Exhaled gas composition for identifying taste sensitivity as well as screening method and application of exhaled gas composition

Abstract

The invention belongs to the technical field of exhaled gas analysis, and particularly relates to an exhaled gas composition for identifying taste sensitivity and a screening method and application thereof. The taste sensitivities of individuals of different age groups are collected by adopting a sensory evaluation method, volatile compound components in exhaled gas are analyzed by adopting high-resolution mass spectrometry, and characteristic exhaled gas markers remarkably related to the taste sensitivities are screened by adopting difference analysis and correlation analysis. Therefore, the exhaled gas marker combination obtained through screening is used for identifying the individual taste sensitivity, and the method has the advantages of being convenient to sample, non-invasive, high in subject acceptability and wide in application range.

Description

Technical Field

[0001] The present invention belongs to the technical field of exhaled gas analysis, and in particular relates to an exhaled gas combination for identifying taste sensitivity, a screening method thereof, and an application thereof. Background Art

[0002] Taste is the core sense for humans to perceive the sensory attributes of food. Differences in taste sensitivity are not only key biomarkers for characterizing individual flavor perception abilities, but also an important scientific basis for constructing a food sensory evaluation system. Abnormal taste sensitivity is closely related to a variety of chronic diseases (such as hypertension, diabetes, Alzheimer's, etc.) and can be used as an early monitoring indicator. Sensory analysis is a method for measuring taste sensitivity specified in national standards, but its experimental process is complex and places high demands on evaluators.

[0003] In recent years, exhaled breath diagnostic analysis, as an emerging in vitro diagnostic method, has been successfully applied in areas such as human health assessment, disease screening, and clinical diagnosis. Endogenous volatile compounds in exhaled breath are metabolites of human tissue. Analyzing the composition and concentration of endogenous markers can non-invasively obtain information about human tissue metabolism and health status, helping to elucidate relevant physiological processes and early disease onset.

[0004] Currently, there are relatively few patents published domestically and internationally that use exhaled breath as a marker, and there is almost no mention of identifying taste sensitivity. Regarding methods for determining taste sensitivity, a patent has been published on methods and compositions for diagnosing and treating loss and / or distortion of taste or smell that is similar: the degree of loss of taste or smell in an individual is diagnosed by measuring the level of Sonic Hedgehog in nasal mucus, saliva, or a combination thereof (the method flow is as follows: Figure 1 The application of exhaled breath as a biomarker primarily focuses on human health, with published patents focusing on depression, yin-yang deficiency syndromes, and health conditions. These applications involve collecting exhaled breath, analyzing the composition and content of volatile compounds in the breath, and performing comparative analysis to assess health status. Currently, there is little research on identifying taste sensitivity.

[0005] Existing methods for identifying taste sensitivity often rely on sensory evaluation and analysis, which are cumbersome and demanding, making them difficult to rapidly identify on a large scale. A patent application titled "Methods and compositions for diagnosing and treating loss and / or distortion of taste or smell" uses sonic hedgehog protein levels to determine sensory loss, using the protein as a marker. While effective, antibody-based ELISA methods are prone to false positives due to cross-reactions and dimers, leaving room for improvement in their accuracy in assessing taste deterioration in large populations. Summary of the Invention

[0006] The patented invention uses exhaled breath as a marker for differentiation, which is convenient for sampling, highly acceptable to subjects, non-invasive and suitable for large-scale rapid identification. Specifically, the invention provides the following technical solutions:

[0007] The first aspect of the present invention is to provide a composition of taste sensitivity-related markers in exhaled breath, wherein the composition comprises butyl acetate, methyl acetate and isoprene.

[0008] Furthermore, the abundance of butyl acetate and methyl acetate was significantly positively correlated with taste threshold;

[0009] Furthermore, isoprene abundance was significantly negatively correlated with taste threshold;

[0010] Furthermore, the taste includes sweetness, saltiness, sourness, bitterness and umami.

[0011] A second aspect of the present invention is to provide a method for detecting a subject's taste sensitivity, the method comprising:

[0012] 1) Collect breath samples from subjects;

[0013] 2) analyzing the relative abundance of the composition described in the first aspect in volatile organic compounds (VOCs) in a breath sample using a GC-MS method;

[0014] 3) Interpretation of results: The abundance of butyl acetate and methyl acetate was significantly positively correlated with the taste threshold; the abundance of isoprene was significantly negatively correlated with the taste threshold.

