Method for identifying stingless bee honey and application

Abstract

The invention relates to the technical field of honey detection, and particularly discloses a method for identifying stingless bee honey and application. The research finds that mannitol is not detected in other honey except the stingless bee honey, and a mannitol standard sample is easy to obtain and suitable for being used as a feature identifier, so that a method for identifying the stingless bee honey is provided, and when the to-be-detected honey contains mannitol, the to-be-detected honey is judged to be stingless bee honey. The method disclosed by the invention provides a new effective method for identifying the stingless bee honey, and has important significance for identifying the quality / authenticity of the stingless bee honey.

Description

Technical Field

[0001] The invention relates to the technical field of honey detection, in particular to a method for identifying stingless bee honey and its application. Background Art

[0002] Stingless bees belong to the order Hymenoptera, family Apidae, tribe Meliponini. They are called stingless bees because their stingers are degenerate. They mainly live in tropical and subtropical areas. Stingless bees are small in size and have low flying ability. They are the main pollinators of small plants. They can collect sap from flowers around their nests, secretions from plants or insects, and store them in spherical jars made of a mixture of propolis and beeswax, which is stingless bee honey. Stingless bee honey tastes sweet and sour, has a high water content, and is mainly composed of sugars. Studies have found that stingless bee honey is rich in nutrients, such as polyphenol compounds, and has significant pharmacological activity and medicinal value, such as anti-oxidation, anti-inflammatory, antibacterial, wound healing, anti-diabetes, treatment of eye diseases, regulation of body metabolism, regulation of intestinal flora, etc., so it is becoming more and more popular among consumers.

[0003] Mannitol is a sugar alcohol, which is an isomer of sorbitol. Its molecular formula and molecular weight are C 6 H 14 O 6 and 182.17 g / mol. The industrial production of mannitol is difficult and expensive, but it can be extracted and obtained from some natural sources, such as glucose metabolism, vegetables, fruits, algae, fungi, nuts and seeds. At present, the use of mannitol in the food, chemical and medical industries has attracted widespread attention. Compared with sucrose and glucose, mannitol has less effect on blood sugar levels and is very suitable as a food ingredient for dieters and diabetic patients. Because mannitol is non-reducing and does not cause the Maillard reaction, it is used in the baking industry to maintain the color appearance of the product. As an efficient dehydrating agent and diuretic, mannitol helps patients undergoing vascular and cardiac surgery to reduce the increase in intracranial pressure and protect the patient's renal function during surgery. As a hexavalent alcohol, the hydroxyl groups of mannitol are easily substituted, esterified and condensed to produce a series of biologically active derivatives or intermediates. Mannitol has excellent biological functions, but there is no report that it can be used as a characteristic marker of a certain type of honey. Summary of the invention

[0004] One of the objects of the present invention is to provide a new method for identifying stingless bee honey.

[0005] The invention provides a method for identifying stingless bee honey. When the honey to be tested contains mannitol, the honey to be tested is determined to be stingless bee honey.

[0006] The present invention has found that mannitol can be detected only in stingless bee honey, but not in other honeys. Therefore, the present invention proposes a new method for identifying stingless bee honey.

[0007] Mannitol is a sugar alcohol with the structural formula: .

[0008] Mannitol standard samples are easy to obtain and suitable as characteristic identifiers.

[0009] The method of the present invention is applicable to the identification of different species of stingless bee honey, and can identify different species of stingless bee honey (including but not limited to a variety of stingless bees such as yellow striped stingless bees). Lepidotrigona flavibasis , stingless bee Lepidotrigona terminata , Black-breasted stingless bee Tetragonula pagdeni , Goethe's stingless bee Tetragonula g give up , Binn's stingless bee Tetrigona binghami , Cassini Tetragonula carbonara , rainbow stingless bee Tetragonula hockingsi , Medog scaleless bee Lepidotrigona arcifera The honey produced is distinguished from other honeys.

[0010] In the method of the present invention, whether mannitol exists in the honey to be tested is detected by HPLC-RID chromatography.

[0011] The present invention establishes a detection method for mannitol in honey by HPLC-RID, and the method can realize rapid analysis of mannitol, thereby being beneficial to identification of stingless bee honey.

