Dairy product freshness index component cis (trans-2-dodecenoic acid, screening method and dairy product freshness evaluation method

Abstract

The invention belongs to the technical field of dairy product detection, and relates to an index component cis (trans-2-dodecenoic acid for dairy product freshness, a screening method and a dairy product freshness evaluation method.According to the method, the index component cis (trans-2-dodecenoic acid for dairy product freshness is screened out from deteriorated dairy products through an electrostatic field orbitrap high-resolution mass spectrometry technology; in the positive ion mode, the precise mass-to-charge ratio range of parent ions is 199.16744 to 199.16944; the cis (trans-2-dodecenoic acid is used as an evaluation index of the freshness of the dairy product and is used for detecting the freshness of the dairy product; the method for detecting the freshness of the dairy products can objectively reflect the freshness of the dairy products and effectively evaluate the freshness of the dairy products, and is high in sensitivity and specificity.

Description

Technical Field

[0001] The invention belongs to the technical field of dairy product detection, and relates to cis(trans)-2-dodecenoic acid, an indicator component of dairy product freshness, a screening method and a dairy product freshness evaluation method. Background Art

[0002] Milk and dairy products are rich in nutrition. With the improvement of people's living standards, they have become one of the important sources of nutrition for people. The consumption is increasing day by day, and their quality and safety are closely related to people's health. Milk and dairy products are a good matrix for the growth of microorganisms. During the production, transportation and storage of raw milk, factors such as the production environment, transportation conditions and storage conditions may cause the raw milk to lose its freshness or even deteriorate; and the storage conditions and storage time of dairy products may also cause the quality of dairy products to change, thus affecting the quality and safety of milk and dairy products.

[0003] Raw milk is the main raw material for processing dairy products, and its quality will directly affect the quality and safety of dairy products. GB19301 "National Food Safety Standard for Raw Milk" clearly stipulates that spoiled milk should not be used as raw milk. However, the identification of spoiled milk is mainly through sensory evaluation, pH value detection, peroxide value detection, lactic acid bacteria counting method, etc., but these methods are not suitable for the identification and detection of spoiled milk mixed into raw milk and the deterioration of dairy products after long-term storage. At present, there is a lack of relevant evaluation and identification technology. The lack of evaluation indicators and identification detection methods for the freshness of milk and dairy products makes it impossible to effectively evaluate the freshness of milk and dairy products, and it is impossible to identify maliciously added spoiled milk or dairy products that have exceeded their validity period, which is not conducive to the quality and safety control of milk and dairy products. Once spoiled milk is consumed, it will seriously threaten the health safety of consumers. Summary of the invention

[0004] In view of the current lack of an effective evaluation index and identification detection method for the freshness of milk and dairy products, which has caused the technical difficulty of being unable to effectively evaluate the freshness of milk and dairy products, the present invention provides an indicator component cis(trans)-2-dodecenoic acid, a screening method and a method for evaluating the freshness of dairy products.

[0005] The first purpose of the present invention is to screen out cis(trans)-2-dodecenoic acid, an indicator component of dairy product freshness, from spoiled dairy products by using electrostatic field orbital trap high-resolution mass spectrometry technology.

[0006] Another object of the present invention is to use cis(trans)-2-dodecenoic acid as an evaluation index for the freshness of dairy products and use it in the detection of the freshness of dairy products; at the same time, a detection method for the freshness of dairy products is constructed, which can objectively reflect the freshness of milk and dairy products and effectively evaluate the freshness of dairy products. The detection method has high sensitivity and strong specificity, and provides a new method for identifying whether dairy products are spoiled.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] Cis (trans)-2-dodecenoic acid, an indicator of the freshness of dairy products, has the structural formula:

[0009] In the positive ion mode, the accurate mass-to-charge ratio range of the precursor ion of cis(trans)-2-dodecenoic acid was 199.16744-199.16944.

[0010] It was further defined that, in the positive ion mode, there were five characteristic secondary mass spectrometry ion fragments of cis(trans)-2-dodecenoic acid, and the precise mass-to-charge ratio (m / z) ranges were 181.15419-181.16419, 129.08614-129.09641, 83.08110-83.09110, 69.06550-69.07550 and 57.06560-57.07560, respectively.

