Method for identifying Chinese mountain sapium sebiferum honey and Italian mountain sapium sebiferum honey based on amino acid ratio
By detecting the difference in the ratio of proline and phenylalanine in honey and using instruments such as liquid chromatography to identify honey from Chinese bees and Italian bees, the problem of high subjectivity of traditional identification methods is solved, and rapid and accurate identification of honey bee species is achieved.
Patent Information
- Application Number
- CN202510943330.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-09
- Publication Date
- 2025-09-30
AI Technical Summary
Existing technologies make it difficult to effectively identify Tung oil honey brewed by Chinese bees and Italian bees, resulting in frequent adulteration in the market. Traditional identification methods are highly subjective and have low accuracy.
By detecting the difference in the ratio of proline and phenylalanine in the total free amino acid content in honey, an identification method based on amino acid ratio is provided, and the detection is carried out using instruments such as liquid chromatography and liquid chromatography-mass spectrometry.
It achieves rapid and accurate identification of honey from Chinese bees and Italian bees, improves the simplicity and accuracy of identification, and supports honey authenticity identification and market supervision.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of honey detection, and particularly relates to a method for identifying Chinese bee mountain tallow tree honey and Italian bee mountain tallow tree honey based on amino acid ratio. Background Art
[0002] Honey, a beloved natural sweet food, is of great concern for its quality and authenticity. While Chinese tallow tree honey boasts a unique flavor and nutritional value, the market value of honey from Apis cerana (Chinese honey bee) and Apis mellifera (Italian honey bee) differs significantly. As a native bee species, Chinese honey produces low yields, a unique flavor, and a higher price than Italian honey bees. However, existing detection technologies struggle to effectively identify the origin of these bees, leading to widespread adulteration in the market. Differences between Chinese and Italian honey bees in nectar collection, the brewing process, and digestive physiology can lead to differences in the composition of their honeys. Traditional identification methods rely primarily on sensory evaluation, such as observing the honey's color, odor, and taste. However, this method is highly subjective, susceptible to environmental factors and individual differences, and has low accuracy. While some scientific testing techniques exist for honey composition analysis, identification of the origins of different bee species in Chinese tallow tree honey remains underdeveloped. Summary of the Invention
[0003] Aiming at the problems that the existing honey bee species identification technology has high cost, complex operation, lack of pertinence, and difficulty in accurately and quickly identifying the bee species source of Sapium sebiferum honey, the present invention provides a method for identifying Sapium sebiferum honey produced by Chinese bees and Sapium sebiferum honey produced by Italian bees.
[0004] The present invention detects and analyzes the components of a large number of Chinese and Italian bee-brewed tung oil honeys from different sources and finds that there are differences in the distribution of free amino acids between Chinese and Italian bee tung oil honeys. Specifically, there are significant differences in the proportion of proline and phenylalanine in the total free amino acid content between Chinese and Italian bee tung oil honeys, which can be used as a basis for distinguishing Chinese and Italian bee tung oil honeys. Based on this, the present invention provides a method for distinguishing Chinese and Italian bee tung oil honeys based on the difference in the ratio of proline and phenylalanine to the free amino acid content.
[0005] Specifically, the present invention provides the following technical solutions.
[0006] In a first aspect, the present invention provides a method for identifying Chinese bee mountain tung oil honey and Italian bee mountain tung oil honey, the method comprising: identifying Chinese bee mountain tung oil honey and Italian bee mountain tung oil honey based on the ratio of proline content to the total content of free amino acids and / or the ratio of phenylalanine content to the total content of free amino acids in the honey sample.
[0007] The present invention detected and analyzed a large number of honey samples, determined the proportion characteristics of proline and phenylalanine in the total free amino acid content of Zhongfeng Mountain Sapium honey and Italian Bee Mountain Sapium honey, and determined the identification basis of Zhongfeng Mountain Sapium honey and Italian Bee Mountain Sapium honey through threshold judgment.
