Method for detecting volatile components in fermented soybean

Abstract

The invention provides a method for detecting volatile components in fermented soybean, and belongs to the technical field of traditional Chinese medicine component analysis. The method comprises the following steps: detecting volatile components in fermented soybean by adopting a comprehensive two-dimensional headspace gas chromatography-mass spectrometry method, artificially analyzing mass spectrum information corresponding to each chromatographic peak according to a gas chromatography-mass spectrometry total ion chromatogram, comparing the mass spectrum information with a standard spectrogram in an NIST 17L spectrum library, and determining the types of the volatile components according to compounds of which the forward matching degrees and the reverse matching degrees are greater than 700. And calculating the relative content of the volatile components by adopting a peak area normalization method. According to the present invention, the volatile components of the fermented soybean samples with different fermentation durations are analyzed by using the comprehensive two-dimensional headspace gas chromatograph-mass spectrometer, 75 volatile components are identified from 10 different fermentation time periods, and the aldehydes are the most abundant; 2 (5H)-furanone, gamma-butyrolactone and n-butyl alcohol are specific components in the post-fermentation stage. O-methoxyphenol, phenol and 2-phenethyl alcohol are specific components in the pre-fermentation stage.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine component analysis, and in particular relates to a method for detecting volatile components in mild fermented black beans. Background Art

[0002] Semem Sojae Prapearatum, also known as fragrant fermented bean curd, is fermented and processed from mature seeds of soybean, a legume plant, and is a traditional Chinese medicine for food therapy. It is mainly made from black seed coat soybeans, and is fermented and processed with Chinese medicines such as mulberry leaves and Artemisia annua. It has the effects of relieving symptoms and relieving restlessness, and dispelling stagnation and heat. The herbal medicines of all dynasties concluded that the quality of Semem Sojae Prapearatum is best when it is "fragrant, beautiful and thick". For example, Tao Hongjing of the Southern and Northern Dynasties summarized in the "Collected Notes on the Classic of Materia Medica" that "the best ones come from Xiangyang and Qiantang, and are fragrant, beautiful and thick". The predecessors established the volatile component fingerprints of Semem Sojae Prapearatum from different origins, and through GC-MS analysis, the transformation of volatile substances in Semem Sojae Prapearatum during the fermentation process was detected. However, the types and contents of volatile components detected in the prior art are few, and the differences and changes in the volatile components of Semem Sojae Prapearatum under different fermentation times are still unclear. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide a method for detecting volatile components in light fermented black beans. The present invention adopts comprehensive two-dimensional gas chromatography-mass spectrometry (GC×GC-MS) to compare the volatile components of light fermented black beans samples with different fermentation times, so as to establish a fast and convenient method for detecting and analyzing the volatile components of light fermented black beans, so as to provide a reference for the establishment of quality standards of light fermented black beans and the research on medicinal value.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for detecting volatile components in fermented black beans, comprising the following steps:

[0006] The volatile components in fermented black beans were detected by comprehensive two-dimensional headspace gas chromatography-mass spectrometry. The mass spectrum information corresponding to each chromatographic peak was manually analyzed according to the total ion current of gas chromatography-mass spectrometry and compared with the standard spectrum in the NIST 17L spectral library. The types of volatile components were determined according to the compounds with both forward and reverse matching degrees greater than 700, and the relative contents of volatile components were calculated by peak area normalization method.

[0007] Preferably, the sample amount of the light fermented black beans is 0.2-0.6 g.

[0008] Preferably, the headspace conditions of the comprehensive two-dimensional headspace gas chromatography-mass spectrometry method are: sample bottle heating temperature 120°C; quantitative loop temperature 130°C; transmission line temperature 140°C; sample bottle equilibration time 10 to 40 min; pressure equilibration time 0.1 min; injection time 0.5 min; injection volume 1 mL.

