qNMR Determination Method of Total Diterpenoids in Coffee and Its Application

Abstract

The present invention provides a qNMR determination method for total coffee diterpenes and its application, belonging to the technical field of food detection. The present invention is a convenient technique for determining total coffee diterpenes by qNMR technology based on the application of the internal standard 3,4,5-trimethoxybenzaldehyde. The present invention can determine the quantitative results of 5 main coffee diterpenes such as cafestol and kahweol, and has great superiority over the previous methods for determining total diterpenes by spectral analysis. By using deuterated chloroform / deuterated water for ultrasonic-assisted extraction to process the analysis sample, the present invention greatly shortens the sample processing flow, and is a fast, simple, efficient and accurate method for determining total coffee diterpenes.

Description

Technical Field

[0001] The present invention belongs to the technical field of food and drug detection, and particularly relates to a qNMR determination method for total diterpenes in coffee and its application. Technical Background

[0002] Coffee diterpenes belong to the ent-kaurane type of diterpenes and are a major class of secondary metabolites in coffee. The two common main components are cafestol and kahweol. At present, the anti-tumor activity of coffee diterpenoids has been relatively deeply studied. In addition to anti-tumor activity, they also have various activities such as antibacterial and antiviral. Recently, the research team of Qiu Minghua discovered more than 40 novel or new ent-kaurane diterpene derivatives from roasted beans of Yunnan coffee. In the activity research, it was found that some novel coffee diterpene compounds showed moderate inhibitory effects on α-glucosidase. The two types of diterpenes with the highest content in coffee, Cafestol and Kahweol, did not show inhibitory effects on α-glucosidase, but interestingly, the dehydration products of the main components of coffee diterpenes, Dehydrocafestol and Dehydrokahweol, showed moderate α-glucosidase inhibitory activity. This phenomenon indicates that the double bond between C-15 and C-16 is beneficial to the exertion of α-glucosidase inhibitory activity. The possible mechanism by which these diterpene derivatives exert their activity was deeply studied through molecular docking experiments.

[0003] Coffee grounds are the main waste after brewing coffee, instant coffee powder, or cold brew coffee extraction. It also has a faint coffee smell. Boiling it continuously with water and collecting the condensed and refluxed liquid also has a fragrant smell, but it is usually directly discarded, which is very wasteful. Some decomposed compounds after rotting will also affect the environment and pollute the environment. In order to improve the comprehensive utilization of coffee grounds and protect the environment, it is very important to construct a content determination technology for total diterpenes in coffee.

[0004] In recent years, quantitative NMR (qNMR) has been widely used to determine the purity and concentration of target compounds. The qNMR method does not require a reference standard of the target compound because in the quantitative detection of most instruments, a known concentration standard is required for calibration. qNMR will be a relatively simple detection method in sample preparation and pretreatment.

[0005] Coffee, as one of the most important cash crops, is remarkable. Coffee diterpenes have also been widely concerned in recent years as tools for studying the origin of plants. The qNMR quantitative analysis technique has naturally been applied to the study of coffee diterpenes. Therefore, it has been reported that the qNMR technique is used to analyze the characteristic component 16-O-methyl caffeol (16-OMC) in coffee diterpenes, which has been proven to be a unique marker for Robusta coffee. By applying its detection and quantitative analysis, the proportion of Robusta in coffee blend beans can be analyzed and judged. Similarly, it has also been reported that roasted coffee is vulnerable to commercial fraud. High-quality Arabica coffee, known as "100% Arabica" or "Highland coffee" beans, is often mixed with cheaper coffee. The quantification of the characteristic diterpene component 16-methoxy caffeol (16-OMC) in Robusta beans helps to monitor the authenticity of products and the content of blended Robusta. By directly esterifying 16-OMC in coffee extracts using the qNMR technique, the detection limit can reach 5 mg / kg, and the quantification limit is 20 mg / kg, which is sufficient to detect the content of less than 0.9% Robusta, being extremely sensitive. This method is a faster, more sensitive, and more reproducible standard method than the German standard method DIN 10779 for determining the content of 16-OMC in roasted coffee beans.

