A method for detecting active components of milk aroma based on enzymatic hydrolysis and olfactory discrimination

Abstract

The present application relates to the technical field of food detection, and more particularly to a milk aroma active ingredient detection method based on enzymatic hydrolysis-smell identification combination, which provides a milk aroma active ingredient detection method, comprising: extracting and enriching volatile components in milk, and detecting aroma active ingredients therein by using gas chromatography-smell-mass spectrometry combination technology; wherein the extraction and enrichment method comprises: first, using streptomyces protease to perform enzymatic hydrolysis treatment on the milk, and then using sequential stirring rod adsorption extraction technology to extract and enrich the volatile components. The method provided by the present application can significantly reduce the detection limit of milk aroma active ingredients, improve the detection sensitivity, and broaden the detection range of aroma active ingredients, and is significantly superior to the prior art in terms of the number and structure type of detected components, and has the advantages of high detection efficiency, short cycle, convenient operation and the like, and has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, and in particular to a method for detecting active aroma components in milk based on enzymatic hydrolysis-olfaction combined. Background Technology

[0002] Current methods for analyzing milk aroma generally employ conventional sample flavor capture techniques and detection pathways. However, milk differs from most conventional samples, which consist of homogeneous solutions. It is a highly complex emulsified system composed of an aqueous phase, fat globules, casein micelles, and whey proteins. Volatile substances undergo complex partitioning between the fat phase, aqueous phase, protein binding sites, and gas phase. Furthermore, the abundant fat globule membranes and protein colloidal structures hinder the diffusion of volatile substances. Consequently, the free state of volatile substances in milk is significantly reduced, directly impacting extraction efficiency and making them more difficult to detect. When consumers drink milk, a large amount of volatile substances are released through both physical and chemical processes during oral consumption, ultimately affecting the overall sensory experience. Therefore, substances missed by conventional detection methods should not be overlooked. However, not all volatile components contribute to sensory perception; their contribution cannot be judged solely by concentration values ​​but rather by the actual aroma intensity of the aroma components. Only by understanding the causal origins of sensory phenomena can off-flavors be traced or optimized.

[0003] Only a few optimized sample pretreatment and capture methods can achieve olfactory identification on the GC-O-MS platform. However, research focuses on moderately volatile, low- to medium-polarity small-molecule esters, aldehydes, and ketones, with limited ability to identify complex aroma active components that are highly polar, thermally unstable, and poorly volatile, which are abundant in the matrix. Existing technologies often only provide a "mainstream aroma profile," with problems such as limited detectable substances, high detection limits, and severe peak overlap. This leads to the omission or inaccurate identification of many components that significantly contribute to key aroma characteristics and even defective flavors, making it impossible to establish a causal relationship between substances and sensory phenomena, thus hindering precise control of milk flavor quality. Furthermore, existing technologies largely remain at the level of "qualitative or semi-quantitative identification of known mainstream components," lacking systematic and scientific identification methods for the comprehensive spectrum of milk aroma active components, especially previously unreported aroma active components. This makes it difficult to support the needs of precise flavor control, quality difference analysis, off-flavor tracing, and high-end product development.

[0004] Specifically, existing methods for extracting and enriching aroma components in milk mainly focus on headspace extraction and solvent extraction. However, headspace extraction (including solid-phase microextraction and dynamic headspace sample preparation) relies heavily on the volatility of the compounds themselves and often promotes volatilization by heating the sample, which prevents the capture of some thermally unstable substances. Solvent extraction (including direct solvent extraction, simultaneous distillation extraction, and solvent-assisted flavor evaporation extraction) mainly utilizes the principle of "like dissolves like" for extraction and enrichment. However, this method is suitable for samples with strong matrix homogeneity. Due to co-extraction interference, most lipophilic compounds in milk are lost, and the solvents used are not environmentally friendly, posing significant risks during operation and requiring a high level of operator skill, making it difficult to promote. A small number of researchers use stir bar adsorption extraction for flavor analysis of plant beverages, fruit juices, and alcoholic beverages, but its application in milk is still very limited. Only a few studies have used it for GC-MS determination of aroma components in milk powder and cheese, and it has rarely been used in the identification of aroma activity intensity of milk flavor substances. Summary of the Invention

[0005] This invention provides a method for detecting active aroma components in milk based on enzymatic hydrolysis-olfaction.

[0006] This invention aims to provide a method that significantly reduces the detection limit of aroma active components in milk, improves detection sensitivity, and broadens the detection range of aroma active components. By introducing a streptococcal protease enzymatic hydrolysis step during sample pretreatment and employing sequential stirring rod adsorption extraction technology in the key stage of aroma component capture, this method overcomes the limitations imposed by the heterogeneity and complexity of the milk matrix on volatile components. It also breaks the technical bias in the field regarding "difficult-to-detect highly polar / thermally unstable components," significantly reducing the detection limit of aroma active components in the complex milk matrix and improving detection sensitivity. This enables stable detection and reliable identification of more aroma active components in milk, especially those not previously reported in conventional technologies, on conventional instrument platforms. In this invention, the detection of aroma active components in milk aims to discover the original aroma of milk, rather than creating aroma; therefore, the choice of enzyme is crucial.

[0007] Specifically, the present invention provides the following technical solutions.

[0008] This invention provides a method for detecting active aroma components in milk. The method includes: extracting and enriching volatile components in milk, and detecting the active aroma components using gas chromatography-olfactometry-mass spectrometry. The extraction and enrichment method includes: first enzymatically hydrolyzing the milk with streptomycin, and then extracting and enriching the volatile components using sequential stir bar adsorption extraction. The amount of streptomycin used is 0.05-0.07% of the mass of milk.

[0009] The enzyme activity of the streptomycin is 6500-7500 U / g.

[0010] Preferably, the enzymatic hydrolysis is performed under stirring conditions for 20-40 minutes.

[0011] Preferably, after adding streptomycin, the method further includes the step of adding 2-nonanol as an internal standard reference for the system.

[0012] In the methods described above, the sequential stirring rod adsorption extraction technology includes: First stirring and adsorption: Stirring and adsorption are performed using a first stirring rod with an adsorption coating. After adsorption is completed, the first stirring rod is removed. Second stirring adsorption: The milk after the first stirring adsorption treatment is mixed with an ionic strength regulator, and then stirred and adsorbed using a second stirring rod with an adsorption coating.

[0013] This invention has found that, compared with other extraction and enrichment methods, sequential stirring rod adsorption extraction technology can better cooperate with the enzymatic hydrolysis step of streptomycin, improve the extraction and enrichment efficiency of aroma active ingredients in milk samples after enzymatic hydrolysis, and thus help increase the number of aroma active ingredients detected.

[0014] Preferably, the adsorption coating is polydimethylsiloxane (PDMS).

[0015] Preferably, the time for both the first and second stirring adsorption is 30-60 minutes. More preferably, the time for the first stirring adsorption is 30-40 minutes, and the time for the second stirring adsorption is 30-60 minutes.

[0016] Preferably, the temperature for adsorption and extraction by the sequential stirring rod is 22-26°C.

[0017] Preferably, the stirring speed is 1000-1400 rpm. More preferably, it is 1100-1300 rpm.

[0018] Preferably, the ionic strength regulator is sodium chloride, and its addition amount is 15%-35% of the mass ratio of milk. More preferably, it is 20%-30%.

[0019] Preferably, before each stirring adsorption begins, a step of placing the stirring rod in the adsorption system for equilibration is included, and the equilibration time is preferably 0.5-2 minutes.

