A sweet orange flavoring based on the olfactory receptor OR2W1 and its design method

Abstract

This invention discloses a sweet orange flavoring based on the olfactory receptor OR2W1 and its design method, belonging to the fields of cell biology and fragrance technology. The method includes: transfecting the olfactory receptor OR2W1 into HEK-293 cells to construct OR2W1 cells; selecting various characteristic aroma substances constituting the sweet orange flavor and preparing a series of concentration test solutions; contacting the test solutions with OR2W1 cells to detect the cAMP signal response within the OR2W1 cells, and determining the optimal response concentration for each characteristic aroma substance to activate OR2W1 cells based on the peak response fold; determining the weight ratio of each characteristic aroma substance in the flavoring formulation based on the optimal response concentration; weighing each aroma ingredient according to the weight ratio, adding it to a base solvent, stirring evenly, and allowing it to mature to obtain the sweet orange flavoring. This invention applies the functional detection results of the olfactory receptor to the flavoring formulation design, achieving precise and scientific design of the sweet orange flavoring, resulting in a product with a pure flavor and harmonious aroma.

Description

Technical Field

[0001] This invention belongs to the field of cell biology and fragrance technology, specifically relating to a sweet orange fragrance based on the olfactory receptor OR2W1 and its design method. Background Technology

[0002] In the food, cosmetics, and fragrance industries, the aroma characteristics and optimal concentration of a single aroma substance are the core basis for product formulation design. Taking sweet orange flavoring as an example, its flavor quality highly depends on the reasonable ratio of various characteristic aroma substances. However, traditional detection techniques, such as gas chromatography-mass spectrometry, while accurately quantifying the content of substances, cannot directly reflect the intensity of the interaction between the substance and the human olfactory system, nor can they scientifically determine its optimal concentration in the flavor formulation. Furthermore, traditional sensory evaluation is heavily influenced by subjective factors, leading to a reliance on experience in flavor formulation design, resulting in low R&D efficiency and poor flavor reproducibility.

[0003] In biological evolution, the olfactory system has developed a highly complex chemical signal recognition mechanism: volatile aromatic substances specifically bind to olfactory receptors (ORs) on the surface of olfactory epithelial cells, triggering a signal transduction cascade and ultimately forming olfactory perception. Therefore, simulating this receptor-ligand interaction process to evaluate aromatic substances at the biological functional level is key to solving the aforementioned problems. However, olfactory receptors, as G protein-coupled receptors (GPCRs), are difficult to functionally express in vitro. OR2W1 is a member of the human olfactory receptor family with broad-spectrum ligand recognition capabilities, specifically responding to a variety of aromatic compounds such as acids, alcohols, phenols, aldehydes, ketones, ethers, and esters, making it an ideal target for constructing a sweet orange flavor detection system. However, currently, there is a lack of a method to systematically apply the functional detection results of the OR2W1 receptor to flavor formulation design. Summary of the Invention

[0004] To address the problems of existing flavor formulation design relying on experience, lacking quantitative basis, and exhibiting poor flavor harmony, this invention provides a sweet orange flavor based on the olfactory receptor OR2W1 and its design method. This invention constructs an in vitro OR2W1 detection system to accurately determine the optimal response concentration of sweet orange characteristic aroma substances activating the receptor, and uses this as a quantitative basis for flavor formulation design. This invention directly applies biological function detection results to flavor formulation design, achieving scientific and precise flavor formulation design.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In one aspect, this invention provides a method for designing sweet orange flavoring based on the olfactory receptor OR2W1, comprising the following steps:

[0007] (1) Construction of OR2W1 receptor expression cells: The olfactory receptor OR2W1 was transfected into HEK-293 cells, and Gαolf and RTP1S cofactor were co-expressed;

[0008] (2) Determination of the optimal response concentration of a single aroma substance: Select a variety of characteristic aroma substances that constitute the flavor of sweet orange, prepare a series of concentration test solutions for each of the characteristic aroma substances, contact the test solutions with the OR2W1 receptor expressing cells, detect the cAMP signal response in the OR2W1 receptor expressing cells, and determine the optimal response concentration of each characteristic aroma substance for activating the OR2W1 receptor expressing cells based on the concentration corresponding to the peak value of the response fold.

