Peptides for identifying stingless bee honey and their applications

Abstract

The present invention discloses peptides for identifying stingless bee honey and their applications, relating to the field of biotechnology. Specifically, the present invention provides characteristic peptides for identifying stingless bee honey. By detecting specific amino acid sequences, it is possible to 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.

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:

[0006] On the one hand, the present invention provides the use of characteristic peptide segments in the identification of stingless bee honey, and the amino acid sequences of the characteristic peptide segments are as shown in SEQ ID NO.2 and / or SEQ ID NO.3.

[0007] 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 as shown in SEQ ID NO.1, and the amino acid sequences of the characteristic peptide segments are as shown in SEQ ID NO.2 and / or SEQ ID NO.3.

[0008] On the second hand, the present invention provides a method for identifying stingless bee honey, which method comprises: detecting a honey sample, and 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 none 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 non-stingless bee honey.

[0009] Furthermore, the method comprises: extracting the protein of the honey sample, enzymatically digesting, 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 none 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 non-stingless bee honey.

[0010] Furthermore, the conditions of the liquid chromatography are as follows:

[0011] 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 acetonitrile-0.1% formic acid aqueous solution, and gradient elution is performed.

[0012] Furthermore, the gradient elution program is as follows:

[0013]

[0014] On the third hand, the present invention provides a kit for identifying stingless bee honey. The kit contains a standard product, and the standard product is glucose dehydrogenase or a characteristic peptide segment. The amino acid sequence of the glucose dehydrogenase is as shown in SEQ ID NO.1, and the amino acid sequences of the characteristic peptide segments are as shown in SEQ ID NO.2 or SEQ ID NO.3.

[0015] Further, the kit also contains dithiothreitol, iodoacetamide, trypsin, formic acid, and an Agilent Bond Elut C18 column.

[0016] Advantages of the present invention:

[0017] 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. Description of the Drawings

[0018] Figure 1 : Mass spectrum of the peptide segment "VGGPLDVER".

[0019] Figure 2 : Mass spectrum of the peptide segment "REVILSAGTINSPK". Detailed Embodiments

[0020] To better understand the technical content of the present invention, the present invention will be further described below in conjunction with specific embodiments and the drawings.

[0021] Example 1 Selection of Specific Peptide Segments

[0022] 1.1 Extraction of Bee Proteins

[0023] Weigh 5 g of honey into a 50 mL centrifuge tube, add 25 μL of the 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 and 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 and 4°C, discard the supernatant; add 10 mL of acetone to wash the precipitate, centrifuge at 8000 rpm and 4°C to discard the supernatant; let the acetone evaporate for 2 - 3 min; add 1.0 mL of 5 M urea, vortex and shake for 10 min to dissolve the protein precipitate; centrifuge at 8000 rpm and 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.

[0024] 1.2 Detection of Proteins by Tandem Liquid Chromatography - Mass Spectrometry

[0025] 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 of 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 and react at 37 °C for 60 min. Next, add 250 μL of 50 mM iodoacetamide (IAA) solution and react in the dark at room temperature for 40 min. Then add 10 μL of 0.2 μg / μL trypsin solution and react at 37 °C overnight for enzymatic digestion. Add 1.0 μL of formic acid solution to terminate the reaction, obtaining the digested polypeptide solution. Desalt this polypeptide solution through an Agilent Bond Elut C18 chromatographic column (Agilent, 12102025).

[0026] The C18 chromatographic column is first activated with 1 mL of pure acetonitrile solution, and then 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 add about 1 mL of the aforementioned polypeptide solution to adsorb the polypeptide onto the C18 chromatographic column. Add 1 mL of 0.1% (v / v) formic acid aqueous solution to elute the salt, and then add 1 mL of 75% (v / v) acetonitrile / 0.1% (v / v) formic acid aqueous solution to elute the 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 onto the machine.

[0027] Peptide identification is carried out by using a liquid chromatography system UPLC3000 (Thermo Fisher Scientific) in tandem with an electrospray source and a mass spectrometer Q-Exactive plus (QEplus, Thermo Fisher Scientific).

[0028] The chromatographic conditions are as follows: the analytical column model is ES-C18 (160 Å, 2.1 mm×50 mm, 2 μm), with 0.1% (v / v) formic acid aqueous solution as mobile phase A and 80% (v / v) acetonitrile / 0.1% (v / v) formic acid aqueous solution as mobile phase B. The injection volume is 5 μL, and the flow rate is 0.3 μL / min; elution is carried out according to the gradient elution program in Table 1.