[0020] In the method described above, after the sequential adsorption extraction by the stirring rod is completed, the first stirring rod and the second stirring rod are subjected to thermal desorption and cold injection; wherein, the heating program for thermal desorption is as follows: initial temperature 40-55℃, hold for 1-2 min, then increase to 295℃-300℃ at a rate of 190-210℃ / min, and hold for 8-12 min.

[0021] Preferably, after each stirring and adsorption is completed, the stirring rod is removed and the surface is rinsed with deionized water, then the surface is wiped clean with clean, odorless, lint-free paper and the surface moisture is absorbed, and then thermal desorption treatment is performed.

[0022] Preferably, the temperature rise procedure for the cold injection is as follows: first, the cold injection system is rapidly cooled to -60℃ to -90℃, and after thermal desorption is completed, the cold injection system is then heated to 290-310℃ at a rate of 8-12℃ / min and maintained for 8-12min.

[0023] The process of rapidly cooling the cold sample introduction system to -60℃ to -90℃ is carried out using ultra-low temperature liquid nitrogen.

[0024] In the method described above, the gas chromatography conditions are as follows: initial temperature 35-45℃, hold for 3-7 min, then increase the temperature to 210℃-220℃ at 2-3℃ / min, hold for 1-2 min, then increase the temperature to 230-235℃ at 8-12℃ / min, hold for 25-30 min, and then run at 240-260℃; separation is performed using a DB-WAX column.

[0025] In the method described above, the odor detector used for smelling has the following parameters: the odor output passes through an uncoated, filled empty chromatographic column, the column temperature is set to 245-255℃, and 3-5 trained evaluators smell the odor through the olfaction port and record the odor characteristics, occurrence time, and odor intensity information; the odor intensity is divided into at least 4 levels, preferably 5 levels: VW: Very Weak, W: Weak, M: Middle, S: Strong, VS: Very Strong.

[0026] In the method described above, the mass spectrometry conditions are as follows: an electron impact (EI) ion source is used, with an electron energy of 68-72 eV, a transfer line temperature of 275-285℃, an ion source temperature of 225-235℃, a quadrupole temperature of 145-155℃, no solvent delay, and a mass scan range of m / z 33-300.

[0027] In the methods described above, the milk includes unprocessed milk such as raw milk, pasteurized milk, ultra-high temperature sterilized milk (UHT milk), and other milk products obtained by sterilizing or sterilizing raw milk.

[0028] The beneficial effects of this invention include at least the following: the method for detecting milk aroma active components based on enzymatic hydrolysis-olfaction provided by this invention can significantly reduce the detection limit of milk aroma active components (the detection limit of more than 90% of aroma active substances is significantly reduced), improve detection sensitivity, and broaden the detection range of aroma active components. It is significantly superior to existing technologies in terms of the number and structural types of detected components, with a more than 5-fold increase in the types of detected aroma active components. Furthermore, this method also has advantages such as high detection efficiency, short cycle time, and convenient operation. Coupled with aroma active ingredient dilution analysis (AEDA), this method can further screen for new aroma active ingredients (such as terpenes, 4-methylphenol, and γ-tetradecyl lactone) that contribute significantly to the characteristic aroma and quality differences of milk. Correlation analysis between the detected new aroma active ingredients and sensory evaluation showed that the correlation coefficients of compounds with lower detection limits were all above 0.8, and more than 70% of these compounds had correlation coefficients above 0.9, indicating a very strong correlation with sensory results. Further recombination and deletion experiments revealed that the absence of these components significantly reduced the sample's reducibility, proving the indispensability of the newly detected components. Using this method helps fill the gap in understanding the causal relationship between milk flavor substances and key sensory qualities, laying a solid foundation for the precise control of milk flavor quality. This method can provide a practical technical tool for optimizing dairy product flavor, establishing raw material and origin flavor fingerprints, quality control, and tracing the source of off-flavor problems.

[0029] Specifically, compared with traditional methods for detecting aroma active ingredients, the method of the present invention has at least the following advantages: (1) Detection of a fundamental extension of the object dimension Traditional methods mainly focus on a few classic esters, aldehydes, ketones, lactones, and other moderately volatile and low-polar substances; the number of aroma active ingredients detected is limited, and very few new components are discovered.

[0030] The method of this invention can systematically detect and identify far more aroma-active components than previously reported; a significant portion of which are substances not previously reported in milk or not reported as aroma-active components; it is not just an increase in the number of detected substance peaks, but an increase in the types of new components that truly contribute to aroma.

[0031] (2) It helps to enhance the interpretability and predictability of product sensory information and improve the accuracy of product flavor detection. Traditional methods: The extremely limited number of aroma active ingredients detected by existing methods (about 10) limits current research on milk flavor, especially in explaining many sensory phenomena (off-flavors, differences in origin / processing) and making it difficult to predict sensory results.

[0032] The method of this invention provides a clearer material explanation of which substances contribute to which flavor and how much each substance contributes; through reconstruction / deletion experiments, it is found that the addition of new components significantly improves the similarity between the model aroma and the original, and the new components are more correlated with sensory phenomena.

[0033] (3) Technical approach designed for difficult-to-detect components Traditional methods: Sample pretreatment, SPME / LLE conditions, and GC procedures mostly employ conventional techniques, primarily serving conventional volatile substances; they are not suitable for the detection of highly polar, thermally unstable, sulfur / nitrogen-containing, or minimally present volatile components that are easily affected by matrix heterogeneity and thus difficult to extract and enrich.

[0034] The method of this invention aims to extract and enrich aroma active ingredients that are difficult or missed due to matrix effects, thus overcoming the limitations of matrix effects on the detection of volatile components and significantly increasing the types of aroma active ingredients that can be detected.

[0035] (4) Higher application value Traditional methods focus on analyzing and controlling a limited number of classic components, which limits basic understanding and forces the selection of key indicators from a low-resolution candidate pool. This approach has significant limitations in tracing off-flavors and implementing targeted regulation, failing to identify the truly target molecules that need to be regulated.

[0036] The method of this invention aims to establish the most complete high-resolution map of flavor substances possible. It can combine sensory association, OAV, statistical modeling, recombination and deletion experiments, etc., to focus on a few key regulatory substances that are truly used for regulation. Other substances serve as background information for explanation, source tracing, and risk assessment. It constructs a complete technical route from basic cognition to application indicators, which can solve problems that were previously unsolvable or difficult to solve (such as odor source tracing) and strongly support product quality control. Detailed Implementation

[0037] In a specific embodiment of the present invention, a method for detecting active components of milk aroma is provided, the method comprising the following steps: (1) Sample pretreatment (extraction and enrichment of aroma active ingredients) Accurately weigh the milk sample to be tested and place it in a headspace container. Add 0.05%-0.07% of the sample mass of streptomycin (CAS No.: 9036-06-0), and then add 2-nonanol as an internal standard reference for the system. Place an uncoated magnetic stir bar in the container and pre-stir at 1100-1300 rpm for 20-40 minutes.

[0038] Place a stir bar coated with polydimethylsiloxane (PDMS) in the headspace container. After equilibration for 0.5-2 minutes, turn on the magnetic stirring function and stir at 1100-1300 rpm for 30-40 minutes, maintaining an adsorption temperature of 22-26℃. After adsorption, remove the stir bar, rinse the surface with deionized water, wipe off any adhering substances with clean, odorless, lint-free paper, and dry the surface. Place the stir bar in a clean, odorless thermal desorption tube. Next, place another stir bar coated with PDMS in the headspace container, add 15%-35% (by weight of sample) of ionic strength modifier (sodium chloride), equilibrate for 0.5-2 minutes, and then stir at 1100-1300 rpm for 30-60 minutes, maintaining an adsorption temperature of 22-26℃. After adsorption, remove the stir bar and wash it. Transfer both stir bars to the thermal desorption tube for thermal desorption.

