CN111208238B

CN111208238B - Sample processing method for detecting volatile substances in minced fillet or minced fillet product and application of sample processing method

Info

Publication number: CN111208238B
Application number: CN202010055537.3A
Authority: CN
Inventors: 安玥琦; 胡杨; 杨姣; 李金铃; 赵钰; 刘茹; 熊善柏
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2021-01-29
Anticipated expiration: 2040-01-17
Also published as: CN111208238A

Abstract

The invention belongs to the technical field of food detection and analysis, and particularly relates to a sample processing method for detecting volatile substances in minced fillet or products of minced fillet and application of the sample processing method. The method comprises the following steps: (1) crushing a sample after quick freezing by liquid nitrogen, performing oscillation extraction by using dichloromethane, filtering and collecting an extracting solution, and performing solvent-assisted distillation extraction; (2) concentrating the extract; (3) the concentrated solution is fractionated by pentane, then the volatile odorous substances are collected by eluting with dichloromethane and concentrated by nitrogen blowing. Aiming at the heat sensitivity of freshwater fish and products thereof, the invention uses liquid nitrogen quick freezing and an extraction and concentration method at the temperature of no more than 50 ℃, avoids the influence of heat action on samples and flavor compounds, and keeps the authenticity of volatile smell extracting solution; the method of organic solvent extraction is used, so that the selective adsorption of methods such as solid phase micro-extraction and the like is avoided; through flavor fractionation, the influence of non-flavor substances is avoided, and the effectiveness of identifying volatile odor components is improved.

Description

Sample processing method for detecting volatile substances in minced fillet or minced fillet product and application of sample processing method

Technical Field

The invention belongs to the technical field of food detection and analysis, and particularly relates to a sample processing method for detecting volatile substances in minced fillet or products of minced fillet and application of the sample processing method.

Background

The fresh water resources in China are rich, and the fresh water fish and the products thereof are popular with consumers due to the unique taste and flavor. However, the off-flavors of grass and earthy smell contained in the freshwater fish are important factors affecting the quality of the freshwater fish and the products thereof. Therefore, the accurate determination of the volatile odor substances of the freshwater fish and the products thereof is very critical.

The gas chromatography-mass spectrometer is a main device for identifying volatile odor substances in food. Before the gas chromatography-mass spectrometry analysis is carried out on the food, the volatile odor substances in the food need to be extracted. At present, headspace-solid phase microextraction is the main means for extracting volatile odor substances of food, for example, Zhang German et al (meat or meat product characteristic volatile flavor substance identification method, patent publication No. CN109696501A) uses solid phase headspace microextraction-gas chromatography-smelling-mass spectrometer, combines with flavor recombination test, and identifies and distinguishes the original volatile flavor substances of meat or meat product. Xuhui qing et al (a detection method for determining volatile sulfur compounds in fermented meat products, patent publication No. CN102944624A) also extract volatile odor substances of samples by a headspace-solid phase microextraction sample pretreatment method, and determine sulfur compounds in fermented meat products by combining gas chromatography-flame photometric detection. Although the headspace-solid phase microextraction has the advantages of rapidness, convenience and the like, the solid phase microextraction cannot accurately measure all flavor substances in a sample due to selective adsorption of an extraction head of the solid phase microextraction and competitive adsorption among volatile compounds.

In order to improve the effect of solid phase microextraction, Routong and the like (a method for measuring volatile flavor substances in turtle meat by an acid-base method, patent publication No. CN105021743A) improve the efficiency of solid phase microextraction by adjusting the pH value of a detection sample, thereby improving the detection sensitivity; forest pine (a method for detecting flavor substances in steamed buns, patent publication No. CN106018630A) adopts an ultrasonic pretreatment method, and improves the extraction efficiency of solid-phase microextraction. However, the solid-phase micro-extraction requires that a detection sample is extracted for a certain time (30-80 min) at a certain temperature (30-60 ℃), and the freshwater fish and products thereof are very sensitive to heat action and are easy to gel under the extraction conditions, so that the odor substances obtained by the solid-phase micro-extraction may not be the true original odor of the freshwater fish or the products thereof.