[0009] (3) Sweet orange flavor formulation design: Based on the optimal response concentration of each characteristic aroma substance, determine the weight ratio of each characteristic aroma substance in the flavor formulation so that each component is within its optimal response concentration range.

[0010] (4) Preparation of sweet orange flavoring: Weigh each flavoring raw material according to the weight ratio, add it to the base solvent, stir evenly and let it stand to mature, and obtain sweet orange flavoring.

[0011] As a further aspect of the present invention: in step (1), the HEK-293 cells are HEK-293 cells with endogenous G protein-coupled receptors knocked out;

[0012] And / or, in step (1), the HEK-293 cells are second-generation cells with a cell density of 80% or higher or a viable cell concentration of ≥1×10⁻⁶. 6 Transfection was performed at a cell / mL concentration, followed by 12-24 hours of culture.

[0013] As a further aspect of the present invention: in step (1), Lipofectamine 3000 reagent is used for plasmid co-transfection, and the transfection system includes Gαolf helper plasmid, RTP1S helper plasmid and OR2W1 receptor plasmid.

[0014] As a further embodiment of the present invention: in step (2), the characteristic aroma substances include: nerol, decanal, limonene, geraniol, citronellol acetate, α-pinene, linalool, ethyl butyrate, octanal, nerolidol, linalool acetate, neroliyl acetate, octyl acetate, phenethyl alcohol and α-terpinene.

[0015] As a further aspect of the present invention: in step (2), the cAMP signal detection employs Glosensor. TM cAMP Assay reagents were used for kinetic detection using the chemiluminescence module of a multi-functional microplate reader.

[0016] As a further aspect of the present invention: in step (2), the formula for calculating the response factor is: response factor = (measured value - negative control value) / baseline value.

[0017] As a further aspect of the present invention: in step (4), the flavoring raw materials are added in order of boiling point from high to low.

[0018] As a further aspect of the present invention: in step (4), the base solvent is food-grade 95% ethanol or food-grade propylene glycol;

[0019] And / or, in step (4), the standing ripening is to stand at room temperature for 24~48h.

[0020] In this invention, the base solvent is usually selected according to the application scenario. For food and beverage applications, food-grade 95% ethanol is used; for cosmetic and daily chemical anhydrous system applications, food-grade propylene glycol is used.

[0021] In another aspect, the present invention provides a sweet orange flavoring, which is prepared by the above-described sweet orange flavoring design method based on the olfactory receptor OR2W1.

[0022] As a further embodiment of the present invention, the following components are composed in parts by weight: 38.0 parts limonene, 18.0 parts octanal, 12.0 parts decanal, 10.0 parts geraniol, 5.0 parts ethyl butyrate, 4.0 parts linalool, 3.0 parts nerol, 2.5 parts α-pinene, 1.8 parts nerolidol, 1.5 parts phenethyl alcohol, 1.2 parts neryl acetate, 1.0 part linalool acetate, 0.8 parts α-terpinene, 0.6 parts octyl acetate, 0.6 parts citronellol acetate, and a base solvent, wherein the amount of the base solvent is 50-80% of the total mass of the sweet orange flavoring.

[0023] Compared with the prior art, the beneficial effects of the present invention are reflected in:

[0024] (1) This invention constructs an in vitro cell system for detecting single aroma substances by transfecting the olfactory receptor OR2W1 into HEK-293 cells and co-expressing Gαolf and RTP1S cofactors. This system uses a HEK-293 cell line with the endogenous G protein-coupled receptor knocked out, which improves the localization efficiency of the OR2W1 receptor on the cell membrane, reduces background signal interference, and increases the detection signal-to-noise ratio. Using Lipofectamine 3000 reagent for plasmid co-transfection, this system can stably express the functional OR2W1 receptor, achieving specific recognition of various aroma substances.

[0025] (2) This invention utilizes Glosensor TMcAMP detection reagents were used to dynamically monitor cAMP signals in transfected cells. After adding different concentrations of a single aroma compound, changes in the chemiluminescence signal were recorded, and the fold change at each concentration was calculated. The concentration corresponding to the peak fold change was taken as the optimal concentration for that aroma compound to activate the OR2W1 receptor. This method can accurately determine the optimal concentration of each aroma compound at the receptor level, providing a quantitative basis for formulation design.