[0011] The screening method for cis(trans)-2-dodecenoic acid, an indicator component of dairy product freshness, comprises the following steps:

[0012] S1. Place the dairy product at room temperature or 37°C for 1 to 7 days and collect samples every day;

[0013] S2. The samples obtained in step S1 are treated as follows: first, water and acetonitrile are added respectively, vortex mixed, ultrasonic extracted, centrifuged, and the supernatant is taken; then filtered to obtain a filtrate; the mass volume ratio of the sample, water and acetonitrile is (0.1-1) g: 1 mL: 2 mL;

[0014] S3, using ultra-high performance liquid chromatography-tandem electrostatic field orbital trap high-resolution mass spectrometry to perform full scan screening on each filtrate;

[0015] S4. Compare and analyze each screening result to determine the component whose mass spectrometer response value increases with the storage time, that is, cis (trans)-2-dodecenoic acid.

[0016] It is further defined that in step S2, the vortex mixing time is 0.5min-2min, the ultrasonic extraction time is 5min-10min, the centrifugal speed is 4000r / min-5000r / min, and the centrifugal time is 5min-10min; and 0.22μm-0.45μm organic filter membrane is used for filtration.

[0017] Further defined, in step S3, the conditions of ultra-high performance liquid chromatography are: the chromatographic column is an Eclipse Plus C18 RRHD column; the mobile phase A is an aqueous solution containing 0.1% formic acid, and the mobile phase B is a methanol solution; the column temperature is 35° C., the flow rate is 0.3 mL / min, the injection volume is 2-5 μL, and the elution gradient is as shown in the following table:

[0018] time Mobile phase A (%) Mobile phase B (%) 0min 95 5 1min 95 5 2min 10 90 4.5min 10 90 5min 95 5 6min 95 5 .

[0019] It is further defined that in step S3, the electrostatic field orbital trap high-resolution mass spectrometry conditions are:

[0020] The mass analyzer was Q Exactive Orbitrap; the ion source was a heated electrospray ion source; the scanning mode was positive ion simultaneous scanning mode; the monitoring mode was parallel reaction monitoring PRM; the spray voltage was 3.0 kV-3.8 kV; the lens voltage was 55 V; the capillary temperature was 300 ° C-350 ° C; the auxiliary gas heating temperature was 300 ° C-350 ° C; the sheath gas was 30 arb-45 arb, and the auxiliary gas was 5 arb-10 arb; the primary resolution was R = 70000 FWHM; the secondary resolution was R = 13500 FWHM; the automatic gain control was 3 × 10 6 , the maximum dwell time is 50ms-100ms.

[0021] As described above, the application of cis(trans)-2-dodecenoic acid, an indicator component of dairy product freshness, as a dairy product freshness evaluation indicator in dairy product freshness detection.

[0022] A method for evaluating the freshness of dairy products comprises the following steps:

[0023] SA, take the dairy product to be tested, and add water and acetonitrile respectively; after vortex mixing, ultrasonic extraction and centrifugation, take the supernatant; then filter to obtain the filtrate; the mass volume ratio of the dairy product, water and acetonitrile is (0.1-1) g: 1 mL: 2 mL;

[0024] SB, full scan of the filtrate using ultra-high performance liquid chromatography coupled to an electrostatic field orbital trap high-resolution mass spectrometer;

[0025] SC, according to the scanning results, determine the mass spectrometry response value of cis (trans)-2-dodecenoic acid in the filtrate;

[0026] SD, when the mass spectrometer response value is 0, it indicates that the tested dairy product is within the shelf life; when the mass spectrometer response value is greater than 0, it indicates that the tested dairy product exceeds the shelf life, thereby completing the freshness evaluation of the dairy product.

[0027] It is further defined that in the step SA, the vortex mixing time is 0.5 min-2 min, the ultrasonic extraction time is 5 min-10 min, the centrifugal speed is 4000 r / min-5000 r / min, and the centrifugal time is 5 min-10 min; and a 0.22 μm-0.45 μm organic filter membrane is used for filtration.

