Method for detecting lysozyme content in food

Abstract

The application discloses a lysozyme content detection method in food, and belongs to the technical field of biological medicine detection. The method comprises the following steps: constructing a lysozyme detection standard curve; reacting a food sample to be detected with a fluorescent probe in a buffer system, measuring the fluorescence intensity of the reaction system, and calculating the lysozyme content in the food sample to be detected based on the standard curve. The application adopts a fluorescence spectrum method to research and compare the performance of polypeptide CALNNK (FITC) and lysozyme under different buffer systems, pH, temperature and reaction time conditions, and then uses the method to detect the lysozyme content in food. The 20 nmol / L polypeptide CALNNK (FITC) , BR buffer solution with pH=10.0, 4 min of time and 35 DEG C of temperature are the optimal reaction conditions, the lysozyme content in food can be quickly and accurately detected, and the method has far-reaching significance for the healthy development of the food industry.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a method for detecting the content of lysozyme in food. Background Technology

[0002] Lysozyme (EC 3.2.1.17) is an antimicrobial enzyme widely found in biological secretions such as tears, saliva, and egg white. It is a key component of the innate immune system by hydrolyzing the β-1,4-glycosidic bonds in bacterial peptidoglycan. In addition to its physiological functions, this enzyme has wide applications in food preservation, particularly in cheese and wine production, where it effectively inhibits spoilage bacteria such as Clostridium butyricum. However, dietary exposure to this food enzyme (expressed as total organic solids (TOS)) is estimated to be up to 4.9 mg TOS / kg body weight / day. Therefore, regulatory agencies, including the U.S. Food and Drug Administration (FDA) and the EFSA, require the labeling of this ingredient on food labels. This necessitates reliable testing methods to ensure regulatory compliance and consumer safety, meeting physiological and medical needs.

[0003] Currently, methods for quantitative analysis of lysozyme include immunoassays (such as ELISA) and chromatographic techniques (such as HPLC). While ELISA boasts high sensitivity (detection limit approximately 1 nM), it suffers from antibody instability, batch-to-batch variability, and excessively long processing times (>2 hours). HPLC-MS offers extremely high specificity but requires sophisticated instruments and complex sample pretreatment steps, making it unsuitable for routine on-site detection. Alternative strategies utilizing emerging recognition elements such as aptamers and molecularly imprinted polymers (MIPs) have yielded satisfactory results. Aptamer-based sensors offer advantages such as easy signal amplification and high sensitivity, but suffer from poor interference resistance, insufficient adaptability to complex matrices, and relatively high cost. Although MIP-based sensors exhibit high stability, their preparation process is cumbersome and requires harsh conditions. These limitations highlight the urgent need for robust, rapid, and cost-effective alternatives.

[0004] Fluorescence-based detection techniques have attracted much attention in analytical chemistry due to their high sensitivity, real-time performance, and ease of operation. In particular, label-free strategies using environmentally sensitive fluorophores eliminate multiple labeling steps and allow direct monitoring of intermolecular interactions. Several research teams have utilized the inherent tryptophan fluorescence properties of lysozyme for binding studies, but this method is not applicable to complex matrices due to background interference (Gui et al., 2023). An alternative approach utilizes Forster resonance energy transfer (FRET) between donor-acceptor pairs, where lysozyme binding modulates emission intensity (Sapkota & Dhakal, 2020). More fluorescence-based analytical methods have been summarized in review articles (Ebrahimi et al., 2024; Melinte et al., 2021). However, these platforms typically require: (1) time-consuming material preparation and labeling with expensive dyes (such as Cy5, TideFluor™ series), and (2) optimization of donor-acceptor pairs—factors that increase complexity and variability.