[0026] (3) This invention uses the optimal response concentration of each aroma substance as a benchmark, combined with the aroma composition characteristics of the natural flavor of sweet orange, to determine the weight ratio of each aroma ingredient in the fragrance formulation. By ensuring that each component in the formulation is within its optimal response concentration range, it ensures that each aroma substance can play its expected flavor contribution in the final product. This design method directly converts biological detection results into formulation parameters, reducing the reliance on experience in the traditional perfumery process.

[0027] (4) This invention establishes an integrated process from detection to formulation, linking receptor response detection, optimal concentration determination, and formulation design into a whole. Based on the scientific evidence of receptor-level biofunctional detection, this process achieves quantitative design of flavor formulations, thereby improving the flavor harmony and aroma purity of the product. This method fills the technological gap in the precise design of flavors from biofunctional detection to flavor development, providing a new technological path for flavor research and development. Attached Figure Description

[0028] Figure 1 This is a response fold curve of different concentrations of octanal over time in Example 1 of the present invention;

[0029] Figure 2 This is a peak response fold curve of different concentrations of the four single aroma substances in Example 1 of the present invention;

[0030] Figure 3 This is a peak response factor curve of different concentrations of 11 single aroma substances in Example 1 of the present invention. Detailed Implementation

[0031] The present invention will now be described in detail with reference to embodiments, providing a clear and complete description of the technical solutions to facilitate understanding of the invention by those skilled in the art. The described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Furthermore, all raw materials mentioned below, unless otherwise specified, are commercially available products; and all process steps or preparation methods not mentioned in detail are process steps or preparation methods known to those skilled in the art.

[0032] Example 1

[0033] A method for designing sweet orange flavoring based on the olfactory receptor OR2W1 includes the following steps:

[0034] 1. Preparation of aroma substance samples

[0035] Fifteen characteristic aroma compounds of sweet orange, including nerol, decanal, limonene, geraniol, citronellol acetate, α-pinene, linalool, ethyl butyrate, octanal, nerolidol, linalyl acetate, neroliyl acetate, octyl acetate, phenethyl alcohol, and α-terpinene, were selected. Dimethyl sulfoxide (DMSO) was used as the solvent to prepare 100-fold concentration stock solutions corresponding to 10 groups of samples with concentration gradients of 10-100 μM and gradient intervals of 10 μM.

[0036] 2. Counting of HEK-293 cells and concentration of co-transfected plasmids

[0037] HEK-293 cells were HEK-293 cells with endogenous G protein-coupled receptor knocked out. Cryopreserved HEK-293 cells were removed from the liquid nitrogen tank, and 1 mL of cryopreservation solution was mixed with 5 mL of complete culture medium. The mixture was centrifuged (1000 rpm, 3 min), the supernatant was discarded, and the cells were resuspended in 1 mL of complete culture medium and transferred to culture dishes. The cells were incubated at 37°C, 5% CO2 for 24 h before passage. Once the cell density reached over 80% under a microscope, plasmid co-transfection was performed. Before preparing the transfection solution, the complete culture medium in the culture dish containing HEK-293 cells was replaced with an equal volume of Opti-MEM medium. The transfection solution was prepared according to the transfection reagent instructions.

[0038] 3. cAMP concentration detection

[0039] After transfection and culturing for 12 hours, trypsin was added to digest the cells. 10 μL of the cell suspension was then mixed with 10 μL of trypan blue cell viability dye. The viable cell concentration was measured using a Countstar Biolab cell counter, requiring a viable cell concentration ≥1 × 10⁻⁶. 6 Cells / mL. After centrifugation to remove the supernatant, 10 mL of detection reagent was added to the remaining cells, mixed well, and transferred to a white, opaque 96-well plate (100 μL per well). The plates were incubated at room temperature in the dark for 2 hours. Full-wavelength kinetic cyclic detection was performed using the chemiluminescence module of a multi-mode microplate reader. Detection parameters included:

[0040] Baseline determination: Detect for 3 minutes (30-second intervals) without added sample;

[0041] Sample detection: Add 1 μL of aroma substance solution (3 replicates) and DMSO negative control to each well. Detect for 15 min (30 s intervals), and record the baseline value and the response value after adding the aroma substance. The response fold = (measured value - negative control value) / baseline value. The negative control is the DMSO control, which was 345 in this example. The baseline value is the background value without added sample. The peak response fold for each concentration is taken as the response fold corresponding to that concentration, and the concentration corresponding to the peak response fold is taken as the optimal response concentration for that aroma substance to activate OR2W1.

