Method for identifying lactone odor active compounds in milk

Abstract

The invention relates to the technical field of substance detection and analysis, in particular to a method for identifying lactone odor active compounds in milk. According to the method for identifying the lactone odor active compounds in the milk, provided by the invention, the pretreatment of the milk sample is performed by combining the thin-film solid-phase microextraction with the sequential stirring rod adsorption method, and the gas chromatography-sniffing-mass spectrometry technology is matched, so that the sniffing detection rate of the lactone odor active compounds in the milk is remarkably increased; the method has the advantages of high efficiency, strong operability, environmental protection and the like, and has important application value in milk lactone odor active compound detection and milk flavor evaluation.

Description

Technical Field

[0001] The present invention relates to the technical field of substance detection and analysis, and in particular to a method for identifying lactone odor-active compounds in milk. Background Art

[0002] Flavor is one of the most important attributes in the quality evaluation of milk. Identifying the key flavor components is crucial for product improvement and new product development. The identification of key flavor components is mainly achieved by identifying odor-active compounds. However, the identification of odor-active compounds cannot be determined by the content of the compounds, but rather by the olfactory stimulation and intensity caused by the compounds. Lactone compounds, as a class of key but weakly aroma-stimulating compounds in milk, often have high olfactory discrimination difficulty. If the lactone compounds cannot be accurately discriminated, it will affect the comprehensive evaluation of milk flavor quality (Wang Wanhou, Mu Zhishen. Effects of UHT heat treatment on flavor substances in milk [J]. China Dairy Industry, 2012, 40(4): 36-38; Chi X, Shao Y, Pan M, et al. Distinction of volatile flavor profiles in various skim milk products via HS-SPME-GC-MS and e-nose [J]. European Food Research and Technology, 2021, 247(6): 1539-1551; Yan Hong, Li Hongliang, Qian Wentao, et al. Effects of direct UHT sterilization on the nutritional components, sensory properties and flavor of milk [J]. Food Industry, 2020, 41(5): 329-332), and thus it is impossible to accurately guide the improvement of milk flavor quality. An appropriate pretreatment method (i.e., an aroma compound extraction and enrichment method) will effectively reduce the olfactory discrimination obstacles brought by the aroma characteristics of the compounds themselves and the food matrix. However, due to the limitations of the existing methods for extracting and enriching milk flavor components, there is no method that can significantly improve the low olfactory discrimination detection rate of lactone aroma-active compounds.

[0003] Existing methods for identifying milk odor components cannot achieve the olfactory discrimination of lactone compounds in milk. There are very few identification methods that can identify 1-3 lactone odor-active compounds (i.e., lactone compounds that can be olfactory discriminated) from other similar dairy product categories other than milk, and the detection rate is relatively low.

[0004] The olfactory detection rate of odor-active compounds depends to a great extent on the extraction and enrichment methods. The existing methods for extracting and enriching the odor-active components of milk mainly focus on two methods: solvent extraction and headspace extraction. Solvent extraction (including direct solvent extraction, simultaneous distillation extraction, solvent-assisted flavor evaporation extraction, etc.) mainly uses the principle of "like dissolves like" for extraction and enrichment. However, the solvent environment used in this method is not friendly, and this method is more suitable for samples with strong matrix homogeneity. The heterogeneity of the milk matrix makes the extraction efficiency of this method for milk flavor substances not high. Headspace extraction mainly utilizes the volatility of flavor substances. Currently, the headspace method of solid-phase microextraction (SPME) is mostly used. However, due to the limited coating area of its fiber head, the adsorption of lactone compounds with weak aroma often fails to meet the requirements of olfactory detection. The headspace method of thin-film solid-phase microextraction (TF-SPME) realizes the enrichment of volatile compounds by increasing the specific surface area of the adsorption phase, and the extraction efficiency is often dozens of times that of the same type of SPME material. However, this method has not only not been applied to the identification of odor-active compounds in milk, but also the effects of the enrichment and olfactory detection rate of lactone odor-active compounds have not been clarified. There are also some flavor analyses of dairy products that are pretreated by using the stir bar sorptive extraction method. Although as an immersion sorption method, it has the advantages of avoiding competitive adsorption, large stationary phase volume, high extraction capacity, and simultaneous extraction and enrichment, etc., the detection rate of lactone odor-active compounds in milk is still low when using the stir bar sorptive extraction method for sample pretreatment.

[0005] Based on the problems existing in the prior art, there is still a need to develop a method that can effectively improve the olfactory detection rate of lactone odor-active compounds in milk. Summary of the Invention

[0006] The present invention provides a method for identifying lactone odor-active compounds in milk.