[0009] Preferably, the chromatographic conditions of the comprehensive two-dimensional headspace gas chromatography-mass spectrometry method are as follows: one-dimensional column: Agilent DB-FFAP chromatographic column, inner diameter × length: 30m × 0.25mm, particle size: 0.25μm; two-dimensional column: W1812 chromatographic column, inner diameter × length: 1.2m × 0.18mm, particle size: 0.18μm; inlet temperature: 250°C; split ratio 20:1, split flow rate: 1mL / min; carrier gas: high-purity helium, purity>99.999%; programmed temperature rise: initial temperature 40°C, maintained for 4min, increased to 110°C at a rate of 10°C, maintained for 3min, increased to 200°C at a rate of 20°C, and then increased to 230°C at a rate of 10°C, maintained for 6min.

[0010] Preferably, the mass spectrometry conditions of the comprehensive two-dimensional headspace gas-mass spectrometry method are: ion source: EI ion source; temperature: 230°C; quadrupole temperature: 150°C; ionization energy: 70 eV; scanning range m / z 40–600, full scan; threshold: 150; solvent delay 2.5 min; scanning speed: 12500.

[0011] Preferably, the modulator conditions of the comprehensive two-dimensional headspace gas chromatography-mass spectrometry method are: SV series modulation column: C5-C30 column; modulator cold zone temperature 9°C, hot zone inlet temperature: 50°C, hot zone outlet temperature: 260°C, column oven bias +30°C; modulation cycle 5s; programmed temperature rise: initial temperature 40°C, maintained for 4min, increased to 110°C at a rate of 10°C, maintained for 3min, increased to 200°C at a rate of 20°C, and then increased to 230°C at a rate of 10°C, and maintained for 6min.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention analyzes the volatile components of light black bean curd samples with different fermentation times by comprehensive two-dimensional headspace-mass spectrometry, and identifies a total of 75 volatile components from 10 different fermentation time periods, including 15 aldehydes, 11 ketones, 10 alcohols, 7 heterocyclic compounds, 7 organic nitrogen-containing compounds, 6 esters, 5 phenols, 5 pyrazine compounds, 3 acids, 2 hydrocarbons, 2 sulfur-containing compounds, and 2 other compounds, with aldehydes being the most abundant. Among them, 20 compounds including isovaleraldehyde, n-valeraldehyde, n-hexanal, 3-furfural, benzaldehyde, 2-phenylacetaldehyde, 2,3-butanedione, 2,3-pentanedione, 3-hydroxy-2-butanone, hydroxyacetone, 2(5H)-furanone, 1-penten-3-ol, isopentanol, n-pentanol, furfuryl alcohol, 2-pentylfuran, 2-methoxy-4-vinylphenol, 2-methylpyrazine, glacial acetic acid, isovaleric acid, and estragole are common components of light fermented black beans with different fermentation times. 2(5H)-furanone, γ-butyrolactone, and n-butanol are unique components of the post-fermentation stage. o-Methoxyphenol, phenol, and 2-phenylethanol are unique components of the pre-fermentation stage. This differential component provides a characteristic indicator for distinguishing the fermentation degree of light fermented black beans.

[0014] The invention adopts a headspace method to obtain volatile components in light fermented black beans, performs qualitative and quantitative detection on the volatile components in the light fermented black beans by using a comprehensive two-dimensional gas chromatography-mass spectrometer, connects two sets of chromatographic columns with different polarities in series, and realizes orthogonal separation of complex components in the light fermented black beans under the action of a modulator, and realizes re-separation of mixed components of co-electrolytes that cannot be separated on a one-dimensional chromatographic column according to the difference in polarity between components on a two-dimensional chromatographic column, thereby greatly improving the detection peak capacity of the volatile components and the separation degree. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the investigation of empty injection conditions (where a is the investigation of the sample weight before fermentation, b is the investigation of the sample weight after fermentation, and c is the investigation of sample equilibrium time);