[0008] Characteristics of Zhongfengshan Tallow Honey: The ratio of phenylalanine content in the total free amino acid content is significantly higher than that of proline, with the proline ratio ranging from 5% to 25% and the phenylalanine ratio ranging from 45% to 75%; Characteristics of Italian bee mountain tung oil honey: the ratio of proline content in the total free amino acid content is significantly higher than that of phenylalanine, the proline ratio is between 45%-80%, and the phenylalanine ratio is between 1%-30%.
[0009] In the above method, if the ratio of proline content in the total free amino acid content is between 5%-25%, and / or the ratio of phenylalanine content in the total free amino acid content is between 45%-75%, it is determined to be Chinese bee mountain tung oil honey; if the ratio of proline content in the total free amino acid content is between 45%-80%, and / or the ratio of phenylalanine content in the total free amino acid content is between 1%-30%, it is determined to be Italian bee mountain tung oil honey.
[0010] Preferably, if the ratio of proline content in the total free amino acid content is between 7%-23%, and / or the ratio of phenylalanine content in the total free amino acid content is between 45%-74%, it is determined to be Chinese bee mountain tung oil honey; if the ratio of proline content in the total free amino acid content is between 45%-80%, and / or the ratio of phenylalanine content in the total free amino acid content is between 1%-28%, it is determined to be Italian bee mountain tung oil honey.
[0011] Preferably, the ratio of proline content to the total free amino acid content and the ratio of phenylalanine content to the total free amino acid content in the honey sample are used as the basis for distinguishing between Chinese bee mountain tallow honey and Italian bee mountain tallow honey. The combined ratio of proline content and phenylalanine content is conducive to improving the accuracy of identification.
[0012] In the above method, the free amino acids include leucine, isoleucine, threonine, proline, valine, serine, alanine, histidine, methionine, glutamic acid, glutamine, aspartic acid, asparagine, lysine, tyrosine, arginine, phenylalanine, tryptophan, glycine and γ-aminobutyric acid.
[0013] The total content of the free amino acids is preferably the sum of the contents of the above 20 amino acids.
[0014] In principle, the present invention has no particular limitation on the method for detecting the content of free amino acids in honey samples. Commonly used methods for detecting amino acid content may be used, such as liquid chromatography, liquid chromatography-mass spectrometry, or ion exchange resin separation-ninhydrin colorimetry.
[0015] In the above method, the content of free amino acids is detected by liquid chromatography, liquid chromatography-mass spectrometry or ion exchange resin separation-ninhydrin colorimetry.
[0016] Specifically, a variety of instrumental methods can be used to detect the free amino acid content, such as automatic amino acid analyzer, liquid chromatograph, liquid chromatography-mass spectrometry, etc.
[0017] As an embodiment, separation is performed using a hydrophilic chromatography column (HILIC-Z or BEH Amide), and then determination is performed using a high performance liquid chromatography-mass spectrometer.
[0018] As an embodiment, phenyl isothiocyanate is used for pre-column derivatization, followed by separation on a reverse phase chromatography column (C18), and detection using an ultraviolet-visible detector.
[0019] As an embodiment, pre-column derivatization is performed with o-phthalaldehyde and 9-fluorenyl methyl chloroformate, followed by separation on an amino acid chromatography column (AdvanceBio AAA), and determination is performed using high performance liquid chromatography-fluorescence detection.
[0020] As an embodiment, an automatic amino acid analyzer is used, the amino acids are separated by an ion exchange resin column, and the amino acids are determined by ninhydrin post-column derivatization.
[0021] In some specific embodiments of the present invention, liquid chromatography-mass spectrometry is used to detect the content of free amino acids.
[0022] The above method further includes the step of pre-treating the honey sample before detecting the free amino acid content; the pre-treatment includes the steps of dissolving, separating and removing insoluble impurities, and filtering with a filter membrane.
[0023] Preferably, the dissolution is carried out using water or an aqueous hydrochloric acid solution (preferably 0.05-0.2 mol / L).
[0024] Preferably, the separation and removal of insoluble impurities is performed by centrifugation.
[0025] Preferably, the membrane filtration uses a 0.22 μm membrane filtration.