[0028] Further defined, in step SB, the conditions of ultra-high performance liquid chromatography are: the chromatographic column is an Eclipse Plus C18 RRHD column; the mobile phase A is an aqueous solution containing 0.1% formic acid, and the mobile phase B is a methanol solution; the column temperature is 35° C., the flow rate is 0.3 mL / min, the injection volume is 2 μL-5 μL, and the elution gradient is as shown in the following table:

[0029] time Mobile phase A (%) Mobile phase B (%) 0min 95 5 1min 95 5 2min 10 90 4.5min 10 90 5min 95 5 6min 95 5 ;

[0030] The electrostatic field orbital trap high-resolution mass spectrometry conditions are as follows: the mass analyzer is Q Exactive Orbitrap; the ion source is a heated electrospray ion source; the scanning mode adopts the positive ion simultaneous scanning mode; the monitoring mode is parallel reaction monitoring PRM; the spray voltage is 3.0 kV-3.8 kV; the lens voltage is 55 V; the capillary temperature is 300°C-350°C; the auxiliary gas heating temperature is 300°C-350°C; the sheath gas is 30 arb-45 arb, and the auxiliary gas is 5 arb-10 arb; the primary resolution R=70000 FWHM; the secondary resolution R=13500 FWHM; the automatic gain control is 3×10 6 , the maximum dwell time is 50ms-100ms.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The present invention screens out cis(trans)-2-dodecenoic acid (also known as cis(trans)-2-dodecenoic acid), an indicator component of dairy freshness, from spoiled dairy products through high-throughput screening analysis of electrostatic field orbital trap high-resolution mass spectrometry, identifies an indicator compound that can reflect the freshness of milk and dairy products, and provides a new idea for the freshness evaluation of milk and dairy products.

[0033] 2. The present invention establishes a method for detecting cis(trans)-2-dodecenoic acid in milk and dairy products, by connecting ultra-high performance liquid chromatography and electrostatic field orbital trap high-resolution mass spectrometry in series. The detection method has high sensitivity and strong specificity, and can be used to identify whether milk and dairy products are deteriorated.

[0034] 3. In the screening and freshness detection of the present invention, the treated samples are first eluted and separated by ultra-high performance liquid chromatography, and the eluted samples are tested by electrostatic field orbital trap high-resolution mass spectrometry. Mass spectrometry analysis is performed while eluting, so that the interference in the samples subjected to mass spectrometry analysis at the same time is small and the analysis accuracy is higher.

[0035] 4. The present invention adopts cis (trans)-2-dodecenoic acid to evaluate the freshness of milk and dairy products, which greatly solves the problem of evaluating the freshness of milk and dairy products, and can quickly and accurately identify whether milk and dairy products are expired or deteriorated, which is of great significance to the quality and safety of milk and dairy products. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The total ion current and mass spectrum fragmentation diagram of cis(trans)-2-dodecenoic acid at m / z=199.169;

[0037] Figure 2 The content changes of the index component cis (trans)-2-dodecenoic acid in pure goat milk after being opened for different days;

[0038] Figure 3 The content changes of the index component cis (trans)-2-dodecenoic acid after fresh goat milk is stored for different days;

[0039] Figure 4 It is the content of cis (trans)-2-dodecenoic acid, an indicator component, in different milk and dairy product samples. DETAILED DESCRIPTION

[0040] The technical solution protected by the present invention is now described in detail in conjunction with the embodiments. However, it is obvious that the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the described embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0041] The invention adopts a method of ultra-high performance liquid chromatography and electrostatic field orbital trap high-resolution mass spectrometry in series to screen out an index component cis(trans)-2-dodecenoic acid from spoiled dairy products, and is used for detecting and evaluating whether the dairy products are spoiled.

[0042] In the present invention, the structural formula of cis(trans)-2-dodecenoic acid is:

[0043]

[0044] In the present invention, cis-(trans)-2-dodecenoic acid refers to cis-2-dodecenoic acid and trans-2-dodecenoic acid which are isomers of each other.

[0045] In the present invention, high-resolution mass spectrometry is an analytical technique for determining the exact mass number of a compound, which can provide better resolution and accuracy than conventional mass spectrometry, thereby more accurately identifying and quantifying the target. A high-resolution mass spectrometer has the function of determining the exact mass number of a compound at ultra-high resolution. The technology has a stronger anti-interference ability and can more accurately quantify the target compound. Common high-resolution mass spectrometers include: time-of-flight mass spectrometry, magnetic mass spectrometry, electrostatic field orbital trap mass spectrometry, etc. Therefore, high-resolution mass spectrometry is used in conjunction with ultra-high performance liquid chromatography, which is suitable for high-throughput detection, identification of unknowns, analysis of complex matrix samples, etc. Ultra-high performance liquid chromatography-tandem electrostatic field orbital trap mass spectrometry (UPLC-Q Exactive) is an effective tool for screening unknowns in complex matrices, and is also a good choice for quantitative analysis of trace components, ensuring accurate analysis results.