[0012] Specifically, the present invention uses a high performance liquid detector equipped with a differential detector to detect the honey sample to be tested. If the obtained chromatogram contains a characteristic peak of mannitol within the corresponding time, it is determined that the honey contains mannitol and the honey sample to be tested is stingless bee honey; conversely, if the characteristic peak is not contained, it is determined that the honey does not contain mannitol and the honey sample to be tested is not stingless bee honey.

[0013] In the method of the present invention, when the HPLC-RID chromatography method is used for detection, the chromatographic column is Hi-Plex Pb and the mobile phase is water.

[0014] In the method of the present invention, when the HPLC-RID chromatography method is used for detection, the column temperature is 50±5° C. and the pressure is 0.5-0.7 kPa.

[0015] In the method of the present invention, when the HPLC-RID chromatography is used for detection, the differential detector is RID-10A and the detection temperature is 50±5°C.

[0016] In the method of the present invention, when the HPLC-RID chromatography method is used for detection, the injection volume is 20-25 μl; the flow rate is 0.4-0.5 ml / min; and the single gradient elution time is 55-60 min.

[0017] Preferably, in the method of the present invention, when the HPLC-RID chromatography is used for detection, the chromatographic column is Hi-Plex Pb; the mobile phase is water; the column temperature is 50°C; the pressure is 0.6 kPa; the differential detector is RID-10A, and the detection temperature is 50°C; the injection volume is 20 μl; the flow rate is 0.4 ml / min; and the single gradient elution time is 60 min.

[0018] The method of the present invention further comprises the step of pre-treating the honey to be tested before the HPLC-RID chromatography is used for detection; the pre-treatment method comprises: mixing the honey to be tested with water and then taking the supernatant to pass through a nylon membrane.

[0019] The detection method of the present invention does not require complicated pre-treatment of the honey to be tested, and only simple dilution and filtration are required to achieve subsequent detection. Preferably, the aqueous solution of the honey to be tested is filtered through a 0.22 μm nylon filter membrane.

[0020] The present invention also provides application of the method in distinguishing the authenticity of stingless bee honey.

[0021] In the application of the present invention, when it is detected that the honey to be tested contains mannitol, the honey to be tested is determined to be true stingless bee honey; when it is detected that the honey to be tested does not contain mannitol, the honey to be tested is determined not to be stingless bee honey.

[0022] The beneficial effects of the present invention are at least: The present invention determines that mannitol can be used as a characteristic identifier for identifying stingless bee honey, and provides a new determination method for the authenticity and quality evaluation system of stingless bee honey. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 HPLC-RID chromatograms of 7 different honeys.

[0024] Figure 2 HPLC-RID chromatograms of stingless bee honey from different bee species. DETAILED DESCRIPTION

[0025] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0026] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0027] Example 1

[0028] This embodiment detects the components in different honeys, as follows: (1) The following pretreatment was performed on stingless bee honey, acacia honey, jujube honey, vitex honey, manuka honey, honeydew honey and Chinese honey (for details, see Table 1): Accurately weigh 1.0 g of sample in a beaker, add 5 ml of water to dissolve, transfer to a 50 ml volumetric flask, rinse the beaker with 3 ml of water, transfer to the same volumetric flask, and make up to volume with pure water. Use a disposable syringe to take 1 ml of the upper clear sample solution and filter it through a 0.22 μm nylon filter membrane into a sampling vial, ready for injection; (2) The conditions for liquid chromatography detection are: Chromatographic column: Hi-Plex Pb, 300×7.7mm, 8μm; Mobile phase: pure water; Column temperature: 50℃; pressure: 0.6kPa.

[0029] The detection conditions of differential refractometry are: The temperature of the differential detector RID-10A was 50°C; Injection volume: 20 μl; flow rate: 0.4 ml / min; single gradient elution time: 60 min.

[0030] Experimental results: The same method as above was used to test stingless bee honey, acacia honey, jujube flower honey, vitex honey, manuka honey, honeydew honey and Chinese honey. The results are shown in Figure 1 , no chromatographic peaks were detected except for stingless bee honey at the corresponding retention time around 44.5 min.

[0031] In this example, non-stingless bee honey samples from different collection areas / collection times / beehive sources were further tested using the same method (see Table 2 for specific information), and it was found that they also had no chromatographic peaks within the retention time corresponding to around 44.5 min.