[0016] Figure 2 This is the two-dimensional gas chromatogram of volatile metabolites of light fermented black beans at 0 days before fermentation;

[0017] Figure 3 This is the two-dimensional gas chromatogram of volatile metabolites of light fermented black beans after 15 days of fermentation;

[0018] Figure 4 It is Table 2. Information table of volatile components of light fermented black beans with different fermentation times;

[0019] Figure 5 It is a stacked bar chart of the percentage content of volatile components in light fermented black beans at different fermentation times;

[0020] Figure 6 is the OPLS-DA model score plot;

[0021] Figure 7 is the permutation test result of OPLS-DA model;

[0022] Figure 8 This is the PCA biplot of 11 volatile components with significant differences before and after fermentation. DETAILED DESCRIPTION

[0023] The present invention provides a method for detecting volatile components in fermented black beans, comprising the following steps:

[0024] The volatile components in fermented black beans were detected by comprehensive two-dimensional headspace gas chromatography-mass spectrometry. The mass spectrum information corresponding to each chromatographic peak was manually analyzed according to the total ion current of gas chromatography-mass spectrometry and compared with the standard spectrum in the NIST 17L spectral library. The types of volatile components were determined according to the compounds with both forward and reverse matching degrees greater than 700, and the relative contents of volatile components were calculated by peak area normalization method.

[0025] In the present invention, the sample amount of the light fermented black beans is preferably 0.2 to 0.6 g, and more preferably 0.3 to 0.5 g.

[0026] In the present invention, the headspace conditions of the comprehensive two-dimensional headspace gas chromatography-mass spectrometry method are: sample bottle heating temperature 120°C; quantitative loop temperature 130°C; transmission line temperature 140°C; sample bottle equilibration time 10 to 40 minutes; pressure equilibration time 0.1 minute; injection time 0.5 minute; injection volume 1 mL.

[0027] In the present invention, the chromatographic conditions of the comprehensive two-dimensional headspace gas chromatography-mass spectrometry method are as follows: one-dimensional column: Agilent DB-FFAP chromatographic column, inner diameter × length is 30m × 0.25mm, particle size is 0.25μm; two-dimensional column: W1812 chromatographic column, inner diameter × length is 1.2m × 0.18mm, particle size is 0.18μm; inlet temperature: 250°C; split ratio 20:1, split flow rate: 1mL / min; carrier gas: high-purity helium, purity>99.999%; programmed temperature rise: initial temperature 40°C, maintained for 4min, increased to 110°C at a rate of 10°C, maintained for 3min, increased to 200°C at a rate of 20°C, and then increased to 230°C at a rate of 10°C, maintained for 6min.

[0028] In the present invention, the mass spectrometry conditions of the comprehensive two-dimensional headspace gas-mass spectrometry method are: ion source: EI ion source; temperature: 230°C; quadrupole temperature: 150°C; ionization energy: 70 eV; scanning range m / z 40-600, full scan; threshold: 150; solvent delay 2.5 min; scanning speed: 12500.

[0029] In the present invention, the modulator conditions of the comprehensive two-dimensional headspace gas chromatography-mass spectrometry method are: SV series modulation column: C5-C30 column; the cold zone temperature of the modulator is 9°C, the inlet temperature of the hot zone is 50°C, the outlet temperature of the hot zone is 260°C, and the column oven is biased at +30°C; the modulation cycle is 5s; the programmed temperature rise is: the initial temperature is 40°C, maintained for 4min, increased to 110°C at a rate of 10°C, maintained for 3min, increased to 200°C at a rate of 20°C, and then increased to 230°C at a rate of 10°C, and maintained for 6min.