[0046] The technical solution of the present invention is described in detail below through several groups of examples. It should be noted that, unless otherwise specified, the chemicals, reagents, etc. used in the following examples are all conventional commercial products in the field; unless otherwise specified, the operating methods used in the following examples are all conventional operations in the field.

[0047] Example 1

[0048] The purpose of this example is to screen out a dairy freshness indicator compound from dairy products.

[0049] The screening method for dairy product freshness indicator compounds provided in this embodiment comprises the following steps:

[0050] (1) Dissolve a batch of milk powder in water at a ratio of 1:6, place in a 37°C constant temperature shaker for 1 to 7 days, and accurately weigh 5 g (accurate to 0.01 g) of the sample each time;

[0051] (2) The samples collected every day were placed in a 50 mL stoppered centrifuge tube, 5 mL of aqueous solution was added, vortexed and mixed, 10 mL of acetonitrile was accurately added, vortexed and mixed for 1 min, ultrasonic extraction was performed for 5 min, and centrifuged at 4500 r / min for 10 min. 2 mL of supernatant was passed through a PRime HLB solid phase extraction column, the front filtrate was discarded, the back filtrate was collected, filtered through a 0.22 μm organic filter membrane, and screened and identified using ultra-high performance liquid chromatography-tandem electrostatic field orbital trap high-resolution mass spectrometry (UPLC-Q Exactive), that is, the filtered solution first entered the ultra-high performance liquid chromatography elution separation, and then the eluate was directly subjected to electrostatic field orbital trap high-resolution mass spectrometry analysis.

[0052] In this implementation, the chromatographic conditions of ultra-high performance liquid chromatography are as follows:

[0053] Chromatographic column: Eclipse Plus C18 RRHD column (150 mm × 3.0 mm, 1.8 μm) from Waters, USA; mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is a methanol solution. Column oven: 35°C, flow rate 0.3 mL / min, injection volume 2 μL. Gradient elution conditions are shown in Table 1.

[0054] Table 1 Mobile phase gradient

[0055] time Mobile phase A (%) Mobile phase B (%) 0 95 5 1 95 5 2 10 90 4.5 10 90 5 95 5 6 95 5

[0056] In this embodiment, the electrostatic field orbital trap high-resolution mass spectrometry mass spectrometry conditions are as follows: the mass analyzer is a quadrupole-electrostatic field orbital trap high-resolution mass spectrometer (Q Exactive Orbitrap, QE); the ion source is a heated electrospray ionization source (H-ElectronSpray Ionization, HESI-Ⅱ); the scanning mode is a positive ion scanning mode (HESI-Ⅱ + ); monitoring mode: PRM scanning mode; spray voltage: 3.5kV; lens voltage: 55V; capillary temperature 260℃; auxiliary gas temperature 260℃; sheath gas (N 2 ): 4.6L / min; auxiliary gas (N 2 ): 5.1L / min; resolution: 35000; maximum residence time: 50ms.

[0057] Figure 1 This is the TIC graph of the parent ion with an accurate mass-to-charge ratio of m / z 199.16972 and a parent ion of 199.16972 in the PRM scanning mode and positive ion mode. Figure 1 A is the total ion current, Figure 1 B is the mass spectrum fragmentation pattern.

[0058] See also Figure 1 A, compared with the first day, a peak with significantly enhanced response appeared in the TIC diagram on the seventh day, and the compound corresponding to the significantly enhanced peak was cis (trans)-2-dodecenoic acid.

[0059] See also Figure 1 B, fragmentation diagram of unknown substance of cis(trans)-2-dodecenoic acid. The accurate mass-to-charge ratio of fragment ions is shown in Table 2. The maximum fragment ion of cis(trans)-2-dodecenoic acid is 181.15919.

[0060] Table 2 High resolution mass spectrometry ion fragment mass-to-charge ratio of compounds

[0061]

[0062] In this embodiment, by multiple use of ultra-high performance liquid chromatography-tandem electrostatic field orbital trap high-resolution mass spectrometry detection, the accurate mass-to-charge ratio (m / z) of the indicative component cis (trans)-2-dodecenoic acid can be detected in the range of 199.16744-199.16944. Within this range, the indicative component cis (trans)-2-dodecenoic acid can be effectively detected.

