradish peroxidase to lipoxidase in the hybrid microspheres is 1:0.5 to 1:

2.

5. A method for preparing the dual-enzyme hybrid microspheres according to any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Dissolve horseradish peroxidase in phosphate buffer to form HRP solution; (2) Add copper salt solution to the HRP solution and react at a temperature below 10°C to pre-chelate HRP with copper ions to form HRP-Cu complex solution; (3) Dissolve lipoxygenase in phosphate buffer to form LOX solution; (4) Add LOX solution to the HRP-Cu complex solution and mix. React at room temperature so that HRP, LOX and copper ions self-assemble in the presence of phosphate to form hybrid microspheres. (5) Separate and purify to remove free copper ions and unreacted enzymes, and collect the solid phase to obtain the dual-enzyme hybrid microspheres.

6. The preparation method according to claim 5, characterized in that, The copper salt mentioned in step (2) is selected from copper sulfate, copper chloride, or copper nitrate; The final concentration of copper ions in the reaction system in step (2) is 2~5 mM.

7. The preparation method according to claim 5, characterized in that, The reaction time in step (2) is 8~16 h; the reaction time in step (4) is 12~36 h.

8. The preparation method according to claim 5, characterized in that, The separation and purification in step (5) is centrifugal separation, with a centrifugation speed of 8000~12000 rpm and a centrifugation time of 5~15 min; the solid phase collection also includes a freeze-drying step.

9. The application of the dual-enzyme hybrid microspheres according to any one of claims 1 to 4 in the detection of unsaturated fatty acid oxidation in fish oil.

10. The application according to claim 9, characterized in that, The fish oil in question is salmon oil, and the application is for identifying adulteration of salmon oil, including the following steps: (a) The dual-enzyme hybrid microspheres were redissolved in water and dispersed by ultrasonication to form a detection solution; (b) Dilute the fish oil sample to be tested with anhydrous ethanol to obtain a diluted oil solution; (c) Add the test solution to the oil sample dilution solution to allow the dual-enzyme hybrid microspheres to catalyze the oxidation of unsaturated fatty acids in the oil sample; (d) By measuring the absorbance values ​​OD at wavelengths of 600 nm and / or 450 nm. 600 and / or OD 450 Fish oil quality can be judged based on differences in turbidity.

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