[0042] 4. Data processing of single aroma compounds

[0043] The curves showing the change in the response factor of octanal at various concentrations over time are shown below. Figure 1 The peak response fold curves for different concentrations of each individual aroma compound are shown in the figure. Figure 2 and Figure 3 .in, Figure 2 These are four substances with a high response factor (>10), including decanal, limonene, geraniol, and octanal; Figure 3 These are the remaining 11 substances with low response folds (<10) and no response. The concentration-response fold peak data for the 15 single aroma substances are shown in Table 1.

[0044] Table 1. Response factor of different concentrations of different substances

[0045]

[0046] As shown in Table 1, OR2W1 exhibits significant specificity in recognizing 15 characteristic aroma compounds of sweet orange. The response patterns of different types of aroma compounds show obvious differences, and the response fold is clearly dependent on the concentration.

[0047] High-response core aroma compounds (response fold > 10): These compounds are key contributors to the sweet orange flavor, and OR2W1 has the strongest affinity for them. Among them, octanal exhibits the most prominent response activity, reaching a peak response value (68.7758) at a concentration of 40 μM. At this concentration, OR2W1 activation efficiency is highest, making it the core source of the fresh aldehyde aroma of sweet orange. Its optimal response concentration is determined to be 40 μM. Limonene, as the main component of sweet orange fruit aroma, shows a continuous increase in response fold with increasing concentration, reaching a peak value (31.2149) at a concentration of 100 μM, with no significant concentration inhibition effect. Its optimal response concentration is 100 μM. Decanal shows a peak response value (12.4729) at a concentration of 80 μM, at which it can fully release its fresh citrus aldehyde aroma. Its optimal response concentration is 80 μM. Geraniol's peak response value appears at a concentration of 90 μM (10.7085), at which point the floral characteristics are most prominent. Its optimal response concentration is determined to be 90 μM.

[0048] Medium-responsive auxiliary aroma compounds (response factor 2.0-10.0): These compounds play an important role in the harmony of sweet orange flavor. Linalool reaches its peak response at 50 μM (3.4963), imparting a soft floral base to the fragrance; the optimal response concentration is 50 μM. Nerol reaches its peak response at 50 μM (2.0809), enhancing the sweetness of sweet orange; the optimal response concentration is 50 μM. Ethyl butyrate's response factor increases with concentration, reaching a peak at 100 μM (3.3677), supplementing the sweet fruity aroma; the optimal response concentration is 100 μM. α-Pinene also shows increased response with increasing concentration, reaching a peak response of 1.5909 (close to 2.0) at 100 μM, laying the foundation for a fresh herbal scent; the optimal response concentration is 100 μM.

[0049] Low-response aroma modifiers (response factor < 2.0): Although these substances have a lower response intensity, they can significantly enhance the flavor profile. The peak responses of nerolidol, phenylethyl alcohol, and nerol acetate all occurred at a concentration of 10 μM (4.6952, 4.0041, and 3.9894, respectively; note: although the response factor here is slightly higher than 2.0, it is still considered a modifier compared to high-response substances, and it works even at low concentrations). At this concentration, they can delicately enhance floral and sweet notes, avoiding abrupt aromas; the optimal response concentration for all of them is 10 μM. The peak responses of linalool acetate, α-terpinene, and octyl acetate occurred at a concentration of 30 μM (2.6368, 2.2076, and 1.9372), respectively, which can adjust the roundness of the aroma; the optimal response concentration is 30 μM. The peak response of citronellol acetate was at a concentration of 10 μM (1.8815), which slightly complements the freshness of the sweet orange flavor; the optimal response concentration is 10 μM.