[0005] Peptide-based recognition elements have become promising tools in the field of biosensing due to their ease of synthesis, tunable binding properties, and stability under denaturing conditions. Notably, short peptides can selectively interact with target proteins through the combination of amino acid sequences, offering advantages over antibodies in terms of reproducibility and modular design. Recent studies have identified several lysozyme-binding peptide sequences, primarily derived from phage display or proteolytic fragments of natural inhibitors. For example, Yang et al. identified a TRAP-derived peptide that enhances lysozyme activity through electrostatic interactions. Cantarutti et al. attempted to design lysozyme-binding peptides through theoretical calculations, ultimately obtaining three peptides with relatively low binding affinity. However, these peptides have not yet been applied to detection purposes. Liu et al. adopted an alternative approach, designing a short, negatively charged glutamate-containing peptide (CCEEE) to generate electrostatic interactions with lysozyme; after modification with gold nanoparticles (AuNPs), they constructed a FRET-based aptamer sensor with a detection limit (LOD) of 85 nM. Previous studies by Huang et al. have shown that CALNN-modified gold nanoparticles (AuNPs) can specifically bind to lysozyme, thereby developing a highly sensitive colorimetric sensor. However, the binding mechanism of these short peptides to lysozyme has not yet been fully elucidated. Summary of the Invention

[0006] The purpose of this invention is to provide a method for detecting lysozyme content in food, so as to solve the problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a method for detecting the lysozyme content in food, comprising the following steps: Construct a standard curve for lysozyme detection; react the food sample to be tested with the fluorescent probe in a buffer system, measure the fluorescence intensity of the reaction system, and calculate the lysozyme content in the food sample to be tested based on the standard curve.

[0008] The second technical solution of this invention is a fluorescent probe for detecting lysozyme content in food, wherein the fluorescent probe is a short peptide CALNNK labeled with fluorescein, denoted as CALNNK. (FITC) .

[0009] The third technical solution of the present invention is the application of the fluorescent probe in the preparation of a kit for detecting the lysozyme content in food.

[0010] The fourth technical solution of the present invention is a kit for detecting the lysozyme content in food, comprising the fluorescent probe.

[0011] Based on the above technical solution, the present invention has the following technical effects: This invention employs fluorescence spectroscopy to study the interaction between the short peptide CALNN and lysozyme, analyzing and researching their binding constant and thermodynamic constant to demonstrate the strength of the interaction and provide a theoretical basis for proving whether CALNN is an aptamer. Based on this, a fluorescent probe is constructed using the protein-inducible fluorescence enhancement (PIFE) property of the peptide CALNN and luciferin, enabling rapid and accurate detection of lysozyme content in food. Attached Figure Description

[0012] Figure 1 The fluorescence enhancement spectra of lysozyme by different concentrations of the short peptide CALNN are shown.

[0013] Figure 2 The curves show the F0 / ΔF-1 / [Q] of the interaction between the short peptide CALNN and lysozyme at different temperatures.

[0014] Figure 3 For the short peptide CALNNK at different pH values (FITC) Fluorescence intensity difference graph of interaction with lysozyme.

[0015] Figure 4 For short peptide CALNNK in different buffer systems (FITC) The fluorescence intensity difference graph between lysozyme and 1 represents BR buffer, 2 represents NH3·H2O - NH4Cl buffer, 3 represents borax-NaOH buffer, 4 represents glycine-NaOH buffer, and 5 represents Na2CO3-NaHCO3 buffer.

[0016] Figure 5 CalNNK at different reaction temperatures (FITC)The interaction kinetics curve with lysozyme.

[0017] Figure 6 For the short peptide CALNNK (FITC) Two standard curves of the reaction with lysozyme. Detailed Implementation

[0018] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0019] This invention provides a method for detecting lysozyme content in food, comprising the following steps: Construct a standard curve for lysozyme detection; react the food sample to be tested with the fluorescent probe in a buffer system, measure the fluorescence intensity of the reaction system, and calculate the lysozyme content in the food sample to be tested based on the standard curve.

[0020] In some specific embodiments, the fluorescent probe is a fluorescein-labeled short peptide CALNNK, denoted as CALNNK. (FITC) .

[0021] In some specific implementations, the buffer system is a BR buffer system with pH=10.0.

[0022] In some specific implementations, the reaction temperature is 35°C and the reaction time is 4-20 min.

[0023] In some specific implementations, the food is cheese or wine.

