Marker for discriminating between apis mellifera honey and apis mellifera ligustica honey and use thereof

Abstract

Biomarkers for distinguishing between Chinese honeybee honey and Italian honeybee honey and their applications. Ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-TQMS) was used for non-targeted identification and comparative analysis of the animal-derived proteomes of Chinese honeybee honey and Italian honeybee honey. Potential characteristic peptides were screened, and then targeted verification of these potential peptides was performed using UPLC-TQMS. Verification yielded one Chinese honeybee-specific peptide (TVAQSDETLQMIVGMK) and five Italian honeybee-specific peptides (SLPILHEWK, SLNVIHEWK, FFDYDFGSDER, YFDYDFGSEER, and VGDGGPLLQPYPDWSFAK). These specific peptides can serve as molecular markers for distinguishing between Chinese honeybee honey and Italian honey, and can be used for animal-derived honey identification, authenticity determination, and quality control.

Description

Technical Field

[0001] This invention relates to the field of honey identification technology, specifically to markers for identifying honey from Chinese honeybees and Italian honeybees and their applications. Background Technology

[0002] Honey is a natural sweet substance produced by bees from plant nectar. It is rich in various bioactive components such as sugars, amino acids, vitamins, minerals, flavonoids, and organic acids, possessing excellent nutritional value and various physiological activities including antibacterial and anti-inflammatory properties. It has been widely used in food processing and healthcare. Based on the bee species that collects the honey, honey can be mainly divided into two categories: Chinese honeybee (Apis cerana, commonly known as Chinese honeybee) honey and Italian honeybee (Apis mellifera, commonly known as Italian honeybee) honey. Chinese honeybee honey, due to its diverse nectar sources, rich aroma, and high content of active ingredients, coupled with its relatively limited production, is often considered a premium honey in the market, and its economic value is significantly higher than that of Italian honeybee honey. However, driven by profit, Italian honey is often adulterated or passed off as Chinese honeybee honey in the market, seriously harming consumer rights and threatening the sustainable development of Chinese honeybee farming to some extent.

[0003] Honey has become the third most adulterated food product globally, after milk powder and olive oil, and its authenticity and quality control are receiving increasing attention. Traditional identification methods mainly rely on physicochemical indicators (such as sugar composition and moisture content), pollen microscopic analysis, and stable isotopes. However, these methods are easily affected by factors such as nectar source plants, geographical environment, and processing and storage conditions, and their specificity and sensitivity in identifying bee species remain limited. On the other hand, in honey traceability studies based on plant-derived markers, screening for characteristic components shared by honey and its nectar source plants is equally important. However, due to the high content of carbohydrates (75–85%) and water (13–20%) in honey, and the fact that non-carbohydrate plant secondary metabolites account for only about 3%, directly isolating and purifying plant-derived characteristic components from honey still faces significant challenges. Therefore, developing an accurate and reliable identification method that can directly reflect the bee species origin of honey is of great importance.

[0004] In recent years, with the rapid development of omics technologies, proteomics has provided a new solution for identifying the authenticity of food products. Honey contains approximately 0.1–0.5% protein, mainly derived from bee secretions and pollen and nectar from nectar-producing plants. Due to differences in physiological structure and metabolism among different bee species, the types and expression levels of their secreted proteins may also differ significantly, thus forming species-specific protein or peptide markers. Existing studies have shown that analyzing differences in protein or peptide components in honey can help identify different honey sources. For example, Ramón-Sierra et al. used SDS-PAGE to identify proteins in stingless bee honey and Italian bee honey, and used protein electrophoresis patterns as an important basis for determining the insect origin of honey. Cai Dongmei et al. screened characteristic peptides of honeybee MRJP1 and honeybee MRJP2 and established quantitative methods. Bong et al. used high-resolution mass spectrometry combined with proteomics technology to identify 17 plant-derived proteins in Manuka honey and established a peptide profiling analysis method. However, systematic comparative proteomic analysis and characteristic peptide targeting verification studies on honey from Chinese honeybees and Italian honeybees are still limited, and there is a lack of standardized methods with high sensitivity and specificity.