[0039] (2) Sample preparation for instrument loading Thermal desorption tubes equipped with stir rods were placed in a thermal desorption system (TDU) for thermal desorption and separated in a cold sample introduction system (CIS). The TDU temperature program was as follows: initial temperature 40-55℃, held for 1 min, then increased to 295-300℃ at 200℃ / min, held for 10 min. The CIS temperature program was as follows: the CIS temperature was first rapidly lowered to -60℃ to -90℃ using ultra-low temperature liquid nitrogen, and after TDU desorption was completed, the system was then increased to 300℃ at 10℃ / min, held for 10 min.

[0040] (3) Separation and identification were performed using gas chromatography-olfactometry-mass spectrometry (GC-O-MS). Gas chromatography (GC) conditions: initial temperature 40℃, hold for 5 min, then increase the temperature to 210℃-220℃ at 2-3℃ / min, hold for 1 min, then increase the temperature to 230℃ at 10℃ / min and hold for 25-30 min, then run at 250℃; separation was performed using a DB-WAX column.

[0041] Odor identification instrument (O) parameters: Odor output is passed through an uncoated packed empty chromatographic column, the column temperature is set to 250°C, and moistened nitrogen gas is simultaneously introduced to prevent the experimenter's nasal cavity from drying out. Three to five trained evaluators smell the odor through the olfactory port and record the odor characteristics, time of occurrence, and odor intensity. Odor intensity is divided into at least four levels, preferably five (Very Weak, Weak, Middle, Strong, Very Strong).

[0042] Mass spectrometry (MS) conditions: Electron impact ion source, electron energy 70 eV, transfer line temperature 280 °C, ion source temperature 230 °C, quadrupole temperature 150 °C, no solvent delay, mass scan range m / z 33-300.

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0044] The streptomycin used in the following examples and comparative examples has the CAS number 9036-06-0 and was purchased from Solarbio Life Science. Its enzyme activity content is 7000 U / g.

[0045] To facilitate comparison of the effectiveness of the methods, the same batch of unprocessed raw milk (i.e., raw milk) was used for analysis in the following examples and comparative examples.

[0046] Example 1 This embodiment provides a method for detecting active components of milk aroma, including the following steps: (1) Sample pretreatment (extraction and enrichment of aroma active ingredients): Accurately weigh 30g of milk and place it in a 40mL headspace vial. Add 0.06% of the sample mass of streptomycin and then add 60μL of 2-nonanol (32ppm) as the internal standard reference of the system. Place an uncoated magnetic stir bar in the vial and pre-stir at 1200 rpm for 25min.

[0047] A stir bar coated with polydimethylsiloxane (PDMS) was placed in a headspace vial. After equilibration for 1 minute, the magnetic stirring function was turned on, and adsorption was carried out at 1200 rpm for 35 minutes. The stir bar was then removed, rinsed with deionized water, and then wiped clean with clean, odorless, lint-free paper to remove any adhering substances and absorb any moisture. The stir bar was then placed in a clean, odorless thermal desorption tube. Another stir bar coated with PDMS was placed in the headspace vial, and sodium chloride (20% of the sample mass) was added. After equilibration for 1 minute, adsorption was carried out at 1200 rpm for 35 minutes. The stir bar was then removed, rinsed with deionized water, and then wiped clean with clean, odorless, lint-free paper to remove any adhering substances and absorb any moisture. Both stir bar were then placed in the same glass thermal desorption tube for thermal desorption. The temperature during the adsorption and stirring process was maintained at room temperature (22-26℃).

[0048] (2) Sample preparation for injection (TDU+CIS method) The thermal desorption tube equipped with a stir bar was placed in the thermal desorption system (TDU) for thermal desorption and then separated in the cold sample introduction system (CIS). The TDU temperature program was as follows: initial temperature 45℃, hold for 1 min, then increase to 300℃ at 200℃ / min and hold for 10 min. The CIS temperature program was as follows: the CIS temperature was first rapidly lowered to -80℃ using ultra-low temperature liquid nitrogen, and after the TDU desorption was completed, the system was then increased to 300℃ at 10℃ / min and held for 10 min.

[0049] (3) Separation and identification were performed using gas chromatography-olfactometry-mass spectrometry (GC-O-MS). Gas chromatography (GC) conditions: initial temperature 40℃, hold for 5 min, then increase to 220℃ at 3℃ / min, hold for 1 min, then increase to 230℃ at 10℃ / min, hold for 28 min, and finally run at 250℃. Separation was performed using a DB-WAX column.

[0050] Odor identification instrument (O) parameters: Odor output passes through an uncoated packed empty chromatographic column, the column temperature is set to 250°C, and moistened nitrogen gas is simultaneously introduced to prevent the experimenter's nasal cavity from drying out. Three to five trained evaluators smell the odor through the olfactory port and record the odor characteristics, time of appearance, and odor intensity. Odor intensity is divided into at least four levels, with five levels commonly used: VW: Very Weak, W: Weak, M: Middle, S: Strong, VS: Very Strong.

[0051] Mass spectrometry (MS) conditions: Electron impact ion source, electron energy 70 eV, transfer line temperature 280 °C, ion source temperature 230 °C, quadrupole temperature 150 °C, solvent-free delay, mass scan range m / z 33~300.

[0052] The detection results of aroma active components in milk are shown in Table 1. The odor intensity is described as follows: VW: Very Weak, W: Weak, M: Middle, S: Strong, VS: Very Strong. In the identification methods, MS indicates consistency between the mass spectrometry results and the Nist 14 library, with a match degree >90% or a positive / negative match degree >800 / 800; RI indicates retention index (qualitative); O indicates odor is detectable. The odor intensity and identification methods in the following examples and comparative examples are explained in the same way.

[0053] Table 1

[0054] Note: "-" indicates that the data cannot be characterized because it has not yet been smelled, and " / " indicates that there are no literature reports on its odor characteristics.

[0055] For the detection results of aroma active components in milk in Table 1, correlation analysis was conducted between the concentration values ​​of the compounds and the sensory attribute scores obtained from descriptive analysis by a professional evaluation panel. Quantitative Descriptive Analysis (QDA) involved a rigorously selected and trained evaluation panel (no fewer than 10 people) systematically and quantitatively evaluating the flavor intensity of the samples using standardized sensory terminology and scoring scales. Statistical analysis was then used to generate a "sensory attribute profile" of the samples, thus transforming subjective perceptions into objective and comparable data. A 10-point scale (0-9 points) was used: 0 points represented "completely imperceptible," 1-3 points represented low intensity, 4-6 points represented medium intensity, and 7-9 points represented high intensity. The correlation analysis results of the 23 newly detected key components are shown in Table 2.

[0056] Table 2

[0057] Analysis of the data in Table 2 shows that among the 23 newly detected compounds by the method of this invention, the correlation coefficients between the compound concentration values ​​and sensory attribute scores are all greater than 0.8, statistically demonstrating a very strong correlation between them. Furthermore, 70% of the substances have correlation coefficients greater than 0.9. This indicates that these newly detected substances play a crucial role in sensory evaluation, rather than having a negligible modifying effect. The detection of these substances is essential for the flavor analysis of milk. Moreover, all 23 components can be detected using the methods of each embodiment, but the detection rate using the comparative method is extremely low.