[0007] To solve the problem of the low olfactory detection rate of lactone odor-active compounds in milk and achieve the extraction, enrichment, and olfactory detection of more lactone compounds, on the basis of trying a variety of different extraction and enrichment methods, the present invention has developed a non-destructive and efficient method for extracting, enriching, and olfactory detecting lactone odor-active compounds in milk. This method combines thin-film solid-phase microextraction (TF-SPME) and sequential stir bar sorptive extraction (Seq-SBSE) to simultaneously extract and enrich lactone compounds in milk, comprehensively utilizes the dual advantages of headspace extraction and immersion sorption, realizes the efficient olfactory analysis of lactone odor-active compounds, reduces the olfactory discrimination difficulty of odor-active substances with weak odor characteristics such as lactone compounds, is conducive to a more comprehensive analysis of milk flavor, and helps to improve the flavor quality of milk.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] The present invention provides a method for identifying lactone odor-active compounds in milk, and the method includes: extracting and enriching lactone compounds in milk, and identifying lactone odor-active compounds by using gas chromatography-olfactometry-mass spectrometry technology; Among them, the extraction and enrichment of lactone compounds in milk adopts the method of thin film solid-phase microextraction combined with sequential stir bar sorptive extraction, and includes: placing a sample to be tested in a headspace container, performing a first stir bar sorptive extraction with a first stir bar having an adsorption coating, taking out the first stir bar after the adsorption is completed and waiting for thermal desorption; in the system of the first stir bar sorptive extraction, adding an ionic strength regulator and installing an adsorption film with a coating used for thin film solid-phase microextraction, and performing a second stir bar sorptive extraction with a second stir bar having an adsorption coating, taking out the second stir bar and the adsorption film after the adsorption is completed, and performing thermal desorption together with the first stir bar.

[0010] Thin film solid-phase microextraction (TF-SPME) is an emerging extraction technology. Compared with the traditional solid-phase microextraction method, the specific surface area of its adsorption phase has increased significantly. However, there is no report on the extraction, enrichment and olfactory discrimination analysis of lactone odor-active compounds by using the TF-SPME method.

[0011] Although the sequential stir bar sorptive extraction method (Seq-SBSE) has been reported for the extraction of volatile substances in dairy products such as milk powder, due to the limitations of the physical and chemical principles it relies on, the enrichment efficiency of the Seq-SBSE method for lactone compounds is relatively limited: the conventional SBSE method extracts in the form of immersion sorption, which has the advantages of avoiding competitive adsorption, large stationary phase volume, high extraction capacity, and simultaneous extraction and enrichment, but there is a disadvantage that it cannot meet the extraction of a variety of different polar compounds. The sequential stir bar sorptive extraction method can broaden the polarity range of the target compounds through method optimization, but the enrichment efficiency of the lactone target compounds is still relatively limited, and the olfactory detection rate of the subsequent lactone odor-active compounds is still relatively low.

[0012] In addition, in the field of food flavor analysis at present, the strategy of selecting a maximum advantage technology and optimizing it is often adopted to achieve the maximum extraction and enrichment of the target compounds. The main reason is that the combined use of many extraction and enrichment methods has difficulties in principle and actual operation. On the one hand, this is because the physical and chemical principles relied on by different extraction and enrichment technologies are different, which is not conducive to maximizing the advantages of both methods at the same time; on the other hand, limited by the actual desorption conditions of the gas chromatograph, the adsorption materials used in different extraction technologies often cannot be thermally desorbed at the same time.

[0013] After a large number of attempts, the present invention unexpectedly discovers that, compared with other extraction and enrichment methods or their combinations, the TF-SPME&Seq-SBSE method obtained by combining TF-SPME and Seq-SBSE has a very good extraction and enrichment effect for the extraction and enrichment of lactone compounds in milk, and can significantly improve the sniffing detection rate of lactone odor-active compounds.

[0014] In the above identification method, the extraction temperature of the thin-film solid-phase microextraction and the adsorption temperature of the sequential stir bar sorptive extraction are both not higher than 35°C.

[0015] Solid-phase microextraction is a typical technique in headspace extraction methods. The headspace method mainly realizes extraction and enrichment by using the volatility principle of the substance to be extracted. In order to achieve the extraction of different volatile target substances, this method usually realizes the comprehensive extraction of target substances by heating the target system. Therefore, its extraction temperature is usually relatively high (50°C or even higher). However, during the research and development process of the present invention, it is found that when pretreating with a conventional heated headspace system, the number of lactone odor-active compounds detected significantly decreases, and the sniffing intensity of some compounds also decreases, and the sniffing detection rate significantly decreases. Moreover, more thiol compounds are detected after heating, resulting in a significant reduction in the accuracy of the extracted flavor substances. The low-temperature extraction method accidentally tried in the present invention has obvious advantages in terms of the sniffing detection rate and accuracy of lactone odor-active compounds compared with the conventional heated extraction method.

[0016] Preferably, the extraction temperature of the thin-film solid-phase microextraction and the adsorption temperature of the sequential stir bar sorptive extraction are both not higher than 30°C.

[0017] Preferably, in addition to adding the sample to be tested, 2-nonanol is added as an internal standard reference substance for the system in the headspace container.

[0018] In the above method, the adsorption coatings of the first stir bar and the second stir bar can be selected from polydimethylsiloxane (PDMS) coatings or polyethylene glycol-dimethylsiloxane (EG) coatings.

[0019] Preferably, the adsorption coatings of the first stir bar and the second stir bar are preferably polydimethylsiloxane (PDMS) coatings. Compared with other coatings, the polydimethylsiloxane coating is beneficial to improving the sniffing detection rate of lactone odor-active compounds.

[0020] In the above method, the adsorption film used in the thin-film solid-phase microextraction is a film with a coating of polydimethylsiloxane / divinylbenzene (PDMS / DVB), polydimethylsiloxane / carbon molecular sieve (PDMS / CAR), or polydimethylsiloxane / poly(divinylbenzene-N-vinyl-pyrrolidone) (PDMS / HLB). The film is installed at the top of the headspace container, above the liquid level.

