Key flavor marker for identifying variety of blue flag milk tofu and application of key flavor marker
By using SPME-GC-QE Orbitrap MS technology, combined with solid phase microextraction and gas chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry, the problem of insufficient research on the differences in flavor substances in milk tofu was solved, and the screening and quality identification of key aroma substances in Zhenglanqi milk tofu were achieved.
Patent Information
- Application Number
- CN202510964617.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-14
- Publication Date
- 2025-09-23
AI Technical Summary
The existing technology lacks research on the differences in flavor substances in different types of milk tofu and the screening of key aroma substances, which affects the in-depth understanding of the flavor mechanism of Zhenglanqi milk tofu and the quality control.
SPME-GC-QE Orbitrap MS technology was used, combined with solid phase microextraction and gas chromatography-quadrupole-orbitrap high-resolution mass spectrometry, to establish a high-throughput non-targeted detection technology for volatile flavor substances and screen out the key aroma substances in milk tofu.
A comprehensive analysis of the volatile substances in different types of milk tofu was achieved, and many previously unreported aroma markers were identified, which enriched the understanding of the flavor of Zhenglanqi milk tofu and provided an important basis for flavor control and quality identification.
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Abstract
Description
Technical Field
[0001] The present application relates to the field of food testing, and in particular to a key flavor marker for identifying the type of Zhenglanqi milk tofu and its application. Background Art
[0002] Milk tofu, known as "Huruda" in Mongolian, is a traditional dairy product in Inner Mongolia Autonomous Region and is widely recognized as a significant source of protein for the human body. Milk tofu is often made from fresh cow's milk, using its own microbial flora and environmental microorganisms to naturally ferment and curdle the milk. It is then processed through whey drainage, kneading, and shaping. Its name stems from its resemblance to tofu. Traditional milk tofu has a unique flavor and can be eaten plain, made into candied milk tofu, or soaked in milk tea. Milk tofu is also rich in protein, amino acids, and beneficial trace elements, making it nutritious and beneficial for boosting immunity and enhancing beauty. The milk tofu from Zhenglan Banner, Xilingol League, is known as "the best milk product in the world from Lanqi," and as a National Geographical Indication product, it is highly sought after by consumers.
[0003] The flavor of milk tofu is a crucial indicator of its quality and a primary sensory criterion for consumers when purchasing and consuming it. Research has shown that non-volatile compounds contribute to milk tofu's flavor, primarily influencing its mouthfeel. Volatile compounds, on the other hand, are the primary flavor contributors to milk tofu's flavor. These diverse volatile compounds contribute to its unique aroma. However, not all volatile compounds contribute significantly to milk tofu's aroma; only a small fraction are crucial for its overall aroma. These characteristic aroma components not only form the foundation of Zhenglanqi milk tofu's unique flavor but also serve as a key distinguishing feature from other similar products. Furthermore, flavors vary among different types of milk tofu due to differences in processing techniques and other factors.
[0004] In recent years, research on traditional Mongolian milk tofu has focused primarily on storage and processing, quality control, production process optimization, strain isolation and identification, and analysis of functional lactic acid bacteria and microbial diversity. While domestic and international researchers have explored the flavor of milk tofu, these studies have primarily focused on the isolation and identification of volatile compounds within a single type of milk tofu. However, research examining the differences in flavor compounds among different milk tofu types and systematic screening of key aroma compounds is lacking. This, to a certain extent, hinders a deeper understanding of the flavor mechanisms of milk tofu and the implementation of scientific quality control. Therefore, exploring the differences in volatile compounds among different milk tofu types and thoroughly screening the characteristic components that determine their flavor is particularly important. This can provide important insights into flavor control, quality identification, and industrial processing of milk tofu in Zhenglan Banner, Xilingol League. Summary of the Invention
[0005] To address these shortcomings and deficiencies, the present invention provides a method for identifying Zhenglan Banner milk tofu varieties based on SPME-GC-QE Orbitrap MS technology. This method establishes a high-throughput, non-targeted detection technique for volatile flavor compounds in milk tofu, enabling qualitative and quantitative analysis of volatile flavor compounds in six representative types of Zhenglan Banner milk tofu. This method, along with screening for differentially expressed volatile compounds and identifying key aroma compounds in milk tofu, provides an important foundation for flavor control, quality identification, and industrial processing of Zhenglan Banner milk tofu in Xilingol League.
[0006] In order to achieve the above object, the first aspect of the present invention provides a method for screening key flavor markers for identifying the types of Zhenglanqi milk tofu, the method comprising the following steps:
[0007] (1) Sample preparation: Weigh different types of milk tofu samples and place them in headspace vials. Solid phase microextraction (SPME) is then used to enrich and extract the volatile flavor compounds in the milk tofu samples.