[0058] To further verify the contribution of these substances to overall sensory experience, a reconstitution-deficient experiment was conducted using molecular sensory science (References: Yang Ping, Song Huanlu, Wang Lijin, et al. Characterization of Key Aroma-Active Compounds in Black Garlic by Sensory-Directed Flavor Analysis[J]. Journal of Agricultural and Food Chemistry, 2019, 67(28): 7926-7934. doi:10.1021 / acs.jafc.9b03269; Buettner, A.; Schieberle, P. Evaluation of key aroma compounds in hand-squeezed grapefruit juice (Citrus paradisi Macfayden) by quantitation and flavor reconstitution experiments. J. Agric. Food Chem. 2001, 49, (1358-1363) The deletion experiments were conducted on the 11 key components (butyric acid, hexanoic acid, hexanal, trans-2-nonanal, δ-decanolide, δ-dodecanolide, 2-cyclohexen-1-one, 4-methylphenol, γ-tetradecanolide, ethyl butyrate, and γ-valerolide) after the final recombination. The results (Table 3) showed that the reduction degree of the sample was greatly reduced after the deletion of the 5 newly detected substances, which once again confirmed the importance and indispensability of the newly detected components.

[0059] Table 3

[0060] Note: In Table 3, circles represent missing components, and √ represents components that are included.

[0061] Example 2 This embodiment provides a method for detecting active components of milk aroma, including the following steps: (1) Sample pretreatment (extraction and enrichment of aroma active ingredients): Accurately weigh 30g of milk and place it in a 40mL headspace vial. Add 0.05% of the sample mass of streptomycin and then add 60μL of 2-nonanol (32ppm) as the internal standard reference of the system. Place an uncoated magnetic stir bar in the vial and pre-stir at 1200 rpm for 20min.

[0062] A stir bar coated with polydimethylsiloxane (PDMS) was placed in a headspace vial. After equilibration for 1 minute, the magnetic stirring function was turned on, and adsorption was carried out at 1200 rpm for 35 minutes. The stir bar was then removed, rinsed with deionized water, and then wiped clean with clean, odorless, lint-free paper to remove any adhering substances and absorb any moisture. The stir bar was then placed in a clean, odorless thermal desorption tube. Another stir bar coated with PDMS was placed in the headspace vial, and sodium chloride (20% of the sample mass) was added. After equilibration for 1 minute, adsorption was carried out at 1200 rpm for 35 minutes. The stir bar was then removed, rinsed with deionized water, and then wiped clean with clean, odorless, lint-free paper to remove any adhering substances and absorb any moisture. Both stir bar were then placed in a glass thermal desorption tube for thermal desorption. The temperature during the adsorption and stirring process was maintained at room temperature (22-26℃).

[0063] (2) Sample preparation for injection (TDU+CIS method) The thermal desorption tube equipped with a stir bar was placed in the thermal desorption system (TDU) for thermal desorption and then separated in the cold sample introduction system (CIS). The TDU temperature program was as follows: initial temperature 45℃, hold for 1 min, then increase to 300℃ at 200℃ / min and hold for 10 min. The CIS temperature program was as follows: the CIS temperature was first rapidly lowered to -80℃ using ultra-low temperature liquid nitrogen, and after the TDU desorption was completed, the system was then increased to 300℃ at 10℃ / min and held for 10 min.

[0064] (3) Separation and identification were performed using gas chromatography-olfactometry-mass spectrometry (GC-O-MS). Gas chromatography (GC) conditions: initial temperature 40℃, hold for 5 min, then increase to 220℃ at 3℃ / min, hold for 1 min, then increase to 230℃ at 10℃ / min, hold for 28 min, and finally run at 250℃. Separation was performed using a DB-WAX column.

[0065] Odor identification instrument (O) parameters: Odor output passes through an uncoated packed empty chromatographic column, the column temperature is set to 250°C, and moistened nitrogen gas is simultaneously introduced to prevent the experimenter's nasal cavity from drying out. Three to five trained evaluators smell the odor through the olfactory port and record the odor characteristics, time of appearance, and odor intensity. Odor intensity is divided into at least four levels, with five levels commonly used: VW: Very Weak, W: Weak, M: Middle, S: Strong, VS: Very Strong.

[0066] Mass spectrometry (MS) conditions: Electron impact ion source, electron energy 70 eV, transfer line temperature 280 °C, ion source temperature 230 °C, quadrupole temperature 150 °C, solvent-free delay, mass scan range m / z 33~300.

[0067] The results of the detection of aroma active components in milk are shown in Table 4.

[0068] Table 4

[0069] Example 3 This embodiment provides a method for detecting active components of milk aroma, including the following steps: (1) Sample pretreatment (extraction and enrichment of aroma active ingredients): Accurately weigh 30g of milk and place it in a 40mL headspace vial. Add 0.07% of the sample mass of streptomycin and then add 60μL of 2-nonanol (32ppm) as the internal standard reference of the system. Place an uncoated magnetic stir bar in the vial and pre-stir at 1200 rpm for 40min.

[0070] A stir bar coated with polydimethylsiloxane (PDMS) was placed in a headspace vial. After equilibration for 1 minute, the magnetic stirring function was turned on, and adsorption was carried out at 1200 rpm for 35 minutes. The stir bar was then removed, rinsed with deionized water, and then wiped clean with clean, odorless, lint-free paper to remove any adhering substances and absorb any remaining moisture. The stir bar was then placed in a clean, odorless thermal desorption tube. Another stir bar coated with PDMS was placed in the headspace vial, and sodium chloride (20% of the sample mass) was added. After equilibration for 1 minute, adsorption was carried out at 1200 rpm for 35 minutes. The stir bar was then removed, rinsed with deionized water, and then wiped clean with clean, odorless, lint-free paper to remove any adhering substances and absorb any remaining moisture. Both stir bar were then placed in a glass thermal desorption tube for thermal desorption. The temperature during the adsorption and stirring process was maintained at room temperature (22-26℃).

[0071] (2) Sample preparation for injection (TDU+CIS method) The thermal desorption tube equipped with a stir bar was placed in the thermal desorption system (TDU) for thermal desorption and then separated in the cold sample introduction system (CIS). The TDU temperature program was as follows: initial temperature 45℃, hold for 1 min, then increase to 300℃ at 200℃ / min and hold for 10 min. The CIS temperature program was as follows: the CIS temperature was first rapidly lowered to -80℃ using ultra-low temperature liquid nitrogen, and after the TDU desorption was completed, the system was then increased to 300℃ at 10℃ / min and held for 10 min.

[0072] (3) Separation and identification were performed using gas chromatography-olfactometry-mass spectrometry (GC-O-MS). Gas chromatography (GC) conditions: initial temperature 40℃, hold for 5 min, then increase to 220℃ at 3℃ / min, hold for 1 min, then increase to 230℃ at 10℃ / min, hold for 28 min, and finally run at 250℃. Separation was performed using a DB-WAX column.

[0073] Odor identification instrument (O) parameters: Odor output passes through an uncoated packed empty chromatographic column, the column temperature is set to 250°C, and moistened nitrogen gas is simultaneously introduced to prevent the experimenter's nasal cavity from drying out. Three to five trained evaluators smell the odor through the olfactory port and record the odor characteristics, time of appearance, and odor intensity. Odor intensity is divided into at least four levels, with five levels commonly used: VW: Very Weak, W: Weak, M: Middle, S: Strong, VS: Very Strong.

[0074] Mass spectrometry (MS) conditions: Electron impact ion source, electron energy 70 eV, transfer line temperature 280 °C, ion source temperature 230 °C, quadrupole temperature 150 °C, solvent-free delay, mass scan range m / z 33~300.

[0075] The results of the detection of aroma active components in milk are shown in Table 5.

