阅读说明：本技术 一种蜂蜜真伪检测方法 (Honey authenticity detection method ) 是由 张斌 黄慧英 何琛 刘瑞琪 林塔祥 梁礼琪 范良 于 2020-07-07 设计创作，主要内容包括：本发明公开了一种蜂蜜真伪检测方法,该方法包括以下步骤：S1：材料仪器准备,准备多份待检测的材料和需要使用的相关仪器；S2：预处理,对多份待检测的样品进行预处理；S3：取样检测,对每份待检测的样品均进行取样检测；S4：样品测试,对S3中的样品进行随机测试；S5：二次测试,对S4中的蜂蜜样品再次测试；S6：数据采集,对S5中的样品测试结果进行数据采集；S7：分析数据；该发明采用核磁共振技术对蜂蜜中多组分进行分析,从获得的多核磁信号中提取出鉴定蜂蜜真伪的标记物,同时结合还原糖含量、蔗糖含量和酸度等理化指标以期为蜂蜜真伪判别提供切实可行的方法。(The invention discloses a method for detecting authenticity of honey, which comprises the following steps: s1: preparing a material instrument, namely preparing a plurality of parts of materials to be detected and related instruments to be used; s2: preprocessing, namely preprocessing a plurality of samples to be detected; s3: sampling and detecting, namely sampling and detecting each sample to be detected; s4: sample testing, random testing is carried out on the sample in S3; s5: a second test, the honey sample in S4 was tested again; s6: data acquisition, namely acquiring data of the sample test result in the S5; s7: analyzing the data; the invention adopts the nuclear magnetic resonance technology to analyze multiple components in the honey, extracts a marker for identifying the authenticity of the honey from the obtained multi-core magnetic signal, and combines physical and chemical indexes such as reducing sugar content, sucrose content and acidity to provide a feasible method for discriminating the authenticity of the honey.)

1. A method for detecting the authenticity of honey is characterized by comprising the following steps:

s1: preparing a material instrument, namely preparing a plurality of parts of materials to be detected and related instruments to be used;

s2: preprocessing, namely preprocessing a plurality of samples to be detected;

s3: sampling and detecting, namely sampling and detecting each sample to be detected;

s4: sample testing, the sample in S3 is tested;

s5: a second test, the honey sample in S4 was tested again;

s6: data acquisition, namely acquiring data of the sample test result in the S5;

s7: the data is analyzed.

2. A method as claimed in claim 1, wherein the method comprises the steps of: in the S2 process, the pretreatment process of the honey sample comprises the following steps: weighing a plurality of equal parts of sample honey to be detected respectively into a centrifuge tube, adding heavy water to prepare an equal-concentration solution, fully dissolving the honey until the system is transparent and clear, then performing centrifugal treatment on the honey sample to ensure that no solution is hung on the wall, then completely transferring the solution in the centrifuge tube into a nuclear magnetic resonance sample tube, and standing to stabilize the configuration of saccharides in the solution.

3. A method as claimed in claim 1, wherein the method comprises the steps of: in the S3 process, the detection modes of the honey sample are all random detection.

4. A method as claimed in claim 1, wherein the method comprises the steps of: in the process of S4, 1H NMR of honey samples was analyzed in a manner of heavy water calibration for all honey samples in S3.

5. A method as claimed in claim 1, wherein the method comprises the steps of: in the S5 procedure, the honey samples from the S4 that were analyzed using the heavy water scale were retested.

6. A method as claimed in claim 1, wherein the method comprises the steps of: in the S4, a noesypr1D pulse sequence is adopted to inhibit a resonance signal of water in honey, a 1H NMR test is carried out on a 600MHz nuclear magnetic resonance spectrometer, the test temperature is 290-310K, and D2O is used for field locking; and before each sample measurement, tuning and shimming are carried out; the signals are accumulated for 10-20 times, the number of empty scanning is 1-3 times, and the number of scanning for inhibiting the resonance signals of the water in the honey by adopting a noesypr1d pulse sequence is 30-50 times.

Technical Field

The invention particularly relates to the technical field of honey detection, and particularly relates to a honey authenticity detection method.

Background

The main components of the natural honey are glucose (25.2-35.3%), fructose (33.3-43.0%), a plurality of amino acids, minerals, vitamins and a plurality of active enzymes. The honey has the reputation of being the most perfect nutritional food in nature, has extremely high nutritional value and health care effect, and is deeply favored by consumers. Because the price of the honey is relatively high, the actually produced honey can not meet the market demand far away, and a possible opportunity of adulteration of the honey is provided for lawbreakers under the drive of huge economic benefits and market demands.

