A method for rapid detection of Amadori compounds in honey
The detection of Amadori compounds in honey by nanoESI-MS technology has solved the problem of difficulty and time-consuming detection in the prior art, achieved rapid and accurate detection results, and can distinguish between natural mature honey and artificial concentrated honey.
Patent Information
- Application Number
- CN202211201764.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-29
- Publication Date
- 2025-03-28
- Estimated Expiration
- 2042-09-29
AI Technical Summary
The prior art is difficult to quickly and accurately detect low levels of Amadori compounds in honey, especially in high sugar complex matrix, which has the problems of complex sample pretreatment and long analysis.
NanoESI-MS technology was used to detect honey samples, and the identification of concentrated honey was achieved by characterizing monosaccharides, amino acids and Amadori compounds, combined with multivariate analysis.
It realizes rapid detection of low-content Amadori compounds in honey, overcomes background interference, and can be effectively applied in complex high-sugar substrates, with simple operation, short detection time, and good application prospects.
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Figure CN115656306B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of detection technology, and in particular to a method for quickly detecting Amadori compounds in honey. Background Art
[0002] Honey is a natural sweet substance that is brewed by bees after collecting nectar or secretions from plants through their own enzymes and special substances. "Compendium of Materia Medica": Honey has five functions as a medicine: clearing away heat and detoxifying, tonifying the middle, removing toxins, moistening dryness, and relieving pain. "Ben Jing": It is used to treat evil spirits in the heart and abdomen, various convulsions, epilepsy, and deficiencies in the five internal organs, replenishing qi and tonifying the middle, relieving pain and detoxifying, and harmonizing all medicines. "Bie Lu": It nourishes the spleen, eliminates irritability, inability to eat or drink, stops diarrhea, muscle pain, mouth sores, and brightens the eyes and ears. Honey is not only a nutritious natural nourishing food, but also widely used in traditional Chinese medicine clinics, Chinese medicine processing, and Chinese medicine preparations as a commonly used Chinese medicine. It has many functions such as regulating the spleen and stomach, relieving pain, moistening the lungs and relieving coughs, moistening the intestines and laxatives, moisturizing the skin and regenerating muscles, and detoxification.
[0003] The conversion from nectar to honey must be completed entirely by bees, and no human intervention is allowed during the maturation process. Thermal processing promotes the accumulation of Maillard reaction products (MRPs), reduces enzyme activity, and loses heat-sensitive components, which seriously affects the quality and safety of honey. Honey is a good matrix for the Maillard reaction. It is rich in monosaccharides (glucose and fructose account for about 75%) and contains a variety of free amino acids. Amadori compounds, as key Maillard reaction products, have been proposed as potential markers for artificial concentrated honey and play an important role in the quality monitoring of a variety of processed foods. Due to the high complexity of the Maillard reaction system, the detection of low-level Amadori compounds in food matrices has always been a difficult problem. The current methods for detecting concentrated honey are mainly based on chromatography-mass spectrometry. Such methods have problems such as complex sample pretreatment and long analysis time, so there are certain limitations in detecting low-content substances in high-sugar matrices. The present invention uses nanoliter electrospray ion source mass spectrometry to quickly detect low-level Amadori compounds in honey, overcomes the background interference generated during sample analysis, and realizes its application in high-sugar complex matrices. Summary of the invention
[0004] In view of the problems mentioned in the background technology, the present invention provides a method for rapid detection of Amadori compounds in honey and identification of concentrated honey based on nanoESI-MS, which specifically adopts the following technical solutions:
[0005] The present invention provides a method for rapidly detecting Amadori compounds in honey and identifying concentrated honey, comprising the following steps:
[0006] Step 1): Prepare the honey to be tested into a honey sample solution for later use;
[0007] Step 2): nanoESI-MS was used to characterize the honey sample solution, detect monosaccharides, amino acids and Amadori compounds, qualitatively identify the substances based on the secondary and tertiary fragment information of the standard, and perform relative quantification based on the response intensity of the secondary fragment ions with higher responses;
[0008] Step 3): Multivariate analysis was performed using mass spectrometry data of m / z 50-500, and a multivariate model was constructed using partial least squares discriminant analysis and orthogonal partial least squares discriminant analysis, and concentrated honey was identified based on the model.
