Peptide fragment for identifying stingless bee honey and application thereof

Abstract

The invention discloses a peptide fragment for identifying stingless bee honey and application of the peptide fragment, and relates to the technical field of biology. The specific peptide fragment for identifying the stingless bee honey is provided, the stingless bee honey and other honey can be accurately distinguished by detecting a specific amino acid sequence, the problems that a traditional identification method is high in subjectivity, physicochemical indexes are prone to interference and the like are solved, and the identification accuracy is greatly improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a peptide segment for identifying stingless bee honey and an application thereof. Background Art

[0002] Stingless bees, belonging to the tribe Meliponini of the family Apidae in the order Hymenoptera, are important pollinating insects in tropical and subtropical regions. Stingless bees collect a wide variety of nectar plants, which also creates the unique flavor and composition characteristics of stingless bee honey. Honey is a natural food that is deeply loved by consumers and has multiple nutritional and health functions. As a type of honey, stingless bee honey has attracted more and more attention in the market due to its unique flavor and potential medicinal value. However, due to the relatively low production of stingless bee honey and the high market price, unscrupulous merchants sell other types of honey as stingless bee honey, which seriously damages the interests of consumers and disrupts the normal order of the honey market.

[0003] At present, the traditional methods for identifying honey types mainly include sensory identification methods and physical and chemical index analysis methods. The sensory identification method mainly relies on human vision, smell, taste and touch to judge the color, smell, taste and state of honey. However, this method is highly subjective, and there are differences in sensory judgments among different people, and it is difficult to accurately identify honey that has been artificially blended or adulterated. The physical and chemical index analysis method is to identify the types of honey by measuring the physical and chemical indicators such as moisture, sugar, acidity, and ash in honey. However, these physical and chemical indicators will also fluctuate in honey from different origins and different collection seasons, and fake honey may adjust its ingredients to meet the requirements of physical and chemical indicators, resulting in inaccurate identification results. It can be seen that the existing methods are difficult to accurately distinguish stingless bee honey from other honeys, and cannot meet the urgent demand for accurate identification of stingless bee honey in the market. In recent years, some protein or peptide-based identification methods have also been reported, but specific identification methods for stingless bee honey are still rare. Therefore, it is of great practical significance to develop a protein or peptide-based method for accurately and reliably identifying stingless bee honey. Summary of the invention

[0004] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a peptide segment for identifying stingless bee honey and its application, which can accurately and quickly determine whether a honey sample is stingless bee honey by detecting a specific amino acid sequence, thereby solving the problems of difficulty in identifying stingless bee honey and low accuracy in the prior art.

[0005] The technical solution of the present invention mainly includes the following contents: On the one hand, the present invention provides the use of a characteristic peptide segment in identifying stingless bee honey, wherein the amino acid sequence of the characteristic peptide segment is shown as SEQ ID NO.2 and / or SEQ ID NO.3.

[0006] Furthermore, the present invention provides the use of a protein containing characteristic peptide segments in the identification of stingless bee honey, the amino acid sequence of the protein is shown in SEQ ID NO.1, and the amino acid sequences of the characteristic peptide segments are shown in SEQ ID NO.2 and / or SEQ ID NO.3.

[0007] In a second aspect, the present invention provides a method for identifying stingless bee honey, the method comprising: detecting a honey sample, if at least one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 is detected, then determining that the honey sample is stingless bee honey, if at least one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 is not detected, then determining that the honey sample is non-stingless bee honey.

[0008] Furthermore, the method comprises: extracting the protein of the honey sample, enzymatic digestion, desalting, and detecting by tandem liquid chromatography-mass spectrometry. If at least one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 is detected, then determining that the honey sample is stingless bee honey, if at least one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 is not detected, then determining that the honey sample is non-stingless bee honey.