[0006] However, it is far from enough to only analyze the content of a characteristic diterpene component (16-OMC) in coffee using the qNMR technique or to judge the proportion of Robusta. Especially after the inventors found a large number of new coffee diterpene components and various biological activities and functions of coffee diterpenes in their research, a reliable analysis technique is needed to determine the total diterpene content in coffee. For this purpose, the inventors aimed to find a new qNMR technique and establish a simple and highly reliable qNMR determination method for coffee diterpenes and total diterpenes. Summary of the Invention

[0007] The object of the present invention is to establish a simple and highly reliable qNMR determination method for coffee diterpenes and total diterpenes in view of the deficiencies existing in the prior art.

[0008] In order to achieve the above object of the present invention, the present invention provides the following technical solutions:

[0009] The 1 1H NMR detection method for total coffee diterpenes, which comprises the following steps:

[0010] 1. Sample preparation method

[0011] Green coffee beans, roasted coffee beans or coffee grounds: The sample to be detected is crushed, passed through an 80-mesh sieve, and dried to obtain a uniform powder.

[0012] Accurately weigh 250.0 mg of each sample, transfer it to a test tube, add 1.5 mL of CDCl3 containing the internal standard (3,4,5-trimethoxybenzaldehyde), perform ultrasonic-assisted extraction in a 40 °C water bath for 15 min, filter the extract through a 1-μm nylon filter, transfer 600 μL to a nuclear magnetic resonance tube for subsequent collection, and test each sample in parallel twice.

[0013] 2. 1 1H NMR experimental method

[0014] Performed at 600 MHz 1 1H NMR and a 30-degree pulse with a spectral width of 12019 Hz (20 ppm). The number of scans is 256 times, and the number of dummy scans is 2 times. The acquisition time is 2.76 s. The relaxation delay (D1) is 10.0 s, which is more than 5 times the maximum longitudinal relaxation time (D1 > T1).

[0015] 3. Selection of internal substance

[0016] In quantitative nuclear magnetic resonance, selecting a suitable internal standard is crucial. By studying the 1H chemical shift values, assignments, multiplicities, and proton numbers (for each signal), the suitability of four different deuterated solvents (D2O, DMSO-d6, CD3OD, CDCl3) as internal standards was investigated, and 25 candidate compounds for effective internal standards were screened. These 25 compounds can provide unique signal chemical shifts, purity, solubility, and ease of use. [5]

[0017] This experiment uses the internal standard: 3,4,5-trimethoxybenzaldehyde, and the quantitative signal: δ 9.88 ppm, 1H.

[0018] 4. Data processing

[0019] Use the TopSpin pair 1 1H NMR spectra for phase adjustment and integration, and use the Lorentz / Gauss function to deconvolute overlapping peaks. The absolute content of the compound is calculated by the following formula.

[0020]

[0021] In the formula: C (mg / 100 g) is the concentration of the test substance in roasted coffee beans. C0 (mol / L) is the concentration of the internal standard. I and I0 represent the integrated areas of the test substance and the internal standard (9.88 ppm 1 1H). V (mL) is the volume of CDCl3 added during extraction. M is the molar mass of the test substance, and m (g) is the mass of the coffee powder.

[0022] 5. Method application

[0023] Based on the above experimental methods, the contents of five types of coffee diterpenes (Cafestol, Kahweol, Dehydrocafestol, Dehydrokahweol, Caffaldehyde) in lightly roasted (LRB), medium roasted (MRB), and dark roasted (DRB) coffee beans were detected. There were 3 replicate samples for each roasting degree, and each sample was detected in parallel twice.