[0030] In the present invention, the preparation method of light fermented black beans comprises the following steps:

[0031] Mix mulberry leaves and Artemisia annua, add 9-11 times of the amount of the raw drug and 7-9 times of the amount of the raw drug in turn, and boil for 0.5-1.5 hours, filter to obtain a filtrate, and combine the two filtrates to obtain a concentrated solution;

[0032] The concentrated liquid and black soybeans are mixed and soaked, then steamed for 1 to 3 hours to obtain steamed black beans. The steamed black beans are placed on mulberry leaf residue and Artemisia annua residue for pre-fermentation for 2 to 6 days until the "yellow skin" is on the surface. The pre-fermented black beans are post-fermented for 3 to 15 days until the aroma overflows, then taken out, steamed in water for 0.2 to 0.8 hours, and dried to obtain light black beans.

[0033] In the present invention, the mass ratio of the mulberry leaves, Artemisia annua and black soybeans is preferably 9:10:80-120, and more preferably 9:10:90-110; the amount of water used for the first decoction is preferably 9.5-10.5 times the amount of the mulberry leaf raw drug; the time of the first decoction is preferably 0.8-1.2 h; the amount of water used for the second decoction is preferably 7.5-8.5 times the amount of the Artemisia annua raw drug; the time of the second decoction is preferably 0.8-1.2 h; the temperature of the front fermentation and the back fermentation is preferably 28-32°C, and more preferably 29-31°C; the humidity of the front fermentation and the back fermentation is preferably 65-75%, and more preferably 68-72%.

[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] The instruments and materials used in this invention are:

[0036] Ultrapure water instrument UPR-II-15TNZP, purchased from Sichuan UPR Ultrapure Technology Co., Ltd.;

[0037] Rotary evaporator CCA-1111CE, purchased from Tokyo EYELA Co.;

[0038] Drug stability test chamber SHH-150SD, purchased from Chongqing Yongsheng Experimental Instrument Factory;

[0039] Desktop centrifuge Centrifuge5424R, purchased from Eppendorf GmbH, Germany;

[0040] Medical ultra-low temperature storage box (-80°C) DW-86L388J, purchased from Qingdao Haier Special Electric Co., Ltd., China;

[0041] Gas chromatography-mass spectrometry 7890B / 5977B, purchased from Agilent Technologies, Inc., USA;

[0042] Headspace sampler 7697A, purchased from Agilent Technologies, USA;

[0043] Solid-state thermal modulator SSM1810, purchased from Snow View Electronic Technology (Shanghai) Co., Ltd., China;

[0044] 1 / 10,000 electronic balance ME20, purchased from METTLER, Switzerland;

[0045] High-speed universal pulverizer FW135, purchased from Tianjin Test Instrument Co., Ltd.;

[0046] Black beans, Artemisia annua, and mulberry leaves were provided by Hebei Chunkai Pharmaceutical Co., Ltd.

[0047] Example 1

[0048] 1. Preparation of light fermented black bean samples with different fermentation times

[0049] Take 90g of mulberry leaves and 100g of Artemisia annua, add 10 and 8 times the amount of raw medicine in water in batches, decoct for 1h, filter, combine the filtrate and concentrate to 1000mL, cool and mix with 1000g of clean black soybeans, soak for 12h, wait for the black soybeans to fully expand and the decoction to be completely absorbed, steam thoroughly for 1.5h, take out, dry slightly, put in the fermentation mold, use the decocted mulberry leaves and Artemisia annua residue as the bottom, put the beans on it, and place it in a constant temperature and humidity incubator with a temperature of (30±2)℃ and a humidity of 70% to ferment for 6d until the "yellow coat is on the top" (pre-fermentation). Take out the sample of the pre-fermentation for 6d, remove the residue, wash it, put it in a ceramic jar, and "stuff it again" in a constant temperature and humidity incubator with a temperature of (30±2)℃ and a humidity of 70% for 15d (post-fermentation). When it is fully fermented and the aroma overflows, take it out, steam it for 0.5h, and dry it. Samples were kept every 2 days during the pre-fermentation period and every 3 days during the post-fermentation period, and stored in a -80°C refrigerator. Three batches of samples with different fermentation times were prepared in parallel.