[0050] Further analysis revealed that OR2W1 exhibited significantly higher recognition activity for aldehydes (octanal, decanal) and terpenes (limonene) than for some esters and alcohols. This aligns closely with the characteristic that aldehydes and terpenes are the core aroma components in the natural flavor of sweet orange, validating the biological relevance of this detection system. Furthermore, the optimal response concentrations for each aroma compound were all within the range of 10 μM–100 μM, with no cases exceeding the cytotoxicity threshold (≤100 μM), ensuring the reliability of the detection results and the safety of subsequent fragrance formulations.

[0051] Step 5): Sweet orange flavor formula and supplementary verification experiment

[0052] Based on the optimal response concentrations of 15 characteristic aroma compounds of sweet orange, combined with the relative contribution of aroma compounds in natural sweet orange fruit, and following the formulation principle of "core aroma compounds as the main component and auxiliary modification compounds as the auxiliary component", the weight proportions of sweet orange flavoring are determined as shown in Table 2.

[0053] Table 2

[0054]

[0055] In this invention, the weight proportions of each fragrance ingredient are not directly calculated from the optimal response concentration. Instead, they are derived by using the optimal response concentration as the core bioavailability quantification benchmark, combined with the OR2W1 receptor response fold, the relative contribution of aroma substances in natural sweet orange fruit, the differences in aroma thresholds of different aroma substances, and the synergistic effect of multi-component concentrations. The optimal response concentration is the actual concentration that each aroma substance needs to achieve in the final fragrance system, and the weight proportions are calculated based on the dilution ratio of the base solvent.

[0056] The specific conversion logic and core steps are as follows: First, based on the typical usage concentration of 0.1% in the end product, the optimal response concentration measured in cell experiments (i.e., the target molar concentration of aroma substances in the terminal aqueous system) is reversed to the target molar concentration of the fragrance stock solution; then, the molar mass of each aroma substance is converted into the target mass concentration in the fragrance stock solution; combined with the 1:1 dilution ratio of fragrance raw materials and base solvent in this embodiment, the preliminary mass proportion of each component in the pure fragrance raw material mixture is calculated; then, four correction coefficients are introduced for precise fine-tuning: OR2W1 receptor competitive binding effect, contribution of natural sweet orange aroma, loss rate of volatile substances, and synergistic effect of multiple components; finally, the corrected mass proportions of all components are normalized to obtain the final formula ratio with a total weight of 100.

[0057] Detailed calculations are performed using the core component octanal as an example:

[0058] 1. Calculating the target molar concentration of the fragrance stock solution: The optimal response concentration of octanal is 40 μM (terminal aqueous phase). With a fragrance addition of 0.1% in the final product, the target molar concentration of octanal in the fragrance stock solution = 40 μM ÷ 0.1% = 40 × 10⁻⁶ -3 mol / L (40mM);

[0059] 2. Conversion to mass concentration: Octal molar mass is 128.21 g / mol, target mass concentration = 40 × 10⁻⁶ g / mol -3 mol / L × 128.21g / mol ≈5.13g / L;

[0060] 3. Calculation of preliminary mass percentage: In this embodiment, the mass ratio of fragrance raw material to 95% ethanol is 1:1, that is, 1L of fragrance contains 500g of pure fragrance raw material. Therefore, the preliminary percentage of octanal in the fragrance raw material = 5.13g / L ÷ 500g / L × 100% ≈ 1.03%;

[0061] 4. Multi-dimensional corrections: ① Receptor competition correction: Other substances in the mixed system will compete for OR2W1 binding, and the core high-response substance needs to be upregulated, correction coefficient 3.2; ② Natural contribution correction: Octaldehyde is the core of the sweet orange fresh aldehyde aroma and has a high contribution to the natural flavor, correction coefficient 2.5; ③ Volatility correction: Octaldehyde has a low boiling point and is easily volatile, with a terminal retention rate of about 30%, correction coefficient 3.3; ④ Synergistic effect correction: It has a significant synergistic effect with limonene, correction coefficient 0.65; Overall correction coefficient = 3.2 × 2.5 × 3.3 × 0.65 ≈ 17.16;

[0062] 5. Corrected percentage: 1.03% × 17.16 ≈ 17.67%, rounded to 18%;

[0063] 6. After all components are calculated using this method, they are normalized to a total weight of 100 to obtain the final formula.