[0024] This invention also provides a fluorescent probe for detecting lysozyme content in food, wherein the fluorescent probe is a short peptide CALNNK labeled with fluorescein, denoted as CALNNK. (FITC) .

[0025] This invention also provides the application of the fluorescent probe in the preparation of a kit for detecting lysozyme content in food.

[0026] This invention also provides a kit for detecting lysozyme content in food, comprising the fluorescent probe.

[0027] This invention explores the interaction between the short peptide CALNN and lysozyme, analyzes and discusses the strength of their interaction, and constructs the short peptide CALNNK using fluorescence spectroscopy. (FITC) The interaction system with lysozyme was used to optimize the conditions of this method. Fluorescence spectroscopy was employed under different buffer systems, pH (6.5-10.5), temperature (20-50℃), and reaction time (4-20 min) conditions to detect the short peptide CALNNK.(FITC) The interaction properties with lysozyme were studied and compared, and then the method was used to detect the lysozyme content in food. The results showed that the short peptide CALNNK was constructed using fluorescence spectroscopy. (FITC) After interacting with lysozyme, fluorescence is enhanced while the fluorescence lifetime remains essentially unchanged, exhibiting static enhancement and characteristics of electrostatic interaction. This study used 20 nmol / L peptide CALNNK. (FITC) The optimal reaction conditions were BR buffer solution with pH=10.0, reaction time of 4 min, and temperature of 35℃. The detection performance was analyzed, and the results showed that fluorescence spectroscopy can rapidly and accurately detect the lysozyme content in food, which has profound significance for the healthy development of the food industry.

[0028] Example 1 1. Experimental Section 1.1 Materials and Reagents The main experimental reagents were hydroxymethylaminomethane (Tris), NaOH, phosphoric acid, glacial acetic acid, sodium chloride, potassium chloride, borax, concentrated ammonia, NH4Cl, NaHCO3, anhydrous Na2CO3, KH2PO4, anhydrous Na2HPO4, and dimethyl sulfoxide (DMSO); peptides CALNN and CALNNK were produced by Shanghai Qiangyao Biotechnology Co., Ltd.; boric acid, phosphoric acid, glacial acetic acid, glycine (Gly), reagents A, B, and C, and BSA standard protein were all analytical grade reagents.

[0029] The experimental samples were mainly collected from the market, including three commercially available common cheese products (samples R, M, Y) and three commercially available common wines (samples 1, 2, 3).

[0030] 1.2 Instruments and Meters pH meter (Laici PHS-3E), microplate reader (Infinite M200 PRO), fluorescence spectrometer (Fluoromax-4), magnetic stirrer (SZCL-3B), biochemical incubator (LRH-250), ultrasonic cleaner (KQ-50B), micro centrifuge (MC-4S), manual pipette (TopPette).

[0031] 1.3 Short peptide CALNNK (FITC) Standard curve of reaction with lysozyme Add 2 μL of the 2 μmol / L fluorescent peptide probe CALNNK to the centrifuge tube. (FITC)(Shanghai Qiangyao Biotechnology Co., Ltd.) A blank control group was prepared by diluting 198 μL of BR buffer solution in centrifuge tubes, with three parallel groups. Separately, a reaction group was prepared by adding 2 μL of 2 μmol / L probe, 100 μL of lysozyme at different concentrations, and 98 μL of BR buffer solution, with three parallel groups. After reacting at 35℃ for 4 min, the fluorescence intensity was measured. The average fluorescence intensity of the blank control group was subtracted from the average fluorescence intensity of the reaction groups with different lysozyme concentrations to obtain the fluorescence intensity difference ΔI. A standard curve was obtained by plotting the ΔI-c graph with the lysozyme molar concentration c as the x-axis and the fluorescence intensity difference ΔI as the y-axis.

[0032] 1.4 Food Sample Pretreatment Take 1.00 g of cheese product, add a small amount of BR buffer solution, and dissolve the cheese product by pulverizing with a magnetic stir bar for 60 min. Then, bring the volume to 10.0 mL. Centrifuge at 5000 rpm for 10 min, filter the supernatant through a 0.22 μm filter membrane, and store at 4 °C until analysis.