[0009] As a further preferred embodiment of the above scheme, the preparation method of the honey sample solution in step 1) is: stirring the honey to be tested at room temperature, adding water to the fixed volume, and centrifuging to obtain the supernatant to obtain the honey sample solution.
[0010] As a further preferred embodiment of the above scheme, the detection conditions of step 2) nanoESI-MS are: using LTQ-XL linear ion trap, the LTQ spray voltage is 2.25 kV (the actual output is 90% of the reading), the capillary temperature is 300°C, and the solvent is pure water; the spectrum is acquired in positive ion mode, and the secondary mass spectrum is acquired by collision induced dissociation (CID).
[0011] As a further preferred embodiment of the above scheme, in step 2) nanoESI-MS, the borosilicate glass capillary at the tip of the nanoESI has the following characteristics: od = 1.5 mm, id = 0.86 mm.
[0012] As a further preferred embodiment of the above scheme, the Amadori compound includes Fru-Pro and Fru-Phe.
[0013] As a further preferred embodiment of the above scheme, the MS of each substance obtained by collision induced dissociation is 2 and MS 3 Mass spectra are compared with references, standards, and databases and used to identify or predict metabolites.
[0014] As a further preference of the above scheme, the obtained MS 2 In the mass spectrometry, m / z 260 and m / z 310 were used as the relative quantitative ions of Fru-Pro and Fru-Phe, respectively.
[0015] Compared with the prior art, the present invention has the following beneficial effects:
[0016] 1. The method of the present invention does not require pretreatment of high-sugar honey, and nanoESI-MS can be used to directly collect mass spectrum data after dissolution and dilution.
[0017] 2. The detection method of the present invention can realize rapid analysis, and the entire mass spectrometry data acquisition process can be completed within 5 minutes.
[0018] 3. The detection method of the present invention has high sensitivity and can detect low-content Amadori compounds in complex honey matrices. It can be combined with multivariate analysis to quickly distinguish natural mature honey from artificial concentrated honey. It is simple to operate and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Optimization diagram of experimental parameters for nanoESI-MS.
[0020] Figure 2 Schematic diagram of the nanoESI-MS device.
[0021] Figure 3 This is the primary mass spectrum of the honey sample collected by nanoESI-MS in positive ion mode.
[0022] Figure 4 The secondary and tertiary mass spectra of Amadori compounds in honey samples.
[0023] Figure 5 This is the changing trend of the Amadori compound content in honey samples with temperature.
[0024] Figure 6 Changes in the content of Amadori compounds in honey from different plant sources under thermal processing conditions at 55°C.
[0025] Figure 7 3D score plots for partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA) to distinguish between natural mature honey and concentrated honey.
[0026] Figure 8 Permutation test plots for PLS-DA and OPLS-DA. DETAILED DESCRIPTION
[0027] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.
[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.
[0029] Example
[0030] 1. Sample collection and processing
[0031] Immature honey (numbered IH 1-IH 4) was collected in the evening of the same day after new honeycombs were placed in the morning; natural mature honey (numbered NH 1-NH 18) was obtained from cooperative beekeepers in different provinces of China in 2021, including acacia honey, mountain tung honey, and citrus honey; artificial concentrated honey (AH) was prepared in the laboratory using a rotary evaporator (immature honey IH 4 was heated and concentrated at 85°C for 2h; rotary evaporator, parameter settings: speed 90rpm, air pressure 100mbar); all samples were stored at -20°C. One hour before analysis, the honey samples were placed at room temperature (25°C) and manually stirred to ensure uniformity; 0.1g honey sample was diluted to 5.0mL, centrifuged at 8000rpm for 10min, and the supernatant was stored at -80°C.