[0021] Preferably, the adsorption film used in the thin-film solid-phase microextraction is a film with a coating of polydimethylsiloxane / divinylbenzene (PDMS / DVB). Compared with other coatings, the polydimethylsiloxane / divinylbenzene (PDMS / DVB) coating is more conducive to improving the olfactory detection rate of lactone odor-active compounds in milk.

[0022] Preferably, a TF-SPME device bracket is installed on the lid of the headspace container (such as a headspace vial) used in the present invention to facilitate the installation of the adsorption film used in TF-SPME.

[0023] In the above method, the ionic strength regulator is preferably sodium chloride, and its addition amount is 20%-40% (preferably 30%-40%) of the mass of the sample to be measured.

[0024] In the above method, the time for the first stirring adsorption and the second stirring adsorption is at least 60 min (preferably 80-130 min, more preferably 90-120 min).

[0025] Preferably, the stirring speed for the first stirring adsorption and the second stirring adsorption is 500-1200 rpm (preferably 700-900 rpm).

[0026] Preferably, the temperature for the thin-film solid-phase microextraction, the first stirring adsorption, and the second stirring adsorption is 22-26 °C.

[0027] Preferably, before the start of the stirring adsorption, equilibration is carried out for 0.5-5 min.

[0028] Preferably, after the adsorption is completed, the surface of the stirring rod is rinsed with deionized water, and then the surface attachments are wiped clean with a clean and odorless lint-free paper, and the surface moisture of the stirring rod is blotted dry, and then thermal desorption is carried out. The adsorption film does not need to be cleaned and is directly subjected to thermal desorption.

[0029] In the above method, the temperature rising program for the thermal desorption is: the initial temperature is 38-42 °C, after maintaining for 1-2 min, it is heated to 245-255 °C at a rate of 190-210 °C / min, and maintained for 8-12 min.

[0030] Preferably, a cold trap injection step is further provided after the thermal desorption. The cold trap injection step is as follows: First, quickly reduce the temperature of the cold trap to -55~-65°C. After the thermal desorption and desorption are completed, the system is then heated to 240-260°C at a rate of 4-6°C / min and maintained for 13-17 min.

[0031] Among them, the rapid reduction of the cold trap temperature to -55~-65°C is achieved by cooling with ultra-low temperature liquid nitrogen.

[0032] In the above method, in the gas chromatography-olfactometry-mass spectrometry coupling technology, the conditions of gas chromatography are as follows: The initial temperature is 38-42°C, maintained for 4-6 min, then heated to 185-195°C at a rate of 2-4°C / min and maintained for 1-2 min, and then heated to 225-235°C at a rate of 9-11°C / min and maintained for 22-28 min, and the post-run temperature is 245-255°C; separation is carried out using a DB-WAX column.

[0033] In the gas chromatography-olfactometry-mass spectrometry coupling technology, the conditions of olfactometry are as follows: The odor output is through an empty chromatographic column filled without coating. The temperature of the chromatographic column is set to 245-255°C. The evaluator sniffs at the sniffing port and records the odor characteristics, appearance time, and odor intensity information detected.

[0034] In the gas chromatography-olfactometry-mass spectrometry coupling technology, the conditions of mass spectrometry are as follows: An electron impact ion source is used, the electron energy is 68-72 eV, the transfer line temperature is 275-285°C, the ion source temperature is 225-235°C, the quadrupole temperature is 145-155°C, there is no solvent delay, and the mass scanning range is m / z 35~350.

[0035] The present invention uses the above-mentioned thin film solid-phase microextraction combined with sequential stir bar sorptive extraction method to pretreat milk samples to achieve the extraction and enrichment of lactone compounds, and performs gas chromatography-olfactometry-mass spectrometry (GC-O-MS) analysis after thermal desorption, significantly improving the olfactory detection rate of lactone odor-active compounds in milk and increasing the detected types of lactone odor-active compounds in milk.

[0036] In the present invention, the sample to be tested can be raw milk and other unprocessed milk, pasteurized milk, ultra-high temperature sterilized milk (UHT sterilized milk), etc., which are milk products obtained by sterilizing or sterilizing raw milk.

[0037] The present invention also provides the application of the above-mentioned identification method in the identification of lactone odor-active compounds in milk.

[0038] Based on the above method for identifying lactone odor-active compounds in milk, the present invention has identified 9 lactone odor-active compounds in milk for the first time, including: δ-hexadecanolide, R-γ-decalactone, jasmonolide, γ-decalactone, γ-octalactone, cis-4-hydroxy-6-dodecenoic acid lactone, δ-dodecalactone, γ-nonalactone, δ-undecalactone. Based on the influence of odor-active compounds on the flavor of milk, the above lactone odor-active compounds can be used to evaluate or improve the flavor quality of milk.

[0039] The present invention also provides any one of the following applications of at least one selected from δ-hexadecanolide, R-γ-decalactone, jasmonolide, γ-decalactone, γ-octalactone, cis-4-hydroxy-6-dodecenoic acid lactone, δ-dodecalactone, γ-nonalactone, δ-undecalactone: (1) Application as an odor-active compound in milk; (2) Application as an odor-active compound for evaluating the flavor quality of milk; (3) Application in improving the flavor quality of milk.