[0063] Example 2

[0064] This example mainly investigates the content variation of cis(trans)-2-dodecenoic acid (index component A) in dairy products after being stored for different days.

[0065] This embodiment uses a batch of pre-packaged pure goat milk with a shelf life of more than half a year for testing, and its detection method is:

[0066] After opening, place it at room temperature (25℃-30℃) for 0 to 15 days. On 0 day, 1 day, 5 days, 10 days and 15 days, take 2.0 g of the sample and place it in a 50 mL stoppered centrifuge tube. Add 5 mL of aqueous solution and vortex mix. Accurately add 10 mL of acetonitrile and vortex mix for 1 min. Ultrasonic extraction for 5 min. Centrifuge at 4500 r / min for 10 min. Take 2 mL of supernatant and pass it through a PRime HLB solid phase extraction column. Discard the front filtrate, collect the back filtrate, and filter with a 0.22 μm organic filter membrane.

[0067] Using ultra-high performance liquid chromatography-tandem electrostatic field orbital trap high-resolution mass spectrometry (see Example 1 for detection conditions), the content of the index component A (cis (trans)-2-dodecenoic acid) of the expired pure goat milk was analyzed when it was placed for 0 days, 1 day, 5 days, 10 days, and 15 days. The change pattern is shown in Figure 2 .

[0068] from Figure 2 It can be seen that expired pure goat milk itself contains indicator component A, and after opening, the longer the storage time, the higher the content of indicator component A. The content of indicator A on the 15th day is 4 times that of the first day.

[0069] Example 3

[0070] The purpose of this study is to investigate the content variation of cis(trans)-2-dodecenoic acid (index component A) in fresh goat milk after being stored for different days.

[0071] This implementation uses a batch of fresh, freshly squeezed pure goat milk for testing, and the testing method is:

[0072] The sample was placed in a constant temperature shaker at 37°C for 0-15 days. On 0, 2, 4 and 15 days, 5 g of the sample was taken and placed in a 50 mL stoppered centrifuge tube. 5 mL of aqueous solution was added and vortexed to mix. Then 10 mL of acetonitrile was added and vortexed to mix for 1 min. Ultrasonic extraction was performed for 5 min. The sample was centrifuged at 4500 r / min for 10 min. 2 mL of the supernatant was passed through a PRime HLB solid phase extraction column. The front filtrate was discarded and the back filtrate was collected and filtered through a 0.22 μm organic filter membrane.

[0073] Ultra-high performance liquid chromatography-tandem electrostatic field orbital trap high-resolution mass spectrometry (detection conditions see Example 1) was used to determine the content of the index component A in the fresh goat milk after 1 day, 2 days, 4 days, and 15 days. The experimental results are shown in Figure 3 show.

[0074] from Figure 3 It can be seen that the goat milk that has been stored for one day does not contain the indicator component A, indicating that it is fresh. However, the longer the storage time, the higher the content of indicator component A. On the 15th day, the indicator component A increased by 5 times.

[0075] Through the above examples, it is found that the mass spectrometry response value of cis(trans)-2-dodecenoic acid increases with the increase of the placement time. After 15 days of placement, its mass spectrometry response value increases by more than 100 times, and the increase at 37°C is more significant than that at room temperature. In this example, in the analysis of the screening results, it is determined that cis(trans)-2-dodecenoic acid is produced during the placement of milk and dairy products. Therefore, cis(trans)-2-dodecenoic acid is used as an indicator component for evaluating the freshness of milk and dairy products.

[0076] Example 4

[0077] This embodiment mainly uses cis (trans)-2-dodecenoic acid as a freshness indicator for the detection of dairy product freshness.

[0078] In this example, the index component cis (trans)-2-dodecenoic acid in raw milk, milk powder, expired milk and pasteurized milk was measured respectively to evaluate the freshness of the dairy products.

[0079] The dairy product samples include: A, pure goat milk powder sold in a certain market (expired for half a year); B, organic pure goat milk sold in a certain market (expired for three months); C, pure goat milk sold in a certain market (within the shelf life); D, children's growing goat milk powder (within the shelf life); E, pure goat milk (expired for three months); F, cow milk powder for middle-aged and elderly people (within the shelf life); G, cow milk powder (expired for half a year).