[0005] Therefore, this invention employs ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-Q-Exactive HF / MS) to systematically analyze the proteomes of honey from Chinese honeybees and Italian honeybees, screening out potential bee-derived characteristic peptides. Furthermore, ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-TQMS) is used to target and verify these characteristic peptides. The aim is to construct a reliable new method for identifying the authenticity of honey, and also to provide new scientific evidence for analyzing the differences between honeys from different bee species from a protein perspective. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides markers for identifying honey from Chinese honeybees and honey from Italian honeybees, and their applications.

[0007] A marker for identifying honey from Chinese honeybees, the marker being a specific peptide, the amino acid sequence of which is shown in SEQ ID No. 1.

[0008] A marker for identifying Italian honeybee honey, the marker being a specific peptide, the amino acid sequence of which is shown in SEQ ID No. 2.

[0009] A marker for identifying Italian honeybee honey, the marker being a specific peptide, the amino acid sequence of which is shown in SEQ ID No. 3.

[0010] A marker for identifying Italian honeybee honey, the marker being a specific peptide, the amino acid sequence of which is shown in SEQ ID No. 4.

[0011] A marker for identifying Italian honeybee honey, the marker being a specific peptide, the amino acid sequence of which is shown in SEQ ID No. 5.

[0012] A marker for identifying Italian honeybee honey, the marker being a specific peptide, the amino acid sequence of which is shown in SEQ ID No. 6.

[0013] Furthermore, the application of the aforementioned markers in the identification of Chinese honeybee honey.

[0014] Furthermore, the application of the aforementioned markers in the identification of Italian honey.

[0015] A method for identifying the source of honey includes the following steps: (1) Protein extraction and enzymatic hydrolysis were performed on the honey sample to be tested to obtain the peptide sample to be tested; (2) The specific peptides in the sample to be tested were detected by liquid chromatography-mass spectrometry. (3) When a specific peptide segment with the amino acid sequence TVAQSDETLQMIVGMK as described in claim 1 is detected, the honey to be tested is determined to be honey from Chinese honeybees; when one or more of the specific peptide segments described in any one of claims 2-6 are detected, the honey to be tested is determined to be honey from Italian honeybees.

[0016] Furthermore, the liquid chromatography-mass spectrometry (LC-MS) technique in step (2) is ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HDMS) or ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-TQMS); the LC-MS technique is UPLC-Q-Exactive HF / MS or UPLC-TQMS; the specific peptide is derived from MRJP1 or MRJP2 of the main royal jelly protein family MRJPs; the method is used for animal-derived identification, authenticity determination, or quality control of Chinese honeybee honey and Italian honeybee honey.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes high-resolution mass spectrometry (Q Exactive HF-X) to systematically analyze animal-derived proteins in honey from Chinese honeybees and Italian honeybees, identifying 127 and 93 proteins, respectively. GO functional annotation and KEGG pathway enrichment analysis revealed that the differentially expressed proteins in the two honey species are mainly involved in ribosome structure and composition, energy metabolism, hydrolytic enzyme activity, and immune defense, showing significant enrichment in pathways such as protein degradation, carbohydrate metabolism, and genetic information processing, indicating significant differences in the protein composition and potential functions among different bee species. Subsequently, UPLC-TQMS was used to target and validate candidate characteristic peptides, ultimately identifying six stable species-specific marker peptides. One peptide (TVAQSDETLQMIVGMK) was consistently detected in Chinese honeybee honey, while five characteristic peptides (SLPILHEWK, SLNVIHEWK, FFDYDFGSDER, YFDYDFGSEER, and VGDGGPLLQPYPDWSFAK) were consistently detected in Italian honeybee honey. Sequence tracing analysis revealed that these characteristic peptides all originated from the MRJPs family (MRJP1 and MRJP2) and exhibited good stability and species specificity in honey samples from different origins, years, and nectar sources. This invention, for the first time, systematically revealed the compositional differences of animal-derived proteins in honey from Chinese honeybees and Italian honeybees at the proteomics level, and identified bee-derived marker peptides with high species specificity, providing reliable molecular markers for identifying the authenticity of honey from animal sources. Furthermore, this research lays an important foundation for constructing a honey traceability detection method based on MRM and for further analysis of the molecular mechanisms underlying the differences in functional proteins among different bee species. Attached Figure Description