[0008] (2) Detection of volatile flavor substances: Based on solid phase microextraction-gas chromatography-tandem high-resolution mass spectrometry technology, the extraction head containing volatile flavor substances is inserted into the inlet of gas chromatography-high-resolution mass spectrometry, and detection and analysis are performed after thermal desorption;
[0009] (3) Qualitative and quantitative analysis of volatile substances: the mass spectrum data of the volatile substances collected in step (2) are compared with the mass spectrum information of the standard substances in the NIST database to identify the volatile substances in the sample; the volatile substances are quantitatively analyzed using a semi-quantitative method;
[0010] (4) Screening of key flavor markers: The semi-quantitative analysis results in step (3) were imported into the software, and an orthogonal partial least squares discriminant analysis (OPLS-DA) model was established through multivariate statistical analysis. The key flavor markers of different types of Zhenglanqi milk tofu were screened out based on the VIP values of the volatile compounds in the OPLS-DA model, and / or the key aroma substances of different types of Zhenglanqi milk tofu were screened out based on the OAV values of the volatile compounds in the OPLS-DA model.
[0011] Preferably, the Zhenglan Banner milk tofu samples in step (1) are Changhong milk tofu, Xibei milk tofu, Tengeng milk tofu, Han Yide milk tofu, Mengyuandu milk tofu and Baqi yurt milk tofu.
[0012] Preferably, the solid phase microextraction method in step (1) is as follows: after the sample is incubated, the extraction head is inserted into the extraction bottle to enrich and extract the volatile compounds in the milk tofu.
[0013] Preferably, the weight of the milk tofu in step (1) is 0.5-2 g. More preferably, the weight of the milk tofu in step (1) is 1 g.
[0014] Preferably, the incubation time in step (1) is 15-30 minutes, and the incubation temperature is 50-70° C. More preferably, the incubation time in step (1) is 25 minutes, and the incubation temperature is 60° C.
[0015] Preferably, the extraction time in step (1) is 30-60 minutes, and the extraction temperature is 50-70° C. More preferably, the extraction time in step (1) is 45 minutes, and the extraction temperature is 60° C.
[0016] Preferably, the SPME extraction head in step (1) is a 50 / 30 μm DVB / CAR / PDMS extraction head.
[0017] Preferably, the solid phase microextraction method in step (1) is as follows: the sample is incubated at 60°C for 25 minutes, an aged 50 / 30 μm DVB / CAR / PDMS extraction head is inserted into an extraction bottle to enrich volatile compounds in milk tofu, the extraction temperature is 60°C, and the extraction time is 45 minutes.
[0018] Preferably, the desorption time in step (2) is 1-10 minutes, and the desorption temperature is 240-260° C. More preferably, the desorption time in step (2) is 5 minutes, and the desorption temperature is 250° C.
[0019] Preferably, the gas chromatography-tandem high-resolution mass spectrometry technique in step (2) is gas chromatography-quadrupole-orbitrap high-resolution mass spectrometry (GC-QE Orbitrap MS).
[0020] Preferably, the gas chromatography conditions in step (2) are: the capillary column is TG-5MS (60m×0.25mm×0.25μm); the carrier gas is high-purity helium; the carrier gas flow rate is 1.2mL / min; and the split ratio is 15:1.
[0021] Preferably, the temperature raising program of the gas chromatography in step (2) is as follows: initial column temperature 40°C, maintained for 2 min, raised to 150°C at 3°C / min, maintained for 2 min, then raised to 250°C at 10°C / min, maintained for 5 min.
[0022] Preferably, the high-resolution mass spectrometry conditions in step (2) are: electron impact ionization source (EI); electron energy of 70 eV; ion source temperature of 280°C; transmission line temperature of 250°C; scan mode of full scan; and mass scan range of m / z 30-550.
[0023] Preferably, the method for identifying the volatile substances in the sample in step (3) is as follows: the mass spectrum of the volatile substances collected in step (2) is preprocessed by data deconvolution, peak extraction, peak filtering, etc. using Trace Finder software; the preprocessed mass spectrum data is compared with the mass spectrum information of the standard substances in the NIST database, and the detected volatile substances are qualitatively analyzed based on the total score value >90, high resolution filter value HRF >90, forward and reverse search index SI, RSI >650, and retention index difference ΔRI <50.
[0024] Preferably, the quantitative analysis method of volatile substances in step (3) is as follows: 2-octanol solution is added to the milk tofu sample as an internal standard before SPME, and the content of each volatile flavor substance in the milk tofu sample is semi-quantitatively determined based on the peak area ratio of the sample peak area and the internal standard peak area.
[0025] Preferably, the multivariate statistical analysis in step (4) uses principal component analysis (PCA) to examine the overall distribution and dispersion of the samples within the group.
[0026] Preferably, in step (4), a volatile compound with a VIP value greater than 1 is selected as the key flavor marker. More preferably, in step (4), a volatile compound with a VIP value greater than 1.10 is selected as the key flavor marker.
[0027] Preferably, in step (4), volatile compounds with an OAV value greater than 1 are selected as key aroma substances.
[0028] Preferably, the key flavor markers screened in step (4) are selected from one or more of the following compounds: vinyl acetate, furfuryl alcohol, 2-heptanone, p-xylene, 2-ethylbutyraldehyde, n-octanol, 2,3-dimethyldecane, isovaleraldehyde, ethylbenzene, 3-methyldecane, 2-methylpentane, butyl acetate, 3-methylundecane, styrene, methyl nonanoate, methyl decanoate, methyl octanoate, 3-ethyloctane, and ethyl trans-4-decenoate.