[0040] The beneficial effects of the present invention at least include: The method for identifying lactone odor-active compounds in milk provided by the present invention uses thin-film solid-phase microextraction combined with sequential stir bar sorptive extraction for the pretreatment of milk samples, and cooperates with gas chromatography-olfactometry-mass spectrometry technology, significantly improving the olfactory detection rate of lactone odor-active compounds in milk, having advantages such as high efficiency, strong operability, and environmental protection, and having important application value in the detection of lactone odor-active compounds in milk and the evaluation of milk flavor. Detailed implementation manners

[0041] In the detailed implementation manners of the present invention, a method for identifying lactone odor-active compounds in milk is provided, which is a thin-film solid-phase microextraction combined with sequential stir bar sorptive extraction-thermal desorption-gas chromatography-olfactometry-mass spectrometry (TF-SPME&Seq-SBSE -TDU-GC-O-MS) method, including first using the thin-film solid-phase microextraction combined with sequential stir bar sorptive extraction method to extract and enrich lactone compounds in milk, and then using gas chromatography-olfactometry-mass spectrometry technology to identify lactone odor-active compounds.

[0042] Specifically, the thin-film solid-phase microextraction combined with sequential stir bar sorptive extraction method includes: (1) Weigh 10 - 20 g of milk and place it in a headspace vial (the small vial lid is equipped with a TF - SPME device bracket for connecting the TF - SPME device), and then add 1 - 5 μL of 2 - nonanol (40 ppm) as the internal standard reference for the system; place the stir bar with a polydimethylsiloxane (PDMS) coating inside the headspace vial; after equilibration for 0.5 - 5 min, turn on the magnetic stirring function and stir for adsorption at a speed of 500 - 1200 rpm for at least 60 min; after completion, take out the stir bar, rinse the surface with water, wipe off the surface attachments, and dry the surface moisture for subsequent thermal desorption treatment; (2) Then take another stir bar with a polydimethylsiloxane (PDMS) coating and place it at the bottom of the headspace vial in step (1), add sodium chloride accounting for 20% - 40% of the sample amount, and at the same time install the film with a polydimethylsiloxane / divinylbenzene (PDMS / DVB) coating on the device bracket of the small vial lid. The film is above the liquid level. After tightening the vial lid and equilibration for 0.5 - 5 min, stir for adsorption at a speed of 500 - 1200 rpm for at least 60 min; after completion, take out the stir bar and the film, rinse the surface of the stir bar with water, wipe off the surface attachments, and dry the surface moisture, and then transfer the stir bar in the above step (1) and the film extracted in step (2) to a thermal desorption tube for thermal desorption together.

[0043] In the above steps (1) and (2), the temperature during the adsorption process is preferably between 22 - 26 °C.

[0044] Specifically, the thermal desorption method includes: placing the thermal desorption tube into a thermal desorption system (TDU) for thermal desorption. The thermal desorption temperature - rising program is: the initial temperature is 40 °C, hold for 1 min, and then rise to 250 °C at a rate of 200 °C / min and hold for 10 min.

[0045] Specifically, after thermal desorption, it also includes separation using a cold injection system (CIS). The CIS temperature - rising program is: first quickly reduce the temperature of the CIS to - 60 °C by ultra - low - temperature liquid nitrogen. After the TDS desorption is completed, the system then rises to 250 °C at a rate of 5 °C / min and holds for 15 min.

[0046] Specifically, the gas chromatography - olfactometry - mass spectrometry technique is carried out using a gas chromatography - olfactometry - mass spectrometry instrument (GC - O - MS).

[0047] In some embodiments of the present invention, the number of lactone - type volatile compounds detected by the above - mentioned identification method reaches an unprecedented 11 kinds, and the number of lactone - type odor - active compounds reaches an unprecedented 9 kinds. The types and olfactory intensities far exceed the current reports in milk. The method of the present invention significantly improves the olfactory detection rate of lactone - type odor - active compounds.

[0048] The 11 detected lactone volatile compounds are shown in Table 1. In the identification method of Table 1, MS refers to the matching of the ion fragments of the unknown substance with those of a known substance in the spectral library; RI (Retention Index) refers to the matching of the retention index of the unknown substance with that of the known substance matched by MS; O (odor characteristic) refers to the ability to distinguish the odor characteristic of the unknown substance, and the accuracy of the qualitative result of the unknown substance can be further verified by the odor characteristic of the known substance corresponding to the unknown substance. By comprehensively qualitatively analyzing various methods, the target compound can be finally accurately locked. The identification method is qualitative analysis (including the matching result of ion fragments in mass spectrometry, the numerical value of the retention index, and the odor characteristic. Since the odor characteristics of different compounds are inconsistent, an additional odor characteristic column is listed in the data column), while the sniffing intensity is quantitative analysis, which is to obtain the contribution degree of the substance to the flavor of the whole sample by distinguishing the sniffing intensity of the target substance.