Another method for extracting volatile odorous substances from food is organic solvent extraction, which is commonly used to determine volatile odorous substances in liquid foods, such as wine, beverages, dairy products, and the like. In the case of solid or semi-solid foods, when the odor substances are extracted with an organic solvent, it is necessary to extract the volatile odor substances after sufficiently crushing the sample. The heat generated during the disruption process may have an effect on the sample and its flavor compounds. In the process of extracting food with organic solvent, most hydrocarbon compounds which do not contribute to volatile smell, such as alkane and alkene substances, also exist in the flavor extracting solution, and the hydrocarbon compounds have larger peaks in a gas chromatogram map, so that the peaks of the substances which contribute to flavor are covered, and the effectiveness of identifying flavor substances is reduced.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a sample treatment method for detecting volatile substances in minced fillet or products thereof and application thereof, and aims to solve part of problems in the prior art or at least alleviate part of problems in the prior art.

The invention is realized in such a way that a sample processing method for detecting volatile substances in minced fillet or products thereof comprises the following steps:

freezing the sample and then crushing to obtain sample powder;

oscillating and extracting organic compounds in the sample powder by using an organic solvent, filtering, and collecting a leaching solution;

extracting the leaching solution by solvent-assisted distillation to obtain volatile organic compound extract;

concentrating the extracting solution to obtain a volatile organic compound extracting concentrated solution;

the volatile organic compound extract concentrate was fractionated with pentane and then eluted with dichloromethane to collect the volatile odorous materials.

Further, the organic solvent is dichloromethane, and the leaching time is 2-24 h.

Further, the mass volume ratio of the sample powder to the organic solvent is (1-100): (2-200). The method comprises the specific steps of putting 10-1000 g of crushed sample powder into a conical flask, adding 20-2000 mL of dichloromethane into the conical flask, sealing the conical flask by using aluminum foil paper, shaking and extracting the mixed sample at 60-300 r/min for 2-24h, collecting extracting solutions through filtration, repeatedly extracting each sample for 3 times, and combining the organic compound extracting solutions.

Further, the conditions of the solvent-assisted distillation extraction are as follows: the temperature is 30-50 ℃, and the vacuum degree is 1.0 multiplied by 10^-3～1.0×10^-2Pa. And (3) separating volatile substances and nonvolatile substances in the extracting solution by solvent-assisted distillation and extraction to obtain a volatile organic compound extracting solution.

Further, the extract is dried and then concentrated. Adding anhydrous sodium sulfate into a volatile organic compound extracting solution until crystals are not formed any more, standing overnight, concentrating an extracting solution to 1-100 mL at room temperature and 50 ℃ by using a K-D concentration device connected with a Vickers fractionating column, and further concentrating a sample to 0.1-10 mL by using micro-flow nitrogen flow blowing and concentration.

Further, the method for concentrating the extract comprises concentrating the extract at 50 deg.C by a K-D concentration device connected to a Weibull fractionation column; also includes using micro-flow nitrogen to purge and concentrate.

Further, the operation of fractionating the volatile organic compound extraction concentrate by using pentane specifically includes: adding the volatile organic compound extraction concentrated solution into pentane, and concentrating; adding the concentrated sample to a silica gel column activated by dichloromethane, and eluting the sample by sequentially using pentane and dichloromethane; collecting the fraction eluted by the dichloromethane, and purging and concentrating the fraction by using micro-flow nitrogen to obtain the volatile odor substance concentrated solution of the sample, wherein the volatile odor substance concentrated solution can be used for subsequent detection. For example, the obtained volatile odorant concentrate is analyzed by a gas chromatography-mass spectrometer/sniffer.

The application of the sample treatment method for detecting the volatile substances of the surimi or the products thereof in detecting the volatile substances of the surimi or the products thereof is disclosed.

Further, the volatile substance includes a polyunsaturated aldehyde substance.

Further, the polyunsaturated aldehyde substance includes at least one of (E, E, E) -2,4, 6-nonatrienol, (E, Z, Z) -2,4, 7-tridecatrienal and (Z, Z, Z) -7,10, 13-hexadecatrienal.

In summary, the advantages and positive effects of the invention are:

(1) through organic solvent extraction and solvent-assisted distillation extraction, selective adsorption and competitive adsorption among substances in the extraction process are avoided, and a method for comprehensively detecting the flavor of the freshwater fish and the product thereof is provided.

