Method for determining peculiar smell of sweet orange essential oil by combining modern molecular sensory technology with molecular docking

Abstract

The invention belongs to a sweet orange essential oil peculiar smell identification technology, and particularly relates to an identification and identification technology for sweet orange essential oil peculiar smell substance generation caused by hot processing treatment. The invention discloses a method for determining peculiar smell of sweet orange essential oil by combining a modern molecular sensory technology with molecular docking. The method comprises the steps of sensory evaluation of the sweet orange essential oil, pretreatment of the sweet orange essential oil, GC-O-MS, smelling sensory, preliminary screening of peculiar smell related compounds, further identification of peculiar smell compounds, pretreatment of molecular docking, molecular docking and the like. The method can solve the problem of limitation of identification of the peculiar smell compound of the sweet orange essential oil in the food field and the aroma field at present.

Description

Technical Field

[0001] The invention belongs to sweet orange essential oil odor identification technology, and in particular relates to an identification technology for generating odor substances in sweet orange essential oil caused by thermal processing. Background Art

[0002] Sweet orange essential oil embodies the unique aroma and flavor of sweet orange fruit. It also has a variety of health-promoting effects such as anti-inflammatory, antioxidant, and anxiety relief. It is an important food additive and aromatherapy product that is popular all over the world. Conventional sweet orange extraction processes include steam distillation extraction (hot extraction) and cold press extraction (non-thermal extraction). The chemical composition of sweet orange essential oil can be mainly divided into two categories: terpenes and oxygenated terpenes. However, terpenes (such as limonene) are not stable. During transportation and storage, sweet orange essential oil is easily oxidized and degraded, resulting in aroma deterioration and poor stability. Therefore, clarifying the flavor substances of sweet orange essential oil and exploring its olfactory perception mechanism is of great significance to the optimization of the sweet orange essential oil extraction process and subsequent purification process, and the control of the quality of sweet orange essential oil.

[0003] Pyrogenic degradation is the main reason for the odor of sweet orange essential oil. According to the previous sensory evaluation, "cooking smell" and "pungent smell" are the main odor attributes of thermally extracted sweet orange essential oil. The generation of odor limits the market development and consumer acceptance of citrus essential oil. However, the current determination methods of odor substances in essential oils are still limited. Most studies still rely on individual subjective evaluation and combine the analysis of the production of comparative difference compounds. For example, some studies have pointed out that limonene and linalool will decompose to produce α-terpinene and D-carvone during thermal processing, which may lead to the generation of odor. However, actual studies have found that the content of linalool in thermally extracted citrus essential oil is higher, and D-carvone is also present in newly extracted citrus essential oil. Therefore, the citrus essential oil industry needs a more precise and systematic method to identify and control odor substances in sweet orange essential oil to improve product quality and market acceptance.

[0004] Modern molecular sensory technology combined with computer simulation technology provides an effective way to identify odor substances. By using human-computer interaction instruments, the complex citrus essential oil system can be further decomposed and the odor properties of each volatile component and its contribution to the overall flavor can be clarified one by one. The development of computer simulation technology provides a valuable tool for analyzing the recognition mechanism between food ingredients and chemical sensory receptors. With the help of molecular docking tools, the binding mechanism of odor molecules and olfactory proteins can be clarified, which helps to develop targeted strategies to reduce odor perception, thereby improving sensory quality and consumer acceptance.

[0005] The main purpose of gas chromatography-olfaction-mass spectrometry (GC-O-MS) measurement technology is to confirm the specific compounds of flavor substances in natural foods / spices. The principle of GC-O / MS technology is to use GC to separate various volatile components in samples or adsorption tubes one by one, and then use MS to objectively analyze various volatile components qualitatively and quantitatively. On the other hand, the human sensitive nose is used to subjectively perceive the odor type and strength of volatile substances, and the subjective perception is compared with objective data to distinguish odor substances from all volatile components, so as to intuitively determine the contribution of odor substances to the overall odor. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a systematic and effective method for detecting odor compounds.