[0029] Table 1 Gradient elution program

[0030]

[0031] 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 resolution of the parent ion scan was 70,000 at 400 m / z, and the mass-to-charge ratio range was 200 - 2000 m / z. For the top 10 parent ions with the highest abundance, fragment ions were generated by high-energy collision-induced dissociation mode. The resolution of the MS / MS scan was 17,500, the collision energy was 30, and dynamic exclusion was performed (excluding those with a charge of 1 or >8; dynamic exclusion: 30 s). The FullMS-PRM method was used to qualitatively and quantitatively analyze the 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 part. The mass spectrometry part collected MS / MS data through Xcalibur software (version 2.2, ThermoFisher Scientific) and saved it as a Raw file.

[0032] 1.3 Data processing

[0033] Download the protein sequences of Apis mellifera ( Apis mellifera ), Apis cerana ( Apis cerana ), and stingless bees from the NCBI website, and use PEAKS 8.5 software to calculate and perform database searching and alignment on the Raw data obtained from mass spectrometry. The parameter settings were as follows: First, de novo sequencing calculation was performed, and the enzyme selected was Trypsin; the fixed modification selected was Carbamidomethyl; the variable modification selected was Oxidation; the mass error range of the parent ion 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 database searching, the search results were screened using two conditions: the false discovery rate (FDR) ≤ 1.0% and the unique peptide in the identified proteins ≥ 1.

[0034] 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 as follows in the table:

[0035] Table 2 Data detected in 6 samples

[0036]

[0037] The sequence of glucose dehydrogenase is shown in SEQ ID NO.1.

[0038] SEQ ID NO.1:

[0039] AGAGGSTVAGRLSEVEKWKVLLIEAGPDEPAGAEIPANLQLYLGSELDWKFETSNEEHACLARDGHCAWPRGRNLGGTTLHHGMAYHRGHPKDYDRWVKEGADGWAWKDVLPYYLKSENNREIKRVGTKYHSVGGPLDVERFPYQPPFAQHILKAAEEVGFGVTEDLVGDKITGFTVAQTISKEGVRTSAVRSYITPVAHRKNLHVAIDAMVTKVNIVDNEATGVHVLMNGETRLIRARREVILSAGTINSPKLLMLSGIGPRDHLKSMKIPVVMNLPGVGENLHNHQSYGLSFTVNEKYYSMLNQNSAEEYLYNQTGPLSSTGLAQVTGLLASNFTDETDPDTQIFFAGYQAICSPKNNIADLTVEDDKMTVMMTSVNVRPMSRGRITLNSNDPLDPPHIWSNDLGTHHDRSVVIQGIRKIQQLSNTQTMKELGLTYVEEHVEQCIDFEYDSDDFWSCIIRWKTRPENHQTGSNKMGPRTNPMAVVSTRLEVHGIKRLRVADASVEPVVVSGNPVASVYMVGERAADFIKQDWGIINL

[0040] In the mass spectrometry data of each sample, the detected peptide sequences of glucose dehydrogenase were screened out, and these sequences were aligned and characterized, and 4 identical characteristic peptides were selected.

[0041] Example 2 Verification of the accuracy of identifying stingless bee honey using characteristic peptides and their related proteins

[0042] Another 10 samples of honey from Western honeybees, 10 samples of honey from Eastern honeybees and 5 samples of honey from stingless bees were selected, and the detection of two peptide segments "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 samples of honey from stingless bees, but not in other honey samples. The response values of the peptide segments "REVILSAGTINSPK" and "VGGPLDVER" were relatively high, and they were stably detected in the verification experiment, and could be used as characteristic peptide segments for the identification of honey from stingless bees.

[0043] Table 3 Response intensities of specific peptide segments in samples of stingless bees

[0044]

[0045] 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 characteristic peptide segments in identifying stingless bee honey, wherein the amino acid sequences of the characteristic peptide segments are as shown in SEQ ID NO.2 and / or SEQ ID NO.

3.

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

3.

3. A method for identifying stingless bee honey, characterized in that, Including: 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, it is determined 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, it is determined that the honey sample is non-stingless bee honey.

4. The method according to claim 3, wherein Including: Extracting the protein of the honey sample, digesting with enzymes, 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, it is determined 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, it is determined that the honey sample is non-stingless bee honey.

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

6. The method according to claim 4, wherein Gradient elution is used for elution in liquid chromatography analysis.

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

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

3.

10. The kit according to claim 9, wherein, The kit also contains dithiothreitol, iodoacetamide, trypsin, formic acid and an Agilent Bond Elut C18 small column.

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