[0018] Figure 1 GO functional annotation of differentially expressed proteins (A) and KEGG pathway (B); Figure 2 MRM profiles of six characteristic peptide segments in different honey samples. Detailed Implementation

[0019] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments: like Figure 1-2 The diagram shows the markers for distinguishing between honey from Chinese honeybees and honey from Italian honeybees, and their applications. Materials and Methods 1.1 Experimental Materials To ensure the authenticity of the samples, honey from Chinese honeybees and Italian honeybees was collected from cooperative demonstration apiaries across the country from 2023 to 2025. The plant origin of all monofloral honeys was determined according to the method of Lutier et al. to ensure their authenticity. After collection, the honey was immediately refrigerated at 4℃ for later use. Sample information is shown in Table 1.

[0020] Table 1 Sample Information ; 1.2 Reagents and Equipment Ammonium bicarbonate, tetraethylammonium borohydride (TEAB), formic acid (Sigma-Aldrich, USA); urea, dithiothreitol (DTT), iodoacetamide (IAM) (Amresco, USA); bovine serum albumin, trypsin, protein quantitative staining solution (Meker, Germany); acetonitrile (Thermo Fisher Scientific, USA). Q-Exactive HF ultra-high electrostatic field orbital trap Fourier transform benchtop mass spectrometer (Thermo Fisher Scientific, USA); G6470 ultra-high performance liquid chromatography-triple quadrupole mass spectrometer (Agilent Technologies, USA).

[0021] 1.3 Experimental Methods 1.3.1 Protein extraction and enzymatic hydrolysis 0.1 g of honey was thoroughly mixed with 2.55 mL of lysis buffer (0.1 mol / L Tris-HCl, 8 mol / L urea, pH 8.5) containing a protease inhibitor, with a lysis buffer:trypsin inhibitor ratio of 50:1. The mixture was sonicated (30 s, 10 s intervals, for a total of 10 min), followed by centrifugation at 12000 × g for 20 min at 4 °C. The supernatant was collected. Protein concentration was determined using the Bradford method and used for subsequent analysis.

[0022] Protein reduction was achieved by adding 100 mmol / L DTT (53 μL) to an appropriate amount of honey lysis solution and incubating at 37°C for 1 h. Alkylation was then performed by adding 100 mmol / L IAM (117 μL) and incubating in the dark for 45 min. The sample was then diluted 4-fold with 25 mmol / L ammonium bicarbonate, and trypsin was added at a protein-to-trypsin mass ratio of 50:1. Digestion was carried out overnight at 37°C. After digestion, formic acid was added to adjust the pH to below 3 to terminate trypsin activity. The digest was desalted and purified using a C18 solid-phase extraction column (Empore Disk Crtdg-C18, 4 mm, 1 mL). The column was first activated with 100% acetonitrile and then equilibrated with 0.1% formic acid aqueous solution. After loading the sample, washing with 0.1% formic acid was used to remove impurities, followed by elution of the target peptide with 70% acetonitrile. The eluent was collected, freeze-dried, reconstituted with 0.1% formic acid aqueous solution, filtered through a 0.22 μm filter membrane, and then used for mass spectrometry analysis.

[0023] 1.3.2 Mass Spectrometry Analysis of Chinese and Italian Honey The complete proteomic identification and analysis of honey from Chinese and Italian bees was performed using an ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-Q-Exactive HF-X, ThermoFisher Scientific, USA) system. Mass spectrometry detection was performed using a Nanospray Flex. The NSI ion source was used with a spray voltage of 2.2 kV and an ion transfer tube temperature of 320 °C. Mass spectrometry employed a data-dependent acquisition mode with a full scan range of m / z 350–1500. The primary mass spectrometry resolution was set to 120,000 (200 m / z), and the automatic gain control (AGC) was 3 × 10⁻⁶. 6 The maximum C-trap injection time was 80 ms. The top 40 precursor ions by ion intensity in the full scan were fragmented using high-energy collisional fragmentation (HCD) and detected by secondary mass spectrometry. The resolution of the secondary mass spectrometry was set to 15000 (200 m / z), and the AGC was 5 × 10⁻⁶. 4 The maximum injection time was 45 ms, and the peptide fragmentation collision energy was 27%, generating raw mass spectrometry data (.raw). Chromatographic separation used mobile phase A (0.1% formic acid aqueous solution) and mobile phase B (0.1% formic acid-80% acetonitrile), with a gradient elution program of: 0-5 min, 8%-12% B; 5-35 min, 12%-30% B; 35-44 min, 30%-40% B; 44-45 min, 40%-95% B; 45-60 min, 95%-95% B. Flow rate: 300 nL / min.