[0032] 2. Fabrication of Nanopipette
[0033] The nanopipette is a pipette with a conical inner diameter in the nanometer range, which is used to load samples and form an emission nozzle for sample electrospray as an ion source. The borosilicate glass capillary is melt-cut and drawn into a nanopipette using a P-97 drawing instrument.
[0034] 3. NanoESI-MS sample detection
[0035] Mass spectrometry (MS) experiments were mainly performed on a linear ion trap (LTQ XL, Thermo Scientific, San Jose, CA). The LTQ spray voltage was 2.25 kV (actual output was 90% of the reading), the capillary temperature was 300 °C, and the solvent was pure water; the spectra were acquired in positive ion mode, and the secondary mass spectra were acquired by collision-induced dissociation (CID) (experimental parameter optimization as shown in Figure 1 The mass spectrometer scan range was m / z 50-500, and the mass spectrometer data were analyzed on the software Thermo Xcalibur Qual Browser 4.0. NanoESI tip borosilicate glass capillary: od = 1.5 mm, id = 0.86 mm; a 2.25 kV direct current (DC) was connected to the rear of the capillary, and the device was as follows Figure 2 shown.
[0036] 4. Qualitative and quantitative analysis of substances
[0037] Collision induced dissociation (CID) MS of each substance 2 and MS 3 The mass spectra were compared with references, standards (Fructose-proline (Fru-Pro), Fructose-phenylalanine (Fru-Phe, purchased from Toronto Research Chemicals, Toronto, Canada)) and databases (Human Metabolome database, HMDB) to identify or predict metabolites. 2 In mass spectrometry, the ion [M+H-H2O] + The peak value was the highest, so m / z 260 and m / z 310 were used as the relative quantitative ions of Fru-Pro and Fru-Phe, respectively, to detect their content changes.
[0038] 5. Primary fingerprint of honey sample
[0039] In this experiment, nanoESI-MS was used to detect immature water honey, artificial concentrated honey and natural mature honey by mass spectrometry. Figure 3 This is the primary mass spectrum of the honey sample (i.e., the supernatant obtained in step 1) collected by nanoESI-MS in positive ion mode. The main ion peak is: m / z 116 ([proline + H] + ), m / z 166([phenylalanine+H] + )、m / z 198([monosaccharide+NH4] + )、m / z 203([monosaccharide+Na] + )、m / z 219([monosaccharide+K] + )、m / z 278([Fru-Pro+H] + )、m / z 328([Fru-Phe+H] + )、m / z 365([disaccharide+Na] + )、m / z 381([disaccharide+K] + ). Monosaccharides and disaccharides are the main components of honey. They exist in various forms of ion adducts in positive ion mode, such as [M+NH4] + 、[M+Na] + and [M+K] + m / z 116 and m / z 166 are the [M+H] +Peak; Two Amadori compounds were detected at m / z 278 (Fructose-proline, Fru-Pro) and m / z 328 (Fructose-phenylalanine, Fru-Phe), which are usually expressed as [M+H] + After a series of dehydration, the corresponding fragment ions are formed. Figure 4 Shown are the secondary and tertiary mass spectra of Amadori compounds in honey samples (Fru-Pro on the top and Fru-Phe on the bottom). The fragment ions of Amadori compounds in honey are consistent with those of the standard.
[0040] In order to study the effect of heating temperature on the level of Amadori compounds, we heated and concentrated sample IH 4 at 25°C, 40°C, 55°C, 70°C, and 85°C (using a rotary evaporator with parameter settings: speed 90 rpm, air pressure 100 mbar; heating and concentration for 2 h). Figure 5 As shown in the figure, with the increase of temperature, Fru-Pro and Fru-Phe in honey samples showed a clear upward trend. It is worth noting that the signal intensity was significantly enhanced at 55°C, which is the commonly used industrial heat processing temperature for honey. This means that Fru-Pro and Fru-Phe can be used as potential markers for whether honey has been heated and concentrated. To further verify the above results, we analyzed three other groups of immature honeys and artificial concentrated honeys from different plants and geographical sources and obtained similar results, that is, the levels of the two Amadori compounds were significantly increased after heat processing at 55°C, such as Figure 6 As shown (Groups 1, 2, 3, and 4 in the figure correspond to immature water honey IH 1-IH 4 and artificial concentrated honey AH 1-AH 4 under 55°C heating conditions (same as the step parameters of 55°C heating as above), among which the left is Fru-Pro and the right is Fru-Phe; * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001, and ns indicates no significant difference).