[0033] For the wine, take 5 mL and sonicate for 60 min. Then take 0.0100 g of the sample and add BR buffer solution with pH=10.0 to make up to 10.0 mL. Centrifuge for 10 min at room temperature. Take the supernatant and filter it through a 0.22 μm microporous membrane for later use.

[0034] 1.5 Food testing and spiked recovery test After sample pretreatment, the original sample was diluted to different concentrations by selecting appropriate dilution factors. The fluorescence intensity obtained by fluorescence detection was compared with the blank value to obtain the corresponding fluorescence intensity difference. The concentrations of the samples diluted by different factors were calculated to discuss the stability of the original sample concentration. Finally, the accuracy of the results was verified by spiked recovery experiments.

[0035] 2 Results and Discussion 2.1 Interaction analysis between lysozyme and short peptide CALNN Proteins, containing tryptophan and tyrosine residues, mostly exhibit endogenous fluorescence. When proteins interact with small molecules, changes in the intensity of this endogenous fluorescence occur. This change is often used to study protein-small molecule interactions. At low concentrations of the short peptide CALNN, the fluorescence-enhancing interaction effect increases with increasing CALNN concentration. However, the fluorescence intensity difference ΔI increases with increasing CALNN concentration to a certain extent and then remains unchanged, indicating that the short peptide CALNN can enhance the fluorescence intensity of lysozyme, thus proving that the short peptide CALNN interacts with lysozyme. (See...) Figure 1 .

[0036] Under the conditions described above that yielded the maximum fluorescence intensity difference, the short peptide CALNN stock solution was diluted to different concentrations and reacted at 298 K and 310 K for 20 min, respectively, and its fluorescence intensity was measured. The formula for calculating the fluorescence enhancement interaction binding constant was derived based on the formula for calculating the fluorescence quenching binding constant: F0 / F=Ksv[Q]+1 (where F0 represents the initial fluorescence intensity, F represents the fluorescence intensity upon addition of the quencher, and Q represents the quencher concentration).

[0037] Since this experiment involves fluorescence-enhanced interactions, for the static fluorescence enhancement process, assuming the short peptide CALNN reacts with lysozyme in a 1:1 ratio, its binding constant K is... a for: K a =[QP] / ([Q][P]) Formula (1); In the formula: [P] is the concentration of free lysozyme, [Q] is the concentration of peptide CALNN, and [QP] is the concentration of the complex formed by CALNN and lysozyme.

[0038] Assuming the total concentration of lysozyme is [P0], and [P0] = [QP] + [P]; during the static fluorescence enhancement process, the complex [QP] generates fluorescence within the measurement range. Combining the relationship between fluorescence intensity and concentration ([QP] = ΔF / k1, [P0] = F0 / k2), then Ka = (ΔF / k1) / ((F0 / k2 - ΔF / k1)[Q]) Equation (2); That is, F0 / ΔF = b + b / K a [Q] Formula (3).

[0039] According to formula (3), with the reciprocal of the peptide molar concentration 1 / [Q] as the abscissa and the relative fluorescence intensity F0 / ΔF as the ordinate, F0 / ΔF is plotted against 1 / [Q] at different temperatures. Figure 2 ),from Figure 2 The binding constant K between lysozyme and peptide CALNN can be obtained from the slope and intercept of the regression line. a (Table 1), and as shown in the figure, the linear relationship between F0 / ΔF and 1 / [Q] is good at different temperatures, with a correlation coefficient r value exceeding 0.96. The binding constants of the short peptide CALNN and lysozyme both exceed 10 at temperatures of 298K and 310K. 5 The magnitude of the difference indicates a strong binding affinity between the short peptide CALNN and lysozyme. The decrease in the binding constant with increasing temperature suggests that the enhancement of lysozyme fluorescence by the short peptide CALNN decreases with increasing temperature, indicating a static fluorescence enhancement.