[0024] The present invention also provides a qNMR determination method for total coffee diterpenes in roasted coffee beans, and this method includes the following steps:

[0025] (1) Crush the roasted coffee bean sample to be detected and pass it through an 80-mesh sieve;

[0026] (2) Accurately weigh 250.0 mg of roasted coffee bean powder, transfer it to a test tube, add 1.5 mL of CDCl3 containing the internal standard 3,4,5-trimethoxybenzaldehyde, ultrasonically extract it in a 40 °C water bath for 15 min, and filter the extract through a 1-μm nylon filter;

[0027] (3) 1 1H qNMR detection: Conducted under a 30-degree pulse of 1H NMR at 600 MHz and a spectral width of 12019 Hz (20 ppm), the number of scans is 256 times, the acquisition time is 2.76 s, and the relaxation delay (D1) is 10.0 s; 1

[0028] (4) Selection of internal substances: Use the internal standard: 3,4,5-trimethoxybenzaldehyde, and the quantitative signal: δ9.88 ppm, 1H; (5) Calculate the quantitative results of Cafestol, Kahweol, Dehydrocafestol, Dehydrokahweol, and Caffaldehyde according to the following formula:

[0029]

[0030]

[0031] In the formula: C (mg / 100 g) is the concentration of the test substance in roasted coffee beans, C0 (mol / L) is the concentration of the internal standard, I and I0 represent the integral areas of the measured substance and the internal standard (9.88 ppm 1 1H), V (mL) is the volume of CDCl3 added during extraction, M is the molar mass of the measured substance, and m (g) is the mass of the coffee powder.

[0032] The present invention also provides a qNMR determination method for total coffee diterpenes in green coffee beans, and this method includes the following steps:

[0033] (1) Crush the green coffee bean sample to be detected and pass it through an 80-mesh sieve;

[0034] (2) Accurately weigh 250.0 mg of green coffee bean powder, transfer it to a test tube, add 1.5 mL of CDCl3 containing the internal standard 3,4,5-trimethoxybenzaldehyde, ultrasonically extract for 15 min in a 40 °C water bath, and filter the extract through a 1-μm nylon filter;

[0035] (3) 1 1H qNMR detection: Conducted at 600 MHz 1 under a 30-degree pulse with a 1H NMR and spectral width of 12019 Hz (20 ppm), the number of scans is 256 times, the acquisition time is 2.76 s, and the relaxation delay (D1) is 10.0 s.

[0036] (4) Selection of internal substances: Use the internal standard: 3,4,5-trimethoxybenzaldehyde, quantitative signal: δ 9.88 ppm, 1H;

[0037] (5) Calculate the quantitative results of cafestol and kahweol according to the following formula,

[0038]

[0039] where: C (mg / 100 g) is the concentration of the test substance in roasted coffee beans, C0 (mol / L) is the concentration of the internal standard, I and I0 represent the integral areas of the test substance and the internal standard (9.88 ppm 1 1H), V (mL) is the volume of CDCl3 added during extraction, M is the molar mass of the test substance, and m (g) is the mass of the coffee powder.

[0040] In addition, the present invention further provides a qNMR determination method for total diterpenes in coffee in instant coffee powder or the residual waste residue after cold brew coffee extraction. This method includes the following steps:

[0041] (1) Crush the instant coffee powder or the residual waste residue after cold brew coffee extraction to be detected and pass it through an 80-mesh sieve to obtain instant coffee powder or the residual waste residue powder after cold brew coffee extraction;

[0042] (2) Accurately weigh 250.0 mg of instant coffee powder or the residual waste residue powder after cold brew coffee extraction, transfer it to a test tube, add 1.5 mL of CDCl3 containing the internal standard 3,4,5-trimethoxybenzaldehyde, ultrasonically extract for 15 min in a 40 °C water bath, and filter the extract through a 1-μm nylon filter;

[0043] (3) 1 1H qNMR detection: Conducted at 600 MHz 11H NMR was carried out with a spectral width of 12019 Hz (20 ppm) under a 30-degree pulse, the number of scans was 256 times, the acquisition time was 2.76 s, and the relaxation delay (D1) was 10.0 s;