[0024] The raw data were processed using Proteome Discoverer software 3.1 (Thermo Fisher Scientific, Massachusetts, USA). The alignment database was the UniProt database (X0049250405_uniprot_20250529). Search parameters were as follows: fixed modification was cysteine ​​aminomethylation (+57.02146 Da), and variable modification was methionine oxidation (+15.99492 Da). The precursor ion mass tolerance was set to 15 ppm, and the fragment ion mass tolerance was set to 0.02 Da. Trypsin was specified as the restriction enzyme, and a maximum of two deletions of cleavage sites were allowed. A reverse sequence decoy strategy was used to control peptide mislabeling, and the detection results were verified using Percolator software. The false detection rate (FDR) was set to 0.01 during protein and peptide profiling.

[0025] 1.3.3 Targeted Analysis of Characteristic Marker Peptides in Chinese and Italian Honey The selected characteristic marker peptides were targeted for analysis using an ultra-high performance liquid chromatography-triple quadrupole mass spectrometry system (Agilent 6470 Triple Quadrupole LC / MS, Agilent Technologies, USA). Sample detection conditions were as follows: peptide solution (5... L) Samples were loaded onto an AdvanceBio peptide column (2.1 × 150 mm, 2.7 μm, Agilent, USA) using electrospray ionization (ESI) in positive ion mode. The scan range was 350–1500 m / z. The primary mass spectrometry resolution was 60,000 m / z (200 m / z), with an AGC of 300% and a maximum C-trap injection time of Custom. The secondary mass spectrometry detection was performed in "TopSpeed" mode, with a resolution of 15,000 m / z (200 m / z), an AGC of 75%, a maximum injection time of 22 ms, and a peptide fragmentation collision energy of 30%. Raw mass spectrometry data (.raw) were generated. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. The gradient elution program is as follows: 0-5 min, 5%-20% B; 5-14.5 min, 20%-30% B; 14.5-22.5 min, 30%-50% B; 22.5-27.5 min, 50%-70% B; 27.5-30 min, 70%-80% B; 30-35 min, 80%-5% B.

[0026] 1.4 Data Processing GO functional annotation and KEGG pathway analysis were performed on the identified differentially expressed proteins using the Uniprot protein database (http: / / www.ebi.uniprot.org). GO annotation included biological processes (BP), molecular functions (MF), and cellular components (CC). Chromatograms of peptides were extracted using MassHunter qualitative analysis software (Agilent, USA). Statistical analysis was performed using Graphpad Prism software (Version 8.0, USA) and Origin software (Version 9.9.0, OriginLab Corporation, USA).