[0007] In order to solve the above technical problems, the present invention provides a method for determining the peculiar smell of sweet orange essential oil, which utilizes modern molecular sensory technology combined with molecular docking to determine the peculiar smell of sweet orange essential oil.

[0008] The technical scheme of the method for measuring the peculiar smell of sweet orange essential oil of the present invention is as follows:

[0009] (1) Sensory evaluation of sweet orange essential oil:

[0010] Based on the results of previous studies, the sensory descriptors of the flavor of sweet orange essential oil were determined. Twelve sensory evaluation panelists were recruited to smell the sweet orange essential oil samples and score them on a 10-point scale, with 0 indicating no odor and 9 indicating the strongest odor.

[0011] (2) Pretreatment of sweet orange essential oil:

[0012] Dissolve the sweet orange essential oil in dichloromethane, add an appropriate amount of internal standard solution, mix well, and pass through a 0.22 μL oil filter membrane.

[0013] (3) GC-O-MS parameter settings:

[0014] The non-polar column DB-5ms was selected as the chromatographic column, the sample port temperature was 240℃, the helium flow rate was 1.5mL / min, and the temperature rise program was set. The injection volume of sweet orange essential oil was 1μL, and the split ratio was 20:1. The MS selected EI source, the ion source temperature was 230℃, the scanning mode was scan, the scanning mass range was 45-350m / z, and the scanning frequency was 4.58scans / s. The sniffing port was connected to the GC-MS, and the GC effluent entered the MS detector and the sniffing port at a ratio of 1:1.

[0015] (4) Olfactory sense: According to the "Introduction and training of sensory analysis methodologies for detecting and identifying odors" (GB / T15549-2022), 3 to 4 sensory evaluators were trained to record the retention time when the odor was smelled, describe the odor characteristics, and score the odor intensity using a 4-point system (1-faint, difficult to identify, 2-weak, recognizable, 3-strong, clear outline, 4-very strong) while the GC procedure was being carried out. Each person repeated it twice.

[0016] (5) Preliminary screening of odor-related compounds: Based on the olfactory results, the NIST spectral library was consulted to determine the odor-active compounds, and the compounds with significant perceived associations with "steaming smell" and "pungent smell" were preliminarily identified as odor-related compounds.

[0017] (6) Further identification of odor compounds: Combined with chemometric methods, further identification of compounds that have a strong correlation with "steaming smell" and "pungent smell".

[0018] (7) Molecular docking pre-processing: Olfactory proteins selected terpenes that widely recognized receptors, and used AlphaFold to predict the three-dimensional structure model and save it in pdb format. The small molecule three-dimensional structure was drawn using Chem 3D and saved in mol2 format.

[0019] (8) Molecular docking: Using Autodock open source software, the ligand and receptor were first dehydrated and hydrogenated and exported to pdbqt format. The specific steps are shown in Figure 1 The conformation with the lowest binding energy was used as the binding site of the olfactory molecule and was used to verify the computer simulation of odor compounds.

[0020] (9) Addition experiment: Using freshly extracted cold-pressed sweet orange essential oil as the matrix, the identified odor-related compounds were added, and the citrus attribute intensity and odor attribute intensity of the added essential oil were sensory evaluated.

[0021] Furthermore, the descriptors selected in step (1) include citrus aroma, floral aroma, sweet aroma, green aroma, fat aroma, earthy aroma, steamed aroma, pungent aroma and overall liking.

[0022] Furthermore, in step (2), the mixing ratio of sweet orange essential oil and dichloromethane is 1:9, and the internal standard solution is 5 μL of 20% cyclohexanone solution (dissolved in dichloromethane).