[0064] The amount of base solvent can be adjusted within the range of 50% to 80% of the total mass of the fragrance, depending on the application scenario. A lower amount results in a richer aroma, while a higher amount results in a lighter aroma. In this example, 95% ethanol is used as the base solvent, and the mass ratio of fragrance raw material to base solvent is 1:1.

[0065] The order of adding ingredients, from lowest to highest volatility (i.e., from highest to lowest boiling point), is as follows:

[0066] Nerolidol → Neroliyl acetate → Citronellol acetate → Geraniol → Nerolidol → Phenethyl alcohol → Linaloyl acetate → Octyl acetate → Decanal → Linalool → Limonene → α-Pineene → Octal → α-Pinene → Ethyl butyrate.

[0067] After each ingredient is added, the mixture is magnetically stirred at 300 rpm for 10 minutes. After all ingredients are added, the mixture is stirred for another 30 minutes until the system is homogeneous and transparent. Then, it is placed in a dark, sealed environment at 25°C and allowed to stand for 48 hours to mature, stirring for 10 minutes every 12 hours to promote the full integration of aroma components.

[0068] Effect Example

[0069] Sensory evaluation verification experiment

[0070] Twenty professionally trained sensory evaluators (10 men and 10 women, aged 20-25) were selected. All of them had no olfactory impairment or taste abnormalities and had received specialized training in sweet orange flavor identification, enabling them to accurately distinguish the characteristic aroma of sweet orange from off-flavors.

[0071] Three commercially available high-quality sweet orange flavorings were selected as control samples: CK1 (Kongque Company 24044), CK2 (Aipu Company 180044), and CK3 (Chuanye Company CYQY015). Simultaneously, a control sample CK4 with an empirically prepared non-optimal response concentration was also included. The preparation process of CK4 was completely consistent with that of S1, with food-grade 95% ethanol as the base solvent, accounting for 50% of the total mass of CK4 fragrance. The total weight of its fragrance raw materials was 100 parts, specifically composed of: 76.0 parts limonene, 9.0 parts octanal, 4.8 parts decanal, 16.0 parts geraniol, 1.5 parts ethyl butyrate, 6.8 parts linalool, 0.6 parts nerol, 4.5 parts α-pinene, 0.72 parts nerol, 2.4 parts phenethyl alcohol, 0.36 parts neryl acetate, 1.7 parts linalool acetate, 0.16 parts α-terpinene, 1.08 parts octyl acetate, and 0.24 parts citronellol acetate. In this formulation, the actual concentration of the core component limonene reaches 200 μM, while the actual concentration of octanal is only 20 μM. The actual concentrations of the remaining 13 auxiliary and modifying components all deviate from the optimal response concentration by more than 50%. The sweet orange flavoring prepared in this invention is sample S1. It is scored on a 10-point scale based on four dimensions: pure sweet orange flavor, aroma harmony, aroma freshness, and aroma persistence. A higher score indicates better performance. Among them:

[0072] Sweet orange flavor purity: Using the natural characteristic flavor of fresh, ripe sweet oranges and freshly squeezed orange juice as a reference, the typicality of the core aroma of the sample sweet orange and its match with the natural flavor are evaluated. The higher the score, the closer the characteristic flavor of the sample sweet orange is to the natural prototype, and the absence of off-flavors, odors, and artificial synthetic notes.

[0073] Aroma Harmony: This evaluates the degree of integration, balance, and layering of various aroma components in the sample, including terpene fruit aromas, aldehyde top notes, alcohol floral aromas, and ester base notes. A higher score indicates that the aroma components in the sample are well-balanced and naturally integrated, without any single aroma being abrupt or flavor fragmentation.

[0074] Aroma Freshness: The freshness and crispness of the aroma of the sample were evaluated based on the top notes of freshly peeled sweet oranges and freshly squeezed orange juice. The higher the score, the closer the aroma of the sample is to the fresh characteristics of fresh sweet oranges, without any unpleasant smells such as stuffiness, cloying sweetness, or pungent odor.

[0075] Aroma persistence: Under standard sensory evaluation conditions, the effective sensory retention time of the sample aroma and the smoothness of aroma release decay are evaluated. The higher the score, the longer the aroma retention time of the sample, the smooth and stable decay process, and the absence of rapid dissipation or aroma gaps.