[0027] 2 Results and Analysis 2.1 Identification and Analysis of Animal-Derived Proteome in Chinese and Italian Honey The protein in honey mainly comes from plant-based pollen, nectar, and the secretions that bees release during the honey-making process. ] Because bee-secreted proteins exhibit significant species specificity, their compositional differences provide potential molecular evidence for identifying the bee species from which honey originates. Therefore, this invention utilizes UPLC-Q-Exactive HF / MS to systematically identify the animal-derived proteomes of honey collected in 2025 from Chinese honeybee (ACH1) and Italian honeybee (AMH1). Based on ProteomeDiscoverer 3.1 software, protein identification was performed using characteristic parameters such as peptide mass-to-charge ratio (m / z), sequence information, fragment ion (b / y ion) analysis, spectral matching score, and false discovery rate (FDR ≤ 1%). 127 proteins (455 peptides) were identified in Chinese honeybee honey, more than the 93 proteins (858 peptides) in Italian honeybee honey, indicating that Chinese honeybees may adapt to complex ecological environments by secreting a wider variety of proteins. The identified peptide lengths are concentrated in the 6–25 amino acid residue range (peak 8–16 residues), consistent with the dynamic range of mass spectrometry detection. The molecular weights of proteins from both Chinese and Italian honeybees are mainly distributed in the 20–90 kDa range, reflecting the conserved physicochemical properties of functional proteins. All identified proteins matched at least one unique peptide, and the more peptides matched, the higher the reliability of the identification results. Furthermore, the average sequence coverage of Italian honeybee proteins (19.42%) was significantly higher than that of Chinese honeybee proteins (9.89%). This difference may be related to the more complete annotation in Italian honeybee databases or differences in enzyme digestion efficiency.

[0028] Next, the proteins identified in ACH1 and AMH1 were functionally classified and compared (Tables 2 and 3). Royal jelly principal proteins (MRJPs) are the most abundant protein family in both types of honey, with MRJP1 being the most abundant in ACH1 (abundance of 5.74 × 10⁻⁶). 9 Among AMH1, MRJP1 has the highest abundance (3.29 × 10⁻⁶). 10 This indicates that MRJP1 is a core protein in both types of honey. Notably, multiple MRJP fragments (such as Q5I223) are present in ACH1, while MRJP8 and MRJP9 are less abundant in AMH1. This may be due to the presence of more variants or degradation products of the MRJP family in Apis cerana. Furthermore, three heat shock proteins (HSPs) were identified in ACH1, compared to only two in AMH1. In particular, HSP70 was more abundant in ACH1 (2.65 × 10⁻⁶). 7This difference may be related to their ability to adapt to environmental stress. Ribosomal proteins are usually closely related to cellular protein synthesis activity. The number of ribosomal proteins identified in ACH1 is significantly higher than that in AMH1, indicating that Chinese honeybee honey has higher protein synthesis-related activity. This difference may be related to the more active secretory activity of the hypopharyngeal or salivary glands of Chinese honeybees. The number and abundance of bee venom-related proteins are much higher in AMH1 than in ACH1. AMH1 contains more than 10 bee venom-related proteins, including phospholipase A1, hyaluronidase, and C1q-like venom protein, which may be related to the stronger defensive behavior and bee venom secretion capacity of Italian honeybees. In addition, ACH1 contains fewer immune-related proteins, while AMH1 contains more immune-related proteins, such as defensin-1, laccase-5, and serine protease inhibitor, indicating that Italian bees have a stronger herd immunity mechanism.

[0029] Table 2. Bee-derived proteins identified in Chinese honeybee honey.

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038] Table 3. Bee-derived proteins identified in Italian honey.

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045]

[0046]

[0047] 2.2 Bioinformatics Analysis of Differential Proteins in Chinese and Italian Honey To further reveal the differences in animal-derived proteomes between honey from Chinese honeybees and honey from Italian honeybees, a T-test was used to analyze the differential expression of identified proteins. p Differentially regulated proteins were screened using thresholds of <0.05 and Fold change (FC) >1.2, ultimately yielding 92 differentially regulated proteins, including 40 upregulated proteins and 52 downregulated proteins. Subsequently, GO functional annotation and KEGG pathway enrichment analysis were performed on the differentially regulated proteins to systematically elucidate their potential biological functions.