[0015] Furthermore, the method for collecting the breath sample of the subject is to collect the breath sample of the subject using a commercial aluminum foil air sampling bag, and then transfer the breath sample to Tenax TA adsorption tubes respectively; preferably, the adsorption tubes are sealed and stored at 4°C, and GC-MS analysis needs to be performed within 3 days.

[0016] Furthermore, the GC-MS method analysis uses a GCMS-TQ8050NX combined with a TD-30R thermal desorption system to detect volatile organic compounds in exhaled breath;

[0017] Preferably, the operation of the thermal desorption system is as follows: VOCs in the adsorption tube are desorbed at 240-260°C for 4-6 minutes at a desorption flow rate of 50-70 mL / min, and are concentrated in a cold trap at -25--18°C; then, desorbed again at 250-270°C for 1-3 minutes, and the VOCs are transferred to the GC inlet;

[0018] Preferably, the adsorption tube is a Tenax TA adsorption tube;

[0019] Preferably, the GC operation in the GC-MS is as follows: chromatographic separation is performed using an SH-Rxi-624MS capillary column, and the column temperature program is as follows: initial temperature 33-37°C, maintained for 3-7 minutes; heating to 140-160°C at 5°C / min; then heating to 250-270°C at 30°C / min, maintained for 4-6 minutes; the carrier gas is helium, with a flow rate of 1-3 mL / min and a split ratio of 8-12:1;

[0020] Preferably, the MS operation in the GC-MS is to use an electron bombardment ion source with an ion source temperature of 250° C. The data acquisition mode is Q3 scan mode, the mass scan range is 30-350 m / z, and the scan speed is 0.3 s / scan.

[0021] Preferably, the result interpretation is that data processing is based on GCMS solution software, qualitative identification of compounds is performed by comparison with the NIST20 mass spectrum database, and compounds with a similarity index higher than 70 are preliminarily identified; retention index (RI) is calculated using C7-C40 normal alkanes for further identification, and compounds with RI value deviation less than 50 are considered to be acceptable qualitative results; peak area normalization is used to semi-quantitatively determine the relative abundance of different compounds in each breath sample.

[0022] Furthermore, the method is a non-disease diagnosis method.

[0023] A third aspect of the present invention is to provide a method for identifying a combination of taste sensitivity markers in a subject's exhaled breath, comprising:

[0024] (1) Taste sensitivity analysis: Based on age differences, we recruited elderly and young subjects and tested their taste sensitivity using sensory evaluation methods.

[0025] (2) Exhaled gas collection and analysis: After collecting human exhaled gas samples, the composition and content of volatile compounds in the exhaled gas are analyzed by high-resolution mass spectrometry;

[0026] (3) Characteristic compound screening: Characteristic compounds were preliminarily screened through differential analysis, and the exhaled gas combination for identifying taste sensitivity was further determined by combining correlation analysis.

[0027] Furthermore, the taste includes one or more of sour, sweet, bitter, salty and umami.

[0028] Furthermore, the sensory evaluation method includes one or more of a three-point forced choice method (3-AFC), a two-point forced choice method (2-AFC), and a rated difference method.

[0029] Furthermore, the taste sensitivity includes one or more of a perception threshold, a recognition threshold, and a difference threshold.

[0030] Furthermore, the exhaled gas collection is to collect the subject's exhaled gas samples using a commercial aluminum foil air sampling bag, and then transfer the exhaled gas samples to Tenax TA adsorption tubes respectively; preferably, the adsorption tubes are sealed and stored at 4°C, and GC-MS analysis needs to be performed within 3 days.

[0031] Furthermore, the analysis is to analyze the exhaled breath sample using GC-MS, specifically: using GCMS-TQ8050NX combined with TD-30R thermal desorption system to detect volatile organic compounds in the exhaled breath;

[0032] Preferably, the operation of the thermal desorption system is as follows: VOCs in the adsorption tube are desorbed at 240-260°C for 4-6 minutes at a desorption flow rate of 50-70 mL / min, and are concentrated in a cold trap at -25--18°C; then, desorbed again at 250-270°C for 1-3 minutes, and the VOCs are transferred to the GC inlet;

[0033] Preferably, the GC operation in the GC-MS is as follows: chromatographic separation is performed using an SH-Rxi-624MS capillary column, and the column temperature program is as follows: initial temperature 33-37°C, maintained for 3-7 minutes; heating to 140-160°C at 5°C / min; then heating to 250-270°C at 30°C / min, maintained for 4-6 minutes; the carrier gas is helium, with a flow rate of 1-3 mL / min and a split ratio of 8-12:1;

[0034] Preferably, the MS operation in the GC-MS is to use an electron bombardment ion source with an ion source temperature of 250° C. The data acquisition mode is Q3 scan mode, the mass scan range is 30-350 m / z, and the scan speed is 0.3 s / scan.