[0023] Furthermore, in step (3), the heating program is as follows: an initial temperature of 40°C, maintained for 2 min, heated to 130°C at 4°C / min, then heated to 160°C at 3°C / min, maintained for 1 min, then heated to 240°C at 10°C / min and maintained for 5 min.

[0024] Furthermore, in step (4), the compound with MF value ≥ 65% is defined as an odor-active compound. The MF value calculation formula is as follows:

[0025] Where DF is the probability of smelling the aroma compound in all trials; AI ave It is the ratio of the average aroma intensity to the maximum aroma intensity (4).

[0026] Furthermore, chemometric methods included PLSR analysis and OPLS-DA analysis.

[0027] Further, the olfactory protein receptors selected are OR1A1, OR2W1, OR1G1, OR5AN1 and OR52D1.

[0028] Furthermore, the docking box parameters are size_x=40, size_y=40, size_z=40, and the center coordinates are (0.100, 1.220, 12.720), (5.700, -7.740, -7.250), (-2.420, -7.100, -10.300), (1.280, -4.880, -10.180), and (10.390, -0.510, 0.330), respectively.

[0029] Furthermore, a semi-flexible docking mode was selected, and the docking parameters and calculation methods were selected as Geneticgorithm, Run 10 times, Output Lamarckian G, and other default parameters were selected.

[0030] The invention can make up for the problem of limitations in the identification of odor compounds of sweet orange essential oil in the current food field and aroma field.

[0031] The technical solution of the present invention has the following advantages:

[0032] (1) High sensitivity and accuracy: This method can detect and analyze odor components at the molecular level, which is more accurate and objective than traditional sensory evaluation.

[0033] (2) Comprehensive analysis of odor substances: This method can not only directly identify odor compounds, but also combine chemometric methods to screen out potential components that cause odor formation and provide a more comprehensive map of odor substances.

[0034] (3) Quantitative analysis: Combined with modern molecular sensory technology, quantitative analysis of odor components can be achieved, providing more comprehensive chemical composition information, which helps to improve product quality.

[0035] (4) Exploration of the mechanism of odor perception: Combined with molecular docking simulation, the interaction between odor molecules and olfactory receptors can be studied to predict the strength of binding between different molecules and receptors, thereby revealing why odor perception is stronger.

[0036] (5) Wide applicability: This method is not only applicable to sweet orange essential oil, but can also be extended to the odor analysis of other essential oils and spice products. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0038] Figure 1 Schematic diagram of molecular docking operation steps;

[0039] Figure 2 Here is the sensory radar chart of blood orange essential oil and navel orange essential oil extracted by steam distillation and cold pressing;

[0040] Figure 3 is OPLS-DA analysis and VIP value;

[0041] Figure 4 It is PLSR analysis; the upper figure shows the PLSR analysis results of all odor descriptors and key odor substances, and the lower figure shows the key odor compounds (and odor-related compounds) associated with cooking, pungent, fatty and earthy smells;

[0042] Note: Figure 4 The numbers in the table correspond to the odor compounds corresponding to the serial numbers in Table 2.

[0043] Figure 5 It is the heat map of the lowest binding energy between odor-related compounds and olfactory proteins;

[0044] Figure 6 These are the results of the recombinant validation experiment. The left picture shows the results of adding the off-flavor related compounds identified to the cold-pressed blood orange essential oil, and the right picture shows the results of adding the off-flavor related compounds identified to the cold-pressed navel orange essential oil. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0046] Example 1: A comparative experiment was conducted on the flavor differences of Zizhong blood orange essential oil extracted by steam and cold pressing, thereby identifying the off-flavor compounds in the blood orange essential oil extracted by steam distillation;

[0047] The test process and results are as follows:

[0048] (1) The blood orange essential oil was extracted from the Zizhong blood orange (peel) using a steam extraction device (solid-liquid ratio of 1:4) and a three-wheel press, respectively, and stored in a 4°C refrigerator for testing.