[0026] In a second aspect, the present invention provides a method for detecting the authenticity of Zhongfengshan Sapium sebiferum honey, the method comprising: judging whether the honey sample is Zhongfengshan Sapium sebiferum honey based on the ratio of proline content to the total content of free amino acids and / or the ratio of phenylalanine content to the total content of free amino acids in the honey sample.
[0027] The above-mentioned detection of the authenticity of Zhongfengshan Tallow Honey includes detecting whether the Zhongfengshan Tallow Honey is adulterated Zhongfengshan Tallow Honey, and the adulterated Zhongfengshan Tallow Honey is adulterated with Yifengshan Tallow Honey.
[0028] In the above method, if the ratio of proline content in the total free amino acid content is between 5%-25%, and / or the ratio of phenylalanine content in the total free amino acid content is between 45%-75%, it is determined to be Zhongfengshan Sapium sebiferum honey.
[0029] Preferably, if the ratio of proline content in the total free amino acid content is between 7%-23%, and / or the ratio of phenylalanine content in the total free amino acid content is between 45%-74%, it is determined to be Zhongfengshan Sapium sebiferum honey.
[0030] Preferably, the authenticity of the Zhongfengshan Tallow Honey is tested based on the ratio of the proline content to the total free amino acid content and the ratio of the phenylalanine content to the total free amino acid content in the honey sample. The combined ratio of the proline content and the phenylalanine content ratio is conducive to improving the accuracy of the test.
[0031] In the above method, the free amino acids include leucine, isoleucine, threonine, proline, valine, serine, alanine, histidine, methionine, glutamic acid, glutamine, aspartic acid, asparagine, lysine, tyrosine, arginine, phenylalanine, tryptophan, glycine and γ-aminobutyric acid.
[0032] In the above method, the content of amino acids is detected by liquid chromatography, liquid chromatography-mass spectrometry or ion exchange resin separation-ninhydrin colorimetry.
[0033] In some specific embodiments of the present invention, liquid chromatography-mass spectrometry is used to detect the content of free amino acids.
[0034] In the above method, before detecting the amino acid content, the step of pre-treating the honey sample is also included; the pre-treatment includes the steps of dissolving, separating and removing insoluble impurities and filtering with a filter membrane.
[0035] Preferably, the dissolution is carried out using water or an aqueous hydrochloric acid solution (preferably 0.05-0.2 mol / L).
[0036] Preferably, the separation and removal of insoluble impurities is performed by centrifugation.
[0037] Preferably, the membrane filtration uses a 0.22 μm membrane filtration.
[0038] The present invention provides at least one beneficial effect: providing a method for distinguishing Chinese and Italian bee Tung honey based on the ratio of proline and phenylalanine content to the total free amino acid content. This method offers the advantages of simplicity, speed, and high accuracy, enabling rapid identification of the bee species of Tung honey. It provides a new method and technical support for honey authenticity testing and market regulation, and is of great significance for promoting the healthy development of the honey industry. BRIEF DESCRIPTION OF THE DRAWINGS
[0039] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0040] Figure 1 The proline ratio in the Sapium sebiferum honey from the standardized bee farm in Example 1 of the present invention is shown.
[0041] Figure 2 This is the phenylalanine ratio in the Sapium sebiferum honey from the standardized bee farm in Example 1 of the present invention.
[0042] Figure 3 It is the proline ratio in the Chinese tallow honey from the main producing area of certain mountain tallow honey in Example 2 of the present invention.
[0043] Figure 4 This is the phenylalanine ratio in the Chinese tallow honey from the main producing area of certain mountain tallow honey in Example 2 of the present invention. DETAILED DESCRIPTION
[0044] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.
[0045] The method for detecting free amino acids in the Chinese bee mountain tallow honey and the Italian bee mountain tallow honey used in the following examples is as follows: 1. Sample pretreatment Weigh an appropriate amount of honey sample, add 0.1 mol / L aqueous hydrochloric acid, and vortex until completely dissolved. Centrifuge to remove insoluble impurities. Filter the supernatant through a 0.22 μm filter membrane and prepare for high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) injection.