[0076] Table 5

[0077] Example 4 This embodiment provides a method for detecting active components of milk aroma, including the following steps: (1) Sample pretreatment (extraction and enrichment of aroma active ingredients): Accurately weigh 30g of milk and place it in a 40mL headspace vial. Add 0.06% of the sample mass of streptomycin and then add 60μL of 2-nonanol (32ppm) as the internal standard reference of the system. Place an uncoated magnetic stir bar in the vial and pre-stir at 1200 rpm for 25min.

[0078] A stir bar coated with polydimethylsiloxane (PDMS) was placed in a headspace vial. After equilibration for 1 minute, the magnetic stirring function was turned on, and adsorption was carried out at 1200 rpm for 40 minutes. The stir bar was then removed, rinsed with deionized water, and then wiped clean with clean, odorless, lint-free paper to remove any adhering substances and absorb any remaining moisture. The stir bar was then placed in a clean, odorless thermal desorption tube. Another stir bar coated with PDMS was placed in the headspace vial, and sodium chloride (20% of the sample mass) was added. After equilibration for 1 minute, adsorption was carried out at 1200 rpm for 60 minutes. The stir bar was then removed, rinsed with deionized water, and then wiped clean with clean, odorless, lint-free paper to remove any adhering substances and absorb any remaining moisture. Both stir bar were then placed in a glass thermal desorption tube for thermal desorption. The temperature during the adsorption and stirring process was maintained at room temperature (22-26℃).

[0079] (2) Sample preparation for injection (TDU+CIS method) The thermal desorption tube equipped with a stir bar was placed in the thermal desorption system (TDU) for thermal desorption and then separated in the cold sample introduction system (CIS). The TDU temperature program was as follows: initial temperature 45℃, hold for 1 min, then increase to 300℃ at 200℃ / min and hold for 10 min. The CIS temperature program was as follows: the CIS temperature was first rapidly lowered to -80℃ using ultra-low temperature liquid nitrogen, and after the TDU desorption was completed, the system was then increased to 300℃ at 10℃ / min and held for 10 min.

[0080] (3) Separation and identification were performed using gas chromatography-olfactometry-mass spectrometry (GC-O-MS). Gas chromatography (GC) conditions: initial temperature 40℃, hold for 5 min, then increase to 220℃ at 3℃ / min, hold for 1 min, then increase to 230℃ at 10℃ / min, hold for 28 min, and finally run at 250℃. Separation was performed using a DB-WAX column.

[0081] Odor identification instrument (O) parameters: Odor output passes through an uncoated packed empty chromatographic column, the column temperature is set to 250°C, and moistened nitrogen gas is simultaneously introduced to prevent the experimenter's nasal cavity from drying out. Three to five trained evaluators smell the odor through the olfactory port and record the odor characteristics, time of appearance, and odor intensity. Odor intensity is divided into at least four levels, with five levels commonly used: VW: Very Weak, W: Weak, M: Middle, S: Strong, VS: Very Strong.

[0082] Mass spectrometry (MS) conditions: Electron impact ion source, electron energy 70 eV, transfer line temperature 280 °C, ion source temperature 230 °C, quadrupole temperature 150 °C, solvent-free delay, mass scan range m / z 33~300.

[0083] The results of the detection of aroma active components in milk are shown in Table 6.

[0084] Table 6

[0085] Example 5 This embodiment provides a method for detecting active components of milk aroma, including the following steps: (1) Sample pretreatment (extraction and enrichment of aroma active ingredients): Accurately weigh 30g of milk and place it in a 40mL headspace vial. Add 0.06% of the sample mass of streptomycin and then add 60μL of 2-nonanol (32ppm) as the internal standard reference of the system. Place an uncoated magnetic stir bar in the vial and pre-stir at 1200 rpm for 25min.

[0086] A stir bar coated with polydimethylsiloxane (PDMS) was placed in a headspace vial. After equilibration for 1 minute, the magnetic stirring function was turned on, and adsorption was carried out at 1200 rpm for 30 minutes. The stir bar was then removed, rinsed with deionized water, and then wiped clean with clean, odorless, lint-free paper to remove any adhering substances and absorb any moisture. The stir bar was then placed in a clean, odorless thermal desorption tube. Another stir bar coated with PDMS was placed in the headspace vial, and sodium chloride (20% of the sample mass) was added. After equilibration for 1 minute, adsorption was carried out at 1200 rpm for 30 minutes. The stir bar was then removed, rinsed with deionized water, and then wiped clean with clean, odorless, lint-free paper to remove any adhering substances and absorb any moisture. Both stir bar were then placed in a glass thermal desorption tube for thermal desorption. The temperature during the adsorption and stirring process was maintained at room temperature (22-26℃).

[0087] (2) Sample preparation for injection (TDU+CIS method) The thermal desorption tube equipped with a stir bar was placed in the thermal desorption system (TDU) for thermal desorption and then separated in the cold sample introduction system (CIS). The TDU temperature program was as follows: initial temperature 45℃, hold for 1 min, then increase to 300℃ at 200℃ / min and hold for 10 min. The CIS temperature program was as follows: the CIS temperature was first rapidly lowered to -80℃ using ultra-low temperature liquid nitrogen, and after the TDU desorption was completed, the system was then increased to 300℃ at 10℃ / min and held for 10 min.

[0088] (3) Separation and identification were performed using gas chromatography-olfactometry-mass spectrometry (GC-O-MS). Gas chromatography (GC) conditions: initial temperature 40℃, hold for 5 min, then increase to 220℃ at 3℃ / min, hold for 1 min, then increase to 230℃ at 10℃ / min, hold for 28 min, and finally run at 250℃. Separation was performed using a DB-WAX column.

[0089] Odor identification instrument (O) parameters: Odor output passes through an uncoated packed empty chromatographic column, the column temperature is set to 250°C, and moistened nitrogen gas is simultaneously introduced to prevent the experimenter's nasal cavity from drying out. Three to five trained evaluators smell the odor through the olfactory port and record the odor characteristics, time of appearance, and odor intensity. Odor intensity is divided into at least four levels, with five levels commonly used: VW: Very Weak, W: Weak, M: Middle, S: Strong, VS: Very Strong.

[0090] Mass spectrometry (MS) conditions: Electron impact ion source, electron energy 70 eV, transfer line temperature 280 °C, ion source temperature 230 °C, quadrupole temperature 150 °C, solvent-free delay, mass scan range m / z 33~300.

[0091] The results of the detection of aroma active components in milk are shown in Table 7.

[0092] Table 7

[0093] Example 6 This embodiment provides a method for detecting active components of milk aroma, including the following steps: (1) Sample pretreatment (extraction and enrichment of aroma active ingredients): Accurately weigh 30g of milk and place it in a 40mL headspace vial. Add 0.06% of the sample mass of streptomycin and then add 60μL of 2-nonanol (32ppm) as the internal standard reference of the system. Place an uncoated magnetic stir bar in the vial and pre-stir at 1200 rpm for 25min.

[0094] A stir bar coated with polydimethylsiloxane (PDMS) was placed in a headspace vial. After equilibration for 1 minute, the magnetic stirring function was turned on, and adsorption was carried out at 1200 rpm for 35 minutes. The stir bar was then removed, rinsed with deionized water, and then wiped clean with clean, odorless, lint-free paper to remove any adhering substances and absorb any moisture. The stir bar was then placed in a clean, odorless thermal desorption tube. Another stir bar coated with PDMS was placed in the headspace vial, and sodium chloride (20% of the sample mass) was added. After equilibration for 1 minute, adsorption was carried out at 1200 rpm for 35 minutes. The stir bar was then removed, rinsed with deionized water, and then wiped clean with clean, odorless, lint-free paper to remove any adhering substances and absorb any moisture. Both stir bar were then placed in a glass thermal desorption tube for thermal desorption. The temperature during the adsorption and stirring process was maintained at room temperature (22-26℃).