[0035] Preferably, the result interpretation is that data processing is based on GCMS solution software, qualitative identification of compounds is performed by comparison with the NIST20 mass spectrum database, and compounds with a similarity index higher than 70 are preliminarily identified; retention index (RI) is calculated using C7-C40 normal alkanes for further identification, and compounds with RI value deviation less than 50 are considered to be acceptable qualitative results; peak area normalization is used to semi-quantitatively determine the relative abundance of different compounds in each breath sample.

[0036] The present invention provides the following beneficial effects:

[0037] 1. Based on a comparative study of young and elderly groups, the screened exhaled gas combination covers a wide range of characteristics of taste sensitivity differences and has good accuracy and universality.

[0038] 2. It adopts a non-invasive sample collection method, which is easy to operate and meets the needs of large-scale population screening.

[0039] 3. Three endogenous volatile compounds in exhaled breath were accurately identified through difference analysis and correlation analysis, all of which were significantly correlated with taste sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Flowchart of methods and compositions for diagnosing and treating loss and / or distortion of taste or smell;

[0041] Figure 2 The overall method and steps of the specific implementation scheme of the method of the present invention;

[0042] Figure 3 Results of sweetness perception and recognition thresholds of people of different ages (***p<0.001, N=40);

[0043] Figure 4 Heat map of the abundance of TOP30 compounds in exhaled breath;

[0044] Figure 5 UMAP plot of exhaled breath compound abundance (left) and UMAP plot of sweet taste perception threshold (right);

[0045] Figure 6 Results of the difference analysis;

[0046] Figure 7 Correlation analysis results (**p<0.01, *p<0.1). DETAILED DESCRIPTION

[0047] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods described are all conventional methods unless otherwise specified. The materials described can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute undue limitations of the present invention. It should be noted that, unless there is a conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0048] Example 1 Screening of taste sensitivity markers

[0049] according to Figure 2 The process design and implementation of the method of the present invention:

[0050] 1. Taste Sensitivity Analysis

[0051] (1) Subject recruitment

[0052] Based on age differences, 30 subjects were recruited from the young group (20-30 years old) and 10 subjects from the elderly group (60-70 years old). Recruitment requirements: The subjects were in good health, had no smoking or heavy drinking habits, had not received specific periodontal treatment, and had not taken antibiotics in the past 3 months. In addition, the subjects were required to have no oral diseases, such as bleeding gums, oral ulcers, or toothaches, on the day of the experiment. All subjects signed the volunteer informed consent form for subsequent research. The information of all subjects is shown in Table 1.

[0053] Table 1 Basic statistics of subjects

[0054]

[0055]

[0056] (2) Sensory evaluation of taste sensitivity

[0057] The differences in individual taste sensitivity in different groups were explored according to the three-point forced choice method (3-AFC). A 15mL taste stimulus sample (food-grade sucrose) of a certain concentration was prepared with pure water and provided to the subjects together with two 15mL reference samples (pure water). The subjects were asked to choose a cup of sample that was different from the other two cups from the three cups of samples presented and evaluate its taste attributes (choose one of seven from sour, sweet, bitter, salty, fresh, water-like taste, and indescribable taste). The test cups were exactly the same tasting cups and were marked with three random numbers. The order of presentation was implemented in an increasing concentration sequence. All samples were tasted at room temperature, and pure water was provided to clean the mouth during the period. In this embodiment, all research subjects who met the inclusion criteria were required not to eat for at least 1 hour before sampling (drinking water was allowed) and to rinse their mouths with pure drinking water before the test. The perception threshold and recognition threshold of sweetness were calculated according to the following formula:

[0058]

[0059] Among them, C is the threshold, C n is the last incorrectly identified concentration, C n+1 is the last concentration that was not correctly identified. Figure 3 As shown, the perception and recognition thresholds for the young adult group were 1.08±0.65g / L and 1.98±1.01g / L, respectively, while those for the elderly group were both 17.14±7.84g / L, indicating that the elderly group had significantly higher sweetness perception and recognition thresholds than the young adult group. Furthermore, the threshold results for both groups showed a wide distribution, effectively covering the broad range of differences in taste sensitivity across different populations.