[0048] GO functional annotation classifies differentially expressed proteins at three levels: biological process, molecular function, and cellular component. Figure 1A shows the top 20 GO functional entries with the highest enrichment. In terms of molecular function, the differentially enriched proteins are mainly concentrated in key functional categories such as the structural constituent of ribosomes and ATP binding, indicating significant differences in the core components of the protein synthesis machinery between Chinese honeybees and Italian honeybees. This difference may stem from the adaptive adjustments of the two bee species to proteins secreted by different nectar sources. ATP binding, along with ATP hydrolysis activity, GTP binding, and GTPase activity, constitutes the core module of energy metabolism and signal transduction. The high abundance differences of ATP / GTP-related proteins reflect the differences in the intensity of energy consumption (such as sucrose conversion and active transport) during honey maturation. Hydrolase activity is also an important enriched functional category, including alpha-amylase activity and glucose-ceramidase activity. α-Amylase is an important enzyme indicator for evaluating honey quality, and its activity level is often used as a key parameter to measure honey quality and freshness. The enrichment of differentially expressed proteins in this function indicates differences in enzyme activity and maturity between the two honey species. Regarding cellular components, the differentially expressed proteins were mainly enriched in the extracellular region, with 17 proteins identified. This result indicates that most of the differentially expressed proteins are secretory proteins, consistent with the characteristic that honey proteins mainly originate from bee secretions. Furthermore, structures such as ribosomes, small ribosomal subunits, and cytosolic small ribosomal subunits were also significantly enriched, further indicating that ribosome-derived proteins are an important marker for distinguishing between the two honey species. These proteins may originate from secretions from head glands such as the bee's pharyngeal glands or via exosome pathways, reflecting systematic differences between the two bee species in protein "production" (ribosome assembly), "secretory packaging," and "degradation and recycling" mechanisms. In terms of biological processes, translation was the most significant functional category for the enrichment of differentially expressed proteins.In addition, a variety of biological processes related to immune defense were significantly enriched, including defense response to bacterium, defense response to Gram-positive / Gram-negative bacteria, innate immune response, and killing of cells of another organism, indicating that there are certain differences in the composition of antimicrobial defense-related proteins between Chinese honeybees and Italian honeybees.

[0049] KEGG analysis was used to identify the most important biochemical metabolic pathways and signal transduction patterns involving proteins. Figure 1B represents the top 20 KEGG analyses with the most annotated proteins. The results showed that differentially expressed proteins were mainly enriched in four functional pathways: (1) protein degradation and recycling systems, such as the proteasome, lysosome, ubiquitin-mediated proteolysis, and other glycan degradation; (2) carbohydrate metabolism-related pathways, such as starch and sucrose metabolism, the pentose phosphate pathway, galactose metabolism, and ascorbate and aldarate metabolism; and (3) cellular signaling and stress response-related pathways, such as oxidative phosphorylation, mitophagy (animal), autophagy (animal), and protein processing in the endoplasmic reticulum. (4) Genetic information processing pathways, including ribosomes, spliceosomes, and drug metabolism (other enzymes). Among them, the proteasome and lysosome pathways were the most enriched, indicating that protein degradation and turnover processes play an important role in the formation of the honey proteome. In addition, the enrichment of pathways such as starch and sucrose metabolism and galactose metabolism also confirmed the differences in enzyme activity and sugar composition between the two types of honey at the molecular level, and also provided evidence for the differences in quality and nutritional value between the two types of honey.

[0050] 2.3 Screening and Target Validation of Specific Peptides in Chinese and Italian Honey Peptide biomarkers are specific peptide sequences that can be used to distinguish different species. This invention is based on the non-targeted identification results of QExactive HF-X and is screened according to the following principles: (1) The peptide is completely consistent with trypsin, that is, it is cleaved only at the C-terminus of the two terminal arginine (R) or lysine (K) residues, with no cleavage omissions and high reproducibility of enzymatic digestion; (2) The peptide is easy to quantify, excluding peptides labeled "Shared" and "No Quan Value"; (3) The peptide length is between 8 and 20 amino acid residues; (4) Preferred selection is based on the abundance of the precursor ion higher than 10. 6(5) The peptides were required to be stably detected in three technical replicates; (6) Based on the Q Exactive HF-X platform, peptides with symmetrical chromatographic peak shapes and no significant overlap between retention time and molecular weight were screened; (7) The candidate peptides were verified by comparison with the UniProtBLAST database to ensure that the candidate peptides had species-specific differences in the proteomes of Chinese honeybees and Italian honeybees. Based on the above screening strategy, a total of 49 potential characteristic peptides were screened from the animal-derived protein hydrolysate peptides identified by high-resolution mass spectrometry to distinguish between Chinese honeybee honey and Italian honeybee honey. Among them, 16 were from Chinese honeybees and 33 were from Italian honeybees.