[0029] More preferably, the key flavor markers screened in step (4) are a combination of vinyl acetate, furfuryl alcohol, 2-heptanone, p-xylene, 2-ethylbutyraldehyde, n-octanol, 2,3-dimethyldecane, isovaleraldehyde, ethylbenzene, 3-methyldecane, 2-methylpentane, butyl acetate, 3-methylundecane, styrene, methyl nonanoate, methyl decanoate, methyl octanoate, 3-ethyloctane and ethyl trans-4-decenoate.
[0030] Preferably, the key aroma substances screened in step (4) are selected from one or more of the following compounds: ethyl acetate, isovaleraldehyde, methyl butyrate, isopentanol, ethyl butyrate, n-hexanol, isopentanol acetate, methyl hexanoate, dimethyl trisulfide, 2-pentylfuran, ethyl hexanoate, methyl heptanoate, phenylacetaldehyde, 2-nonanone, methyl octanoate, 2-undecanone, methyl decanoate, ethyl decanoate, heptanal, 2-heptanone, styrene, and ethyl octanoate.
[0031] More preferably, the key aroma substances screened in step (4) are a combination of ethyl acetate, isovaleraldehyde, methyl butyrate, isopentanol, ethyl butyrate, n-hexanol, isopentanol acetate, methyl hexanoate, dimethyl trisulfide, 2-pentylfuran, ethyl hexanoate, methyl heptanoate, phenylacetaldehyde, 2-nonanone, methyl octanoate, 2-undecanone, methyl decanoate, ethyl decanoate, heptanal, 2-heptanone, styrene and ethyl octanoate.
[0032] Further preferably, the key aroma substance screened in step (4) is a combination of ethyl acetate, isoamyl alcohol and dimethyl trisulfide.
[0033] A second aspect of the present invention provides a method for identifying the type of Zhenglanqi milk tofu based on key flavor markers, the method comprising the following steps:
[0034] (1) Sample preparation: Weigh a sample of the Zhenglanqi milk tofu of the type to be tested and place it in a headspace vial. Use solid phase microextraction to enrich and extract the volatile flavor compounds in the sample of the Zhenglanqi milk tofu of the type to be tested;
[0035] (2) Detection of volatile flavor substances: Based on solid phase microextraction-gas chromatography-tandem high-resolution mass spectrometry technology, the extraction head containing volatile flavor substances is inserted into the inlet of gas chromatography-high-resolution mass spectrometry, and detection and analysis are performed after thermal desorption;
[0036] (3) Quantitative analysis of key flavor markers: The mass spectrometry data of the volatile substances collected in step (2) are used to quantitatively analyze the key flavor markers in the test type Zhenglanqi milk tofu using a semi-quantitative method;
[0037] (4) Determination of milk tofu type: The semi-quantitative analysis results in step (3) were introduced into the OPLS-DA discriminant model established by the above screening method, and the type of Zhenglanqi milk tofu was determined based on the score of the sample to be tested by the discriminant model.
[0038] Preferably, the types of Zhenglanqi milk tofu to be tested in step (1) are Changhong milk tofu, Xibei milk tofu, Tengeng milk tofu, Hanyide milk tofu, Mengyuandu milk tofu and Baqi felt-house milk tofu.
[0039] Preferably, the solid phase microextraction method in step (1) is as follows: after the sample is incubated, the extraction head is inserted into the extraction bottle to enrich and extract the volatile compounds in the milk tofu.
[0040] Preferably, the weight of the milk tofu in step (1) is 0.5-2 g. More preferably, the weight of the milk tofu in step (1) is 1 g.
[0041] Preferably, the incubation time in step (1) is 15-30 minutes, and the incubation temperature is 50-70° C. More preferably, the incubation time in step (1) is 25 minutes, and the incubation temperature is 60° C.
[0042] Preferably, the extraction time in step (1) is 30-60 minutes, and the extraction temperature is 50-70° C. More preferably, the extraction time in step (1) is 45 minutes, and the extraction temperature is 60° C.
[0043] Preferably, the SPME extraction head in step (1) is a 50 / 30 μm DVB / CAR / PDMS extraction head.
[0044] Preferably, the solid phase microextraction method in step (1) is as follows: the sample is incubated at 60°C for 25 minutes, an aged 50 / 30 μm DVB / CAR / PDMS extraction head is inserted into an extraction bottle to enrich volatile compounds in milk tofu, the extraction temperature is 60°C, and the extraction time is 45 minutes.
[0045] Preferably, the desorption time in step (2) is 1-10 minutes, and the desorption temperature is 240-260° C. More preferably, the desorption time in step (2) is 5 minutes, and the desorption temperature is 250° C.
[0046] Preferably, the gas chromatography-tandem high-resolution mass spectrometry technique in step (2) is gas chromatography-quadrupole-orbitrap high-resolution mass spectrometry (GC-QE Orbitrap MS).
[0047] Preferably, the gas chromatography conditions in step (2) are: the capillary column is TG-5MS (60m×0.25mm×0.25μm); the carrier gas is high-purity helium; the carrier gas flow rate is 1.2mL / min; and the split ratio is 15:1.
[0048] Preferably, the temperature raising program of the gas chromatography in step (2) is as follows: initial column temperature 40°C, maintained for 2 min, raised to 150°C at 3°C / min, maintained for 2 min, then raised to 250°C at 10°C / min, maintained for 5 min.