[0044] (4) Selection of internal substance: Internal standard: 3,4,5-trimethoxybenzaldehyde was used, and the quantitative signal was δ 9.88 ppm, 1H;

[0045] (5) Calculate the quantitative results of cafestol, kahweol, dehydrocafestol, dehydrokahweol and caffaldehyde according to the following formula,

[0046]

[0047] where: C (mg / 100 g) is the concentration of the test substance in roasted coffee beans, C0 (mol / L) is the concentration of the internal standard, I and I0 represent the integration areas of the test substance and the internal standard (9.88 ppm 1 H), V (mL) is the volume of CDCl3 added during extraction, M is the molar mass of the test substance, and m (g) is the mass of coffee powder.

[0048] The present invention also provides the application of the qNMR determination method of the total coffee diterpenes in the detection of the contents of five types of coffee diterpenes, namely cafestol, kahweol, dehydrocafestol, dehydrokahweol, and caffaldehyde, in light-roasted LRB, medium-roasted MRB, and dark-roasted DRB coffee beans.

[0049] In addition, the application of the qNMR determination method of the total coffee diterpenes in roasted coffee beans in the detection of the contents of five types of coffee diterpenes, namely cafestol, kahweol, dehydrocafestol, dehydrokahweol, and caffaldehyde, in light-roasted LRB, medium-roasted MRB, and dark-roasted DRB coffee beans.

[0050] In addition, the application of the qNMR determination method of the total coffee diterpenes in green coffee beans in the detection of the contents of cafestol and kahweol in green coffee beans.

[0051] Furthermore, the application of the qNMR determination method for total coffee diterpenes in the residual coffee dregs after instant coffee powder or cold brew coffee extraction in the detection of the contents of five types of coffee diterpenes, namely Cafestol, Kahweol, Dehydrocafestol, Dehydrokahweol, and Caffaldehyde, in lightly roasted LRB, medium roasted MRB, and dark roasted DRB coffee beans.

[0052] Compared with the prior art, the present invention has the following advantages:

[0053] 1. The present invention provides a qNMR determination technique for total coffee diterpenes in coffee products, which is different from the previous method of using a diterpene structure with a similar structure as a standard and determining the total content of coffee diterpenes through a standard curve.

[0054] 2. The present invention provides a qNMR determination technique for total coffee diterpenes in coffee products. In the past, the quantification of 16 - methoxycafestol (16 - OMC) was helpful for monitoring the content of blended Robusta in products and was used as a method for identifying Robusta beans. The present invention is based on the application of the internal standard 3,4,5 - trimethoxybenzaldehyde and a convenient technique for determining total coffee diterpenes using qNMR technology.

[0055] 3. The coffee total diterpene determination technique provided by the present invention can determine the quantitative results of main coffee diterpenes such as Cafestol and Kahweol. It has great superiority compared with the previous method of using spectroscopic analysis to determine total diterpenes.

[0056] 4. By using deuterated water ultrasonic - assisted extraction to process the analysis samples, the present invention greatly shortens the sample - processing process and is a fast, simple, efficient, and accurate method for determining total diterpenes.

[0057] 5. The detection method of the present invention is simple, fast, accurate, highly sensitive, has good repeatability, and reliable results. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 is the 1 1H qNMR spectrum for detecting total coffee diterpenes and the characteristic signal assignment diagram, which is the quantitative signal and characteristic signal assignment for the five types of coffee diterpenes in the present invention. In the figure: the 1 1H NMR signal assignment of coffee diterpene characteristics (CDCl3), C: cafestol; Cb: isokahweol; Cd: dehydrocafestol; K: kahweol; Ka: caffaldehyde; Kd: secokahweol; Ke: dehyrdokahweol, internal standard: 3,4,5 - trimethoxybenzaldehyde (quantitative signal, δ 9.88 ppm, 1H).