[0049] Table 1

[0050] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0051] Example 1 This example provides a method for identifying lactone odor-active compounds in milk, which includes the following steps: (1) Pretreatment (extraction and enrichment): Thin film solid phase microextraction combined with sequential stir bar adsorption extraction (TF-SPME&Seq-SBSE method) was used to pretreat the samples to be tested, as follows: 15 g of sample was accurately weighed and placed in a 40 mL headspace bottle (the vial cap was equipped with a TF-SPME device holder for connecting the TF-SPME device), and then 1 μL of 2-nonanol (40 ppm) was added as the internal standard reference of the system. A stir bar with polydimethylsiloxane (PDMS) coating was placed in the headspace bottle, and after 1 min of equilibration, the magnetic stirring function was turned on, and the stir bar was adsorbed at 800 rpm for 90 min; then the stir bar was carefully removed, the surface was rinsed with deionized water, and then the surface attachments were wiped clean with clean, odorless, lint-free paper, and the surface moisture was absorbed and left for subsequent thermal desorption treatment. Then, take another stirring rod containing polydimethylsiloxane (PDMS) coating and place it in the headspace bottle, add sodium chloride with 30% of the sample amount, and install the film with polydimethylsiloxane / divinylbenzene (PDMS / DVB) coating on the device bracket of the vial cap at the same time. The film is above the liquid surface. After balancing for 1 minute, the stirring rod is adsorbed at a speed of 800 rpm for 90 minutes; then take out the stirring rod and the film, rinse the surface of the stirring rod with deionized water, wipe the surface attachment with clean and odorless lint-free paper, and absorb the surface moisture; transfer the above two stirring rods and a film together to a glass thermal desorption tube for thermal desorption; the temperature of the above extraction and adsorption processes is room temperature (24°C).

[0052] (2) Thermal desorption (TDU) and cold injection (CIS) treatment: The stirring rod and film obtained by the two adsorptions in step (1) were subjected to thermal desorption and cold injection treatment using a TDU+CIS system (model: Gerstel TDU2), as follows: a thermal desorption tube equipped with two stirring rods and a film was placed in a thermal desorption system (TDU) for thermal desorption, and then separated in a cold injection system (CIS). The TDS temperature program was as follows: the starting temperature was 40°C, maintained for 1 min, then increased to 250°C at 200°C / min, and maintained for 10 min. The CIS temperature program was as follows: the CIS temperature was first rapidly reduced to -60°C by ultra-low temperature liquid nitrogen, and after the TDS desorption was completed, the system was then increased to 250°C at 5°C / min and maintained for 15 min.

[0053] (3) Isolation, analysis and identification: The extract obtained in step (2) was identified using gas chromatography-olfaction-mass spectrometry (GC-O-MS), as follows: Gas chromatography (GC, model: Agilent 7890B) condition parameters: The initial temperature is 40 °C, held for 5 min, then heated at 3 °C / min to 190 °C, held for 1 min, then heated at 10 °C / min to 230 °C and held for 25 min, and the post-run temperature is 250 °C; separation is carried out using a DB-WAX column.

[0054] Olfactometer (O, model: Gerstel ODP4) condition parameters: Odor output is through an empty chromatographic column filled without coating. The chromatographic column temperature is set at 250 °C, and humid nitrogen is introduced simultaneously to prevent the nasal cavity of the appraisers from drying. Three well-trained appraisers sniff at the sniffing port respectively and record the odor characteristics, appearance time, and odor intensity information smelled. The odor intensity is at least divided into 4 levels, and usually 5 levels (very weak, weak, middle, strong, very strong) are used.

[0055] Mass spectrometry (MS, model: Agilent 5977A) condition parameters: Use an electron impact (EI) 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 35 - 350.

[0056] Example 2 This example provides a method for identifying lactone odor-active compounds in milk, which includes the following steps: (1) Pretreatment (extraction and enrichment): The thin-film solid-phase microextraction combined with sequential stir bar sorptive extraction method (TF-SPME&Seq-SBSE method) was used to pretreat the sample to be measured as follows: Accurately weigh 15 g of the sample and place it in a 40 mL headspace vial (the vial cap is equipped with a TF-SPME device holder for connecting the TF-SPME device), and then add 1 μL of 2-nonanol (40 ppm) as the internal standard reference substance for the system. Place the stir bar coated with polydimethylsiloxane (PDMS) in the headspace vial. After equilibration for 1 min, turn on the magnetic stirring function and let the stir bar adsorb for 120 min at a speed of 800 rpm; then carefully take out the stir bar, rinse the surface with deionized water, wipe off the surface attachments with a clean and odorless lint-free paper, and dry the surface moisture; leave it for subsequent thermal desorption treatment. Then, take another stir bar coated with polydimethylsiloxane (PDMS) and place it in the headspace vial, add sodium chloride accounting for 40% of the sample amount, and at the same time install the thin film with a PDMS / DVB coating on the device holder of the vial cap. The thin film is above the liquid level. After equilibration for 1 min, let the stir bar adsorb for 120 min at a speed of 800 rpm; then take out the stir bar and the thin film, rinse the surface of the stir bar with deionized water, wipe off the surface attachments with a clean and odorless lint-free paper, and dry the surface moisture; transfer the above two stir bars and a thin film to a glass thermal desorption tube for thermal desorption; the temperature of the above extraction and adsorption processes is room temperature (between 22 - 26 °C).

[0057] (2) Thermal desorption (TDU) and cold injection (CIS) treatment: The TDU + CIS system (model: Gerstel TDU2) was used to perform thermal desorption and cold injection treatment on the stir bars and thin film obtained from the two adsorptions in step (1) as follows: Place the thermal desorption tube containing two stir bars and a thin film into the thermal desorption system (TDU) for thermal desorption and separate in the cold injection system (CIS). Among them, the TDS temperature programming is: the initial temperature is 40 °C, hold for 1 min, and then rise to 250 °C at a rate of 200 °C / min and hold for 10 min. The CIS temperature programming is: first quickly lower the temperature of the CIS to -60 °C through ultra-low temperature liquid nitrogen. After the TDS desorption is completed, the system then rises to 250 °C at a rate of 5 °C / min and holds for 15 min.