[0080] The determination method is as follows: take (0.5-5) g of the dairy product to be tested, add 5 mL of water and 10 mL of acetonitrile respectively; vortex mix for 1 min, ultrasonic extract for 5 min, centrifuge at 4500 r / min for 10 min, take the supernatant; then filter through a 0.22 μm organic filter membrane to obtain a filtrate. Use ultra-high performance liquid chromatography-tandem electrostatic field orbital trap high-resolution mass spectrometry (detection conditions refer to Example 1) to fully scan the filtrate; the scanning results are as follows Figure 4 shown.

[0081] See also Figure 4 The experiment found that the indicative component cis (trans)-2-dodecenoic acid was not detected in the samples within the shelf life, while cis (trans)-2-dodecenoic acid was detected in the samples after the shelf life.

[0082] It can be seen that in this embodiment, the mass spectrometer response value of cis (trans)-2-dodecenoic acid in the filtrate is determined; and the freshness of the dairy product is evaluated based on the mass spectrometer response value. Specifically, when the mass spectrometer response value is 0, it indicates that the tested dairy product is within the shelf life; when the mass spectrometer response value is greater than 0, it indicates that the tested dairy product has exceeded the shelf life.

[0083] Furthermore, the screening method and detection method adopted by the present invention are verified.

[0084] The main purpose is to investigate and verify the detection method of the indicator component cis (trans)-2-dodecenoic acid.

[0085] Verification of ultra-high performance liquid chromatography conditions: Chromatographic column: C18 chromatographic column (150 mm×3.0 mm, 1.8 μm); mobile phase: methanol-water solution containing 0.1% formic acid or methanol-ammonium acetate solution containing 0.2 mmol / L, gradient elution; injection volume: 2 μL-5 μL.

[0086] Mass spectrometry conditions verification: mass analyzer is Q Exactive Orbitrap; ion source: heatable electrospray ion source (HESI-Ⅱ); scanning mode: positive ion simultaneous scanning mode (HESI-Ⅱ+); monitoring mode: parallel reaction monitoring PRM (high resolution secondary full scan); spray voltage: 3.0-3.8 kV (ESI+); lens voltage 55 V; capillary temperature 300-350 °C; auxiliary gas heating temperature 300-350 °C; sheath gas is 30-45 arb (4×10 7 Pa), auxiliary gas is 5-10arb (1×10 7 Pa); primary resolution R = 70000 FWHM; secondary resolution R = 13500 FWHM; automatic gain control (AGC) is 3×10 6, maximum residence time: 50-100ms. The above conditions are for Thermo Fisher Electrostatic Field Orbital Trap High Resolution Mass Spectrometer. Other mass spectrometers with equivalent performance can be used for reference.

[0087] Verification of pretreatment conditions: The sample to be tested is weighed at 0.5g-5.0g. After adding water and acetonitrile, the vortex mixing time is 0.5min-2min, the ultrasonic extraction time is 5min-10min, the centrifugal speed is 4000r / min-5000r / min, and the centrifugal time is 5min-10min; 0.22μm-0.45μm organic filter membrane is used for filtration.

[0088] It has been verified that within the above-mentioned detection conditions, the indicator component cis (trans)-2-dodecenoic acid in milk and dairy products can be effectively detected.

[0089] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A cis (trans)-2-dodecenoic acid which is an indicator of the freshness of dairy products, characterized in that: The structural formula is: In the positive ion mode, the accurate mass-to-charge ratio range of the precursor ion of cis(trans)-2-dodecenoic acid was 199.16744-199.16944.

2. The cis(trans)-2-dodecenoic acid, an indicator of dairy product freshness, according to claim 1, characterized in that: In the positive ion mode, there are five characteristic secondary mass spectrometry ion fragments of cis(trans)-2-dodecenoic acid, and the accurate mass-to-charge ratio ranges are 181.15419-181.16419, 129.08614-129.09641, 83.08110-83.09110, 69.06550-69.07550 and 57.06560-57.07560, respectively.