[0050] Table 1 Sampling time and number of mild fermented black bean samples

[0051]

[0052] Example 2

[0053] Comprehensive two-dimensional headspace gas chromatography-mass spectrometry method

[0054] (1) Preparation of test samples

[0055] The sample was crushed and passed through a No. 2 sieve, and 0.3 g of powder was weighed as dry weight, placed in a 20 ml headspace injection bottle, and sealed with a cover.

[0056] (2) Headspace conditions

[0057] The sample bottle heating temperature was 120°C; the quantitative loop temperature was 130°C; the transfer line temperature was 140°C; the sample bottle equilibration time was 30 min; the pressure equilibration time was 0.1 min; the injection time was 0.5 min; and the injection volume was 1 mL.

[0058] (3) Chromatographic conditions

[0059] One-dimensional column: Agilent DB-FFAP column (30m×0.25mm, 0.25μm); two-dimensional column: W1812 column (1.2m×0.18mm, 0.18μm); inlet temperature: 250℃; split ratio 20:1, split flow rate: 1mL / min; carrier gas: high-purity helium (purity>99.999%); programmed temperature: initial temperature 40℃, maintained for 4min, increased to 110℃ at a rate of 10℃, maintained for 3min, increased to 200℃ at a rate of 20℃, then increased to 230℃ at a rate of 10℃, maintained for 6min.

[0060] (4) Mass spectrometry conditions

[0061] Ion source: EI ion source; temperature: 230°C; quadrupole temperature: 150°C; ionization energy: 70 eV; scan range m / z 40–600, full scan; threshold: 150; solvent delay 2.5 min; scan speed: 12500.

[0062] (5) Modulator conditions

[0063] SV series modulation column: C5–C30 column; the cold zone temperature of the modulator is 9°C, the inlet temperature of the hot zone is 50°C, the outlet temperature of the hot zone is 260°C, and the column oven is biased at +30°C; the modulation cycle is 5s; the program temperature is set the same as the chromatographic conditions.

[0064] (6) Data processing

[0065] According to the NIST 17L library search results, matching degree (both forward matching degree and reverse matching degree were greater than 700) and references, the volatile components detected by HS-GC-MS×MS were qualitatively analyzed, and the relative content of each component was determined by peak area normalization method. Origin 2021 and SIMCA 14.1 software were used to perform orthogonal partial least squares-discriminant analysis (OPLS-DA) on the volatile components, and the differential volatile components were screened based on the principle of variable importance projection (VIP)>1. Origin2021 software was used to perform correlation analysis between the differential volatile components and the fermentation period.

[0066] Example 3

[0067] HS-GC-MS×MS analysis of light fermented black bean samples with different fermentation times

[0068] 1. Investigation of headspace analysis conditions

[0069] The main factors affecting the efficiency of automated static headspace analysis include sample volume and equilibration time. The amount of sample will affect the concentration of volatile components in the headspace bottle and the detection effect; too long an equilibration time may lead to reactions such as thermal degradation or cause some unstable volatile components to undergo thermal decomposition, oxidation and other reactions, which will reduce the analytical sensitivity. Therefore, the two conditions of headspace injection volume and equilibration time were optimized. In order to eliminate the influence of sample heterogeneity, the total peak area of ​​volatile substances detected under each optimization project was used as the evaluation index.

[0070] Experimental results: Figure 1 shown.

[0071] The results of headspace sampling analysis showed that when the sample amount in the headspace bottle was 0.2-0.4g in the pre-fermentation stage, the amount of volatile index components in the headspace was positively correlated with the sample amount; when the sample amount was greater than 0.4g, the volatile components in the headspace reached supersaturation. When the sample amount was 0.5g in the post-fermentation stage, the amount of volatile index components in the headspace was positively correlated with the sample amount; when the sample amount was greater than 0.6g, the volatile components in the headspace reached supersaturation. Considering the two stages comprehensively, the sample amount of 0.3g dry weight was selected as the optimal sample amount, and the volatile components of light fermented black beans were determined in a 20mL headspace bottle.