[0049] Preferably, the high-resolution mass spectrometry conditions in step (2) are: electron impact ionization source (EI); electron energy of 70 eV; ion source temperature of 280°C; transmission line temperature of 250°C; scan mode of full scan; and mass scan range of m / z 30-550.
[0050] Preferably, the semi-quantitative analysis method of the key flavor markers in step (3) is as follows: before SPME, a 2-octanol solution is added to the milk tofu sample as an internal standard, and the content of each volatile flavor substance in the milk tofu sample is semi-quantitatively determined based on the peak area ratio of the sample peak area and the internal standard peak area.
[0051] Preferably, the key flavor marker in step (3) is selected from one or more of the following compounds: vinyl acetate, furfuryl alcohol, 2-heptanone, p-xylene, 2-ethylbutyraldehyde, n-octanol, 2,3-dimethyldecane, isovaleraldehyde, ethylbenzene, 3-methyldecane, 2-methylpentane, butyl acetate, 3-methylundecane, styrene, methyl nonanoate, methyl decanoate, methyl octanoate, 3-ethyloctane, and ethyl trans-4-decenoate.
[0052] More preferably, the key flavor marker in step (3) is a combination of vinyl acetate, furfuryl alcohol, 2-heptanone, p-xylene, 2-ethylbutyraldehyde, n-octanol, 2,3-dimethyldecane, isovaleraldehyde, ethylbenzene, 3-methyldecane, 2-methylpentane, butyl acetate, 3-methylundecane, styrene, methyl nonanoate, methyl decanoate, methyl octanoate, 3-ethyloctane and ethyl trans-4-decenoate.
[0053] Preferably, the standard for judging the type of Zhenglan Banner milk tofu according to the score value of the sample to be tested in step (4) is as follows: calculate the score values of the sample to be tested in the Changhong milk tofu area, Xibei milk tofu area, Tengeng milk tofu area, Hanyide milk tofu area, Mengyuandu milk tofu area and Baqi felt house milk tofu area respectively, and the area corresponding to the highest score among the 6 areas is the Zhenglan Banner milk tofu type of the sample to be tested.
[0054] The third aspect of the present invention provides a key flavor marker for identifying the type of Zhenglanqi milk tofu, wherein the key flavor marker is selected from one or more of the following compounds: vinyl acetate, furfuryl alcohol, 2-heptanone, p-xylene, 2-ethylbutyraldehyde, n-octanol, 2,3-dimethyldecane, isovaleraldehyde, ethylbenzene, 3-methyldecane, 2-methylpentane, butyl acetate, 3-methylundecane, styrene, methyl nonanoate, methyl decanoate, methyl octanoate, 3-ethyloctane, and ethyl trans-4-decenoate.
[0055] Preferably, the key flavor marker is a combination of vinyl acetate, furfuryl alcohol, 2-heptanone, p-xylene, 2-ethylbutyraldehyde, n-octanol, 2,3-dimethyldecane, isovaleraldehyde, ethylbenzene, 3-methyldecane, 2-methylpentane, butyl acetate, 3-methylundecane, styrene, methyl nonanoate, methyl decanoate, methyl octanoate, 3-ethyloctane and ethyl trans-4-decenoate.
[0056] A fourth aspect of the present invention provides an application of a key flavor marker in simultaneously identifying different types of Zhenglanqi milk tofu, wherein the key flavor marker is selected from one or more of the following compounds: vinyl acetate, furfuryl alcohol, 2-heptanone, p-xylene, 2-ethylbutyraldehyde, n-octanol, 2,3-dimethyldecane, isovaleraldehyde, ethylbenzene, 3-methyldecane, 2-methylpentane, butyl acetate, 3-methylundecane, styrene, methyl nonanoate, methyl decanoate, methyl octanoate, 3-ethyloctane, and ethyl trans-4-decenoate.
[0057] Preferably, the key flavor marker is a combination of vinyl acetate, furfuryl alcohol, 2-heptanone, p-xylene, 2-ethylbutyraldehyde, n-octanol, 2,3-dimethyldecane, isovaleraldehyde, ethylbenzene, 3-methyldecane, 2-methylpentane, butyl acetate, 3-methylundecane, styrene, methyl nonanoate, methyl decanoate, methyl octanoate, 3-ethyloctane and ethyl trans-4-decenoate.
[0058] Preferably, the different types of Zhenglanqi milk tofu are Changhong milk tofu, Xibei milk tofu, Tengeng milk tofu, Hanyide milk tofu, Mengyuandu milk tofu and Eight Banners Yurt House milk tofu.
[0059] Compared with the prior art, the present invention has the following beneficial effects:
[0060] This study combines solid-phase microextraction (SPME) with gas chromatography-quadrupole-orbitrap high-resolution mass spectrometry (GC-QE) to investigate the differences between different types of Zhenglanqi milk tofu and identify key aroma compounds. SPME requires no complex sample pretreatment, offers high sensitivity, and offers excellent reproducibility, enabling comprehensive detection of volatile compounds. GC-QE Orbitrap MS coupled with mass spectrometry provides rich and accurate fragment ion information, effectively capturing and accurately identifying volatile compounds in milk tofu.