[0051] Furthermore, a good biomarker should also possess characteristics such as universality and the ability to perform targeted quantitative analysis. Therefore, this invention further employs UPLC-TQMS to target and validate the aforementioned candidate biomarkers, and expands the honey sample sources (including 8 honeys from Chinese honeybees collected from different regions and years, and 8 honeys from 8 monofloral Italian honeybees from 5 different plant sources) for cross-validation. Due to the complex composition of honey matrix, containing a large amount of carbohydrates, volatile substances, flavonoids, and small molecules such as organic acids, and the high homology between the protein sequences of Chinese and Italian honeybees, as well as the high coexistence ratio of peptides released by enzyme decomposition, some candidate peptides exhibit overlapping chromatographic retention times and ion inhibition in UPLC-TQMS detection, thus reducing the signal-to-noise ratio and making effective differentiation difficult. Through targeted mass spectrometry validation, 6 species-specific peptides with stable detection characteristics were finally screened. Among them, the peptide TVAQSDETLQMIVGMK (m / z 875.9393, z=2) was stably detected in all eight types of Chinese honeybee honey without interference from other peaks, but was not detected in any Italian honeybee honey. Meanwhile, five peptides, SLPILHEWK (m / z 561.8189, z=2), SLNVIHEWK (m / z 563.3062, z=2), FFDYDFGSDER (m / z 699.2859, z=2), YFDYDFGSEER (m / z 714.2911, z=2), and VGDGGPLLQPYPDWSFAK (m / z 973.9860, z=2), were only stably present in Italian honeybee honey, and were not detected in Chinese honeybee honey. Therefore, these six peptides can serve as potential characteristic marker peptides to distinguish between honey from Chinese honeybees and Italian honeybees. Their identification information on QExactive HF-X is shown in Table 4, and the MRM targeting detection method for characteristic peptides on UPLC-TQMS is shown in Table 5. The MRM maps of the target peptides in the honey samples are shown below. Figure 2 As shown.

[0052] Table 4. Identification information of specific peptide markers on Q Exactive HF-X

[0053] Table 5 MRM parameters of specific peptide markers

[0054] By comparing the UniProt BLAST database and tracing protein sequences, this invention discovered that characteristic peptides in honey from Chinese honeybees and Italian honeybees are located in different MRJPs family members, revealing the differentiation characteristics of the two bee species in key functional genes from a molecular evolutionary perspective. The Chinese honeybee-specific biomarker peptide TVAQSDETLQMIVGMK uniquely corresponds to MRJP1 (serial number: Q8MMJ1) from Chinese honeybees. This peptide was consistently detected in honey samples from Chinese honeybees collected from different geographical locations and years, demonstrating the high conservation of the sequence encoding this peptide in the MRJP1 gene within the Chinese honeybee population. As the most abundant functional protein in royal jelly, MRJP1 directly participates in bee-level typological differentiation. Its species-level sequence uniqueness suggests inherent differences between Chinese honeybees and Italian honeybees at the molecular level in the regulation of social behavior. The stable presence of this peptide makes it an ideal target for tracing the origin of Chinese honeybee honey. The specific marker peptides SLPILHEWK, FFDYDFGSDER, and VGDGGPLLQPYPDWSFAK in Italian bee honey are traced back to the Italian bee animal-derived protein MRJP1 (sequence number: O18330), while SLNVIHEWK and YFDYDFGSEERK correspond to the Italian bee animal-derived protein MRJP2 (sequence number: O77061). Notably, two pairs of peptides with highly similar sequences (SLPILHEWK / SLNVIHEWK and FFDYDFGSDER / YFDYDFGSEERK) exist among the markers from Italian bees. This may be related to gene duplication and sequence differentiation that occurred during the evolution of the MRJP gene family. Although MRJP1 and MRJP2 belong to the same royal jelly main protein family and have high sequence homology, substitutions at specific amino acid residues (such as Ile→Val, Asp→Glu, etc.) result in different characteristic peptides after trypsin digestion, thus forming molecular markers that can be used for species identification.