[0059] Figure 2 This is a diagram showing the changes in diterpene content and PLS-DA model analysis for coffee beans with different roasting degrees in the present invention. In the figure, A - E: Changes in diterpene content of coffee with different roasting degrees. F: Score plot of the PLS-DA model based on the diterpene content of coffee bean samples; G: Loading plot of the PLS-DA model based on the diterpene content of samples. Specific Embodiments

[0060] The following combines the accompanying drawings and uses the embodiments of the present invention to further illustrate the substantial content of the present invention, but does not limit the present invention thereto.

[0061] Example 1

[0062] I. 1 1H NMR detection method for the total diterpenes of coffee in the present invention.

[0063] 1. Sample preparation method

[0064] Green coffee beans, roasted coffee beans or coffee grounds: The sample to be detected is crushed, passed through an 80-mesh sieve, and dried to obtain a uniform powder.

[0065] Accurately weigh 250.0 mg of each sample, transfer it to a test tube, add 1.5 mL of CDCl3 containing an internal standard (3,4,5-trimethoxybenzaldehyde), perform ultrasonic-assisted extraction in a 40 °C water bath for 15 min, filter the extract through a 1-μm nylon filter, transfer 600 μL into a nuclear magnetic resonance tube for subsequent collection, and perform parallel tests twice for each sample.

[0066] 2. 1 1H NMR experimental method

[0067] Performed under 1H NMR at 600 MHz and a 30-degree pulse with a spectral width of 12019 Hz (20 ppm). The number of scans is 256 times, and the number of dummy scans is 2 times. The acquisition time is 2.76 s. The relaxation delay (D1) is 10.0 s, which is more than 5 times the maximum longitudinal relaxation time (D1 > T1). 1 Performed under 1H NMR at 600 MHz and a 30-degree pulse with a spectral width of 12019 Hz (20 ppm). The number of scans is 256 times, and the number of dummy scans is 2 times. The acquisition time is 2.76 s. The relaxation delay (D1) is 10.0 s, which is more than 5 times the maximum longitudinal relaxation time (D1 > T1).

[0068] 3. Selection of internal substances

[0069] In quantitative nuclear magnetic resonance, the selection of a suitable internal standard is crucial. By studying the 1H chemical shift values, assignments, multiplicities, and proton numbers (for each signal), the suitability of four different deuterated solvents (D2O, DMSO-d6, CD3OD, CDCl3) as internal standards was screened, and 25 candidate compounds for effective internal standards were selected. These 25 compounds can provide unique signal chemical shifts, purity, solubility, and ease of use.

[0070] In this experiment, 3,4,5-trimethoxybenzaldehyde was used as the internal standard for coffee diterpenes, and the quantitative signal was δ9.88 ppm, 1H.

[0071] 4. Data processing

[0072] The upper spin pair was used 1 The 1H NMR spectrum was phase-adjusted and integrated, and the overlapping peaks were deconvoluted using the Lorentz / Gauss function. The absolute content of the compound was calculated by the following formula (1).

[0073]

[0074] In the formula: C (mg / 100 g) is the concentration of the test substance in roasted coffee beans. C0 (mol / L) is the concentration of the internal standard. I and I0 represent the integrated areas of the test substance and the internal standard (9.88 ppm 1 1H). V (mL) is the volume of CDCl3 added during extraction. M is the molar mass of the test substance, and m (g) is the mass of the coffee powder.

[0075] II. qNMR determination and method of total coffee diterpenes in roasted coffee beans, instant coffee powder or residual waste after cold brew coffee extraction.