[0095] (2) Sample preparation for injection (TDU+CIS method) A thermal desorption tube equipped with a stir bar was placed in a thermal desorption system (TDU) for thermal desorption and then separated in a cold sample introduction system (CIS). The TDU temperature program was as follows: initial temperature 45℃, held for 1 min, then increased to 300℃ at 200℃ / min and held for 10 min. The CIS temperature program was as follows: the CIS temperature was first rapidly lowered to -60℃ using ultra-low temperature liquid nitrogen, and after TDU desorption was completed, the system was then increased to 300℃ at 10℃ / min and held for 10 min.

[0096] (3) Separation and identification were performed using gas chromatography-olfactometry-mass spectrometry (GC-O-MS). Gas chromatography (GC) conditions: initial temperature 40℃, hold for 5 min, then increase to 220℃ at 3℃ / min, hold for 1 min, then increase to 230℃ at 10℃ / min, hold for 28 min, and finally run at 250℃. Separation was performed using a DB-WAX column.

[0097] Odor identification instrument (O) parameters: Odor output passes through an uncoated packed empty chromatographic column, the column temperature is set to 250°C, and moistened nitrogen gas is simultaneously introduced to prevent the experimenter's nasal cavity from drying out. Three to five trained evaluators smell the odor through the olfactory port and record the odor characteristics, time of appearance, and odor intensity. Odor intensity is divided into at least four levels, with five levels commonly used: VW: Very Weak, W: Weak, M: Middle, S: Strong, VS: Very Strong.

[0098] Mass spectrometry (MS) conditions: Electron impact ion source, electron energy 70 eV, transfer line temperature 280 °C, ion source temperature 230 °C, quadrupole temperature 150 °C, solvent-free delay, mass scan range m / z 33~300.

[0099] The results of the detection of aroma active components in milk are shown in Table 8.

[0100] Table 8

[0101] Example 7 This embodiment provides a method for detecting active components of milk aroma, including the following steps: (1) Sample pretreatment (extraction and enrichment of aroma active ingredients): Accurately weigh 30g of milk and place it in a 40mL headspace vial. Add 0.06% of the sample mass of streptomycin and then add 60μL of 2-nonanol (32ppm) as the internal standard reference of the system. Place an uncoated magnetic stir bar in the vial and pre-stir at 1200 rpm for 25min.

[0102] A stir bar coated with polydimethylsiloxane (PDMS) was placed in a headspace vial. After equilibration for 1 minute, the magnetic stirring function was turned on, and adsorption was carried out at 1200 rpm for 35 minutes. The stir bar was then removed, rinsed with deionized water, and then wiped clean with clean, odorless, lint-free paper to remove any adhering substances and absorb any moisture. The stir bar was then placed in a clean, odorless thermal desorption tube. Another stir bar coated with PDMS was placed in the headspace vial, and sodium chloride (20% of the sample mass) was added. After equilibration for 1 minute, adsorption was carried out at 1200 rpm for 35 minutes. The stir bar was then removed, rinsed with deionized water, and then wiped clean with clean, odorless, lint-free paper to remove any adhering substances and absorb any moisture. Both stir bar were then placed in a glass thermal desorption tube for thermal desorption. The temperature during the adsorption and stirring process was maintained at room temperature (22-26℃).

[0103] (2) Sample preparation for injection (TDU+CIS method) A thermal desorption tube equipped with a stir bar was placed in a thermal desorption system (TDU) for thermal desorption and then separated in a cold sample introduction system (CIS). The TDU temperature program was as follows: initial temperature 45℃, held for 1 min, then increased to 300℃ at 200℃ / min and held for 10 min. The CIS temperature program was as follows: the CIS temperature was first rapidly lowered to -90℃ using ultra-low temperature liquid nitrogen, and after TDU desorption was completed, the system was then increased to 300℃ at 10℃ / min and held for 10 min.

[0104] (3) Separation and identification were performed using gas chromatography-olfactometry-mass spectrometry (GC-O-MS). Gas chromatography (GC) conditions: initial temperature 40℃, hold for 5 min, then increase to 220℃ at 3℃ / min, hold for 1 min, then increase to 230℃ at 10℃ / min, hold for 28 min, and finally run at 250℃. Separation was performed using a DB-WAX column.

[0105] Odor identification instrument (O) parameters: Odor output passes through an uncoated packed empty chromatographic column, the column temperature is set to 250°C, and moistened nitrogen gas is simultaneously introduced to prevent the experimenter's nasal cavity from drying out. Three to five trained evaluators smell the odor through the olfactory port and record the odor characteristics, time of appearance, and odor intensity. Odor intensity is divided into at least four levels, with five levels commonly used: VW: Very Weak, W: Weak, M: Middle, S: Strong, VS: Very Strong.

[0106] Mass spectrometry (MS) conditions: Electron impact ion source, electron energy 70 eV, transfer line temperature 280 °C, ion source temperature 230 °C, quadrupole temperature 150 °C, solvent-free delay, mass scan range m / z 33~300.

[0107] The results of the detection of aroma active components in milk are shown in Table 9.

[0108] Table 9

[0109] Example 8 This embodiment provides a method for detecting active components of milk aroma, including the following steps: (1) Sample pretreatment (extraction and enrichment of aroma active ingredients): Accurately weigh 30g of milk and place it in a 40mL headspace vial. Add 0.06% of the sample mass of streptomycin and then add 60μL of 2-nonanol (32ppm) as the internal standard reference of the system. Place an uncoated magnetic stir bar in the vial and pre-stir at 1200 rpm for 25min.

[0110] A stir bar coated with polydimethylsiloxane (PDMS) was placed in a headspace vial. After equilibration for 1 minute, the magnetic stirring function was turned on, and adsorption was carried out at 1200 rpm for 35 minutes. The stir bar was then removed, rinsed with deionized water, and then wiped clean with clean, odorless, lint-free paper to remove any adhering substances and absorb any moisture. The stir bar was then placed in a clean, odorless thermal desorption tube. Another stir bar coated with PDMS was placed in the headspace vial, and sodium chloride (20% of the sample mass) was added. After equilibration for 1 minute, adsorption was carried out at 1200 rpm for 35 minutes. The stir bar was then removed, rinsed with deionized water, and then wiped clean with clean, odorless, lint-free paper to remove any adhering substances and absorb any moisture. Both stir bar were then placed in a glass thermal desorption tube for thermal desorption. The temperature during the adsorption and stirring process was maintained at room temperature (22-26℃).

[0111] (2) Sample preparation for injection (TDU+CIS method) The thermal desorption tube equipped with a stir bar was placed in the thermal desorption system (TDU) for thermal desorption and then separated in the cold sample introduction system (CIS). The TDU temperature program was as follows: initial temperature 45℃, hold for 1 min, then increase to 300℃ at 200℃ / min and hold for 10 min. The CIS temperature program was as follows: the CIS temperature was first rapidly lowered to -80℃ using ultra-low temperature liquid nitrogen, and after the TDU desorption was completed, the system was then increased to 300℃ at 10℃ / min and held for 10 min.

[0112] (3) Separation and identification were performed using gas chromatography-olfactometry-mass spectrometry (GC-O-MS). Gas chromatography (GC) conditions: initial temperature 40℃, hold for 5 min, then increase to 210℃ at 2℃ / min, hold for 1 min, then increase to 230℃ at 10℃ / min, hold for 25 min, and finally run at 250℃. Separation was performed using a DB-WAX column.