[0049] The extraction of the water vapor extraction device is as follows: the crushed Zizhong blood orange peel is mixed with deionized water in a weight ratio of 1:4, placed in a distillation flask, the heating temperature is controlled at 120-135°C, and the water-extracted blood orange essential oil is collected by condensation reflux. When the essential oil in the collector no longer increases, the heating is stopped. After dehydration with anhydrous sodium sulfate, blood orange essential oil 1 is obtained;

[0050] The three-wheel press extraction is as follows: soak the Zizhong blood orange peel in a 1.0% calcium chloride solution for 4 hours (make sure the Zizhong blood orange peel is always immersed during the soaking process), rinse with deionized water, and then squeeze it with a three-wheel press. The resulting mixed solution is allowed to stand for 20 minutes, the upper layer mixture is centrifuged at 6000r / min for 20 minutes, the upper layer essential oil is collected and dried with anhydrous sodium sulfate to obtain blood orange essential oil II.

[0051] Blood orange essential oil 1 and blood orange essential oil 2 are subjected to the following steps (2) to (8):

[0052] (2) Recruiting 12 sensory evaluation panelists to smell the blood orange essential oil obtained in step (1) and record the sensory scores. The sensory description vocabulary and scoring criteria are shown in Table 1, where 0 means no smell is felt and 10 means the smell is the strongest.

[0053] Table 1. Sensory descriptors and scoring criteria

[0054]

[0055] (3) Accurately pipette 100 μL of blood orange essential oil, dissolve it in 900 μL of dichloromethane (as a solvent), add 5 μL of 20% (volume %) cyclohexanone solution (as an internal standard, dissolved in dichloromethane), vortex and oscillate, and filter through a 0.22 μm organic filter to obtain the diluted blood orange essential oil. Take 1 μL of the diluted blood orange essential oil as an injection sample for GC-O-MS analysis in the following step (4).

[0056] (4) GC-O-MS parameter setting: The non-polar column DB-5ms was selected as the chromatographic column, the sample port temperature was 240°C, the helium flow rate was constant at 1.5 mL / min, and the heating program was set as follows: initial temperature 40°C, maintained for 2 min, heated to 130°C at 4°C / min, then heated to 160°C at 3°C / min, maintained for 1 min, then heated to 240°C at 10°C / min and maintained for 5 min.

[0057] The injection volume of diluted blood orange essential oil was 1 μL, and the split ratio was 20:1. The MS used EI source, the ion source temperature was 230°C, the scanning mode was scan, the scanning mass range was 45-350 m / z, and the scanning frequency was 4.58 scans / s. The sniffing port was connected to the GC-MS, and the GC effluent entered the MS detector and the sniffing port at a ratio of 1:1.

[0058] Run the GC-O-MS program, and the smeller records the retention time when the smell is smelled at the sniff port, describes the smell characteristics, and scores the smell intensity using a 4-point system. Specifically, according to the "Introduction and Training of Evaluators in Sensory Analysis Methodology Detection and Identification of Odors" (GB / T 15549-2022), 3 sensory evaluators are trained to record the retention time when the smell is smelled, describe the smell characteristics (which are the smell characteristics given subjectively by the sensory evaluators themselves) while the GC program is running, and score the smell intensity using a 4-point system (1-faint, difficult to identify, 2-weak, recognizable, 3-strong, clear outline, 4-very strong). Each person repeats twice.

[0059] (5) The calibrated frequency method (MF) was used to conduct a preliminary evaluation of the compound olfactory results, and the odor-active compounds were determined based on the compound MF value ≥ 65%.

[0060] The MF value calculation formula is as follows:

[0061] Where DF is the probability of smelling the aroma compound in all trials, that is, the probability that the three sensory evaluators in step (4) smell a certain substance in two repetitions (a total of 6 times); AI ave It is the ratio of the average aroma intensity (i.e. the average aroma intensity of a substance smelled) to the maximum aroma intensity (i.e. 4).