[0076] Based on the above experimental method in (I): The contents of five types of coffee diterpenes (Cafestol, Kahweol, Dehydrocafestol, Dehydrokahweol, Caffaldehyde) in roasted coffee beans with different roasting degrees (light, medium, dark) were detected. There were 3 replicate samples for each roasting degree, and each sample was detected in parallel 2 times. The specific experimental steps are as follows:

[0077] 1) Crush the roasted coffee bean samples to be detected, pass through an 80-mesh sieve to obtain roasted coffee bean powder;

[0078] Or crush the instant coffee powder or residual waste after cold brew coffee extraction to be detected, pass through an 80-mesh sieve to obtain instant coffee powder or residual waste powder after cold brew coffee extraction;

[0079] 2) Accurately weigh 250.0 mg of roasted coffee bean powder, instant coffee powder or residual waste powder after cold brew coffee extraction, transfer it to a test tube, add 1.5 mL of CDCl3 containing the internal standard (3,4,5-trimethoxybenzaldehyde), and ultrasonically extract for 15 min in a 40 °C water bath. The extract was filtered through a 1-μm nylon filter.

[0080] 3) 1 1H qNMR detection: At 600 MHz 11H NMR was carried out with a spectral width of 12019 Hz (20 ppm) and a 30-degree pulse. The number of scans was 256. The acquisition time was 2.76 s. The relaxation delay (D1) was 10.0 s.

[0081] 4) An internal standard: 3,4,5-trimethoxybenzaldehyde was used, and the quantitative signal: δ 9.88 ppm, 1H;

[0082] 5) The quantitative results of cafestol, kahweol, dehydrocafestol, dehydrokahweol and caffaldehyde were calculated according to formula (1).

[0083] 5. Method application

[0084] Based on the above experimental method, the contents of five types of coffee diterpenes (Cafestol, Kahweol, Dehydrocafestol, Dehydrokahweol, Caffaldehyde) in lightly roasted (LRB), medium roasted (MRB), and dark roasted (DRB) coffee beans were detected. There were 3 replicate samples for each roasting degree, and each sample was detected in parallel 2 times. The designation of the quantitative signals of the above 5 types of coffee diterpenes is as Figure 1 shown.

[0085] The analysis and detection results of the above 5 types of coffee diterpenes are shown in Table 1 and Table 2.

[0086] Table 1 Detection results of coffee diterpenes in coffee beans roasted to different degrees

[0087]

[0088]

[0089] Table 2 Detection results of coffee diterpenes in instant coffee powder or cold brew coffee roasted to different degrees

[0090]

[0091] Example 2

[0092] qNMR determination of total coffee diterpenes in green coffee beans.

[0093] Based on the above experimental method in (1): The contents of five types of coffee diterpenes (Cafestol, Kahweol, Dehydrocafestol, Dehydrokahweol, Caffaldehyde) in green coffee beans were detected. There were 3 replicate samples for each roasting degree, and each sample was detected in parallel 2 times. The specific experimental steps are as follows:

[0094] 1) Pulverize the green coffee bean sample to be detected, sieve it through a 80-mesh sieve to obtain green coffee bean powder;

[0095] 2) Accurately weigh 250.0 mg of green coffee bean powder, transfer it to a test tube, add 1.5 mL of CDCl3 containing the internal standard (3,4,5-trimethoxybenzaldehyde), and ultrasonically extract it in a 40 °C water bath for 15 min. The extract is filtered through a 1-μm nylon filter.

[0096] 3) 1 1H qNMR detection: Conducted under a 30-degree pulse with 1H NMR at 600 MHz and a spectral width of 12019 Hz (20 ppm). The number of scans is 256 times. The acquisition time is 2.76 s. The relaxation delay (D1) is 10.0 s. 1 H NMR and a spectral width of 12019 Hz (20 ppm) at a 30-degree pulse. The number of scans is 256 times. The acquisition time is 2.76 s. The relaxation delay (D1) is 10.0 s.

[0097] 4) Use the internal standard: 3,4,5-trimethoxybenzaldehyde, quantitative signal: δ9.88 ppm, 1H;

[0098] 5) Calculate the quantitative results of cafestol and kahweol in the green coffee beans according to the following formula (Table 3).