[0009] Furthermore, the conditions of the liquid chromatography are as follows: The chromatographic column is an ES-C18 chromatographic column, mobile phase A is an aqueous solution of 0.1% (v / v) formic acid, mobile phase B is an aqueous solution of acetonitrile-0.1% formic acid, and gradient elution is performed.

[0010] Furthermore, the gradient elution program is as follows:

[0011] In a third aspect, the present invention provides a kit for identifying stingless bee honey, the kit contains a standard product, the standard product is glucose dehydrogenase or a characteristic peptide segment, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO.1, and the amino acid sequences of the characteristic peptide segments are shown in SEQ ID NO.2 or SEQ ID NO.3.

[0012] Furthermore, the kit also contains dithiothreitol, iodoacetamide, trypsin, formic acid and an Agilent Bond Elut C18 small column.

[0013] The beneficial effects of the present invention: The present invention provides proteins and characteristic peptide segments for identifying stingless bee honey. By detecting specific amino acid sequences, it can accurately distinguish stingless bee honey from other honeys, avoiding problems such as strong subjectivity and easy interference of physical and chemical indicators in traditional identification methods, and greatly improving the accuracy of identification. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Mass spectrum of the peptide segment "VGGPLDVER".

[0015] Figure 2 : Mass spectrum of the peptide segment "REVILSAGTINSPK". DETAILED DESCRIPTION OF THE INVENTION

[0016] To better understand the technical content of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings.

[0017] Example 1 Selection of Specific Peptide Segments 1.1 Extraction of Bee Proteins Weigh 5 g of honey into a 50 mL centrifuge tube, add 25 μL of internal standard working solution (rMRJP1 protein content is 10 μg / μL), add 3 mL of water, shake for 5 min to fully dissolve, add 20 mL of pre-cooled 10% (v / v) trichloroacetic acid (TCA) acetone solution, shake for 5 min to mix evenly, precipitate at -20 °C for > 2 h; centrifuge at 8000 rpm at 4 °C, discard the supernatant; add 10 mL of pre-cooled 20% (v / v) TCA aqueous solution, shake for 5 min to mix evenly; centrifuge at 8000 rpm at 4 °C, discard the supernatant; add 10 mL of acetone to wash the precipitate, centrifuge at 8000 rpm at 4 °C and discard the supernatant; let it stand for 2 - 3 min to volatilize the acetone; add 1.0 mL of 5 M urea, vortex and shake for 10 min to dissolve the protein precipitate; centrifuge at 8000 rpm at 4 °C, take the supernatant protein solution into a new 1.5 mL centrifuge tube, and detect the protein concentration using a Bradford protein quantification kit. Store the sample solution at -20 °C.

[0018] 1.2 Detection of Proteins by Tandem Liquid Chromatography-Mass Spectrometry Take 100 μL of the protein solution extracted in 1.1 dissolved in 5 M urea (>200 μg), and take 100 μL of the rMRJP1 reference protein solution dissolved in 5 M urea (concentration: 1 μg / μL). Add 400 μL of 40 mM ammonium bicarbonate solution to each and mix well. Then add 50 μL of 0.1 M dithiothreitol (DTT) solution, react at 37 °C for 60 min, add 250 μL of 50 mM iodoacetamide (IAA) solution, react in the dark at room temperature for 40 min, add 10 μL of 0.2 μg / μL trypsin solution, react and digest overnight at 37 °C, add 1.0 μL of formic acid solution to terminate the reaction, and obtain the digested polypeptide solution. Desalt this polypeptide solution through an Agilent Bond Elut C18 chromatographic column (Agilent, 12102025).

[0019] The C18 chromatographic column is first activated with 1 mL of pure acetonitrile solution, successively added with 1 mL of 75% (v / v) acetonitrile / 0.1% (v / v) formic acid aqueous solution and 0.1% (v / v) formic acid aqueous solution for equilibration, then added with about 1 mL of the aforementioned polypeptide solution to adsorb the polypeptide onto the C18 chromatographic column, added 1 mL of 0.1% (v / v) formic acid aqueous solution to elute salts, and then added 1 mL of 75% (v / v) acetonitrile / 0.1% (v / v) formic acid aqueous solution to elute peptide segments. Freeze-dry the final eluate. For the dried polypeptide, add 50 μL of 0.1% (v / v) formic acid aqueous solution to redissolve and wait for injection.