[0113] Odor identification instrument (O) parameters: Odor output passes through an uncoated packed empty chromatographic column, the column temperature is set to 250°C, and moistened nitrogen gas is simultaneously introduced to prevent the experimenter's nasal cavity from drying out. Three to five trained evaluators smell the odor through the olfactory port and record the odor characteristics, time of appearance, and odor intensity. Odor intensity is divided into at least four levels, with five levels commonly used: VW: Very Weak, W: Weak, M: Middle, S: Strong, VS: Very Strong.

[0114] Mass spectrometry (MS) conditions: Electron impact ion source, electron energy 70 eV, transfer line temperature 280 °C, ion source temperature 230 °C, quadrupole temperature 150 °C, solvent-free delay, mass scan range m / z 33~300.

[0115] The results of the detection of aroma active components in milk are shown in Table 10.

[0116] Table 10

[0117] Example 9 This embodiment provides a method for detecting active components of milk aroma, including the following steps: (1) Sample pretreatment (extraction and enrichment of aroma active ingredients): Accurately weigh 30g of milk and place it in a 40mL headspace vial. Add 0.065% of the sample mass of streptomycin and then add 60μL of 2-nonanol (32ppm) as the internal standard reference of the system. Place an uncoated magnetic stir bar in the vial and pre-stir at 1200 rpm for 30min.

[0118] A stir bar coated with polydimethylsiloxane (PDMS) was placed in a headspace vial. After equilibration for 1 minute, the magnetic stirring function was turned on, and adsorption was carried out at 1200 rpm for 38 minutes. The stir bar was then removed, rinsed with deionized water, and then wiped clean with clean, odorless, lint-free paper to remove any adhering substances and absorb any surface moisture. The stir bar was then placed in a clean, odorless thermal desorption tube. Another stir bar coated with PDMS was placed in the headspace vial, and sodium chloride (30% of the sample mass) was added. After equilibration for 1 minute, adsorption was carried out at 1200 rpm for 50 minutes. The stir bar was then removed, rinsed with deionized water, and then wiped clean with clean, odorless, lint-free paper to remove any adhering substances and absorb any surface moisture. Both stir bar were then placed in a glass thermal desorption tube for thermal desorption. The temperature during the adsorption and stirring process was maintained at room temperature (22-26℃).

[0119] (2) Sample preparation for injection (TDU+CIS method) A thermal desorption tube equipped with a stir bar was placed in a thermal desorption system (TDU) for thermal desorption and then separated in a cold sample introduction system (CIS). The TDU temperature program was as follows: initial temperature 50℃, held for 1 min, then increased to 295℃ at 200℃ / min and held for 10 min. The CIS temperature program was as follows: the CIS temperature was first rapidly lowered to -70℃ using ultra-low temperature liquid nitrogen, and after TDU desorption was completed, the system was then increased to 300℃ at 10℃ / min and held for 10 min.

[0120] (3) Separation and identification were performed using gas chromatography-olfactometry-mass spectrometry (GC-O-MS). Gas chromatography (GC) conditions: initial temperature 40℃, hold for 5 min, then increase to 210℃ at 3℃ / min, hold for 1 min, then increase to 230℃ at 10℃ / min, hold for 30 min, and finally run at 250℃. Separation was performed using a DB-WAX column.

[0121] Odor identification instrument (O) parameters: Odor output passes through an uncoated packed empty chromatographic column, the column temperature is set to 250°C, and moistened nitrogen gas is simultaneously introduced to prevent the experimenter's nasal cavity from drying out. Three to five trained evaluators smell the odor through the olfactory port and record the odor characteristics, time of appearance, and odor intensity. Odor intensity is divided into at least four levels, with five levels commonly used: VW: Very Weak, W: Weak, M: Middle, S: Strong, VS: Very Strong.

[0122] Mass spectrometry (MS) conditions: Electron impact ion source, electron energy 70 eV, transfer line temperature 280 °C, ion source temperature 230 °C, quadrupole temperature 150 °C, solvent-free delay, mass scan range m / z 33~300.

[0123] The results of the detection of aroma active components in milk are shown in Table 11.

[0124] Table 11

[0125] Comparative Example 1 This comparative example provides a method for detecting active aroma components in milk, which differs from the method in Example 1 only in that the sample pretreatment in step (1) uses headspace solid-phase microextraction (HS-SPME). The specific method is as follows: HS-SPME is used to extract volatile compounds from milk. In a 40 mL headspace vial, 15 mL of milk sample, 20% sodium chloride by sample mass, and a magnetic stir bar are added, followed by 30 μL of 2-nonanol (32 ppm) as an internal standard reference. The sample is stirred at 30 rpm using a magnetic stir bar and equilibrated at 40 °C for 20 minutes. A 2 cm long polydimethylsiloxane-divinylbenzene-carbon molecular sieve (PDMS / DVB / CAR) fiber-coupled extraction head (where the PDMS and CAR membranes are 30 μm thick and the DVB membrane is 50 μm thick) is used to extract volatile substances from the milk sample. The fiber is inserted into the headspace vial and positioned 5 mm-1 cm above the sample matrix, and allowed to stand for 30 minutes.

[0126] The results of the detection of aroma active components in milk are shown in Table 12.

[0127] Table 12

[0128] Analysis of the results of the detected components in Table 12 reveals that the number of olfactory substances has significantly decreased, and the method failed to detect all the newly detected substances that significantly contribute to sensory perception in the examples, resulting in a large number of omissions. The method has obvious limitations in providing a complete sensory analysis.

[0129] Comparative Example 2 This comparative example provides a method for detecting active ingredients of milk aroma, which differs from the method in Example 1 only in that the enzymatic hydrolysis step of streptomycin is removed in the sample pretreatment of step (1).

[0130] The results of the detection of aroma active components in milk are shown in Table 13.

[0131] Table 13

[0132] Analysis of the results of the detected components in Table 13 reveals that the number of olfactory substances has significantly decreased, and the method only detects 17% of the substances that make significant contributions to sensory perception in the examples, indicating a large number of omissions. The method has obvious limitations in providing a complete analysis of sensory perception.

[0133] Comparative Example 3 This comparative example provides a method for detecting active components of milk aroma, which differs from the method in Example 1 only in that: in the sample pretreatment of step (1), the streptococcal protease in the enzymatic hydrolysis step is replaced with proteinase K (CAS No. 39450-01-6, purchased from Solarbio Life Science, with an enzyme activity content of 30000U / g) with an added amount of enzyme activity units (U).

[0134] The results of the detection of aroma active components in milk are shown in Table 14.

[0135] Table 14

[0136] Analysis of the results of the detected components in Table 14 reveals that the number of olfactory substances has significantly decreased, and the method only detects 26% of the newly detected substances that make significant contributions to sensory perception in the examples, indicating a large number of omissions. The method has obvious limitations in providing a complete analysis of sensory perception.

[0137] Comparative Example 4 This comparative example provides a method for detecting active ingredients in milk aroma. The only difference between this method and the method in Example 1 is that in the sample pretreatment step (1), the streptococcal protease in the enzymatic hydrolysis step is replaced with Bacillus subtilis protease (CAS No. 9014-01-1, purchased from Shanghai Yuanye Biotechnology Co., Ltd., with an enzyme activity content of 150,000 U / g) with an added amount of enzyme activity units (U).

[0138] The results of the detection of aroma active components in milk are shown in Table 15.

[0139] Table 15

[0140] Analysis of the results of the detected components in Table 15 reveals that the number of olfactory substances has significantly decreased, and the method only detects 8% of the substances that make significant contributions to sensory perception in the examples, indicating a large number of omissions. The method has obvious limitations in providing a complete analysis of sensory perception.