[0099]

[0100] Table 3 Quantitative results of cafestol and kahweol in green coffee beans

[0101]

[0102] Because the contents of cafestol and kahweol in the green bean sample are relatively high, and most of the diterpenes in roasted beans are generated from the thermal reactions of these two types of compounds, these two components can be used as the total diterpene content of green coffee beans.

[0103] Structures of the main components of coffee diterpenes:

[0104]

Claims

1. qNMR determination method of total diterpenes in coffee, characterized in that The method comprises the following steps: (1) Sample preparation: Take the green coffee beans, roasted coffee beans or coffee grounds to be tested, grind them, pass through an 80-mesh sieve, and dry them to obtain a uniform powder; accurately weigh 250.0 mg of each sample, transfer them to a test tube, add 1.5 mL of CDCl3 containing the internal standard 3,4,5-trimethoxybenzaldehyde, and perform ultrasonic-assisted extraction in a 40°C water bath for 15 min. The extract is filtered through a 1 μm nylon filter, and 600 μL is transferred to a nuclear magnetic resonance tube for subsequent collection. Each sample is tested twice in parallel; (2) 1 H NMR experiments: at 600 MHz 1 H NMR was performed with a spectral width of 12019 Hz, a 30-degree pulse at 20 ppm, 256 scans, 2 dummy scans, an acquisition time of 2.76 s, and a relaxation delay D1 of 10.0 s; (3) Selection of internal substances: internal standard: 3,4,5-trimethoxybenzaldehyde, quantitative signal: δ9.88ppm,1H; (4) Data processing: using spin-up 1 The H NMR spectrum was phase adjusted and integrated, and the overlapping peaks were deconvoluted using the Lorentz / Gauss function. The absolute content of the compound was calculated using the following formula: Where C (mg / 100g) is the concentration of the test substance in roasted coffee beans, C0 (mol / L) is the internal standard concentration, and I and I0 represent the integrated area of the test substance and the internal standard (9.88ppm 1 H), V (mL) is the volume of CDCl3 added during extraction, M is the molar mass of the substance being measured, and m (g) is the mass of coffee powder; (5) Application of the method: Based on the above experimental method, the contents of five types of coffee diterpenes, namely cafestol, kahweol, dehydrocafestol, dehydrokahweol, and caffaldehyde, were detected in lightly roasted LRB, medium roasted MRB, and dark roasted DRB coffee beans. There were three replicate samples for each roasting degree, and each sample was tested twice in parallel.

2. qNMR determination method for total coffee diterpenes in roasted coffee beans, characterized in that The method comprises the following steps: (1) Grinding the roasted coffee bean sample to be tested and passing it through an 80-mesh sieve to obtain roasted coffee bean powder; (2) Accurately weigh 250.0 mg of roasted coffee bean powder, transfer it to a test tube, add 1.5 mL of CDCl3 containing the internal standard 3,4,5-trimethoxybenzaldehyde, and extract by ultrasonication in a 40°C water bath for 15 min. The extract is filtered through a 1 μm nylon filter; (3) 1 H qNMR detection: at 600 MHz 1 H NMR was performed with a 30-degree pulse and a spectral width of 12019 Hz (20 ppm), 256 scans, an acquisition time of 2.76 s, and a relaxation delay (D1) of 10.0 s; (4) Selection of internal substances: internal standard: 3,4,5-trimethoxybenzaldehyde, quantitative signal: δ9.88ppm,1H; (5) Calculate the quantitative results of cafestol, kahweol, dehydrocafestol, dehydrokahweol and caffealdehyde according to the following formula: Where: C (mg / 100g) is the concentration of the test substance in roasted coffee beans, C0 (mol / L) is the internal standard concentration, I and I0 represent the integrated area of the test substance and the internal standard (9.88ppm 1 H), V (mL) is the volume of CDCl3 added during extraction, M is the molar mass of the substance being measured, and m (g) is the mass of coffee powder.