[0076] The overall score is the arithmetic mean of the four dimensions mentioned above. The statistical results of the sensory evaluation are shown in Table 3.

[0077] Table 3

[0078]

[0079] The results showed that the sweet orange flavoring prepared by this invention was significantly higher than the commercially available control sample in terms of sweet orange flavor purity, aroma harmony, and overall score (P<0.05). Among them, the flavor purity was the best, and it was highly consistent with the flavor of fresh sweet orange. The aroma freshness score was 8.6 points, which was higher than the control samples CK2 and CK3, and only slightly lower than the control sample CK1, which is still at an excellent level in the industry. The aroma persistence score was 8.8 points, which was higher than the control samples CK1 and CK2, and on par with the control sample CK3, corresponding to an effective fragrance retention time of about 7.5 hours. In this experiment, aroma persistence and fragrance retention time were directly positively correlated. The effective fragrance retention time was defined as the longest duration during which a professional evaluator could clearly perceive the aroma after equal amounts of the test sample were dropped onto standardized qualitative filter paper. This was combined with the stability of aroma release decay for comprehensive evaluation. Simultaneous measurements showed that the effective fragrance retention times for control samples CK1, CK2, and CK3 were 7.2 h, 6.8 h, and 7.5 h, respectively. Referring to the QB / T 1505-2017 "Edible Flavors" industry standard and the general evaluation specifications for food flavors in China, an effective fragrance retention time ≥7 h and an aroma persistence score ≥8.0 points are considered excellent in the industry. The sample of this invention fully meets this standard and satisfies the practical application needs of food, daily chemical, and other scenarios. Evaluators reported that the sweet orange flavor prepared by this invention has no pungent aldehyde aroma or abrupt floral scent; the fruity, aldehyde, and floral aromas are distinct and naturally blended, exhibiting typical characteristics of fresh sweet orange aroma. To further verify the necessity of designing formulations based on the optimal response concentration of the OR2W1 receptor, this experiment added an empirical control sample CK4 with a non-optimal response concentration. The preparation process of this sample was completely identical to that of S1, except that the fragrance ingredient ratio was not based on the optimal response concentration. The actual concentration of the core component limonene reached 200 μM, while the actual concentration of octanal was only 20 μM. The concentrations of other auxiliary and modifying components deviated from the optimal values ​​by more than 50%. CK4 scored significantly lower than S1 and the commercially available control samples CK1-CK3, with a total score of only 6.1. This sample exhibits significant flavor defects due to a severe deviation of the core component concentration from the optimal response concentration of the OR2W1 receptor. The excessively high limonene concentration exceeds the optimal response range, resulting in a prominent astringent taste of terpenes and a dull, murky fruit flavor that masks the fresh aldehyde aroma. The insufficient concentration of octanal fails to reach the optimal concentration for receptor activation, thus failing to release the core fresh aldehyde aroma. Furthermore, the imbalance in the concentrations of various components leads to extremely poor aroma integration and chaotic layers. The aroma of the modified components is abrupt. Although the aroma persistence reaches 7.0 points, the flavor decays unevenly during the longevity process. Initially, only a strong astringent taste can be perceived, while later, there is no obvious sweet orange characteristic aroma, resulting in poor longevity.

[0080] Based on the optimal response concentration, this invention follows the "core-auxiliary-modification" principle, scientifically formulating a sweet orange flavoring based on the contribution of natural sweet orange aroma. By optimizing the order of ingredient addition and the maturation process, it ensures that each component is at its optimal concentration, achieving a natural fusion of fruity, aldehyde, and floral aromas. Sensory evaluation verification shows that this flavoring is significantly superior to commercially available products in terms of flavor purity and aroma harmony, possessing a typical fresh sweet orange aroma. Its storage stability and safety meet relevant standards, satisfying the needs of multiple application fields.

[0081] The integrated "detection-quantification-formulation-verification" process established in this invention fills the technological gap from chemical composition analysis to biological function evaluation and precise flavor design, promoting the deep integration of olfactory receptor biology and flavor engineering. Its detection methods are standardized and reproducible, and its formulations are both scientific and practical, making it a promising core R&D tool in the flavor field. It provides standardized solutions for flavor innovation in the food and daily chemical industries, possessing both significant scientific value and broad industrial application prospects.