[0046] 2. Instrumental analysis The specific method of LC-MS / MS detection is as follows: (1) High performance liquid chromatography separation Column: Waters BEH Amide 1.7 μm, 2.1 × 100 mm; Column temperature: 40°C; Flow rate: 0.3 mL / min; Injection volume: 2 μL; Mobile phase: Phase A: water (0.1% formic acid-2mmol ammonium acetate); Phase B: 90% acetonitrile (0.1% formic acid-2 mmol ammonium acetate); The elution gradient is shown in Table 1.
[0047] Table 1
[0048] (2) Mass spectrometry detection Ion source type: ESI source, positive ion mode scanning. The mass spectrometry detection information of 20 free amino acids is shown in Table 2.
[0049] Table 2
[0050] 3. Data processing The ratios (i.e., proportions) of proline and phenylalanine in the total content of 20 free amino acids (leucine, isoleucine, threonine, proline, valine, serine, alanine, histidine, methionine, glutamic acid, glutamine, aspartic acid, asparagine, lysine, tyrosine, arginine, phenylalanine, tryptophan, glycine, and γ-aminobutyric acid) in honey were calculated respectively.
[0051] Example 1 In a standardized bee farm in the main production area of Sapium sebiferum honey, both Chinese and Italian bee colonies were placed, and new honey combs were used in the beehives. Sapium sebiferum honey was collected in the same environment and at the same time. After the collection was completed, 30 samples of Sapium sebiferum honey brewed by Chinese and Italian bees were obtained. After the samples were dissolved, centrifuged, and filtered, the free amino acids were quantified and analyzed using the aforementioned LC-MS / MS method. The results are as follows Figure 1 and Figure 2As shown in the results, the ratio of proline content to the total free amino acid content of 20 in Zhongfengshan Tallow Honey ranged from 7% to 19%, and the ratio of phenylalanine content to the total free amino acid content ranged from 45% to 60%. In Yifengshan Tallow Honey, the ratio of proline content to the total free amino acid content ranged from 54% to 80%, and the ratio of phenylalanine content to the total free amino acid content ranged from 1% to 6%. The differences were highly significant (p < 0.01) as determined by the t-test, confirming the reliability of the method for distinguishing Zhongfengshan Tallow Honey from Yifengshan Tallow Honey using the ratio of proline to phenylalanine content to the total free amino acid content.
[0052] Example 2 In a certain Sapium sebiferum honey source area in southern China (different from the production area in Example 1), 9 samples of Sapium sebiferum honey brewed by Chinese bees and Italian bees were obtained. The samples were pre-treated, tested and analyzed by LC-MS / MS according to the above method. The results are as follows: Figure 3 and Figure 4 As shown in the table, the ratio of proline content to the total content of 20 free amino acids in Chinese bee mountain tung oil honey ranged from 8% to 21%, and the ratio of phenylalanine content to the total content of 20 free amino acids ranged from 56% to 74%. In Italian bee mountain tung oil honey, the ratio of proline content to the total content of 20 free amino acids was 45% to 62%, and the ratio of phenylalanine content to the total content of 20 free amino acids was 18% to 28%. The differences were highly significant (p < 0.01) as determined by a t-test.
[0053] Example 3 Sapium sebiferum honey and Apis mellifera honey produced during the same period from a major Sapium sebiferum honey producing area (different from the producing area in Example 1) were randomly collected. The samples were pretreated, tested, and analyzed by LC-MS / MS according to the aforementioned methods. The results are shown in Table 3. The proline content of the Sapium sebiferum honey from Apis mellifera ranged from 15% to 21% of the total free amino acids, and the phenylalanine content ranged from 51% to 65% of the total free amino acids. The proline content of the Sapium sebiferum honey from Apis mellifera ranged from 58% to 63% of the total free amino acids, and the phenylalanine content ranged from 3% to 7% of the total free amino acids.