[0060] 2. Exhaled Gas Collection and Analysis

[0061] 1) Breath samples were collected from subjects using commercial aluminum foil air sampling bags. An MP-W5P pump was used to transfer 0.8 L of breath sample from each bag to a Tenax TA sorbent tube at a constant flow rate of 0.15 L / min. The tubes were sealed and stored at 4°C. GC-MS analysis was performed within 3 days.

[0062] 2) Volatile organic compounds (VOCs) in exhaled breath were detected using a GCMS-TQ8050NX coupled with a TD-30R thermal desorption system. In the thermal desorption system, VOCs were desorbed from the adsorption tube at 250°C for 5 minutes at a flow rate of 60 mL / min and concentrated in a -20°C cold trap. Subsequently, desorption was continued at 260°C for another 2 minutes, and the VOCs were transferred to the GC inlet.

[0063] 3) Chromatographic separation was performed using an SH-Rxi-624MS capillary column with the following column temperature program: initial temperature 35°C, hold for 5 min; increase the temperature at 5°C / min to 150°C; then increase the temperature at 30°C / min to 260°C, hold for 5 min. The carrier gas was helium at a flow rate of 2 mL / min, with a split ratio of 10:1.

[0064] 4) Mass spectrometry detection uses an electron bombardment ion source with an ion source temperature of 250°C. The data acquisition mode is Q3 scan mode, the mass scan range is 30-350m / z, and the scan speed is 0.3s / scan. Data processing is based on GCMSsolution software, and the qualitative identification of the compounds is compared with the NIST20 mass spectrum database. Compounds with a similarity index higher than 70 are preliminarily identified. In addition, the retention index (RI) of C7-C40 normal alkanes is calculated for further identification. Compounds with an RI value deviation of less than 50 are considered acceptable qualitative results. Peak area normalization is used to semi-quantitatively determine the relative abundance of different compounds in each breath sample.

[0065] 3. Screening of characteristic compounds

[0066] A total of 116 compounds were detected in oral exhaled air. Among them, the top 30 compounds with the highest abundance accounted for 87.1% of the total abundance and were used for subsequent analysis (results as shown in Figure 4 The UMAP dimensionality reduction algorithm was used to observe the distribution of exhaled gas compound abundance information and sweetness sensitivity information between the two groups (the results are shown in Figure 5 The results showed that the distribution of exhaled gas compounds in the elderly and young groups could be clearly distinguished, and the classification pattern was highly consistent with the sweet taste perception threshold, showing strong indicative properties.

[0067] Based on differential abundance analysis, it was found that 7 compounds were significantly down-regulated and 2 compounds were significantly up-regulated in the elderly group (results as shown in Figure 6Further correlation analysis revealed that among the nine differentially expressed compounds, the abundance of butyl acetate and methyl acetate was significantly positively correlated with the sweetness threshold, while the abundance of isoprene was significantly negatively correlated with the sweetness threshold (results shown in Figure 7 ). Specifically, the abundance of butyl acetate and methyl acetate was significantly negatively correlated with sweet taste sensitivity, while the abundance of isoprene was significantly positively correlated with sweet taste sensitivity. This suggests that the characteristic oral exhaled breath identified by this method can be used to determine an individual's sweet taste sensitivity.

[0068] Example 2: Salty taste sensitivity verification

[0069] The only difference from Example 1 is that food-grade sodium chloride was used as the taste stimulus sample in the taste sensitivity analysis and the salty taste threshold was calculated. Correlation analysis showed that among the three characteristic exhaled gas combinations, the abundance of butyl acetate and methyl acetate was significantly positively correlated with the salty taste threshold, while the abundance of isoprene was significantly negatively correlated with salty taste sensitivity (see the results). Figure 7 That is, the abundance of butyl acetate and methyl acetate was significantly negatively correlated with salty taste sensitivity, while the abundance of isoprene was significantly positively correlated with salty taste sensitivity. This suggests that the characteristic oral exhaled breath identified by this method can be used to determine individual salty taste sensitivity.