In the prior art, thin-layer chromatography is an effective method for determining high-fructose starch syrup. The content of reducing sugar, sucrose and maltose in honey is measured by a high performance liquid chromatography differential refraction method, the method can detect maltose syrup doped in the honey, and the method is ineffective for the honey doped with the fructose-glucose syrup.

The existing detection method is developed aiming at the existing adulteration and adulteration technology and is inevitably lagged behind the development of the adulteration and adulteration technology. The defects of the existing detection means provide a multiplier for adulteration of honey for lawbreakers. In conclusion, a set of sensitive, efficient and accurate method for identifying the authenticity of honey is urgently established.

Disclosure of Invention

The invention aims to solve the defect that the adulteration of honey cannot be fully detected in the prior art, and provides a method for detecting the authenticity of honey.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for detecting authenticity of honey comprises the following steps:

s1: preparing a material instrument, namely preparing a plurality of parts of materials to be detected and related instruments to be used;

s2: preprocessing, namely preprocessing a plurality of samples to be detected;

s3: sampling and detecting, namely sampling and detecting each sample to be detected;

s4: sample testing, random testing is carried out on the sample in S3;

s5: a second test, the honey sample in S4 was tested again;

s6: data acquisition, namely acquiring data of the sample test result in the S5;

s7: the data is analyzed.

Further, in the S2 process, the honey sample pretreatment process includes: weighing a plurality of equal parts of sample honey to be detected respectively into a centrifuge tube, adding heavy water to prepare an equal-concentration solution, fully dissolving the honey until the system is transparent and clear, then performing centrifugal treatment on the honey sample to ensure that no solution is hung on the wall, then completely transferring the solution in the centrifuge tube into a nuclear magnetic resonance sample tube, and standing to stabilize the configuration of saccharides in the solution.

On the basis of the scheme, in the S3 process, the detection modes of the honey sample are all random detection.

As a still further aspect of the present invention, in the S4 process, 1H NMR of honey samples was analyzed by heavy water calibration for all honey samples in S3.

Further, in the S5 procedure, the honey samples were retested using the overhydrated scale analysis of S4.

On the basis of the scheme, in S5, a noesypr1D pulse sequence is adopted to inhibit a resonance signal of water in honey, a 1H NMR test is carried out on a 600MHz nuclear magnetic resonance spectrometer, the test temperature is 290-310K, and D2O is used for field locking; and before each sample measurement, tuning and shimming are carried out; the signals are accumulated for 10-20 times, the number of empty scanning is 1-3 times, and the number of scanning for inhibiting the resonance signals of the water in the honey by adopting a noesypr1d pulse sequence is 30-50 times.

The invention has the beneficial effects that:

according to the invention, multiple components in honey are analyzed by adopting a nuclear magnetic resonance technology, a marker for identifying the authenticity of the honey is extracted from an obtained multi-core magnetic signal, and a feasible method is provided for the authenticity identification of the honey by combining physical and chemical indexes such as reducing sugar content, sucrose content and acidity, so that the phenomenon that the honey doped with high fructose syrup cannot be detected in the prior art is avoided.

Drawings

FIG. 1 shows the configuration of glucose, fructose and sucrose in heavy water according to the present invention;

FIG. 2 is 1H-NMR of honey 1 as a sample proposed by the present invention;

FIG. 3 shows 1H-NMR fingerprints of honey samples 2 according to the invention, which are tested in parallel 6 times;

FIG. 4 is the 1H-NMR fingerprint of class 4 honey proposed by the present invention;

FIG. 5 shows the attribution of the characteristic H of common saccharides in 1H-NMR fingerprint of honey provided by the invention;

FIG. 6 shows 1H-NMR 4-6ppm finger-prints of honey according to the invention

Fig. 7 is a schematic structure diagram of the process proposed by the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.

Examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are exemplary only for the purpose of explaining the present patent and are not to be construed as limiting the present patent.

In the description of this patent, it is noted that unless otherwise specifically stated or limited, the terms "mounted," "connected," and "disposed" are to be construed broadly and can include, for example, fixedly connected, disposed, detachably connected, disposed, or integrally connected and disposed. The specific meaning of the above terms in this patent may be understood by those of ordinary skill in the art as appropriate.