[0054] Table 3 Proline ratio and phenylalanine ratio of randomly sampled Sapium sebiferum honey
[0055] Example 4 Seventeen samples of Sapium sebiferum honey were collected from Chinese bee farms in different regions. After dissolution, centrifugation, and filtration, the free amino acid content was quantified and analyzed using the aforementioned LC-MS / MS method. As shown in Table 4, the ratios of proline and phenylalanine to the total content of 20 free amino acids ranged from 16% to 23% and 50% to 60%, respectively, both of which meet the characteristics of Sapium sebiferum honey determined by the present invention.
[0056] Fifteen samples of Sapium sebiferum honey were collected from Italian bee farms in different regions. After dissolution, centrifugation, and filtration, the free amino acid content was quantified and analyzed using the aforementioned LC-MS / MS method. As shown in Table 4, the ratios of proline and phenylalanine to the total content of 20 free amino acids ranged from 50% to 70% and 1% to 21%, respectively, both meeting the characteristics of Sapium sebiferum honey determined by the present invention.
[0057] Table 4 Proline ratio and phenylalanine ratio of Sapium sebiferum honey from different regions
[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.
Claims
1. A method for distinguishing Chinese bee mountain tallow tree honey and Italian bee mountain tallow tree honey, characterized in that: The method comprises: using the ratio of proline content to the total content of free amino acids and / or the ratio of phenylalanine content to the total content of free amino acids in the honey sample as a basis to identify Chinese bee mountain tallow honey and Italian bee mountain tallow honey.
2. The method according to claim 1, characterized in that If the ratio of proline content to the total free amino acid content is between 5%-25%, and / or the ratio of phenylalanine content to the total free amino acid content is between 45%-75%, it is determined to be Zhongfengshan Tallow Honey; If the ratio of proline content in the total free amino acid content is between 45%-80%, and / or the ratio of phenylalanine content in the total free amino acid content is between 1%-30%, it is determined to be Italian bee mountain tung oil honey.
3. The method according to claim 1, characterized in that The free amino acids include leucine, isoleucine, threonine, proline, valine, serine, alanine, histidine, methionine, glutamic acid, glutamine, aspartic acid, asparagine, lysine, tyrosine, arginine, phenylalanine, tryptophan, glycine and gamma-aminobutyric acid.
4. The method according to any one of claims 1 to 3, characterized in that In the method, liquid chromatography, liquid chromatography-mass spectrometry or ion exchange resin separation-ninhydrin colorimetry is used to detect the content of free amino acids.
5. The method according to claim 4, characterized in that Before detecting the free amino acid content, the method also includes the step of pre-treating the honey sample; the pre-treatment includes the steps of dissolving, separating and removing insoluble impurities and filtering with a filter membrane.
6. A method for detecting the authenticity of Chinese Fengshan Sapium sebiferum honey, characterized in that: The method comprises: judging whether the honey sample is Chinese bee mountain tallow tree honey based on the ratio of proline content to total free amino acid content and / or the ratio of phenylalanine content to total free amino acid content in the honey sample.
7. The method according to claim 6, characterized in that If the ratio of proline content in the total free amino acid content is between 5%-25%, and / or the ratio of phenylalanine content in the total free amino acid content is between 45%-75%, it is determined to be Zhongfengshan Sapium sebiferum honey.
8. The method according to claim 6, characterized in that The free amino acids include leucine, isoleucine, threonine, proline, valine, serine, alanine, histidine, methionine, glutamic acid, glutamine, aspartic acid, asparagine, lysine, tyrosine, arginine, phenylalanine, tryptophan, glycine and gamma-aminobutyric acid.
9. The method according to any one of claims 6 to 8, characterized in that In the method, liquid chromatography, liquid chromatography-mass spectrometry or ion exchange resin separation-ninhydrin colorimetry is used to detect the content of free amino acids.
10. The method according to claim 9, characterized in that Before detecting the free amino acid content, the method also includes the step of pre-treating the honey sample; the pre-treatment includes the steps of dissolving, separating and removing insoluble impurities and filtering with a filter membrane.