[0061] 2. The present invention establishes a high-throughput non-targeted detection technology for volatile flavor substances in milk tofu, comprehensively analyzes the volatile substances in different types of Zhenglan Banner milk tofu, identifies many aroma markers that have not been reported, greatly enriches the understanding of its volatile substances, clarifies the differences in volatile substances in different types of milk tofu and the key aroma substances that play a decisive role in the flavor of milk tofu, and provides an important basis for the flavor control, quality identification and industrial processing of Zhenglan Banner milk tofu in Xilingol League. BRIEF DESCRIPTION OF THE DRAWINGS
[0062] Figure 1 The optimization results of key parameters in the pre-treatment process of Zhenglanqi milk tofu are shown in Figure 2. Figure 1 A is the incubation time screening, Figure 1 B is the extraction time screening, Figure 1 C is the desorption time screening, Figure 1 D is sample weight screening;
[0063] Figure 2 This is a classification diagram of 92 volatile compounds detected in Zhenglanqi milk tofu;
[0064] Figure 3 This is the PCA score diagram of volatile compounds in different types of Zhenglanqi milk tofu;
[0065] Figure 4 This is the OPLS-DA score diagram of volatile substances in different types of Zhenglanqi milk tofu;
[0066] Figure 5 This is a displacement test diagram of volatile substances in different types of Zhenglanqi milk tofu;
[0067] Figure 6 The following is a classification diagram of 42 different volatile compounds in different types of Zhenglanqi milk tofu;
[0068] Figure 7 This is the cluster heat map of 42 differential volatile compounds in different types of Zhenglanqi milk tofu;
[0069] Figure 8 This is an OAV display of 22 key aroma compounds in different types of Zhenglanqi milk tofu;
[0070] Figure 9 A milk tofu type discrimination model based on 19 key flavor markers;
[0071] Figure 10 This is the volatile matter replacement test chart for the Zhenglanqi milk tofu verification sample;
[0072] Figure 11 This is the prediction result diagram of the discriminant model for the Zhenglanqi milk tofu verification sample. DETAILED DESCRIPTION
[0073] The present invention will be further described by way of examples, but the present invention is not limited to the following examples.
[0074] Test Example 1: Screening test of solid phase microextraction (SPME) conditions for Zhenglanqi milk tofu
[0075] The HS-SPME experimental process is easily affected by subtle factors, which can lead to poor reproducibility. Therefore, it is necessary to determine the optimal SPME extraction conditions in advance. This experiment used peak intensity and peak number as evaluation criteria, and investigated the optimal conditions for incubation time, extraction time, desorption time, and sample weight.
[0076] 1. Incubation time
[0077] Weigh 1.0g of Changhong milk tofu sample and place it in a 20mL headspace injection bottle, add 2-octanol as the internal standard substance (dissolved in methanol), mix well and seal. The sample was incubated at 60℃ for different times (20min, 25min, 30min, 35min), and the aged 50 / 30μm DVB / CAR / PDMS extraction head was inserted into the extraction bottle to enrich the volatile compounds in the milk tofu. The extraction temperature was 60℃ and the extraction time was 20min. The desorption temperature was 250℃ and the desorption time was 7min. The instrument and detection conditions of gas chromatography-tandem high-resolution mass spectrometry were the same as those in Experiment 2.
[0078] The changes in total peak intensity and peak number at different incubation times were investigated. Figure 1 As shown in Figure A, the peak intensities at the four incubation times are 9.16E8, 1.10E9, 1.20E9, and 1.30E9, respectively. Although both the total peak intensity and the total number of peaks increase with increasing incubation time, the difference is not significant. Therefore, 25 min was determined to be the optimal incubation time.
[0079] 2. Extraction time
[0080] The incubation time was fixed at 25 min, and the other experimental conditions were referred to the above "1. Incubation time" section, and the changes in total peak intensity and peak number under different extraction times (25 min, 30 min, 35 min, 40 min, 45 min) were investigated respectively. Figure 1 As shown in Figure 2, the total peak intensity reaches its highest at an extraction time of 25 minutes, decreases slightly at 30 minutes, and then increases with increasing extraction time. Furthermore, the number of peaks also increases with extraction time. To obtain more volatile compounds, the extraction time was set to 45 minutes.
[0081] 3. Desorption time
[0082] The incubation time was fixed at 25 min, the extraction time was fixed at 45 min, and the other experimental conditions were referred to the above "1. Incubation time" section, and the changes in total peak intensity and peak number at different desorption times (5 min, 7 min, 9 min and 11 min) were investigated respectively. Figure 1It can be seen from C that when the desorption time is 5 min, the total peak intensity and the number of peaks reach the maximum value. With the increase of desorption time, the total peak intensity and the number of peaks gradually decrease. Therefore, 5 min is determined to be the optimal desorption time.
[0083] 4. Sample weighing
[0084] The incubation time was fixed at 25 min, the extraction time was fixed at 45 min, and the desorption time was fixed at 5 min. The other experimental conditions were referred to the above "1. Incubation time" section, and the effects of different sample weights (1 g, 2 g, and 3 g) on the total peak intensity and peak number were investigated. Figure 1 D shows that under the conditions investigated, the changes in the total peak intensity and peak quantity of the sample weight are not obvious, so the sample weight is determined to be 1g.