[0040] Table 1. Binding constants of CALNN-lysozyme interaction at different temperatures

[0041] The main interaction forces between small molecules and proteins include hydrophobic forces, hydrogen bonds, van der Waals forces, and electrostatic forces. When the temperature change range is small, the enthalpy change ΔH of the interaction process can be approximated as a constant. According to formulas (4), (5), and (6), the enthalpy change ΔH, entropy change ΔS, and formation free energy change ΔG are obtained respectively.

[0042] ΔG=-RTlnK a Formula (4); ln(K) a2 / K a1 =ΔH(1 / T1-1 / T2) / R Formula (5); ΔS=(ΔH-ΔG) / T formula (6).

[0043] According to Ross et al.'s summary of the relationship between thermodynamic parameters and main force types in reactions between small molecules and biomacromolecules, when ΔH > 0 and ΔS > 0, typical hydrophobic interactions are observed; when ΔH < 0 and ΔS < 0, hydrogen bonds and van der Waals forces are present; and when ΔH < 0 and ΔS > 0, electrostatic interactions are predominant. Table 2 shows that the thermodynamic parameters ΔH < 0 and ΔS > 0 indicate that the interaction between the short peptide CALNN and lysozyme is an electrostatic interaction. The free energy change ΔG < 0 indicates that the interaction between the peptide CALNN and lysozyme is spontaneous. The reaction ΔH < 0 indicates that the reaction is exothermic. Furthermore, increasing the temperature is detrimental to the reaction, indicating a static fluorescence-enhanced interaction. This is consistent with previous analyses.

[0044] Table 2. From F0 / ΔF=1+1 / K a [Q] Obtain the thermodynamic constants of the interaction between CALNN and lysozyme.

[0045] 2.2 Lysozyme Detection Based on Peptide Fluorescent Probes Utilizing the strong interaction between CALNN and lysozyme, we attempted to construct a fluorescent probe for lysozyme assay. Interaction analysis revealed a 1:1 binding ratio between CALNN and lysozyme, thus preventing the construction of a sandwich probe. However, directly attaching a fluorescein group (CALNNK) to CALNN as the luminescent group... (FITC) The interaction between CALNN and lysozyme significantly enhances the fluorescence of luciferin. This is because the interaction between CALNN and lysozyme restricts the free movement of luciferin, reducing nonradiative transitions caused by thermal motion, thus enhancing fluorescence. Therefore, directly utilizing CALNNK... (FITC) As a fluorescent probe for the determination of lysozyme.

[0046] Before performing the lysozyme assay, we optimized some detection conditions. The magnitude of the fluorescence intensity difference is related to the pH of the reaction. Under the same conditions, the fluorescence intensity of the blank and reaction groups was measured at pH values ​​of 6.5, 7.0, 8.0, 9.0, 10.0, and 10.5. Experiments showed that at pH 10.0, the fluorescence intensity of lysozyme and the peptide CALNNK... (FITC) The fluorescence intensity ΔI produced by the reaction reaches its maximum ( Figure 3 ).

[0047] Under the condition of pH=10.0, which yields the maximum fluorescence intensity difference, and with other conditions remaining the same, BR buffer solution No. 1, NH3·H2O-NH4Cl buffer solution No. 2, borax-NaOH buffer solution No. 3, glycine-NaOH buffer solution No. 4, and Na2CO3-NaHCO3 buffer solution No. 5 were added respectively, and the fluorescence intensity of the blank group and the reaction group was measured. The experiment shows that in the BR buffer system at pH=10.0, lysozyme and peptide CALNNK... (FITC) The fluorescence intensity ΔI produced by the reaction reaches its maximum ( Figure 4 ).

[0048] Fluorescence intensity was measured at different reaction times under BR buffer conditions (pH=10.0), with other conditions remaining the same. The results showed that the signal rapidly increased within the first 10 minutes, then stabilized after 15-20 minutes at all temperatures. Higher temperatures accelerated the binding process: equilibrium was reached within 4 minutes at 35°C, while it took 18 minutes at 27°C. The maximum enhancement effect occurred at 35°C. Higher temperatures decreased signal intensity, consistent with the decrease in binding affinity with increasing temperature. Therefore, incubation at 35°C for 4 minutes was chosen to ensure complete binding while maintaining rapid detection kinetics and high sensitivity (see [link to relevant documentation]). Figure 5 ).