[0072] The results of headspace sampling analysis showed that with the extension of headspace equilibrium time, the volatile components released by light fermented black beans increased. Before 35 minutes, the abundance of volatile components increased with the extension of time; after 35 minutes, the volatile components did not change much. Considering the effect and efficiency comprehensively, 30 minutes was selected as the optimal headspace equilibrium time for light fermented black beans.

[0073] 2. Identification of volatile components of mild fermented black bean samples with different fermentation times

[0074] According to the method of Example 2 above, the volatile metabolite components of light fermented black beans with different fermentation times were analyzed by headspace comprehensive two-dimensional gas chromatography-mass spectrometry to obtain comprehensive two-dimensional gas chromatograms of volatile components, as shown in FIG. Figure 2 , Figure 3 shown.

[0075] Through mass spectrometry analysis, a total of 75 volatile components were identified from 10 different fermentation time periods, as shown in Table 2. The specific content of the volatile component information table of light fermented black beans at different fermentation times is shown in Table 2. Figure 4 (Note: “ / ” has not yet been accurately named in Chinese; “–” was not detected; “+” was detected). Among them, there were 15 aldehydes, 11 ketones, 10 alcohols, 7 heterocyclic compounds, 7 organic nitrogen compounds, 6 esters, 5 phenols, 5 pyrazine compounds, 3 acids, 2 hydrocarbons, 2 sulfur compounds, and 2 other compounds. Aldehydes were the most abundant. 32, 46, 50, 56, 44, 51, 45, 46, 43, and 46 volatile components were identified in the light fermented black bean samples of pre-fermentation 0d, pre-fermentation 2d, pre-fermentation 4d, pre-fermentation 6d, post-fermentation 0d, post-fermentation 3d, post-fermentation 6d, post-fermentation 9d, post-fermentation 12d, and post-fermentation 15d, respectively. Among them, 20 compounds including isovaleraldehyde, n-valeraldehyde, n-hexanal, 3-furfural, benzaldehyde, 2-phenylacetaldehyde, 2,3-butanedione, 2,3-pentanedione, 3-hydroxy-2-butanone, hydroxyacetone, 2(5H)-furanone, 1-penten-3-ol, isopentanol, n-pentanol, furfuryl alcohol, 2-pentylfuran, 2-methoxy-4-vinylphenol, 2-methylpyrazine, glacial acetic acid, isovaleric acid, and estragole are common components of light fermented black beans with different fermentation times. 2(5H)-furanone, γ-butyrolactone, and n-butanol are unique components of the post-fermentation stage. o-Methoxyphenol, phenol, and 2-phenylethanol are unique components of the pre-fermentation stage.

[0076] 3. Semi-quantitative analysis of volatile components of fermented black bean samples with different fermentation times

[0077] According to the method described in Example 1, light fermented black bean samples with different fermentation times were prepared respectively, and the volatile components in 6 batches of light fermented black bean samples with different fermentation times were detected at the same time, and semi-quantitative analysis of the components was performed based on the peak area normalization method. The relative contents are shown in Table 3.

[0078] Table 3 Analysis of relative contents of volatile components in fermented black beans at different fermentation times (n=6)

[0079]

[0080]

[0081]

[0082]

[0083] Note: “–”, not detected

[0084] The stacked bar chart of the percentage of volatile components at different fermentation times is shown in Figure 5 shown.