[0085] Experimental Example 2: Screening of Volatile Compounds in Zhenglanqi Milk Tofu Based on SPME-GC-QE Orbitrap MS Technology
[0086] 1. Preparation of Zhenglanqi milk tofu samples
[0087] Milk tofu samples were collected from six representative brands of Zhenglan in Xilingol League (Changhong Milk Tofu (CH), Xibei Milk Tofu (HBXT), Tengeng Milk Tofu (TNG), Hanyide Milk Tofu (HYD), Mengyuandu Milk Tofu (MYD), and Baqi Feel House Milk Tofu (BQZF)). Three samples of each milk tofu type were collected, with two replicates per sample, for a total of six samples of each type. 1.0 g of each milk tofu sample was weighed and placed into a 20 mL headspace vial. 2-octanol (dissolved in methanol) was added as an internal standard, mixed, and sealed. The sample was incubated at 60°C for 25 minutes. A conditioned 50 / 30 μm DVB / CAR / PDMS extraction tip was then inserted into the vial to enrich the volatile compounds in the milk tofu. The extraction temperature was 60°C for 45 minutes, and the desorption temperature was 250°C for 5 minutes.
[0088] 2. Volatile flavor substance detection
[0089] Sample data were collected using solid phase microextraction-gas chromatography-tandem high-resolution mass spectrometry (SPME-GC-QE Orbitrap MS). The instrument conditions used for the collection were as follows:
[0090] Gas chromatography conditions: capillary column: TG-5MS (60 m × 0.25 mm × 0.25 μm); carrier gas: high-purity helium; carrier gas flow rate: 1.2 mL / min; split ratio: 15:1; temperature program: initial column temperature: 40°C, hold for 2 min, increase to 150°C at 3°C / min, hold for 2 min, then increase to 250°C at 10°C / min, hold for 5 min.
[0091] The high-resolution mass spectrometry conditions were as follows: electron impact ionization source (EI); electron energy of 70 eV; ion source temperature of 280°C; transfer line temperature of 250°C; full scan scan mode; and mass scan range of m / z 30-550.
[0092] 3. Qualitative and quantitative analysis of volatile substances
[0093] The mass spectra of volatile compounds collected from Zhenglanqi milk tofu were preprocessed using Trace Finder software, including data deconvolution, peak extraction, and peak filtering. Peaks containing Si, peaks with non-normal distribution patterns, and anomalous fragment ions were excluded before putative identification. The preprocessed mass spectral data were compared with mass spectra of reference materials in the NIST database. Qualitative analysis of the detected volatile compounds was performed using screening criteria including a total score >90, a high-resolution filter (HRF) >90, forward and reverse search indices (SI) and RSI >650, and a retention index difference (ΔRI) <50. Before SPME, a 2-octanol solution was added to the milk tofu sample as an internal standard. The content of volatile flavor compounds in the milk tofu sample was semi-quantified based on the peak area ratio between the sample peak area and the internal standard peak area.
[0094] By optimizing the detection conditions, this study established a high-throughput non-targeted detection method for volatile compounds in milk tofu. By analyzing 6 representative types of milk tofu samples, a total of 92 volatile compounds ( Figure 2 These compounds are mainly divided into 8 categories, including 9 alcohols, 4 aromatic hydrocarbons, 10 acids, 5 aldehydes, 10 ketones, 33 alkanes, 15 esters and 6 others (including furans, sulfides, lactones and alkenes, etc.), among which alkanes, esters, ketones and acids are the main volatile compounds.
[0095] 4. Screening and optimization of key flavor markers
[0096] The quantitative results of milk tofu volatile compounds were imported into SIMCA-P software, and the overall distribution and dispersion of each sample in the group were examined by unsupervised principal component analysis (PCA). Figure 3 As shown in the PCA score diagram, different types of milk tofu samples can be clearly distinguished, indicating that there are certain differences in the volatile compounds of different types of milk tofu samples.
[0097] In order to further quantify the degree of difference between different groups and reduce the intra-group error, the supervised orthogonal partial least squares discriminant analysis (OPLS-DA) was used to distinguish the overall differences of volatile substances between groups, and the OPLS-DA model ( Figure 4 ). The six groups of milk tofu samples showed a clear separation trend, and each milk tofu group had a good clustering effect, further indicating that there were significant differences in the volatile substances of different types of milk tofu. In the OPLS-DA model, R2X and R2Y represent the model's explanatory power for the variables X and Y matrices, and Q2 represents the model's predictability. The closer R2 and Q2 are to 1, and the smaller the difference (difference < 0.3), the better the model's accuracy and better explanatory and predictive capabilities. In this experimental case, R2X = 0.917, R2Y = 0.927, and Q2 = 0.846, indicating that the discriminant analysis results of the model are highly accurate.
[0098] Important variables (VIPs) are variable weights for variables in the OPLS-DA model, measuring the strength of a metabolite's influence on the ability to classify and interpret each sample group. Based on the VIP values provided by the OPLS-DA analysis, compounds with a VIP greater than 1 were selected as differentially expressed volatile compounds. Ultimately, 42 volatile compounds were identified as flavor markers for different types of milk tofu (see Table 1).