[0062] (6) Preliminary screening of odor-related compounds:

[0063] According to the characteristics and retention time of the odor substances smelled in step (4), the reference substances with a matching degree ≥ 80% are given by the NIST20.0 spectral library. Combined with the odor description of the compounds (refer to relevant literature), the odor-active compounds are qualitatively identified, and the compounds with significant perceived odors such as "steaming taste" and "pungent taste" are preliminarily identified as odor-related compounds.

[0064] (7) Further identification of odor compounds:

[0065] The compounds with strong correlation with "cooking smell" and "pungent smell" preliminarily screened out in step (6) were further identified by chemometrics. The chemometrics included OPLS-DA analysis and PLSR analysis, as follows:

[0066] 7.1) The odor compounds detected were quantified using the internal standard method and single factor analysis was performed using SPSS, with P < 0.05 being considered significant. The calculation formula for the internal standard method is as follows:

[0067]

[0068] Where C is the concentration of volatile compounds, Ai is the internal standard peak area, and Ci is the cyclohexanone concentration (i.e. 0.947×10 - 3 mg / mL), At is the peak area of ​​the corresponding compound; such data are obtained from step (4).

[0069] 7.2) OPLS-DA analysis was performed using SIMCA software. The odor compounds and corresponding concentrations calculated in the above step (7.1) were imported into the SIMCA software, different group names were set, and the supervised model OPLS-DA analysis was selected. "Autofit" and "Scores" were clicked in turn to obtain a scatter plot. After 200 permutation tests (i.e., click Analysis-Permutations), the importance projection value (VIP) of the predictor variable was calculated, and the differential aroma components in blood orange essential oil were screened under the conditions of P < 0.05 and VIP ≥ 1.

[0070] 7.3) PLSR analysis was performed using The Unscrambler X software. The odor compounds and corresponding concentrations calculated in the above step (7.1) were imported into The Unscrambler X software, and the sensory evaluation results (sensory evaluation scores) in step (2) were added. Tasks-Analysis-PLSR analysis was selected, and flavor substances were used as independent variables. The sensory description (i.e., the sensory evaluation results in step (2)) was used as the dependent variable. After "A / (SDev+B)" standardization, random cross-validation and Kernel PLS regression algorithm were selected to obtain a PLSR correlation analysis graph. The closer the flavor substances and sensory descriptions in the correlation analysis graph are, the stronger the correlation is. The compounds with a strong correlation with "steaming taste" and "pungent taste" were further identified.

[0071] (8) Based on the above-screened odor compounds, molecular docking simulations were performed on the selected olfactory proteins to obtain the binding site information and minimum binding energy data of different compounds with olfactory proteins. According to the "Spatiotemporal Coding Hypothesis of Olfactory Information" (published in Science magazine in 2018), compounds with stronger perception first bind to olfactory protein receptors to inhibit the perception of other compounds with weaker affinity. Therefore, compounds with stronger odor perception should have lower binding energy with olfactory receptors.

[0072] Description: Molecular docking experiment is a method to corroborate odor substances. According to the above, it is used to verify the molecular sensory results.

[0073] The details are as follows:

[0074] 8.1) Molecular docking pre-processing: According to existing literature reports, terpenoid compounds with affinity to olfactory protein receptors were selected, including OR1A1, OR2W1, OR1G1, OR5AN1 and OR52D1. AlphaFold was used to predict the three-dimensional structure model and save it in pdb format. The small molecule three-dimensional structure was drawn using Chem 3D and saved in mol2 format.

[0075] 8.2) Molecular docking: Using Autodock open source software, first dehydrate and hydrogenate the odor difference components (ligands) and olfactory proteins (receptors) screened in step 7.2) (using Autodock software), and export them to pdbqt format. For the subsequent specific steps, see Figure 1 .