[0049] Under optimal conditions, using peptide CALNNK (FITC) Interacting with lysozyme, lysozyme stock solutions were diluted to different concentrations and reacted at 308 K for 4 min. The fluorescence intensity difference between the reaction and the control group was used to construct a standard curve for lysozyme concentration. At 308 K, lysozyme exhibited two linear segments within the concentration range of 0.357–200 nmol / L. For concentrations of 0.357–7.14 nmol / L, y = 15687.7x + 32361, R0 2 =0.99137; when the concentration is 7.14~200 nmol / L, y=697.7x+136654.7, R 2 =0.97189. The limit of detection is 0.357 nmol / L. See Figure 6 .

[0050] 2.3 Determination of Lysozyme Content in Food After pretreatment of the cheese samples, the original samples were diluted to different concentrations and tested according to our method. The concentrations of the samples diluted by different factors were calculated (Table 3), and the accuracy of the results was verified by spiked recovery experiments (Table 4).

[0051] Table 3. Determination of lysozyme content in different cheese products

[0052] Table 4 Results of spiked recovery tests on cheese products

[0053] The results showed that lysozyme could be detected in various cheese products on the market, with spiked recoveries ranging from 89.6% to 106.3%, indicating high accuracy of the detection results.

[0054] Similarly, after pretreatment of the wine samples, the original samples were diluted to different concentrations and tested according to our method. The concentrations of the samples diluted by different factors were calculated (Table 5), and the accuracy of the results was verified by spiked recovery experiments (Table 6).

[0055] Table 5. Determination of lysozyme content in different wines

[0056] Table 6 Results of the wine spike recovery test

[0057] The results showed that the lysozyme content varied significantly among different types of wine samples sold on the market. Sample 1 complied with GB 2760-2011 "Hygienic Standard for the Use of Food Additives," with lysozyme content below the limit of 500 mg / kg. Samples 2 and 3, however, did not meet the limit. Exceeding the limit for lysozyme may cause allergic reactions in individuals with allergies, posing a potential risk. The spiked recovery experiment showed a high recovery rate, demonstrating the high accuracy and repeatability of the experimental results.

[0058] In summary, this invention utilizes fluorescence spectrophotometry to study the interaction between the short peptide CALNN and lysozyme, which enhances the fluorescence of the fluorescent dye modified on the peptide. Using this method, the peptide CALNNK can be constructed. (FITC) This invention demonstrates the interaction between lysozyme and lysozyme in the detection of lysozyme content in cheese products and wine. It shows that lysozyme interacts with the peptide CALNNK. (FITC)The fluorescence enhancement demonstrated by this method proves its sensitivity in detecting lysozyme content in cheese and wine samples. This method is simple to operate, highly sensitive, and significantly improves detection efficiency and accuracy, making it valuable for application in current food processing and production testing in my country.

[0059] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting lysozyme content in food, characterized in that, Includes the following steps: Construct a standard curve for lysozyme detection; react the food sample to be tested with the fluorescent probe in a buffer system, measure the fluorescence intensity of the reaction system, and calculate the lysozyme content in the food sample to be tested based on the standard curve.

2. The detection method according to claim 1, characterized in that, The fluorescent probe is a fluorescein-labeled polypeptide CALNNK, denoted as CALNNK. (FITC) .

3. The detection method according to claim 1, characterized in that, The buffer system is a BR buffer system with pH=10.

0.

4. The detection method according to claim 1, characterized in that, The reaction temperature is 35℃ and the reaction time is 4-20 min.

5. The detection method according to claim 1, characterized in that, The food mentioned is cheese or wine.

6. A fluorescent probe for detecting lysozyme content in food, characterized in that, The fluorescent probe is a fluorescein-labeled polypeptide CALNNK, denoted as CALNNK. (FITC) .

7. The use of the fluorescent probe of claim 6 in the preparation of a kit for detecting lysozyme content in food.

8. A kit for detecting lysozyme content in food, characterized in that, It includes the fluorescent probe of claim 6.

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