[0099] Table 1 42 different flavor markers in different types of Zhenglanqi milk tofu
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[0102] Figure 6 and Figure 7 The classification and content changes of volatile compounds in different types of Zhenglanqi milk tofu are presented. The differential compounds are mainly divided into eight categories, including 11 alkanes, 9 esters, 4 ketones, 6 acids, 2 aldehydes, 3 aromatic hydrocarbons, 5 alcohols, and 2 other categories. Among them, alkanes, esters, and acids are the main differential compounds.
[0103] Quantification of differentially expressed compounds revealed significant differences in the volatile compounds found in different types of milk tofu. Among the five alcohols, n-octanol was not detected in Teng'eng milk tofu, while n-hexanol was not detected in either Mengyuandu or Teng'eng milk tofu. Among the two aldehydes, 2-ethylbutyraldehyde was not detected in Han'ide milk tofu, and isovaleraldehyde was not detected in either Mengyuandu or Teng'eng milk tofu. Among the six acids, isovaleric acid was not detected in Xibei milk tofu, heptanoic acid was not detected in Changhong, Xibei, Han'ide, or Teng'eng milk tofu, and 2-hydroxy-2-methylmalonic acid was not detected in Changhong, Xibei, or Han'ide milk tofu. Among the 11 alkanes, 2,3-dimethyldecane was only detected in Baqi Yufang milk tofu, while 3-methylundecane was not detected in either Baqi Yufang or Mengyuandu milk tofu. Among the 9 esters, ethyl trans-4-decenoate was not detected in Changhong, Xibei and Hanyide milk tofu, and isoamyl acetate was not detected in Xibei and Hanyide milk tofu.
[0104] In order to further identify the most critical flavor compounds in different types of milk tofu, the screening threshold was increased to VIP>1.10, and a total of 19 key flavor markers that can be used to distinguish different types of Zhenglanqi milk tofu were screened out (see compounds numbered 1-19 in Table 1 for details).
[0105] 5. Exploration of key aroma substances
[0106] The odor activity value (OAV) was used to evaluate the contribution of each volatile flavor compound to the overall aroma of Zhenglanqi milk tofu. Volatile flavor compounds with OAV>1 were screened as key aroma compounds, and the key characteristic aroma compounds of milk tofu were further explored.
[0107] The OAV values of volatile flavor compounds in milk tofu samples were calculated based on the threshold values of compounds in water as specified in the "Compendium of Olfactory Thresholds of Compounds" using the following formula: OAVx = Cx / Tx, where OAVx represents the odor activity value of volatile flavor compound x, Cx represents the content of volatile flavor compound x in milk tofu, and Tx represents the odor threshold value of volatile flavor compound x. The results showed that 22 volatile compounds had an OAV value greater than 1 (see Table 2 for details). These compounds contribute significantly to the flavor of Zhenglanqi milk tofu and are considered key aroma compounds in the milk tofu. Key aroma compounds shared by the six milk tofu samples were further selected as key characteristic aroma compounds of milk tofu. The results revealed that three compounds (ethyl acetate, isopentanol, and dimethyl trisulfide) were key characteristic aroma compounds in Zhenglanqi milk tofu.
[0108] Table 2 Threshold values and aroma activity values of aroma volatile flavor substances
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[0111] Test Example 3: Verification test of different flavor markers of different types of Zhenglanqi milk tofu
[0112] To further validate the accuracy of the selected key markers in distinguishing different types of milk tofu, the semi-quantitative data (including sample names and sample contents) for the 19 markers (see compounds numbered 1-19 in Table 1) were imported into SIMCA-P software. After log transformation and UV scaling, an OPLS-DA model was established (six types of milk tofu, five samples each). The model prediction parameters (R² = 0.973, R² = 0.897, Q² = 0.825) and the results of 200 permutation tests (R² = 0.942, Q² = 0.880) indicated that the established model was stable and reliable, and the data were not overfitted.
[0113] In addition, the accuracy of the model was further verified using real milk tofu samples. The semi-quantitative results of six known types of milk tofu were used as validation samples, and the results were imported into the OPLS-DA discriminant model established using 19 key markers. The validation sample points accurately fell into the corresponding milk tofu type region. The model's scoring table for the predicted samples (Table 3) also shows that validation sample 1 scored the highest in the Eight Banners Yurt area and can be considered to be milk tofu from the Eight Banners Yurt area; validation sample 2 scored the highest in the Changhong area and can be considered to be milk tofu from Changhong; validation samples 3-6 scored the highest in the Xibei, Hanyide, Mengyuandu, and Tengeng areas, respectively, and can be considered to be milk tofu from Xibei, Hanyide, Mengyuandu, and Tengeng, respectively.
[0114] Table 3. Scoring values of the discriminant model for validation samples
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[0117] The results of the above-mentioned real verification samples further confirmed that the combination of 19 key flavor markers of the present invention can be used to accurately identify different types of Zhenglanqi milk tofu.
[0118] Although specific embodiments of the present invention have been described, it will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the scope or spirit of the present invention. Therefore, the present invention is intended to cover all such changes and modifications that fall within the scope of the appended claims and their equivalents.