[0076] Set the docking box parameters to size_x=40, size_y=40, size_z=40, and the center coordinates to (0.100, 1.220, 12.720), (5.700, -7.740, -7.250), (-2.420, -7.100, -10.300), (1.280, -4.880, -10.180), and (10.390, -0.510, 0.330). Save as a .gpf file and run Autogrid4. After the run is finished, select the semi-flexible docking mode, select the Genetic algorithm, run 10 times, and OutputLamarckian G in the calculation method, and select the default parameters for other parameters. After completing the above processing, run Autodock, and when the run is finished, analyze the results. The conformation with the lowest binding energy is taken as the binding site of the odorant and the olfactory protein. According to the "temporal coding hypothesis of olfactory information" - the more strongly perceived compounds (and odor compounds) first bind to the olfactory protein receptors and show lower binding energy to verify the molecular sensory results.

[0077] (9) Back-addition experiment verification: Blood orange essential oil 2 was selected as the matrix, and the odor-related compounds identified in steps 7.2 and 7.3 were added in sequence (based on the quantitative results of step 7.1). According to the sensory evaluation method in step (2), the odor and citrus flavor perception intensity of blood orange essential oil 1 was set to 10, and the odor intensity and citrus aroma intensity ratio of the back-added solution to blood orange essential oil 1 was calculated to verify the odor contribution of the identified compounds.

[0078] Example 2: A comparative experiment was conducted on the flavor differences of Gannan navel orange essential oil extracted by steam and cold pressing, thereby identifying the odorous compounds in the navel orange essential oil extracted by steam distillation. The experimental process and results are as follows:

[0079] (1) Blood orange essential oil was extracted from Gannan navel orange (peel) using a steam extraction device (solid-liquid ratio 1:4) and a three-wheel press, respectively, and stored in a 4°C refrigerator for testing.

[0080] The extraction of the water vapor extraction device is as follows: the peel of Gannan navel orange is mixed with deionized water in a weight ratio of 1:4, then crushed and placed in a distillation flask, the heating temperature is controlled at 120-135°C, and the water-extracted blood orange essential oil is collected by condensation reflux. When the essential oil in the collector no longer increases, the heating is stopped. After dehydration with anhydrous sodium sulfite, navel orange essential oil 1 is obtained;

[0081] The three-wheel press extraction is as follows: soak the Gannan navel orange peel in a 1.0% calcium chloride solution for 4 hours, rinse with deionized water, and then squeeze it with a three-wheel press. The resulting mixed solution is allowed to stand for 20 minutes, the upper layer mixture is centrifuged at 6000r / min for 20 minutes, the upper layer essential oil is collected and dried with anhydrous sodium sulfate to obtain navel orange essential oil II.

[0082] The navel orange essential oil 1 and the navel orange essential oil 2 are subjected to the same steps as those described in (2) to (8) in Example 1;

[0083] (9) Add back the experiment to verify:

[0084] Select navel orange essential oil 2 as the matrix, add the odor-related compounds identified in steps 7.2 and 7.3 of Example 2 (based on the quantitative results of step 7.1) in sequence, refer to the sensory evaluation method in step (2), set the odor and citrus flavor perception intensity of navel orange essential oil 1 to 10, calculate the odor intensity and citrus aroma intensity ratio of the added essential oil to navel orange essential oil 1, and verify the odor contribution of the identified compounds.

[0085] The experimental results of the above-mentioned Example 1 and Example 2 are as follows:

[0086] Sensory evaluation: The sensory evaluation results are as follows: Figure 2 As shown in Figure 2, steam-extracted blood orange and navel orange essential oils had higher off-flavor attributes and lower overall liking.