[0020] Use a liquid chromatography system UPLC3000 (Thermo Fisher Scientific) to identify peptide segments by tandem instrument method through an electrospray source and a mass spectrometer Q-Exactive plus (QEplus, Thermo Fisher Scientific).

[0021] The chromatographic conditions are as follows: the analytical column model is ES-C18 (160 Å, 2.1 mm × 50 mm, 2 μm), 0.1% (v / v) formic acid aqueous solution is used as mobile phase A, 80% (v / v) acetonitrile / 0.1% (v / v) formic acid aqueous solution is used as mobile phase B, the injection volume is 5 μL, and the flow rate is 0.3 μL / min; elute according to the gradient elution program in Table 1.

[0022] Table 1 Gradient elution program

[0023] The mass spectrometry conditions were as follows: First, FullMS-MS / MS was used to collect all peptide information: ion signals were collected in data-dependent mode; the precursor ion scan resolution was 70,000 at 400 m / z, the mass-to-charge ratio range was 200 - 2000 m / z, and the top 10 precursor ions with the highest abundance were fragmented by high-energy collision-induced dissociation mode. The MS / MS scan resolution was 17,500, the collision energy was 30, and dynamic exclusion (excluding ions with a charge of 1 or >8; dynamic exclusion: 30 s) was performed. The FullMS-PRM method was used for qualitative and quantitative analysis of target peptide ions: the data acquisition mode and parameters were basically the same as those in the full-scan mode, and the accurate m / z value of the target peptide was filled in the inclusion information section. The MS data were collected by Xcalibur software (version 2.2, ThermoFisher Scientific) and saved as Raw files.

[0024] 1.3 Data processing Download the protein sequences of Apis mellifera Apis mellifera ), Apis cerana Apis cerana ), and stingless bees from the NCBI website. The PEAKS 8.5 software was used to calculate and perform database searching and alignment on the Raw data from the mass spectrometry. The parameter settings were as follows: First, de novo sequencing was performed; the enzyme selected was Tryspin; the fixed modification selected was Carbamidomethyl; the variable modification selected was Oxidation; the precursor ion mass error range was 15.0 ppm; the fragment ion error range was 0.05 Da; each peptide was allowed to have a maximum of 2 missed cleavage sites; each peptide was allowed to have a maximum of 3 post-translational modifications. After the database search was completed, the search results were screened using two conditions: the false discovery rate (FDR) ≤ 1.0% and the unique peptide in the identified proteins ≥ 1.

[0025] Peptide detection and database alignment analysis were performed on Apis mellifera honey samples W1 - W10, Apis cerana honey samples A1 - A10, and 6 stingless bee honey samples. The proteins that were not detected in Apis cerana honey and Apis mellifera honey but were identified in stingless bee honey were selected as glucose dehydrogenase (Glucose dehydrogenase, gi|925682191gb|KOX78881.1), and its identification information is shown in the following table: Table 2 Data detected in 6 samples

[0026] The sequence of glucose dehydrogenase is shown as SEQ ID NO.1.