[0055] This invention utilizes high-resolution mass spectrometry (Q Exactive HF-X) to systematically analyze the animal-derived proteomes of honey from Chinese honeybees and Italian honeybees, identifying 127 and 93 proteins, respectively. GO functional annotation and KEGG pathway enrichment analysis revealed that the differentially expressed proteins in the two honey species are mainly involved in ribosome structure, energy metabolism, hydrolytic enzyme activity, and immune defense, showing significant enrichment in pathways such as protein degradation, carbohydrate metabolism, and genetic information processing, indicating significant differences in honey protein composition and potential functions among different bee species. Subsequently, UPLC-TQMS was used to target and validate candidate characteristic peptides, ultimately identifying six stable species-specific marker peptides. One peptide (TVAQSDETLQMIVGMK) was consistently detected in Chinese honeybee honey, while five characteristic peptides (SLPILHEWK, SLNVIHEWK, FFDYDFGSDER, YFDYDFGSEER, and VGDGGPLLQPYPDWSFAK) were consistently detected in Italian honeybee honey. Sequence tracing analysis revealed that these characteristic peptides all originated from the MRJPs family (MRJP1 and MRJP2) and exhibited good stability and species specificity in honey samples from different origins, years, and nectar sources. This invention, for the first time, systematically revealed the compositional differences of animal-derived proteins in honey from Chinese honeybees and Italian honeybees at the proteomics level, and identified bee-derived marker peptides with high species specificity, providing reliable molecular markers for identifying the authenticity of honey from animal sources. Furthermore, this research lays an important foundation for constructing a honey traceability detection method based on MRM and for further analysis of the molecular mechanisms underlying the differences in functional proteins among different bee species.

[0056] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions conceived without inventive effort should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A marker for identifying honey from Chinese honeybees, characterized in that, The marker is a specific peptide, and the amino acid sequence of the specific peptide is shown in SEQ ID No.

1.

2. A marker for identifying Italian bee honey, characterized in that, The marker is a specific peptide, and the amino acid sequence of the specific peptide is shown in SEQ ID No.

2.

3. A marker for identifying Italian bee honey, characterized in that, The marker is a specific peptide, and the amino acid sequence of the specific peptide is shown in SEQ ID No.

3.

4. A marker for identifying Italian bee honey, characterized in that, The marker is a specific peptide, and the amino acid sequence of the specific peptide is shown in SEQ ID No.

4.

5. A marker for identifying Italian bee honey, characterized in that, The marker is a specific peptide, and the amino acid sequence of the specific peptide is shown in SEQ ID No.

5.

6. A marker for identifying Italian bee honey, characterized in that, The marker is a specific peptide, and the amino acid sequence of the specific peptide is shown in SEQ ID No.

6.

7. The application of the marker described in claim 1 in the identification of honey from Chinese honeybees.

8. The application of the markers described in any one of claims 2-6 in the identification of Italian honey.

9. A method for identifying the source of honey, characterized in that, Includes the following steps: (1) Protein extraction and enzymatic hydrolysis were performed on the honey sample to be tested to obtain the peptide sample to be tested; (2) The specific peptides in the sample to be tested were detected by liquid chromatography-mass spectrometry. (3) When a specific peptide segment with the amino acid sequence TVAQSDETLQMIVGMK as described in claim 1 is detected, the honey to be tested is determined to be honey from Chinese honeybees; when one or more of the specific peptide segments described in any one of claims 2-6 are detected, the honey to be tested is determined to be honey from Italian honeybees.

10. The method according to claim 9, characterized in that, The liquid chromatography-mass spectrometry (LC-MS) technique mentioned in step (2) is ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-Q-Exactive HF / MS) or ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-TQMS); the liquid chromatography-mass spectrometry technique is UPLC-Q-Exactive HF / MS or UPLC-TQMS; the specific peptide is derived from MRJP1 or MRJP2 of the main royal jelly protein family MRJPs; the method is used for animal-derived identification, authenticity determination, or quality control of Chinese honeybee honey and Italian honeybee honey.

Images