[0141] Comparative Example 5 This comparative example provides a method for detecting active aroma components in milk, which differs from the method in Example 1 only in that the sample pretreatment in step (1) uses headspace solid-phase microextraction (HS-SPME). The specific method is as follows: HS-SPME is used to extract volatile compounds from milk. In a 40 mL headspace vial, 15 mL of milk sample, 20% sodium chloride (by weight of sample), 0.06% streptomycin (by weight of sample), and a magnetic stir bar are added, followed by 30 μL of 2-nonanol (32 ppm) as an internal standard reference. The sample is stirred at 1200 rpm using a magnetic stir bar and pre-stirred at 40 °C for 25 minutes. A 2 cm long polydimethylsiloxane-divinylbenzene-carbon molecular sieve (PDMS / DVB / CAR) fiber-coupled extraction head (where the PDMS and CAR membranes are 30 μm thick and the DVB membrane is 50 μm thick) is used to extract volatile substances from the milk sample. Insert the fiber into the headspace vial and position it 5 mm to 1 cm above the sample matrix, and let it stand for 30 minutes.

[0142] The results of the detection of aroma active components in milk are shown in Table 16.

[0143] Table 16

[0144] Analysis of the results of the detected components in Table 16 reveals that the number of olfactory substances has significantly decreased. Furthermore, the method failed to detect the number of substances that were newly detected in the examples and made significant contributions to sensory perception even after treatment with streptomycin protease, indicating a large number of omissions. The method has obvious limitations in providing a complete sensory analysis.

[0145] Comparative Example 6 This comparative example provides a method for detecting active components of milk aroma, which differs from the method in Example 1 only in that: in the sample pretreatment of step (1), the streptococcal protease in the enzymatic hydrolysis step is replaced with an amount of lipase with an equal enzyme activity unit (U) (product model is Palatase 20000 L, purchased from Novo in Denmark, with an enzyme activity content of 2000U / g).

[0146] The results of the detection of aroma active components in milk are shown in Table 17.

[0147] Table 17

[0148] Analysis of the results of the detected components in Table 17 reveals that the number of olfactory substances has significantly decreased, and the method only detects 4% of the substances that make significant contributions to sensory perception in the examples, indicating a large number of omissions. The method has obvious limitations in providing a complete analysis of sensory perception.

[0149] Comparative Example 7 This comparative example provides a method for detecting active components of milk aroma. The only difference between this method and the method in Example 1 is that the streptococcal protease in the enzymatic hydrolysis step is replaced in the sample pretreatment step (1) with a compound flavor complex enzyme with an equal amount of added enzyme activity units (U) (purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd., with an enzyme activity content of 30000U / g).

[0150] The results of the detection of aroma active components in milk are shown in Table 18.

[0151] Table 18

[0152] Analysis of the results of the detected components in Table 18 reveals that the number of olfactory substances has significantly decreased, and the method only detects 34% of the newly detected substances that make significant contributions to sensory perception in the examples, indicating a large number of omissions. The method has obvious limitations in providing a complete analysis of sensory perception.

[0153] Comparative Example 8 This comparative example provides a method for detecting active components of milk aroma, which differs from the method in Example 1 only in that: the sample pretreatment in step (1) uses headspace solid-phase microextraction (HS-SPME), and the streptomycin is replaced with a compound flavor enzyme (purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd., with an enzyme activity of 30,000 U / g) at an equal enzyme activity unit (U) addition amount. The specific method is as follows: HS-SPME is used to extract volatile compounds in milk. In a 40 mL headspace vial, 15 mL of milk sample, 20% sodium chloride (sample mass), compound flavor enzyme (purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd., with an enzyme activity of 30,000 U / g), and a magnetic stir bar are added respectively. Then, 30 μL of 2-nonanol (32 ppm) is added as an internal standard reference for the system. The sample is stirred at 1200 rpm using a magnetic stir bar and pre-stirred at 40°C for 25 minutes. Volatile substances in milk samples were extracted using an extraction head coupled with a 2 cm long polydimethylsiloxane-divinylbenzene-carbon molecular sieve (PDMS / DVB / CAR) fiber (with PDMS and CAR membranes 30 μm thick and DVB membrane 50 μm thick). The fiber was inserted into the headspace vial and positioned 5 mm–1 cm above the sample matrix, and allowed to stand for 30 minutes.

[0154] The results of the detection of aroma active components in milk are shown in Table 19.

[0155] Table 19

[0156] Analysis of the results of the detected components in Table 19 reveals that only three substances were detected, and their odor intensity was weak. Furthermore, the detection rate of the newly detected substances that significantly contribute to sensory perception in the examples was only 17% after the addition of compound flavor protease, indicating a significant omission. The method has obvious limitations in providing a complete sensory analysis.

[0157] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting active ingredients in milk aroma, characterized in that, include: The volatile components in milk were extracted and enriched, and the aroma active components were detected by gas chromatography-olfactometry-mass spectrometry. The extraction and enrichment method includes: first, enzymatically hydrolyzing milk with streptomycin, and then extracting and enriching the volatile components therein using sequential stirring rod adsorption extraction technology. The amount of streptomycin used is 0.05-0.07% of the mass of milk; In the gas chromatography-olfactometry-mass spectrometry (GC-MS) technique, the GC conditions are as follows: initial temperature 35-45℃, hold for 3-7 min, then increase the temperature at 2-3℃ / min to 210℃-220℃, hold for 1-2 min, then increase the temperature at 8-12℃ / min to 230-235℃, hold for 25-30 min, and finally run at 240-260℃; separation is performed using a DB-WAX column. The mass spectrometry conditions were as follows: electron impact ion source with electron energy of 68-72 eV, transfer line temperature of 275-285℃, ion source temperature of 225-235℃, quadrupole temperature of 145-155℃, no solvent delay, and mass scan range of m / z 33-300.

2. The method for detecting active components of milk aroma according to claim 1, characterized in that, The enzymatic hydrolysis process is carried out under stirring conditions for 20-40 minutes.

3. The method for detecting active ingredients of milk aroma according to claim 1 or 2, characterized in that, The sequential stirring bar adsorption extraction technology includes: First stirring and adsorption: Stirring and adsorption are performed using a first stirring rod with an adsorption coating. After adsorption is completed, the first stirring rod is removed. Second stirring adsorption: The milk after the first stirring adsorption treatment is mixed with an ionic strength regulator, and then stirred and adsorbed using a second stirring rod with an adsorption coating.

4. The method for detecting active components of milk aroma according to claim 3, characterized in that, The adsorption coating is polydimethylsiloxane; And / or, the time for the first and second stirring adsorptions is 30-60 min each.

5. The method for detecting active ingredients of milk aroma according to claim 3, characterized in that, The temperature for sequential stirring rod adsorption extraction is 22-26℃; And / or, the stirring speed is 1000-1400 rpm.

6. The method for detecting active components of milk aroma according to claim 3, characterized in that, The ionic strength regulator is sodium chloride, and its addition amount is 15%-35% of the mass of milk.

7. The method for detecting active ingredients of milk aroma according to claim 3, characterized in that, After sequential stir bar adsorption extraction is completed, the first and second stir bars are subjected to thermal desorption and cold injection. The heating program for thermal desorption is as follows: starting temperature 40-55℃, hold for 1-2 min, then increase to 295℃-300℃ at a rate of 190-210℃ / min, hold for 8-12 min; And / or, the temperature rise program for the cold injection is as follows: first, the cold injection system is rapidly cooled to -60℃ to -90℃, and after thermal desorption is completed, the cold injection system is then heated to 290-310℃ at a rate of 8-12℃ / min and held for 8-12min.

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