[0058] (3) Separation, analysis and identification: A gas chromatography-olfactometry-mass spectrometry (GC-O-MS) was used to identify the extract obtained in step (2) as follows: Gas chromatography (GC, model: Agilent 7890B) condition parameters: Initial temperature is 40 °C, held for 5 min, then heated at 3 °C / min to 190 °C, held for 1 min, then heated at 10 °C / min to 230 °C and held for 25 min, and the post-run temperature is 250 °C; separation is carried out using a DB-WAX column.

[0059] Olfactometer (O, model: Gerstel ODP4) condition parameters: Odor output is through an empty chromatographic column filled without coating. The column temperature is set at 250 °C, and humid nitrogen is introduced simultaneously to prevent the nasal cavity of the appraisers from drying. Three well-trained appraisers sniff at the sniffing port respectively and record the odor characteristics, appearance time, and odor intensity information they smell. The odor intensity is at least divided into 4 levels, and 5 levels are commonly used (very weak, weak, middle, strong, very strong).

[0060] Mass spectrometry (MS, model: Agilent 5977A) condition parameters: Use an electron impact (EI) ion source, electron energy is 70 eV, transfer line temperature is 280 °C, ion source temperature is 230 °C, quadrupole temperature is 150 °C, no solvent delay, and the mass scanning range is m / z 35 - 350.

[0061] Comparative Example 1 This comparative example provides a method for identifying lactone odor-active compounds in milk, and the difference from the identification method of Example 1 is only that: for the sample pretreatment (extraction and enrichment) in step (1), the dynamic headspace sampling extraction method (DHS method) is used, and the DHS method is as follows: Accurately weigh 250 g of the sample and place it in a 40 mL headspace bottle, then add 10 μL of 2-nonanol (40 ppm) as the internal standard reference in the system. The system temperature is set at 40 °C, and the magnetic stirrer is adjusted to 800 rpm. The system is stably balanced under this condition for 20 min. Then, nitrogen (purity = 99.9992%) is introduced at one end of the two outlets on both sides of the bottle body, and an adsorption column containing Tenax adsorption material is inserted at the other end. Nitrogen purges the system at a flow rate of 150 mL / min for 90 min, and the system temperature remains at 40 °C. After the volatile compounds in the sample are adsorbed onto the Tenax TA thermal desorption tube, it is also necessary to use nitrogen with a gentle flow rate to remove the water vapor remaining on the glass tube wall.

[0062] Comparative Example 2 This comparative example provides a method for identifying lactone odor-active compounds in milk, which is only different from the identification method in Example 2 in that: for the sample pretreatment (extraction and enrichment) in step (1), the stir bar sorptive extraction method (SBSE method) is used. The SBSE method is as follows: Accurately weigh 30 g of the sample and place it in a 40 mL headspace vial, and then add 1 μL of 2-nonanol (40 ppm) as the internal standard reference in the system. Place the stir bar coated with polydimethylsiloxane (PDMS) in the headspace vial. After equilibration for 1 min, turn on the magnetic stirring function and let the stir bar adsorb at a speed of 800 rpm for 120 min; then carefully take out the stir bar, rinse the surface with deionized water, wipe off the surface attachments with a clean and odorless lint-free paper, and absorb the surface moisture; then place the stir bar in a clean and odorless thermal desorption tube; the temperature during the adsorption process is room temperature (24 °C).

[0063] Comparative Example 3 This comparative example provides a method for identifying lactone odor-active compounds in milk, which is only different from the identification method in Example 2 in that: for the sample pretreatment (extraction and enrichment) in step (1), the thin-film solid-phase microextraction method (TF-SPME) is used. The TF-SPME method is as follows: Accurately weigh 15 g of the sample and place it in a 40 mL headspace vial, and then add 1 μL of 2-nonanol (40 ppm) as the internal standard reference in the system. Place an uncoated magnetic stir bar at the bottom of the headspace vial. The small vial cap is equipped with a TF-SPME device holder for connecting the TF-SPME device. Add sodium chloride accounting for 30% of the sample amount. At the same time, install a thin film coated with polydimethylsiloxane / divinylbenzene (PDMS / DVB) on the device holder of the small vial cap. The thin film is above the liquid level, and stir at the same time. After equilibration for 1 min, stir at a speed of 800 rpm, and the thin film adsorbs for 90 min; after completion, take out the thin film and transfer the thin film to a glass thermal desorption tube for thermal desorption; the temperature during the adsorption process is room temperature (24 °C).

[0064] Comparative Example 4 This comparative example provides a method for identifying lactone odor-active compounds in milk, which differs from the identification method of Example 1 only in that the sample pretreatment (extraction and enrichment) of step (1) uses the sequential stir bar adsorption extraction method (Seq-SBSE method), and the Seq-SBSE method is as follows: 15 g of sample is accurately weighed and placed in a 40 mL headspace bottle, and then 1 μL of 2-nonanol (40 ppm) is added as the internal standard reference of the system. A stir bar with a polydimethylsiloxane (PDMS) coating is placed in the headspace bottle, and after balancing for 1 min, the magnetic stirring function is turned on, and the stir bar is adsorbed at a speed of 800 rpm for 90 min; then the stir bar is carefully removed, the surface is rinsed with deionized water, and then the surface attachments are wiped clean with clean, odorless, lint-free paper, and the surface moisture is absorbed; it is reserved for subsequent thermal desorption treatment. Then, take another stirring bar with polydimethylsiloxane (PDMS) coating and place it in the headspace bottle, add sodium chloride with a volume of 30% of the sample, balance for 1 minute, and then adsorb the stirring bar at a speed of 800 rpm for 90 minutes; then take out the stirring bar and wash it, and transfer the magnetic particles used for the two extractions to a glass thermal desorption tube for thermal desorption; the temperature of the adsorption process is room temperature (24°C).

