A method for identifying buckwheat honey

Abstract

The present invention relates to a method for identifying buckwheat honey, the method comprising: (1) extracting norleucine (Nle) in a honey sample and adding phenyl isothiocyanate for derivatization reaction; (2) using a high performance liquid chromatography-ultraviolet detector to quantitatively detect norleucine derivatives in the honey sample; (3) performing comparative analysis on the norleucine content in the honey sample, and determining the type of the sample to be tested according to the norleucine content; when norleucine is detected in the honey sample to be tested, it is determined that the sample to be tested contains buckwheat honey.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and specifically relates to a method for identifying buckwheat honey. Background Art

[0002] Honey is a natural sweet substance that bees collect from plants, secretions or honeydew, mix with their own secretions, and then fully brew. Honey contains nutrients such as sugars and amino acids. At the same time, it has certain effects in antibacterial, enhancing immune function, relieving cough and moistening lungs, accelerating the repair of traumatic tissues, and anti-tumor. It is now one of the important foods for people's daily consumption. China has a vast territory and abundant nectar plants. However, honey from different nectar sources has certain differences in nutritional content, functional characteristics, quality and price.

[0003] Buckwheat honey is made from buckwheat, which is mostly produced in Inner Mongolia, Ningxia and other places in China. Buckwheat honey is dark amber in color, viscous in texture, and has micro-crystallization. It contains a lot of mineral elements and phenolic substances, has antibacterial and antioxidant activities, and has a certain effect on the treatment of colitis and regulating gastrointestinal function.

[0004] Amino acids are important biologically active substances, and some amino acids are essential components of the human body. The content of amino acids in honey accounts for only about 1%, but it is one of the most important nutrients in honey. Studies have found that amino acids in honey have a certain effect on free radical scavenging, and the content of amino acids in honey affects the antioxidant activity of honey. Since the amino acids in honey mainly come from pollen, there are huge differences in the content and types of amino acids in honey from different nectar plants. Therefore, the amino acid content in honey can be used as a potential basis for distinguishing honey species. The most widely used amino acid determination method is currently pre-column derivatization liquid chromatography. For example, the applicant's prior application CN202011116872.6 involves a method and step for identifying fennel honey using leucine and isoleucine.

[0005] Currently, there is no method for identifying buckwheat honey by amino acid composition, especially by the content of norleucine. Based on this, the present invention is proposed. Summary of the invention

[0006] The present invention firstly relates to a method for identifying buckwheat honey, the method comprising the following steps:

[0007] (1) Norleucine (Nle) in honey samples was extracted and phenyl isothiocyanate was added for derivatization reaction.

[0008] (2) High performance liquid chromatography-ultraviolet detector was used to quantitatively detect the norleucine derivatives in honey samples.

[0009] (3) performing comparative analysis on the norleucine content in the honey samples, and determining the type of the sample to be tested according to the norleucine content;

[0010] The derivatization reaction in step (1) is as follows: the norleucine in the honey sample to be tested is derivatized into phenylaminothiocarboxylic norleucine by using phenyl isothiocyanate and triethylamine;

[0011] The type of honey sample to be tested in step (3) is determined as follows:

[0012] 1) When norleucine is detected in the honey sample to be tested, it is determined that the sample to be tested contains buckwheat honey.

[0013] Preferably, the derivatization reaction in step (1) is:

[0014] 1) Weigh 1 g of honey sample, add 25 mL of 0.1 mol / L hydrochloric acid solution, and vortex for 5 min until the sample is completely dissolved;

[0015] 2) Take 1 mL of the diluted solution in step 1), add 0.5 mL each of 1.2% phenyl isothiocyanate solution and 14% triethylamine solution, mix well and react at room temperature for 1 hour;

[0016] 3) Add 0.1 mL of 2% acetic acid solution, add 2 mL of n-hexane and extract for 2 minutes. After standing for stratification, draw the lower layer solution through a 0.22 μL filter membrane.