Referring to fig. 1-6, a method for detecting the authenticity of honey comprises the following steps:

s1: preparing a material instrument, namely preparing a plurality of parts of materials to be detected and related instruments to be used, specifically selecting sample honey from 4 for detection:

sample 1: garden agriculture brand jujube honey, production area: xiangyang city, Hubei province; the production date is as follows: year 2017, 03 month 01; shelf life: 2 years;

sample 2: forest bee garden brand basswood honey, production area: shanghai Fengxian district; the production date is as follows: 2016, 04, 29 months; shelf life: 2 years;

sample 3: loquat honey purchased from supermarkets, production area: fujian province, Fuzhou city; the production date is as follows: 2016, 12 months, 01 days; shelf life: 2 years;

sample 4: guanshengyuan brand honey, production area: shanghai Fengxian district; the production date is as follows: 2016, month 01, and day 21; shelf life: 1.5 years;

the following are specific instruments to be used in this embodiment:

bruker Ultrashield Plus-600 nuclear magnetic resonance apparatus;

a 5mm dual core z-gradient probe;

5mm NMR sample tube;

topspin 2.3 test control and data processing software;

a high speed centrifuge;

centrifuging the tube;

an ultrasonic instrument;

an analytical balance;

a deuterated reagent: ALDRICH heavy water (deuteration degree is 99.9%),

potassium hydrogen phthalate.

S2: preprocessing, namely preprocessing a plurality of samples to be detected; for 4 samples in the S1 process, the 4 samples need to be preprocessed before detection to be in the same state, so that the two most basic advantages are achieved, the first is to place each sample in a state which is most convenient for detection, and the second is to avoid the problem of deviation of detection results caused by different states; in 4 detection samples, uniformly stirring the honey samples without crystallization; placing a sample with crystals in a water bath at a temperature of not more than 60 ℃ under a closed condition for warming and oscillating, stirring uniformly after the sample is completely melted, rapidly cooling to room temperature, and separating out 0.5kg of the sample; placing the prepared sample in a centrifuge tube, sealing, marking, and storing at room temperature; the specific operation is as follows:

weighing 1 honey sample into a 1.5mL centrifuge tube in three equal parts respectively, adding heavy water to prepare an equal concentration solution (20mg/mL), fully dissolving honey by using an ultrasonic instrument until the system is transparent and clear, using a high-speed centrifuge to enable the rotation speed to reach 5000rpm, centrifuging for 5min to ensure that no solution is hung on the wall, then completely transferring the solution in the centrifuge tube into a 5mm nuclear magnetic resonance sample tube by using a liquid transfer gun, and standing for 15min to enable the configuration of saccharides in the solution to be stable.

Weighing three equal parts of sample 2 honey into 1.5mL centrifuge tubes respectively, adding heavy water to prepare an equal concentration solution (20mg/mL), fully dissolving the honey by using an ultrasonic instrument until the system is transparent and clear, using a high-speed centrifuge to enable the rotation speed to reach 5000rpm, centrifuging for 5min to ensure that no solution is hung on the wall, then completely transferring the solution in the centrifuge tubes into 5mm nuclear magnetic resonance sample tubes by using a liquid transfer gun, and standing for 15min to enable the configuration of saccharides in the solution to be stable.

Weighing three equal parts of 3 honey of a sample into a 1.5mL centrifuge tube respectively, adding heavy water to prepare an equal concentration solution (20mg/mL), fully dissolving the honey by using an ultrasonic instrument until the system is transparent and clear, using a high-speed centrifuge to enable the rotating speed to reach 5000rpm, centrifuging for 5min to ensure that no solution is hung on the wall, then completely transferring the solution in the centrifuge tube into a 5mm nuclear magnetic resonance sample tube by using a liquid transfer gun, and standing for 15min to enable the configuration of saccharides in the solution to be stable.

Weighing three equal parts of sample 4 honey into 1.5mL centrifuge tubes respectively, adding heavy water to prepare an equal concentration solution (20mg/mL), fully dissolving the honey by using an ultrasonic instrument until the system is transparent and clear, using a high-speed centrifuge to enable the rotating speed to reach 5000rpm, centrifuging for 5min to ensure that no solution is hung on the wall, completely transferring the solution in the centrifuge tubes into 5mm nuclear magnetic resonance sample tubes by using a liquid transfer gun, and standing for 15min to enable the configuration of saccharides in the solution to be stable;

s3: sampling and detecting, namely sampling and detecting each sample to be detected; the detection modes of the honey sample are random detection so as to avoid instrument errors possibly generated in the analysis process;

s4: sample testing, the sample in S3 is tested; analyzing 1H NMR of the honey samples in S3 by adopting a heavy water scaling mode;