[0027] SEQ ID NO.1: AGAGGSTVAGRLSEVEKWKVLLIEAGPDEPAGAEIPANLQLYLGSELDWKFETSNEEHACLARDGHCAWPRGRNLGGTTLHHGMAYHRGHPKDYDRWVKEGADGWAWKDVLPYYLKSENNREIKRVGTKYHSVGGPLDVERFPYQPPFAQHILKAAEEVGFGVTEDLVGDKITGFTVAQTISKEGVRTSAVRSYITPVAHRKNLHVAIDAMVTKVNIVDNEATGVHVLMNGETRLIRARREVILSAGTINSPKLLMLSGIGPRDHLKSMKIPVVMNLPGVGENLHNHQSYGLSFTVNEKYYSMLNQNSAEEYLYNQTGPLSSTGLAQVTGLLASNFTDETDPDTQIFFAGYQAICSPKNNIADLTVEDDKMTVMMTSVNVRPMSRGRITLNSNDPLDPPHIWSNDLGTHHDRSVVIQGIRKIQQLSNTQTMKELGLTYVEEHVEQCIDFEYDSDDFWSCIIRWKTRPENHQTGSNKMGPRTNPMAVVSTRLEVHGIKRLRVADASVEPVVVSGNPVASVYMVGERAADFIKQDWGIINL In the mass spectrometry data of each sample, the detected peptide sequences of glucose dehydrogenase were screened out, and these sequences were aligned and screened for characteristics, and 4 identical characteristic peptides were selected.

[0028] Example 2 Verification of the accuracy of identifying stingless bee honey using characteristic peptides and their related proteins Another 10 samples of Western honeybee honey, 10 samples of Eastern honeybee honey and 5 samples of stingless bee honey were selected, and the detection of two peptides, "REVILSAGTINSPK" (SEQ ID NO.2) and "VGGPLDVER" (SEQ ID NO.3), was verified. The experimental method was referred to Example 1. The same results showed that glucose dehydrogenase (gi|925682191 gb|KOX78881.1) could be detected in all stingless bee honey samples, but not in other honey samples. The response values of the peptides "REVILSAGTINSPK" and "VGGPLDVER" were relatively high, and they were stably detected in the verification experiment, and could be used as characteristic peptides for the identification of stingless bee honey.

[0029] Table 3 Response intensities of specific peptide segments in stingless bee samples

[0030] The above are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims

1. Application of a characteristic peptide segment in identifying stingless bee honey, wherein the amino acid sequence of the characteristic peptide segment is shown in SEQ ID NO.2 and / or SEQ ID NO.

3.

2. Application of a protein containing a characteristic peptide segment in identifying stingless bee honey, wherein the amino acid sequence of the protein is shown as SEQ ID NO.1, and the amino acid sequence of the characteristic peptide segment is shown as SEQ ID NO.2 and / or SEQ ID NO.

3.

3. A method for identifying stingless bee honey, characterized in that: include: The honey sample is tested. If at least one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 is detected, the honey sample is determined to be stingless bee honey. If at least one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 is not detected, the honey sample is determined to be non-stingless bee honey.

4. The method according to claim 3, characterized in that include: The protein of the honey sample is extracted, enzymatically digested, and desalted, and the honey sample is detected by a tandem liquid chromatography-mass spectrometry method. If at least one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 is detected, the honey sample is determined to be stingless bee honey. If at least one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 is not detected, the honey sample is determined to be non-stingless bee honey.

5. The method according to claim 4, characterized in that The analytical column used in the liquid chromatography is an ES-C18 chromatographic column.

6. The method according to claim 4, characterized in that In liquid chromatography analysis, gradient elution was used.

7. The method according to claim 6, characterized in that The mobile phase A for gradient elution was 0.1% (v / v) formic acid in water, and the mobile phase B was acetonitrile-0.1% formic acid in water.

8. The method according to claim 7, characterized in that The gradient elution program was: 。 9. A kit for identifying stingless bee honey, characterized in that: The kit contains a standard substance, which is glucose dehydrogenase or a characteristic peptide segment. The amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO.1, and the amino acid sequence of the characteristic peptide segment is shown in SEQ ID NO.2 or SEQ ID NO.

3.

10. The kit according to claim 9, characterized in that The kit also contains dithiothreitol, iodoacetamide, trypsin, formic acid and an Agilent Bond Elut C18 cartridge.

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