[0032] In this embodiment, the substance discharged during the detection of stingless bee honey at a retention time corresponding to about 44.5 min was collected and subjected to nuclear magnetic resonance spectroscopy analysis. 1 H-NMR and 13 C-NMR spectra were compared to confirm that the unique component contained in stingless bee honey is mannitol, which can be used as a characteristic identifier of stingless bee honey.

[0033] Table 1

[0034] Table 2

[0035] Example 2

[0036] In this example, chromatographic detection was performed on stingless bee honey of 7 different bee species, as follows: Test samples: A. 5 mg / mL mannitol standard, B. stingless bee honey from Lepidotrigona flavibasis 、C. Stingless bee honey comes from Lepidotrigona terminata , D. Stingless bee honey comes from Tetragonula pagdeni 、E. Stingless bee honey comes from Tetragonula g give up 、F. Stingless bee honey comes from Tetragonula binghami 、G. Stingless bee honey comes from Tetragonula carbonaria and H. stingless bee honey from Tetragonula hockingsi (See Table 3 for specific information on each honey).

[0037] The chromatographic detection method is the same as in Example 1.

[0038] Experimental results: The method of Example 1 was used to Lepidotrigona flavibasis , Lepidotrigona finished , Tetragonula pagdeni , Tetragonula g give up , Tetrigona binghami , Tetragonula carbonaria and Tetragonula hockingsi A total of 7 different species of stingless bee honey were tested, and the results are shown in Figure 2 At the retention time of 44.5min, the chromatographic peaks of all bee species' stingless bee honeys were detected to be consistent with the peak position of the mannitol standard. This indicates that mannitol can be used as a marker for stingless bee honey.

[0039] Table 3

[0040] Example 3

[0041] In this example, the stingless bee honey from different collection areas / times / beehives was further chromatographically tested according to the method described in Example 1. The specific test sample information is shown in Table 4. The results showed that all stingless bee honeys detected a chromatographic peak consistent with the peak position of the mannitol standard at a retention time corresponding to around 44.5 minutes.

[0042] Table 4

[0043] Example 4

[0044] In this example, according to the method described in Example 1, the mannitol content of other honeys and stingless bee honey was further tested by external standard method according to the peak area (three parallel tests for each sample), and the specific test sample information and test results are shown in Table 5. The experimental results show that mannitol content was detected in all stingless bee honeys, but not in other honeys.

[0045] Table 5

[0046] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for identifying stingless bee honey, characterized in that: When the honey to be tested contains mannitol, the honey to be tested is determined to be stingless bee honey.

2. The method according to claim 1, characterized in that The presence of mannitol in the honey to be tested is detected by HPLC-RID chromatography.

3. The method according to claim 2, characterized in that When the HPLC-RID chromatography method is used for detection, the chromatographic column is Hi-Plex Pb and the mobile phase is water.

4. The method according to claim 3, characterized in that When the HPLC-RID chromatography method is used for detection, the column temperature is 50±5°C and the pressure is 0.5-0.7 kPa.

5. The method according to claim 2, characterized in that: When the HPLC-RID chromatography method is used for detection, the differential detector is RID-10A and the detection temperature is 50±5°C.

6. The method according to any one of claims 2 to 5, characterized in that: When the HPLC-RID chromatography method is used for detection, the injection volume is 20-25 μl; the flow rate is 0.4-0.5 ml / min; and the single gradient elution time is 55-60 min.

7. The method according to claim 6, characterized in that When HPLC-RID chromatography was used for detection, the chromatographic column was Hi-Plex Pb; the mobile phase was water; the column temperature was 50°C; the pressure was 0.6 kPa; the differential detector was RID-10A, and the detection temperature was 50°C; the injection volume was 20 μl; the flow rate was 0.4 ml / min; and the single gradient elution time was 60 min.

8. The method according to claim 7, characterized in that Before the HPLC-RID chromatography is used for the detection, the method further comprises the step of pre-treating the honey to be detected; The pretreatment method is: mixing the honey to be tested with water and then taking the supernatant to pass through a nylon membrane.

9. Use of the method according to any one of claims 1 to 8 in determining the authenticity of stingless bee honey.

10. The use according to claim 9, characterized in that: When it is detected that the honey to be tested contains mannitol, the honey to be tested is determined to be true stingless bee honey; when it is detected that the honey to be tested does not contain mannitol, the honey to be tested is determined not to be stingless bee honey.

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