[0065] Comparative Example 5 This comparative example provides a method for identifying lactone odor-active compounds in milk, which differs from the identification method of Example 1 only in that the sample pretreatment (extraction and enrichment) in step (1) uses a heated system, and the headspace bottle is placed in a metal bath vessel at 50°C, that is, the temperature of the extraction and adsorption process is 50°C.

[0066] Experimental Example 1 The identification methods of Example 1 and Comparative Example 2 were used to identify lactone odor active compounds in raw milk, respectively, and the same batch of raw milk samples were used for the different methods.

[0067] The identification results of the method of Example 1 are shown in Table 2. In the following test results, the olfactory intensity is described as follows: VW - Very Weak (very weak), W - Weak (weak), M - Middle (medium), S - Strong (strong), VS - Very Strong (very strong).

[0068] Table 2 Detection results of lactone odor-active compounds in raw milk (1)

[0069] The results showed that in the raw milk samples with a lighter flavor, lactone odor-active compounds could still be identified using the method of Example 1 of the present invention. A total of 4 different lactone compounds were co-sniffed, and the sniffing intensity was obvious and easy to distinguish.

[0070] The identification results of the method of Comparative Example 2 are shown in Table 3.

[0071] Table 3 Detection results of lactone odor-active compounds in raw milk (2)

[0072] The results showed that for the same batch of raw milk samples, the types of lactone odor-active compounds identified by the method of Comparative Example 2 were significantly fewer than those of the method of Example 1.

[0073] Experimental Example 2 The identification methods of Example 1 and Example 2 and Comparative Examples 1, 3, 4, and 5 were respectively used to identify lactone odor-active compounds in commercially available UHT milk, and the same batch of commercially available UHT milk samples were used for different methods.

[0074] Among them, the identification results of the method of Example 1 are shown in Table 4.

[0075] Table 4 Detection results of lactone odor-active compounds in commercially available UHT milk (1)

[0076] The results showed that using the method of Example 2 of the present invention, there were as many as 9 lactone odor-active compounds, and the types and sniffing intensity far exceeded the reports of predecessors in white milk.

[0077] The identification results of the method of Example 2 are shown in Table 5.

[0078] Table 5 Detection results of lactone odor-active compounds in commercially available UHT milk (2)

[0079] The results showed that the method of Example 2 extended the adsorption time, but the number of detected lactone odor-active compounds did not increase compared with Example 1. Although the sniffing intensity of some compounds changed, it did not affect the judgment of lactone odor-active compounds. However, due to the extension of the adsorption time, the experimental time cost of the pretreatment increased. Therefore, the optimal adsorption time was 90 minutes for a single adsorption.

[0080] The identification results of the method of Comparative Example 1 are shown in Table 6.

[0081] Table 6 Detection results of lactone odor-active compounds in commercially available UHT milk (3)

[0082] The results showed that the method of Comparative Example 1 detected only one lactone odor-active compound, and the sniffing intensity was lower than that detected by the method of the Examples.

[0083] The identification results of the method of Comparative Example 3 are shown in Table 7.

[0084] Table 7 Detection results of lactone odor-active compounds in commercially available UHT white milk (4)

[0085] The results showed that the number of lactone odor-active compounds obtained by the method of Comparative Example 3 using thin-film solid-phase microextraction for pretreatment was reduced to 2, and compared with the Examples, the sniffing intensity of the compounds decreased, and the sniffing detection rate decreased significantly.

[0086] The identification results of the method of Comparative Example 4 are shown in Table 8.

[0087] Table 8 Detection results of lactone odor-active compounds in commercially available UHT white milk (5)

[0088] The results showed that for Comparative Example 4, using the Seq-SBSE method reported in the literature for pretreatment, the number of detected lactone odor-active compounds was reduced to 5, and compared with the Examples, the sniffing intensity of all compounds decreased, and the sniffing detection rate decreased significantly.

[0089] The identification results of the method of Comparative Example 5 are shown in Table 9.

[0090] Table 9 Detection results of lactone odor-active compounds in commercially available UHT white milk (6)

[0091] The results showed that for Comparative Example 5, using a conventional heated headspace system for pretreatment, the number of detected lactone odor-active compounds was reduced by 3, and compared with the Examples, the sniffing intensity of some compounds decreased, and the sniffing detection rate decreased significantly. In addition, during the data analysis process, it was found that compared with the non-heated method, the heated method also detected more thiol compounds. This may be because the long-term heating of the sample under the condition of adding an ionic strength agent promoted the structural denaturation of proteins, thus changing the milk system, resulting in a significant impact on the accuracy of the flavor substances extracted under this heated system.