[0087] GC-O-MS results: The results are shown in Table 2. 20 and 29 odor-active compounds were detected in blood orange essential oil and navel orange essential oil, respectively. Among them, terpinene-4-ol, α-terpinene, α-terpineol, calendula, dihydrocarveol, trans-carveol and unknown compound 3 (the content was below the GC-MS detection limit and was not detected) all smelled obvious odors related to odors, so it was speculated that the above substances contributed to the formation of odors. Further, given that the MF values ​​of α-terpinene, calendula, dihydrocarveol and unknown compound 3 in cold-pressed and water-extracted essential oils were similar or less than 65%, while the MF values ​​of β-citronellol and geraniol in water-extracted sweet orange essential oils were higher than those in cold-pressed essential oils, it was preliminarily inferred that terpinene-4-ol, α-terpineol, trans-carveol, β-carveol and geraniol were odor-related compounds.

[0088] Chemometric results: Based on OPLS-DA analysis combined with VIP values, 11 compounds were identified that contributed significantly to the flavor differences of citrus essential oils. Figure 3 As shown. According to the screening conditions of "P<0.05, VIP≥1", five odor substances, terpinene-4-ol, α-terpinene, trans-carveol, geraniol and β-citronellol, were screened out, which are both odor-related compounds and key differential components of sweet orange essential oil. PLSR analysis results ( Figure 4 ) showed that terpinene-4-ol, α-terpineol and trans-carveol were most correlated with the odors such as "cooking taste" and "pungent taste". Therefore, it was inferred that terpinene-4-ol, α-terpineol and trans-carveol were the main odor compounds identified in sweet orange essential oil.

[0089] Molecular docking results: The lowest binding energy is used as the basis for judging the affinity between the compound and the olfactory protein. Figure 5 Terpinen-4-ol, α-terpineol, and trans-carveol, which have off-flavor properties, showed lower binding energy to olfactory proteins than β-carveol and geraniol.

[0090] Verification results of the back-addition experiment: For Example 1, β-citronellal, terpinene-4-ol, α-terpineol, and trans-carveol were added to blood orange essential oil one in sequence, and it was found that the odor perception intensity in blood orange essential oil one increased, while the citrus aroma attribute decreased, thereby verifying that β-citronellal, terpinene-4-ol, α-terpineol, and trans-carveol are odor-related compounds in blood orange essential oil. For Example 2, β-citronellal, geraniol, terpinene-4-ol, α-terpineol, and trans-carveol were added to navel orange essential oil one in sequence, and it was found that the odor perception intensity in blood orange essential oil one increased, while the citrus aroma attribute decreased, thereby verifying that β-citronellal, geraniol terpinene-4-ol, α-terpineol, and trans-carveol are odor-related compounds in navel orange essential oil.

[0091] Table 2. Identification and odor characterization of odor-active compounds

[0092]

[0093]

[0094]

[0095] Note: / indicates not detected.

[0096] MF>65% was determined to contribute to odor.

[0097] Comparative Example 1: The method for identifying odorous substances in sweet orange essential oil specially designed by the present invention in Example 1 is changed to identifying odorous substances in sweet orange essential oil only from the perspective of smell as described in conventional technology, and the rest can refer to Example 1.

[0098] The results obtained are: terpinene-4-ol, α-terpinene, α-terpineol, calendula, dihydrocarveol, and trans-carveol are identified as compounds related to odor. Comparison with the results obtained in Example 1 of the present invention shows that the comparative example cannot well screen out odor substances caused by thermal processing. Therefore, it proves the importance of combining chemometric means with computer simulation verification in the present invention.

[0099] Comparative Example 2: The docking box setting method of the present invention in Example 1 is changed to set the size of the docking box to wrap the entire olfactory protein as described in conventional technology, and the rest is the same as Example 1.

[0100] The results obtained are: the binding site of the odorant in the olfactory protein is located in the extracellular region. Compared with the results obtained in Example 1 of the present invention, it can be seen that the binding energy obtained in Comparative Example 2 has no reference value (the odorant does not bind to the catalytic pocket of the olfactory protein).

[0101] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A method for measuring the peculiar smell of sweet orange essential oil, characterized in that: Determination of off-flavors of sweet orange essential oil using modern molecular sensory technology combined with molecular docking.