[0041] 6. Differentiation between NH and AH based on multivariate models
[0042] Depend on Figure 7 (PLS-DA on the top, OPLS-DA on the bottom) It can be seen that NH and AH are well distinguished in the 3D PLS-DA score graph and the 3DOPLS-DA score graph. Figure 8 is the permutation test diagram of PLS-DA and OPLS-DA, such as Figure 8As shown in the figure, the model did not show overfitting after 200 permutation tests, indicating that the model constructed by the present invention has good stability and reliability. The above results show that nanoESI-MS can be used to quickly detect low-content Amadori compounds in complex honey matrices, and can be combined with multivariate analysis to quickly distinguish natural mature honey from artificial concentrated honey.
[0043] The embodiments described above only express several preferred embodiments of the present invention, and the descriptions thereof are relatively specific and detailed, but are not intended to limit the present invention. It should be noted that for those skilled in the art, the present invention may also have various changes and modifications, and any modifications, equivalent substitutions, improvements, etc. made within the concept and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for rapidly detecting Amadori compounds in honey and identifying concentrated honey, characterized in that: The following steps are involved: Step 1): Prepare the honey to be tested into a honey sample solution for later use; Step 2): The honey sample solution was characterized by using nano-electrospray ion source mass spectrometry to detect monosaccharides, amino acids and Amadori compounds, the substances were qualitatively identified based on the secondary and tertiary fragment information of the standard, and relative quantification was performed based on the response intensity of the secondary fragment ions with higher responses; Step 3): Multivariate analysis was performed using mass spectrometry data of m / z 50-500, and a multivariate model was constructed using partial least squares discriminant analysis and orthogonal partial least squares discriminant analysis, and concentrated honey was identified based on the model.
2. The method for rapid detection of Amadori compounds in honey and identification of concentrated honey according to claim 1, characterized in that: Step 1) The preparation method of the honey sample solution is as follows: stir the honey to be tested at room temperature, add water to make up the volume, and centrifuge to obtain the supernatant to obtain the honey sample solution.
3. The method for rapid detection of Amadori compounds in honey and identification of concentrated honey according to claim 1, characterized in that: Step 2) The detection conditions of nano-electrospray ion source mass spectrometry are as follows: using LTQ-XL linear ion trap, LTQ spray voltage is 2.25 kV, capillary temperature is 300° C., and solvent is pure water; the spectrum is acquired in positive ion mode, and the secondary mass spectrum is acquired by collision induced dissociation.
4. The method for rapid detection of Amadori compounds in honey and identification of concentrated honey according to claim 1, characterized in that: Step 2) In the nano-electrospray ion source mass spectrometry, the tip of the nano-electrospray ion source has a borosilicate glass capillary: od = 1.5 mm, id = 0.86 mm.
5. The method for rapid detection of Amadori compounds in honey and identification of concentrated honey according to claim 1, characterized in that: The Amadori compounds include Fru-Pro and Fru-Phe.
6. The method for rapid detection of Amadori compounds in honey and identification of concentrated honey according to claim 3, characterized in that: The collision-induced dissociation was used to obtain the MS of each substance. 2 and MS 3 Mass spectra are compared with references, standards, and databases and used to identify or predict metabolites.
7. The method for rapid detection of Amadori compounds in honey and identification of concentrated honey according to claim 6, characterized in that: The obtained MS 2 In the mass spectrometry, m / z 260 and m / z 310 were used as the relative quantitative ions of Fru-Pro and Fru-Phe, respectively.