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for designing sweet orange flavoring based on the olfactory receptor OR2W1, characterized in that, Includes the following steps: (1) Construction of OR2W1 receptor expression cells: The olfactory receptor OR2W1 was transfected into HEK-293 cells, and Gαolf and RTP1S cofactor were co-expressed; (2) Determination of the optimal response concentration of a single aroma substance: Select a variety of characteristic aroma substances that constitute the flavor of sweet orange, prepare a series of concentration test solutions for each of the characteristic aroma substances, contact the test solutions with the OR2W1 receptor expressing cells, detect the cAMP signal response in the OR2W1 receptor expressing cells, and determine the optimal response concentration of each characteristic aroma substance for activating the OR2W1 receptor expressing cells based on the concentration corresponding to the peak value of the response fold. (3) Sweet orange flavor formulation design: Based on the optimal response concentration of each characteristic aroma substance, determine the weight ratio of each characteristic aroma substance in the flavor formulation so that each component is within its optimal response concentration range. (4) Preparation of sweet orange flavoring: Weigh each flavoring raw material according to the weight ratio, add it to the base solvent, stir evenly and let it stand to mature, and obtain the sweet orange flavoring.

2. The method for designing sweet orange flavoring based on the olfactory receptor OR2W1 according to claim 1, characterized in that, In step (1), the HEK-293 cells are HEK-293 cells with endogenous G protein-coupled receptor knocked out; And / or, in step (1), the HEK-293 cells are second-generation cells with a cell density of 80% or higher or a viable cell concentration of ≥1×10⁻⁶. 6 Transfection was performed at a cell / mL concentration, followed by 12-24 hours of culture.

3. The method for designing sweet orange flavoring based on the olfactory receptor OR2W1 according to claim 1, characterized in that, In step (1), plasmid co-transfection was performed using Lipofectamine 3000 reagent. The transfection system included Gαolf helper plasmid, RTP1S helper plasmid and OR2W1 receptor plasmid.

4. The method for designing sweet orange flavor based on olfactory receptor OR2W1 according to claim 1, characterized in that, In step (2), the characteristic aroma substances include: nerol, decanal, limonene, geraniol, citronellol acetate, α-pinene, linalool, ethyl butyrate, octanal, nerolidol, linalool acetate, neroliyl acetate, octyl acetate, phenethyl alcohol, and α-terpinene.

5. The method for designing sweet orange flavoring based on olfactory receptor OR2W1 according to claim 1, characterized in that, In step (2), the cAMP signal detection uses Glosensor. TM cAMP Assay reagents were used for kinetic detection using the chemiluminescence module of a multi-functional microplate reader.

6. The method for designing sweet orange flavor based on olfactory receptor OR2W1 according to claim 1, characterized in that, In step (2), the formula for calculating the response fold is: response fold = (measured value - negative control value) / baseline value.

7. The method for designing sweet orange flavoring based on olfactory receptor OR2W1 according to claim 1, characterized in that, In step (4), the flavoring ingredients are added in order of boiling point from high to low.

8. The method for designing sweet orange flavoring based on olfactory receptor OR2W1 according to claim 1, characterized in that, In step (4), the base solvent is food-grade 95% ethanol or food-grade propylene glycol; And / or, in step (4), the standing ripening is to stand at room temperature for 24~48h.

9. A sweet orange flavoring, characterized in that, It is prepared by the sweet orange flavoring design method based on the olfactory receptor OR2W1 as described in any one of claims 1 to 8.

10. The sweet orange flavoring according to claim 9, characterized in that, It is composed of the following components in parts by weight: 38.0 parts limonene, 18.0 parts octanal, 12.0 parts decanal, 10.0 parts geraniol, 5.0 parts ethyl butyrate, 4.0 parts linalool, 3.0 parts nerol, 2.5 parts α-pinene, 1.8 parts nerolidol, 1.5 parts phenethyl alcohol, 1.2 parts neryl acetate, 1.0 part linalool acetate, 0.8 parts α-terpinene, 0.6 parts octyl acetate, 0.6 parts citronellol acetate, and a base solvent, wherein the amount of the base solvent is 50-80% of the total mass of the sweet orange flavoring.

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