[0017] Preferably, the steps of the liquid chromatography method described in step (2) are as follows: the liquid chromatography parameters include:

[0018] Chromatographic column: reverse phase C18 column;

[0019] Mobile phase A: acetonitrile,

[0020] Mobile phase B: 5% acetonitrile solution containing 10 mmol / L ammonium acetate;

[0021] Elution was performed using a gradient method;

[0022] Detector: UV detector; detection wavelength: 254nm.

[0023] More preferably, the steps of the liquid chromatography method in step (2) are as follows: the liquid chromatography parameters include:

[0024] Chromatographic column: reverse phase C18 column (ACQUITY UPLC BEH C18, 100 mm × 2.1 mm, 1.7 μm);

[0025] Flow rate: 0.3 mL / min;

[0026] Injection volume: 2 μL;

[0027] Mobile phase A: acetonitrile,

[0028] Mobile phase B: 5% acetonitrile solution containing 10 mmol / L ammonium acetate;

[0029] The mobile phase gradient was: 0-1 min, 99% mobile phase B, 1-9 min, 82% mobile phase B, 9-13 min, 5% mobile phase B, 13-17 min, 99% mobile phase B.

[0030] Detector: UV detector; detection wavelength: 254nm.

[0031] Preferably, the quantitative detection method in step (2) is an external standard quantitative method; more preferably, the external standard standard curve equation used in the external standard quantitative method is: Y=12100X-2370, and the linear correlation coefficient R2=0.9993.

[0032] In the above external standard curve equation, X is the concentration of the target substance, and Y is the peak area of ​​the target substance.

[0033] The basis for determining the type of the sample to be tested in step (3) further includes:

[0034] (1) When the norleucine content in the honey sample to be tested is ≥15.3 mg / kg, the sample to be tested is determined to be pure buckwheat honey;

[0035] (2) When the norleucine content in the tested honey sample is less than 15.3 mg / kg, it is judged to be adulterated buckwheat honey or mixed buckwheat honey.

[0036] The beneficial effects of the present invention are:

[0037] (1) The present invention determines for the first time the characteristic amino acid marker of buckwheat honey, and buckwheat honey can be effectively identified based on the norleucine content in honey; in particular, it is confirmed that the norleucine content of buckwheat honey is in the range of 15.3 to 58.2 mg / kg;

[0038] (2) The present invention establishes a method for determining norleucine in honey by high performance liquid chromatography, which can achieve accurate separation and rapid analysis of norleucine in honey, and is of great significance for the identification of buckwheat honey. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1UPLC chromatograms of norleucine in representative honey samples: A buckwheat honey, B fennel honey, C acacia honey, D linden honey, E vitex honey, F jujube honey, G rape honey, H litchi honey, I nut honey, G longan honey, K sunflower honey, L alfalfa honey, M astragalus honey, N vetch honey, O wolfberry honey, P tung honey, Q lavender honey, R Schefflera honey, S Amorpha honey, T rubber honey. (Nle: norleucine).

[0040] Figure 2 , the content of norleucine in different types of honey samples.

[0041] Figure 3 , norleucine content in commercial buckwheat honey samples. DETAILED DESCRIPTION

[0042] Instruments and reagents:

[0043] ultra-high pressure liquid chromatograph equipped with UV detector (Waters);

[0044] Vortex-Genie2 vortex mixer (Scientific Industries);

[0045] SA 300 multifunctional oscillator (Yomato);

[0046] Mettler 05XS205DU electronic analytical balance;

[0047] Amino acid standards: Norleucine (Nle) purity is ≥99%;

[0048] Phenyl isothiocyanate (PITC), purity ≥99%;

[0049] Triethylamine, purity ≥99%;

[0050] Ammonium acetate, acetonitrile and n-hexane were all chromatographically pure; the water used in the experiments was ultrapure water.

[0051] Example 1 Determination of norleucine content in honey samples

[0052] Step 1: Sample extraction and derivatization:

[0053] (1) Sample extraction: Weigh 1 g of honey sample, add 25 mL of 0.1 mol / L hydrochloric acid solution, and vortex for 5 min until the sample is completely dissolved.