[0085] Depend on Figure 5 It can be seen that the relative content of volatile components fluctuates significantly in different samples. The volatile components of light fermented black beans at different fermentation periods are complex, but they are mainly composed of acids and aldehydes. Acid compounds are mainly produced by the metabolism of sugars and lipid compounds, have a certain pungent smell, and play a harmonizing role in the flavor of fermented black beans. Acetic acid is an acid substance common to all fermentation periods and is the acid with the highest content. It may be produced by biochemical reactions of organic acids produced during the fermentation process, followed by isobutyric acid and isovaleric acid. Aldehydes account for a proportion second only to acids, mainly accumulated in the koji making stage, and are related to the degradation of amino acids and the metabolism of proteins. Aldehydes have a strong aroma, mostly floral and fruity. Isovaleraldehyde, n-valeraldehyde, n-hexanal, 3-furfural, benzaldehyde, and 2-phenylacetaldehyde are aldehydes present at all fermentation times. Among them, benzaldehyde and 2-phenylacetaldehyde are important flavoring substances in fermented black beans. They can also be detected in soy sauce and can provide caramel, cocoa, coffee and other aromas. The relative content is relatively high in light black bean samples with different fermentation times. Relevant studies have shown that n-hexanal, 2-pentylfuran, 1-octen-3-ol and other substances are related substances that produce beany odor. The relative content of these three volatile components in the post-fermentation stage is lower than that in the pre-fermentation stage. 2,3-Pentanedione has the aroma of sweet butter, cream, and caramel, and has a nutty base aroma. As the fermentation time increases, the relative content increases. Ethyl linoleate is a higher fatty acid that can produce a fruity aroma. This substance can make the aroma of light black beans after post-fermentation more coordinated and rich. 3-Penten-2-one has a strong pungent smell, such as spicy, ethereal, pepper, garlic, mustard, onion, etc., and only exists in the pre-fermentation process of light black beans.

[0086] 4. Study on the differences of volatile components of light fermented black beans at different fermentation times

[0087] In order to effectively distinguish the fermentation period of light fermented soybeans and screen the compounds that contribute most to light fermented soybeans of different fermentation times, OPLS-DA was used to analyze the relative contents of volatile components of light fermented soybeans at 10 different fermentation periods. The explanatory power R2Y of the model was 0.811, and the predictive power Q2 was 0.507, both greater than 0.5, proving that the model has good stability and predictability. From the score scatter plot ( Figure 6) It can be seen that the sample information falls within the 95% confidence interval and can be divided into three categories, of which pre-fermentation 0d to pre-fermentation 4d can be classified into one category, pre-fermentation 6d is one category, and post-fermentation 0d to post-fermentation 15d can be classified into one category. In order to avoid the overfitting phenomenon when the OPLS-DA model can effectively distinguish samples between groups but cannot effectively predict new sample data sets, the OPLS-DA model is subjected to a permutation test ( Figure 7 ), the permutation test results [R2=(0.0, 0.364), Q2=(0.0, -0.867)], indicating that the model has a good general explanation rate. The VIP diagram can intuitively reflect the weight of each component on the difference between groups. VIP>1 indicates that the influence rate of the component on the weight is higher than 50%, which is a difference marker. According to the VIP value of the compound and combined with the t-test to screen the difference marker, based on VIP>1.0, P<0.05, the differences between the groups of acetic acid, ethyl linoleate, benzaldehyde, 5-methyl-2-furfural, isovaleric acid, 2,5-dimethylpyrazine, isobutyric acid, furfuryl alcohol, 3-furfural, isovaleronitrile, and 2-methylpyrazine in the light fermented black bean samples at different fermentation times are statistically significant. The VIP values ​​of the 11 volatile components are shown in Table 4.

[0088] Table 4 VIP values ​​of 11 differential markers

[0089]

[0090] 5. The relationship between the odor and specific components of light fermented black bean samples with different fermentation times

[0091] In order to study the association between different fermentation periods and differential components, PCA biplot analysis was performed.