[0092] Based on the above results, the olfactory detection rate of lactone compounds in the same commercially available white milk using the identification method of the present invention has been increased by at least 50% - 800%. TF-SPME and Seq-SBSE jointly and sufficiently adsorb and enrich lactone compounds with different physicochemical properties. This method abandons the conventional idea that the extraction and enrichment method relies on a heating system, not only avoiding the by-products generated by long-term heating after the addition of ionic strength agents, but also effectively improving the olfactory detection rate of the target compounds. On this basis, by optimizing the extraction time, the system works under an equilibrium state, further ensuring the sensitivity and precision. Therefore, the identification method of the present invention restores the original flavor of the sample to the greatest extent, can effectively solve the problem of difficult olfactory discrimination of lactone odor-active compounds in milk such as raw milk and white milk, and significantly improves the olfactory detection rate of lactone odor-active compounds. More accurate and sufficient olfactory discrimination analysis of lactone odor-active compounds will contribute to more precise dissection of the flavor of white milk at the molecular level, contribute to the upstream and downstream research of the flavor of white milk, and play a crucial role in the control and improvement of the flavor quality of white milk.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for identifying lactone odor active compounds in milk, characterized in that: The method comprises: extracting and enriching lactone compounds in milk, and identifying lactone odor active compounds by using gas chromatography-olfaction-mass spectrometry technology; Among them, the method for extracting and enriching lactone compounds in milk by using thin film solid phase microextraction combined with sequential stirring rod adsorption includes: placing the sample to be tested in a headspace container, using a first stirring rod containing an adsorption coating to perform a first stirring adsorption, and after the adsorption is completed, taking out the first stirring rod and waiting for thermal desorption; in the system of the first stirring adsorption, adding an ionic strength regulator and installing an adsorption film with a coating used for thin film solid phase microextraction, and using a second stirring rod containing an adsorption coating to perform a second stirring adsorption, after the adsorption is completed, taking out the second stirring rod and the adsorption film, and performing thermal desorption together with the first stirring rod.

2. The identification method according to claim 1, characterized in that: The extraction temperature of the thin film solid phase microextraction and the adsorption temperature of the sequential stirring rod adsorption are both no higher than 35°C.

3. The identification method according to claim 1, characterized in that: The adsorption coating of the first stirring rod and the second stirring rod is a polydimethylsiloxane coating or a polyethylene glycol-dimethylsiloxane coating; And / or, the adsorption film used in the thin film solid phase microextraction is a film with a polydimethylsiloxane / divinylbenzene, polydimethylsiloxane / carbon molecular sieve or polydimethylsiloxane / poly(divinylbenzene-N-vinyl-pyrrolidone) coating, and the film is installed on the top of the headspace container, above the liquid surface.

4. The identification method according to any one of claims 1 to 3, characterized in that: The ionic strength regulator is sodium chloride, and the mass ratio of its addition amount to the sample to be tested is 20%-40%.

5. The identification method according to any one of claims 1 to 3, characterized in that: The time for the first stirring adsorption and the second stirring adsorption is at least 60 minutes; And / or, the stirring speed of the first stirring adsorption and the second stirring adsorption is 500-1200 rpm; And / or, the temperature of thin film solid phase microextraction, the first stirring adsorption and the second stirring adsorption is 22-26°C.

6. The identification method according to any one of claims 1 to 3, characterized in that: The heating program of the thermal desorption is as follows: the starting temperature is 38-42°C, maintained for 1-2 min, then increased to 245-255°C at a rate of 190-210°C / min, and maintained for 8-12 min.

7. The identification method according to claim 6, characterized in that: A cold trap injection step is also provided after the thermal desorption, and the cold trap injection step is: firstly, the cold trap temperature is rapidly reduced to -55--65°C, and after the thermal desorption is completed, the system is raised to 240-260°C at 4-6°C / min and maintained for 13-17 min.

8. The identification method according to any one of claims 1 to 3 and 7, characterized in that: In the gas chromatography-olfaction-mass spectrometry technique, the gas chromatography conditions are as follows: starting temperature 38-42°C, maintained for 4-6 min, then heated to 185-195°C at 2-4°C / min, maintained for 1-2 min, then heated to 225-235°C at 9-11°C / min, maintained for 22-28 min, and then operated at 245-255°C; using a DB-WAX column for separation; And / or, in the gas chromatography-olfaction-mass spectrometry technique, the sniffing conditions are as follows: the odor output passes through an empty chromatographic column filled with no coating, the temperature of the chromatographic column is set to 245-255° C., the evaluator sniffs at the sniffing port, and records the characteristics of the smell, the time of occurrence, and the odor intensity information; And / or, in the gas chromatography-olfaction-mass spectrometry technology, the mass spectrometry conditions are as follows: using an electron bombardment ion source, an electron energy of 68-72eV, a transmission line temperature of 275-285°C, an ion source temperature of 225-235°C, a quadrupole temperature of 145-155°C, no solvent delay, and a mass scan range of m / z 35~350.

9. Use of the identification method according to any one of claims 1 to 8 in the identification of lactone odor-active compounds in milk.

10. Any one of the following uses of at least one selected from the group consisting of butyl hexadecanoic acid lactone, R-γ-decanoic acid lactone, jasmine lactone, γ-decanoic acid lactone, γ-octanolactone, cis-4-hydroxy-6-dodecenoic acid lactone, δ-dodecalactone, γ-nonalactone, and δ-undecalactone: (1) Application as an odor-active compound in milk; (2) Application as an odor active compound in the flavor quality evaluation of milk; (3) Application in improving the flavor quality of milk.

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