s5: a second test, the honey sample in S4 was tested again; retesting the honey sample subjected to the calibration analysis of the heavy water in the S4, inhibiting a resonance signal of the water in the honey by adopting a noesypr1D pulse sequence, performing 1H NMR test on a 600MHz nuclear magnetic resonance spectrometer at the test temperature of 290-310K, specifically 298K, and performing field locking by using D2O; and before each sample measurement, tuning and shimming are carried out; the number of signal accumulations is 10-20, the number of empty scanning is 1-3, the number of signal accumulations is 16 in the embodiment, the number of empty scanning is 2, the number of scanning for inhibiting the resonance signal of water in honey by adopting a noesypr1d pulse sequence is 30-50, the specific number is 40 in the embodiment, wherein parameters in Topspin software are set as PULPROG (Noesypr 1 d), AQ _ mod (DQD), TD (32K), NS (16N), DS (2), and the number of signal accumulations is 40;

s6: data acquisition, namely acquiring data of the sample test result in the S5;

s7: analyzing the data;

according to the known literature, Hades and politics, and NMR quantitative analysis research on glucose, fructose and sucrose in honey. Food science, 2009, vol.30, no14. it is known that glucose exists in honey in both α -D-glucopyranose and β -D-glucopyranose configurations. The natural glucose, whether free or combined with a mean D type, has very little chain glucose content in aqueous solution, mainly exists in an epoxy pyran type configuration, and two configurational isomers of alpha and beta realize dynamic equilibrium through the chain glucose. Similarly, fructose in honey is known to have three configurations, beta-D-fructofuranose, beta-D-fructopyranose and alpha-D-fructofuranose. Wherein the highest content is beta-D-fructopyranose, the second content is beta-D-fructofuranose, and the lowest content is alpha-D-fructofuranose. The sucrose in honey has only one configuration in heavy water, the 1-C on the alpha-D-glucopyranose ring and the 8-C of the beta-D-fructofuranose are connected through oxygen atoms, and the configurations of the glucose, the fructose and the sucrose in the heavy water are shown in figure 1.

The assignment of the characteristic H in the spectrum is briefly analyzed by taking 1H-NMR of the sample 1 honey as an example.

As shown in fig. 2, δ 5.38 is assigned to sucrose 1H, and splits into d-peaks due to coupling by sucrose 2H, with a coupling constant of about 3JH-H ═ 4 Hz; δ 5.21 from 1-H for α -D-glucopyranose; δ 4.63 from 1-H for β -D-glucopyranose; delta 4.1 is ascribed to 3-H and 4-H of beta-D-fructopyranose 3-H and beta-D-fructofuranose; delta 4.02 is assigned as one peak of two peaks of one proton on the beta-D-fructopyranose 6-H; delta 3.99 is assigned as the other peak of two peaks of one proton on beta-D-fructopyranose 6-H and one peak of two peaks of alpha-D-fructofuranose 4-H; delta 3.98 is assigned to the other of the two peaks, beta-D-fructopyranose 5-H and alpha-D-fructofuranose 4-H.

Selecting one part of honey in the sample 2 to perform parallel determination for 6 times, scanning for 16 times, sequentially numbering Desktop/1, 12, 13, 14, 15, 16, 1H-NMR,

as shown in fig. 3; the 6-time parallel measurement spectrograms of the same sample are almost the same, the coincidence degree of the characteristic peaks is perfect, and the intensities are almost the same, so that the concentration of the selected sample and the actual information of the sample can be reliably reflected by a nuclear magnetic resonance spectrometer, and the method can be used as a sensitive, efficient and accurate test means for quantitative analysis of the content of the honey.

Similarly, the percentage of glucose, fructose and sucrose in honey 2-1 was calculated and the mean, standard deviation and relative standard deviation were calculated and the results are shown in Table 1. As can be seen from table 1: the relative standard deviation of the glucose, fructose and sucrose measurement results is 1.54%, 1.28% and 3.28% respectively, the multiple measurement results are approximate, and the relative standard deviation is also in an allowable range, which shows that the accuracy and the reproducibility of the quantitative analysis of the percentage content of the glucose, the fructose and the sucrose in the honey by using a nuclear magnetic resonance spectrometer are high.

Table 1: precision experiment

And performing baseline correction and phase correction on the obtained one-dimensional 1H NMR spectrum. The nuclear magnetic resonance signals of the samples are subjected to Fourier transform by using Topspin software, and typical 1H NMR fingerprints of the honey samples, adulterated honey and syrup samples are obtained (as shown in figure 4). As can be seen from FIG. 4, it is difficult to visually see the significant difference spectrum peaks among the 4 kinds of honey through a typical fingerprint spectrum. Therefore, all the data obtained must be subjected to chemometric analysis to extract from the subtle differences the biomarkers that judge the authenticity of honey.