3. A qNMR method for determining total diterpenes in green coffee beans, characterized in that: The method comprises the following steps: (1) Grinding the green coffee bean sample to be tested and passing it through an 80-mesh sieve to obtain green coffee bean powder; (2) Accurately weigh 250.0 mg of green coffee bean powder, transfer it to a test tube, add 1.5 mL of CDCl3 containing the internal standard 3,4,5-trimethoxybenzaldehyde, and extract by ultrasonication in a 40°C water bath for 15 min. The extract is filtered through a 1 μm nylon filter; (3) 1 H qNMR detection: at 600 MHz 1 H NMR was performed with a 30-degree pulse and a spectral width of 12019 Hz (20 ppm), 256 scans, an acquisition time of 2.76 s, and a relaxation delay (D1) of 10.0 s; (4) Selection of internal substances: internal standard: 3,4,5-trimethoxybenzaldehyde, quantitative signal: δ9.88ppm,1H; (5) Calculate the quantitative results of cafestol and kahweol according to the following formula: Where: C (mg / 100g) is the concentration of the test substance in roasted coffee beans, C0 (mol / L) is the internal standard concentration, I and I0 represent the integrated area of the test substance and the internal standard (9.88ppm 1 H), V (mL) is the volume of CDCl3 added during extraction, M is the molar mass of the substance being measured, and m (g) is the mass of coffee powder.

4. qNMR determination method for total coffee diterpenes in residual waste after extraction of instant coffee powder or cold brew coffee, characterized in that The method comprises the following steps: (1) Grinding the instant coffee powder or the residual waste residue after cold brew coffee extraction to be tested, and passing it through an 80-mesh sieve to obtain instant coffee powder or the residual waste residue powder after cold brew coffee extraction; (2) Accurately weigh 250.0 mg of instant coffee powder or residual waste powder after cold brew coffee extraction, transfer it to a test tube, add 1.5 mL of CDCl3 containing the internal standard 3,4,5-trimethoxybenzaldehyde, and extract by ultrasonication in a 40°C water bath for 15 min. The extract is filtered through a 1 μm nylon filter; (3) 1 H qNMR detection: at 600 MHz 1 H NMR was performed with a 30-degree pulse and a spectral width of 12019 Hz (20 ppm), 256 scans, an acquisition time of 2.76 s, and a relaxation delay (D1) of 10.0 s; (4) Selection of internal substances: internal standard: 3,4,5-trimethoxybenzaldehyde, quantitative signal: δ9.88ppm,1H; (5) Calculate the quantitative results of cafestol, kahweol, dehydrocafestol, dehydrokahweol and caffealdehyde according to the following formula: Where: C (mg / 100g) is the concentration of the test substance in roasted coffee beans, C0 (mol / L) is the internal standard concentration, I and I0 represent the integrated area of the test substance and the internal standard (9.88ppm 1 H), V (mL) is the volume of CDCl3 added during extraction, M is the molar mass of the substance being measured, and m (g) is the mass of coffee powder.

5. Application of the qNMR determination method for total coffee diterpenes according to claim 1 in detecting the contents of five types of coffee diterpenes, namely cafestol, kahweol, dehydrocafestol, dehydrokahweol, and caffaldehyde, in lightly roasted LRB, medium roasted MRB, and dark roasted DRB coffee beans.

6. Use of the qNMR determination method for total coffee diterpenes in roasted coffee beans according to claim 2 in detecting the content of five types of coffee diterpenes: cafestol, kahweol, dehydrocafestol, dehydrokahweol, and caffaldehyde in light roasted LRB, medium roasted MRB, and dark roasted DRB coffee beans.

7. Use of the qNMR determination method of total coffee diterpenes in green coffee beans according to claim 3 in detecting the content of cafestol and kahweol in green coffee beans.

8. Use of the qNMR determination method for total coffee diterpenes in residual waste grounds after extraction of instant coffee powder or cold brew coffee according to claim 4 in detecting the total content of five types of coffee diterpenes, namely cafestol, kahweol, dehydrocafestol, dehydrokahweol, and caffaldehyde, in coffee grounds.

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