3. The screening method for cis(trans)-2-dodecenoic acid, an indicator component of dairy product freshness, as claimed in claim 1, characterized in that: The screening method comprises the following steps: S1. Place the dairy product at room temperature or 37°C for 1 to 7 days and collect samples every day; S2. The samples obtained in step S1 are treated as follows: first, water and acetonitrile are added respectively, vortex mixed, ultrasonic extracted, centrifuged, and the supernatant is taken; then filtered to obtain a filtrate; the mass volume ratio of the sample, water and acetonitrile is (0.1-1) g: 1 mL: 2 mL; S3, using ultra-high performance liquid chromatography-tandem electrostatic field orbital trap high-resolution mass spectrometry to perform full scan screening on each filtrate; S4. Compare and analyze each screening result to find out the component whose mass spectrometry response value increases with the storage time, and determine that the component is cis (trans)-2-dodecenoic acid.

4. The screening method according to claim 3, characterized in that: The screening method comprises the following steps: in step S2, the vortex mixing time is 0.5min-2min, the ultrasonic extraction time is 5min-10min, the centrifugal speed is 4000r / min-5000r / min, and the centrifugal time is 5min-10min; and filtration is performed using a 0.22μm-0.45μm organic filter membrane.

5. The screening method according to claim 3, characterized in that: The screening method comprises the following steps: In step S3, the conditions of ultra-high performance liquid chromatography are: the chromatographic column is an Eclipse Plus C18RRHD column; the mobile phase A is an aqueous solution containing 0.1% formic acid, and the mobile phase B is a methanol solution; the column temperature is 35°C, the flow rate is 0.3mL / min, the injection volume is 2μL-5μL, and the elution gradient is as shown in the following table: 。 6. The screening method according to claim 3, characterized in that: The screening method comprises the following steps: In step S3, the electrostatic field orbital trap high-resolution mass spectrometry conditions are: The mass analyzer was Q Exactive Orbitrap; the ion source was a heated electrospray ion source; the scanning mode was positive ion simultaneous scanning mode; the monitoring mode was parallel reaction monitoring PRM; Spray voltage: 3.0kV-3.8kV; lens voltage 55V; capillary temperature 300-350℃; auxiliary gas heating temperature 300-350℃; sheath gas 30-45arb, auxiliary gas 5-10arb; primary resolution R=70000FWHM; secondary resolution R=13500FWHM; automatic gain control 3×10 6 , the maximum dwell time is 50-100ms.

7. Use of cis(trans)-2-dodecenoic acid, an indicator component of dairy product freshness as claimed in claim 1, as an evaluation index of dairy product freshness in the detection of dairy product freshness.

8. A method for evaluating the freshness of dairy products, characterized in that: The following steps are involved: SA, take the dairy product to be tested, and add water and acetonitrile respectively; after vortex mixing, ultrasonic extraction and centrifugation, take the supernatant; then filter to obtain the filtrate; the mass volume ratio of the dairy product, water and acetonitrile is (0.1-1) g: 1 mL: 2 mL; SB, full scan of the filtrate using ultra-high performance liquid chromatography coupled to an electrostatic field orbital trap high-resolution mass spectrometer; SC, according to the scanning results, determine the mass spectrometry response value of cis (trans)-2-dodecenoic acid in the filtrate; SD, when the mass spectrometer response value is 0, it indicates that the tested dairy product is within the shelf life; when the mass spectrometer response value is greater than 0, it indicates that the tested dairy product exceeds the shelf life, thereby completing the freshness evaluation of the dairy product.

9. The method for evaluating the freshness of dairy products according to claim 8, characterized in that: In the step SA, the vortex mixing time is 0.5 min-2 min, the ultrasonic extraction time is 5 min-10 min, the centrifugal speed is 4000 r / min-5000 r / min, and the centrifugal time is 5 min-10 min; and a 0.22 μm-0.45 μm organic filter membrane is used for filtration.

10. The method for evaluating the freshness of dairy products according to claim 8, characterized in that: In the step SB, the electrostatic field orbital trap high-resolution mass spectrometry conditions are: the mass analyzer is Q Exactive Orbitrap; the ion source is a heated electrospray ion source; the scanning mode adopts the positive ion simultaneous scanning mode; the monitoring mode is parallel reaction monitoring PRM; the spray voltage is 3.0kV-3.8kV; the lens voltage is 55V; the capillary temperature is 300℃-350℃; the auxiliary gas heating temperature is 300℃-350℃; the sheath gas is 30arb-45arb, and the auxiliary gas is 5arb-10arb; the primary resolution R=70000FWHM; the secondary resolution R=13500FWHM; Automatic gain control is 3×10 6 , the maximum dwell time is 50ms-100ms.

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