[0092] Experimental results: Figure 8 As shown. Figure 8 It can be seen that C3 (benzaldehyde), C9 (3-furfural), C1 (glacial acetic acid), C4 (5-methyl-2-furfural) point to the pre-fermentation stage, and C7 (isobutyric acid), C2 (ethyl linoleate), C10 (isovaleronitrile), C8 (furfuryl alcohol), C6 (2,5-dimethylpyrazine), and C5 (isovaleric acid) point to the post-fermentation period. The above differential compounds play an important role in distinguishing the pre- and post-fermentation of light fermented black beans, and can be used as characteristic components to distinguish the fermentation degree of light fermented black beans.

[0093] It can be seen from the above embodiments that the present invention achieves the separation of complex components in light fermented black beans through optimized HS-GC-MS×MS conditions, greatly improves the detection peak capacity of volatile components in light fermented black beans and improves the separation degree, performs qualitative and quantitative detection of volatile components in light fermented black beans, and can accurately detect the differences in types and contents of volatile components in light fermented black beans with different fermentation times, providing a reference for the establishment of quality standards for light fermented black beans and the study of their medicinal value.

[0094] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting volatile components in light fermented black beans, characterized in that: The following steps are involved: The volatile components in fermented black beans were detected by comprehensive two-dimensional headspace gas chromatography-mass spectrometry. The mass spectrum information corresponding to each chromatographic peak was manually analyzed according to the total ion current of gas chromatography-mass spectrometry and compared with the standard spectrum in the NIST 17L spectral library. The types of volatile components were determined according to the compounds with both forward and reverse matching degrees greater than 700, and the relative contents of volatile components were calculated by peak area normalization method.

2. The detection method according to claim 1, characterized in that: The sample amount of the light fermented black beans is 0.2-0.6 g.

3. The detection method according to claim 1, characterized in that: The headspace conditions of the comprehensive two-dimensional headspace gas chromatography-mass spectrometry method are as follows: sample bottle heating temperature 120°C; quantitative loop temperature 130°C; transmission line temperature 140°C; sample bottle equilibration time 10-40min; pressure equilibration time 0.1min; injection time 0.5min; injection volume 1mL.

4. The detection method according to claim 1, characterized in that: The chromatographic conditions of the comprehensive two-dimensional headspace gas chromatography-mass spectrometry method are as follows: one-dimensional column: Agilent DB-FFAP chromatographic column, inner diameter × length: 30m × 0.25mm, particle size: 0.25μm; two-dimensional column: W1812 chromatographic column, inner diameter × length: 1.2m × 0.18mm, particle size: 0.18μm; injection port temperature: 250°C; split ratio: 20:1, split flow rate: 1mL / min; Carrier gas: high-purity helium, purity>99.999%; program Heating: Initial temperature 40℃, hold for 4 min, increase to 110℃ at a rate of 10℃, hold for 3 min, increase to 200℃ at a rate of 20℃, then increase to 230℃ at a rate of 10℃, hold for 6 min.

5. The detection method according to claim 1, characterized in that: The mass spectrometry conditions of the comprehensive two-dimensional headspace gas chromatography-mass spectrometry method are as follows: ion source: EI ion source; Temperature: 230 °C; quadrupole temperature: 150 °C; ionization energy: 70 eV; scan range m / z 40–600, full scan; Threshold: 150; solvent delay 2.5 min; scan speed: 12500.

6. The detection method according to claim 1, characterized in that: The modulator conditions of the comprehensive two-dimensional headspace gas chromatography-mass spectrometry method are as follows: SV series modulation column: C5-C30 column; the cold zone temperature of the modulator is 9°C, the inlet temperature of the hot zone is 50°C, the outlet temperature of the hot zone is 260°C, and the column oven is biased at +30°C; the modulation cycle is 5s; the programmed temperature rise is as follows: the initial temperature is 40°C, maintained for 4min, increased to 110°C at a rate of 10°C, maintained for 3min, increased to 200°C at a rate of 20°C, and then increased to 230°C at a rate of 10°C, and maintained for 6min.

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