2. The method for measuring the peculiar smell of sweet orange essential oil according to claim 1, wherein: The mixing ratio (volume ratio) of sweet orange essential oil and dichloromethane as a solvent was 1:9, and the injection volume was 1 μL.

3. The method for measuring the peculiar smell of sweet orange essential oil according to claim 1 or 2, characterized in that The following steps are involved: (1) Sensory evaluation of sweet orange essential oil: Determine sensory descriptors for the flavor of sweet orange essential oil; (2) Pretreatment of sweet orange essential oil: Dissolve sweet orange essential oil in dichloromethane, add internal standard solution, mix well, and filter through a 0.22 μL oil filter membrane; (3) GC-O-MS parameter setting; (4) Olfactory sense: While the GC program was running, the retention time when the odor was detected was recorded, the odor characteristics were described, and the odor intensity was scored using a 4-point scale; (5) Preliminary screening of odor-related compounds: The set odors are cooking and pungent; (6) Further identification of odor compounds: Further identification of compounds that are strongly associated with "cooking smell" and "pungent smell"; (7) Pre-molecular docking processing; (8) Molecular docking: The conformation with the lowest binding energy is taken as the binding site of the olfactory molecule and used to verify the computer simulation of odor compounds.

4. The method for measuring the peculiar smell of sweet orange essential oil according to claim 3, characterized in that The method further comprises the following step (9): (9) Addition experiment: Using freshly extracted cold-pressed sweet orange essential oil as the matrix, the identified odor-related compounds were added, and the citrus attribute intensity and odor attribute intensity of the added essential oil were sensory evaluated.

5. The method for measuring the peculiar smell of sweet orange essential oil according to claim 3 or 4, characterized in that Step 3) is: The GC-O-MS temperature program was as follows: initial temperature 40 °C, hold for 2 min, increase to 130 °C at 4 °C / min, increase to 160 °C at 3 °C / min, hold for 1 min, increase to 240 °C at 10 °C / min and hold for 5 min; The GC-O-MS used a DB-5ms column, an inlet temperature of 240 °C, a constant helium flow rate of 1.5 mL / min, and a split ratio of 20:1; The MS used EI source, the ion source temperature was 230°C, the scanning mode was scan, the scanning mass range was 45-350 m / z, and the scanning frequency was 4.58 scans / s.

6. The method for measuring the peculiar smell of sweet orange essential oil according to claim 5, characterized in that: The step 5) is: Based on the olfactory results, the NIST spectral library was consulted to determine the odor-active compounds, and the compounds with significant perceived "cooking smell" and "pungent smell" were preliminarily identified as odor-related compounds; The step 6) is: Combined with chemometrics, compounds with strong correlations with "cooking smell" and "pungent smell" were further identified; The step 7) is: the olfactory protein selects a terpene compound broadly recognized receptor, uses AlphaFold to predict the three-dimensional structure model, and saves it in pdb format; the small molecule three-dimensional structure is drawn using Chem 3D and saved in mol2 format; The step 8) is: using Autodock open source software, first dehydrating and hydrogenating the ligand and receptor, exporting them into pdbqt format, and taking the conformation with the lowest binding energy as the olfactory molecule binding site for verification of computer simulation of odor compounds.

7. The method for measuring the peculiar smell of sweet orange essential oil according to claim 6, characterized in that: The olfactory protein receptors are OR1A1, OR2W1, OR1G1, OR5AN1 and OR52D1.

8. The method for measuring the peculiar smell of sweet orange essential oil according to claim 7, characterized in that: The docking box setting parameters are size_x=40, size_y=40, size_z=40, and the center coordinates are (0.100, 1.220, 12.720), (5.700, -7.740, -7.250), (-2.420, -7.100, -10.300), (1.280, -4.880, -10.180) and (10.390, -0.510, 0.330).

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