[0054] (2) Sample derivatization treatment: Take 1 mL of the sample dilution to be tested, add 0.5 mL each of 1.2% phenyl isothiocyanate solution and 14% triethylamine solution, mix well and react at room temperature for 1 h, add 0.1 mL of 2% acetic acid solution, add 2 mL of n-hexane to extract for 2 minutes, let stand for stratification, and then aspirate the lower layer solution to pass through a 0.22 μL filter membrane for testing.

[0055] Step 2: Standard solution preparation and derivatization:

[0056] (1) Preparation of standard solution: Accurately weigh 0.1g of norleucine standard into a 10ml volumetric flask, dissolve it with 0.1mol / L hydrochloric acid and dilute to the mark to prepare a 10000mg / L standard stock solution, and store it at 4°C. Transfer 0.1mL of the single standard stock solution into a 10ml volumetric flask, dissolve it with 0.1mol / L hydrochloric acid and dilute to the mark to prepare a 100mg / L mixed standard solution, and then dilute it with 0.1mol / L hydrochloric acid solution to obtain a mixed standard working solution with concentrations of 0.2mg / L, 1mg / L, 2mg / L, 5mg / L, 10mg / L, and 20mg / L, respectively.

[0057] (2) Derivatization of standard solution: Same as described in step 1 (2).

[0058] Step 3: Ultra-high pressure liquid chromatography:

[0059] Preferred liquid phase conditions:

[0060] ACQUITY UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm) column;

[0061] 0.3mL / min flow rate;

[0062] Injection volume: 2 μL;

[0063] UV detector, detection wavelength 254nm;

[0064] Mobile phase A was acetonitrile, and mobile phase B was 5% acetonitrile solution (containing 10 mmol / L ammonium acetate).

[0065] The mobile phase gradient is:

[0066] 0-1min, 99% mobile phase B,

[0067] 1-9min, 82% mobile phase B,

[0068] 9-13min, 5% mobile phase B,

[0069] 13-17 min, 99% mobile phase B.

[0070] Step 4: Data processing

[0071] By comparing with the standard, the retention time of norleucine was determined to be 7.41min. The external standard method was used to accurately quantify norleucine in honey.

[0072] The standard curve equation is: norleucine: Y=12100X-2370, and the linear correlation coefficient R2=0.9993.

[0073] Example 2: Establishment of norleucine as a characteristic marker of buckwheat honey

[0074] Sample source: The honey samples used in this study were collected during the flowering and nectar flow seasons of various nectar plants. All samples were obtained directly from beekeepers, including: 17 buckwheat honey samples, 10 acacia honey, 10 linden honey, 10 vitex honey, 10 jujube honey, 10 rape honey, 10 litchi honey, 10 nut honey, 10 longan honey, 10 sunflower honey, 10 alfalfa honey, 10 astragalus honey, 10 vetch honey, 10 wolfberry honey, 10 tung honey, 10 lavender honey, 10 Schefflera honey, 10 Amorpha honey, 10 rubber honey. They were kept at 4°C before testing.

[0075] Test procedure: The test procedure and test conditions are the same as in Example 1.

[0076] The method of the present invention was used to determine the norleucine in honey samples from 20 different nectar plants. The representative sample chromatograms are shown in FIG. Figure 1 The analysis of the norleucine content in all honey samples revealed that there was a significant difference in the norleucine content between buckwheat honey and other honey types ( Figure 2 ), norleucine was only detected in buckwheat honey, so the content of norleucine can be used as the basis for the identification of buckwheat honey.

[0077] The basis for distinguishing buckwheat honey finally determined by the test values ​​of the above research samples is: when norleucine is detected in the honey sample and its content is between 15.3 and 58.2 mg / kg, it is judged to be pure buckwheat honey; if the sample does not contain norleucine, it is judged to be non-buckwheat honey; if norleucine is detected in the sample but its content is not within the above range, it is judged to be adulterated buckwheat honey or mixed buckwheat honey.