[0070] Example 3: Sour taste sensitivity verification

[0071] The only difference from Example 1 is that food-grade sodium citrate monohydrate was used as the taste stimulus sample in the taste sensitivity analysis and the sour taste threshold was calculated. Correlation analysis showed that among the three characteristic exhaled gas combinations, the abundance of butyl acetate and methyl acetate was significantly positively correlated with the sour taste threshold, while the abundance of isoprene was significantly negatively correlated with sour taste sensitivity (see the results). Figure 7 That is, the abundance of butyl acetate and methyl acetate was significantly negatively correlated with sour sensitivity, while the abundance of isoprene was significantly positively correlated with sour sensitivity. This suggests that the characteristic oral exhaled breath identified by this method can be used to determine individual sour sensitivity.

[0072] The embodiments described above are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present invention.

Claims

1. A composition of taste sensitivity-related markers in exhaled breath, characterized in that: The composition includes butyl acetate, methyl acetate and isoprene; the abundance of butyl acetate and methyl acetate is significantly positively correlated with the taste threshold; and the abundance of isoprene is significantly negatively correlated with the taste threshold.

2. The marker composition according to claim 1, characterized in that The taste includes one or more of sweetness, saltiness, sourness, bitterness and umami.

3. A method for detecting a subject's taste sensitivity, characterized in that: The method includes: 1) Collect breath samples from subjects; 2) analyzing the relative abundance of the composition according to claim 1 or 2 in volatile organic compounds (VOCs) in a breath sample using a GC-MS method; 3) Interpretation of results: The abundance of butyl acetate and methyl acetate was significantly positively correlated with the taste threshold; the abundance of isoprene was significantly negatively correlated with the taste threshold.

4. A method for identifying a combination of taste sensitivity markers in a subject's exhaled breath, characterized in that: The method specifically includes: (1) Taste sensitivity analysis: Based on age differences, we recruited elderly and young subjects and tested their taste sensitivity using sensory evaluation methods. (2) Collect breath samples from subjects; (3) analyzing the relative abundance of the composition of claim 1 or 2 in the volatile organic compounds (VOCs) in the breath sample using a GC-MS method; (4) Interpretation of results: differential analysis was used to preliminarily screen characteristic compounds, and correlation analysis was combined to further determine the exhaled gas combination for identifying taste sensitivity; Among them, the taste includes one or more of sour, sweet, bitter, salty, and umami; the sensory evaluation method includes one or more of the three-point forced choice method 3-AFC, the two-point forced choice method 2-AFC, and the rated difference method; the taste sensitivity includes one or more of the perception threshold, the recognition threshold, and the difference threshold.

5. The method according to claim 3 or 4, characterized in that The method for collecting the breath sample of the subject is to use a commercial aluminum foil air sampling bag to collect the breath sample of the subject, and then transfer the breath sample to Tenax TA adsorption tubes respectively.

6. The method according to claim 3 or 4, wherein: The GC-MS method analysis uses a GCMS-TQ8050NX combined with a TD-30R thermal desorption system to detect volatile organic compounds in exhaled breath.

7. The method according to claim 6, characterized in that The operation of the thermal desorption system is as follows: VOCs in the adsorption tube are desorbed at 240-260°C for 4-6 minutes at a desorption flow rate of 50-70 mL / min, and are enriched in a cold trap at -25--18°C; then, desorbed at 250-270°C for another 1-3 minutes, and the VOCs are transferred to the GC inlet.

8. The method according to claim 3 or 4, characterized in that The GC operation in the GC-MS is as follows: chromatographic separation is performed using an SH-Rxi-624MS capillary column, and the column temperature program is as follows: initial temperature 33-37°C, maintained for 3-7 minutes; heated to 140-160°C at 5°C / min; then heated to 250-270°C at 30°C / min, maintained for 4-6 minutes; the carrier gas is helium, with a flow rate of 1-3 mL / min and a split ratio of 8-12:

1.

9. The method according to claim 3 or 4, wherein the MS in the GC-MS is operated using an electron bombardment ion source, an ion source temperature of 250° C., a data acquisition mode of Q3 scan mode, a mass scan range of 30-350 m / z, and a scan speed of 0.3 s / scan.

10. The method according to claim 3 or 4 is preferably characterized in that the result interpretation is data processing based on GCMS solution software, qualitative identification of compounds is performed using the NIST20 mass spectrum database for comparison, and compounds with a similarity index greater than 70 are preliminarily identified; retention indices (RIs) are calculated using C7-C40 normal alkanes for further identification, and compounds with RI value deviations less than 50 are considered acceptable qualitative results; peak area normalization is used to semi-quantitatively determine the relative abundance of different compounds in each breath sample.

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