Claims
1. A method for screening key flavor markers for identifying the types of Zhenglanqi milk tofu, characterized in that: The method comprises the following steps: (1) Sample preparation: Weigh different types of milk tofu samples and place them in headspace vials. Solid phase microextraction (SPME) is then used to enrich and extract the volatile flavor compounds in the milk tofu samples. (2) Detection of volatile flavor substances: Based on solid phase microextraction-gas chromatography-tandem high-resolution mass spectrometry technology, the extraction head containing volatile flavor substances is inserted into the inlet of gas chromatography-high-resolution mass spectrometry, and detection and analysis are performed after thermal desorption; (3) Qualitative and quantitative analysis of volatile substances: the mass spectrum data of the volatile substances collected in step (2) are compared with the mass spectrum information of the standard substances in the NIST database to identify the volatile substances in the sample; the volatile substances are quantitatively analyzed using a semi-quantitative method; (4) Screening of key flavor markers: The semi-quantitative analysis results in step (3) were imported into the software, and an orthogonal partial least squares discriminant analysis (OPLS-DA) model was established through multivariate statistical analysis. The key flavor markers of different types of Zhenglanqi milk tofu were screened out based on the VIP values of the volatile compounds in the OPLS-DA model, and / or the key aroma substances of different types of Zhenglanqi milk tofu were screened out based on the OAV values of the volatile compounds in the OPLS-DA model.
2. The screening method according to claim 1, wherein The Zhenglan Banner milk tofu samples in the step (1) are Changhong milk tofu, Xibei milk tofu, Tengeng milk tofu, Khan Yide milk tofu, Mengyuandu milk tofu and Eight Banners Yurt House milk tofu.
3. The screening method according to claim 1, wherein The incubation time in step (1) is 15-30 minutes, and the incubation temperature is 50-70°C.
4. The screening method according to claim 1, wherein The extraction time in step (1) is 30-60 min, and the extraction temperature is 50-70°C.
5. The screening method according to claim 1, wherein The gas chromatography conditions in step (2) are as follows: the capillary column is TG-5MS (60m×0.25mm×0.25μm); the carrier gas is high-purity helium; The carrier gas flow rate was 1.2 mL / min, and the split ratio was 15:
1.
6. The screening method according to claim 1, wherein The high-resolution mass spectrometry conditions in step (2) are as follows: electron impact ionization source (EI); electron energy of 70 eV; ion source temperature of 280° C.; transfer line temperature of 250° C.; full scan scan mode; and mass scan range of m / z 30-550.
7. The screening method according to claim 1, wherein The key flavor markers screened in step (4) are selected from one or more of the following compounds: vinyl acetate, furfuryl alcohol, 2-heptanone, p-xylene, 2-ethylbutyraldehyde, n-octanol, 2,3-dimethyldecane, isovaleraldehyde, ethylbenzene, 3-methyldecane, 2-methylpentane, butyl acetate, 3-methylundecane, styrene, methyl nonanoate, methyl decanoate, methyl octanoate, 3-ethyloctane, and ethyl trans-4-decenoate.
8. A method for identifying the type of Zhenglanqi milk tofu based on key flavor markers, characterized in that: The method comprises the following steps: (1) Sample preparation: Weigh a sample of the Zhenglanqi milk tofu of the type to be tested and place it in a headspace vial. Use solid phase microextraction to enrich and extract the volatile flavor compounds in the sample of the Zhenglanqi milk tofu of the type to be tested; (2) Detection of volatile flavor substances: Based on solid phase microextraction-gas chromatography-tandem high-resolution mass spectrometry technology, the extraction head containing volatile flavor substances is inserted into the inlet of gas chromatography-high-resolution mass spectrometry, and detection and analysis are performed after thermal desorption; (3) Quantitative analysis of key flavor markers: The mass spectrometry data of the volatile substances collected in step (2) are used to quantitatively analyze the key flavor markers in the test type Zhenglanqi milk tofu using a semi-quantitative method; (4) Determination of the type of milk tofu: The semi-quantitative analysis results in step (3) are introduced into the OPLS-DA discriminant model established by the screening method according to any one of claims 1 to 7, and the type of Zhenglanqi milk tofu is determined based on the score of the sample to be tested by the discriminant model.
9. A key flavor marker for identifying the type of Zhenglanqi milk tofu, characterized by: The key flavor marker is selected from one or more of the following compounds: vinyl acetate, furfuryl alcohol, 2-heptanone, p-xylene, 2-ethylbutyraldehyde, n-octanol, 2,3-dimethyldecane, isovaleraldehyde, ethylbenzene, 3-methyldecane, 2-methylpentane, butyl acetate, 3-methylundecane, styrene, methyl nonanoate, methyl decanoate, methyl octanoate, 3-ethyloctane, and ethyl trans-4-decenoate.
10. Application of a key flavor marker in simultaneously identifying different types of Zhenglanqi milk tofu, characterized in that: The key flavor marker is selected from one or more of the following compounds: vinyl acetate, furfuryl alcohol, 2-heptanone, p-xylene, 2-ethylbutyraldehyde, n-octanol, 2,3-dimethyldecane, isovaleraldehyde, ethylbenzene, 3-methyldecane, 2-methylpentane, butyl acetate, 3-methylundecane, styrene, methyl nonanoate, methyl decanoate, methyl octanoate, 3-ethyloctane, and ethyl trans-4-decenoate.