Therefore, sucrose, α -D-glucopyranose and β -D-glucopyranose, β -D-fructofuranose, β -D-fructopyranose and α -D-fructofuranose three configuration features H are first assigned in a 1H-NMR fingerprint (see for details the confirmation of the 1H-NMR characteristic peaks for 2.2 glucose, fructose and sucrose in heavy water) as shown in FIG. 5.

Meanwhile, the spectrum is processed by using the method in the auxiliary material, the proportions of sucrose, reducing sugar and water in the honey sample are quantitatively analyzed, and the average value is obtained as shown in table 2.

At present, national standards for honey clearly indicate that the content of sucrose is not more than 5%, the content of reducing sugar (glucose + fructose) is not less than 60%, and the content of water is not more than 23%. From the analysis results of the honey components in table 2, it is seen that the contents of sucrose, reducing sugar and water in the 4 brands of honey basically meet the national standard, which indicates that the 4 brands of honey are all likely to be true by using the national standard as an index for measuring the authenticity of honey.

Table 2: sucrose, reducing sugar and water content in honey

The natural honey contains a trace amount of oligosaccharide, the oligosaccharide is a natural sweet substance which is prepared by fully brewing nectar, secretion or honeydew of a honey collection plant after being mixed with self secretion, and commercial honey obtained by artificial adulteration does not contain the oligosaccharide, so the oligosaccharide can be used as a fundamental standard for distinguishing the authenticity of the honey. The characteristic peaks of these oligosaccharides are mainly concentrated at 4-6ppm in 1H-NMR, so the fingerprint of 4-6ppm in 1H-NMR of four kinds of honey is analyzed, as shown in FIG. 6, and the positions marked by arrows (black, red, blue) are the characteristic peaks of oligosaccharides specific to natural honey.

As can be seen from the figure, the characteristic peaks of the oligosaccharides of the honeys of the samples 1 and 2 are obvious, the characteristic peak signal of the oligosaccharide of the honey of the sample 4 is incomplete, and the characteristic peak signal of the oligosaccharide of the honey of the sample 3 is completely absent. Therefore, the loquat honey (sample 3 honey) purchased from the supermarket is judged to be fake honey, and because the sugar content and the water content of the loquat honey basically meet the national standard, the sample 3 honey is guessed to be a poor product possibly adulterated with the fructose-glucose syrup, and the authenticity identification reliability of the honey by using the nuclear magnetic resonance technology is further proved to be high.

The nuclear magnetic resonance technology can nondestructively detect all small molecular compounds with the content within the detection limit, the detection sensitivity of each compound in a sample is the same under a specific condition, the method is an unbiased analysis means, multiple components in 4 kinds of honey are analyzed by the unbiased analysis means of the nuclear magnetic resonance, the attribution of key H of each sugar component in 1H-NMR of the honey is determined, the percentage ratio of different sugar component contents and water contents in each kind of honey is obtained by quantitative calculation by a chemometrics analysis method, the goodness of fit with the national standard is very high by comparison, wherein the honey of a sample 3 basically meets the national standard, and the authenticity of the honey cannot be judged by simply analyzing the percentage ratio of the different sugar component contents and the water contents.

In conclusion, the contents of reducing sugar, sucrose and moisture are simply and quantitatively analyzed by the nuclear magnetic resonance technology. 4-6ppm signals in 1H-NMR are emphatically analyzed, and it is found that honey (loquat honey purchased from a supermarket) in a sample 3 does not have signals of key oligosaccharide characteristic H in the fingerprint spectrum, and the oligosaccharides are natural sweet substances which are obtained by fully brewing nectar, secretion or honeydew of honey collection plants after being mixed with self secretion, and are the fundamental standard for distinguishing the authenticity of the honey. Therefore, the honey of sample 3 can be judged to be a poor product mixed with the high fructose corn syrup. While sample 1 and sample 2 are true honey, sample 4 has incomplete oligosaccharide characteristic H signal, weak intensity, and low starch value content due to long-term storage, and is not recommended to be eaten continuously.

The research shows that: sample 1, sample 2 and sample 4 are true honey, but the oligosaccharide characteristic H signal of sample 4 honey is incomplete, weak in intensity, and low in starch value due to long-term storage, and no continuous eating is recommended. Sample 3 was mock honey. Therefore, the combination of the nuclear magnetic resonance unbiased analysis means and various physical and chemical index measuring means can be proved to be used as a method for distinguishing the authenticity of the honey, and the method is sensitive, efficient and high in reliability.

In the description of this patent, it is to be understood that the terms "center," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like are used in the orientations and positional relationships indicated in the drawings for the convenience of describing the patent and for the simplicity of description, and are not intended to indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and are not to be considered limiting of the patent.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.