[0078] Example 3: Actual sample detection and identification

[0079] Sample source: 10 batches of commercial buckwheat honey samples were purchased from an online e-commerce platform.

[0080] The content of norleucine in the commercial buckwheat honey sample was determined by ultra-high pressure liquid chromatography, and the test steps and conditions were the same as those in Example 1.

[0081] The contents of norleucine in 10 batches of commercial buckwheat honey samples ( Figure 3 ) were analyzed, and the results showed that norleucine was detected in all 10 batches of buckwheat honey, with a content range of 8.4-51.5 mg / kg. According to the method of the present invention, only 50% of the samples (No.1-No.5) had a norleucine content of 20.5-51.6 mg / kg, which was within the identification range determined in the above Example 2 and was identified as pure buckwheat honey; the other 50% of the samples (No.6-No.10) had a norleucine content that was not within the above identification range and were identified as non-pure buckwheat honey, adulterated or mixed buckwheat honey, indicating that there is a relatively serious adulteration or mixed buckwheat honey phenomenon in the market. The above test results show that the method of the present invention can be effectively applied to the identification of buckwheat honey in actual samples.

[0082] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention and are not used to limit the protection scope of the present invention.

Claims

1. A method for identifying buckwheat honey, the method comprising the following steps: (1) extracting norleucine from a honey sample and adding phenyl isothiocyanate for derivatization reaction; (2) Quantitative detection of norleucine derivatives in honey samples using high performance liquid chromatography-ultraviolet detector; (3) performing comparative analysis on the norleucine content in the honey samples, and determining the type of the honey sample to be tested according to the norleucine content; in, The derivatization reaction in step (1) is: The norleucine in the honey sample to be tested is derivatized to phenylaminothiocarboxylic norleucine by phenyl isothiocyanate and triethylamine, and the derivatization reaction steps are as follows: 1) Weigh 1 g of honey sample, add 25 mL of 0.1 mol / L hydrochloric acid solution, and vortex for 5 min until the sample is completely dissolved; 2) Take 1 mL of the diluted solution in step 1), add 0.5 mL each of 1.2% phenyl isothiocyanate solution and 14% triethylamine solution, mix well and react at room temperature for 1 hour; 3) Add 0.1 mL of 2% acetic acid solution, add 2 mL of n-hexane and extract for 2 minutes. After standing for stratification, draw the lower layer solution through a 0.22 μL filter membrane; The type of honey sample to be tested in step (3) is determined as follows: When norleucine is detected in a honey sample and its content is between 15.3 and 58.2 mg / kg, it is judged to be pure buckwheat honey; if the sample does not contain norleucine, it is judged to be non-buckwheat honey; if norleucine is detected in the sample but its content is not within the above range, it is judged to be adulterated buckwheat honey or mixed buckwheat honey.

2. The method according to claim 1, characterized in that The liquid chromatography parameters of step (2) include: Chromatographic column: reverse phase C18 column; Flow rate: 0.3 mL / min; Injection volume: 2 μL; Mobile phase A: acetonitrile, Mobile phase B: 5% acetonitrile solution containing 10 mmol / L ammonium acetate; Elution was performed using a gradient method; The mobile phase gradient is: 0-1min, 99% mobile phase B, 1-9min, 82% mobile phase B, 9-13min, 5% mobile phase B, 13-17 min, 99% mobile phase B; Detector: UV detector; detection wavelength: 254nm.

3. The method according to claim 1 or 2, characterized in that: The quantitative detection method described in step (2) is an external standard quantitative method.

4. The method according to claim 3, characterized in that The external standard standard curve equation used in the external standard quantitative method is: Y = 12100X-2370, and the linear correlation coefficient R 2 =0.9993; in the above external standard curve equation, X is the concentration of the target substance, and Y is the peak area of ​​the target substance.

Images