(2) In the process of extracting and concentrating the flavor, the influence of heat action on the sample and flavor compounds of the sample is avoided by quick freezing with liquid nitrogen and assisting the processes of distilling, extracting and distilling with a solvent at the temperature of not more than 50 ℃, so that the flavor extraction concentrate keeps the authenticity of the original sample flavor.

(3) Through flavor fractionation, the influence of compounds which do not contribute to volatile odor is removed, and the accuracy and the effectiveness of flavor analysis are improved. The invention links a Weibull fractionating column in the traditional K-D concentrator, and can reduce the loss of volatile substances to the maximum extent in the distillation concentration process.

(4) The traditional fractionation test steps are simplified, the process only needs to detect the fraction eluted by dichloromethane, and the traditional control reagents such as ether and the like are not used in the process, so that the pretreatment simplicity of the volatile odor substance is improved.

Drawings

FIG. 1 is a GC-MS total ion spectrum of an unfractionated sample of example 1;

FIG. 2 is a GC-MS total ion spectrum of a pentane fractionated sample of example 1;

FIG. 3 is a GC-MS total ion spectrum of a sample fractionated in methylene chloride in example 1;

FIG. 4 is a schematic view of a K-D concentration apparatus connected to a Vickers fractionation column;

figure 5 is a schematic of a flavor fractionation analysis.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

The invention discloses a sample treatment method for detecting volatile substances in minced fillet or products thereof and application thereof, and particularly relates to the following examples.

The sample which is not subjected to liquid nitrogen quick freezing has high viscosity and cannot be crushed into powder, and the sample can only be cut into blocks for extraction, but the extraction is not complete, so that the taste of the sample does not need to be measured for incomplete extracting solution in experimental design. Therefore, the following examples of the present invention are experimental protocols subjected to liquid nitrogen freezing treatment.

Example 1 preparation of volatile odorant extract from frozen silver carp surimi and analysis of the result

(1) Cutting the frozen silver carp surimi into about 0.5-2 cm³The block of (2) is quickly frozen with liquid nitrogen and then crushed into powder with a crusher.

(2) Weighing 200g of sample powder in a conical flask, adding 300mL of dichloromethane, sealing with aluminum foil paper, shaking and extracting the mixed sample at 150r/min for 2h, collecting the extracting solution by filtering, repeatedly extracting for 3 times, and combining the dichloromethane extracting solutions.

(3) And (2) carrying out solvent-assisted distillation extraction on the collected dichloromethane extracting solution, and separating volatile substances and nonvolatile substances in the extracting solution, wherein the extraction conditions are as follows: the temperature is 45 ℃, and the vacuum degree is 9.0 multiplied by 10^-3Pa。

(4) Anhydrous sodium sulfate was added to the collected volatile organic compound extract until no crystals formed, and after standing overnight, the extract was concentrated to 5mL at 46 ℃ using a K-D concentration device (see fig. 4 for structural diagram) connected to a wechsler fractionation column, and the sample was further concentrated to 1mL using micro-flow nitrogen purge concentration.

(5) The 1mL of the VOC extraction concentrate obtained in step 4 was divided into 2 portions, and 0.5mL of the concentrate was further concentrated to 0.1 mL. The concentrated samples were analyzed using an Agilent 7890A Gas Chromatography (GC) system with an Agilent 5975 Mass Selective Detector (MSD) with a sample size of 1 μ L, a GC inlet temperature of 250 deg.C, helium as carrier gas, a flow rate of 2mL/min, and a DB-wax column (60m × 0.32mm × 0.25 μm). The temperature-raising procedures are respectively as follows: after keeping the temperature at 40 ℃ for 4min, the temperature is increased to 230 ℃ at 4 ℃/min and kept for 5 min. The electron impact energy is 70eV, the ion source temperature is 230 ℃, and enhanced ChemStation software is adopted for system software control and data management/analysis. Mass spectra of the compounds were compared to the NIST database. The Retention Index (RI) was calculated according to the modified Kov-ts method. Compounds were identified by comparing standard mass spectra and RI. The total ion spectrum of the obtained sample without fractionation test is shown in fig. 1.

(6) And (3) continuously concentrating the other 0.5mL of volatile organic compound extraction concentrated solution obtained in the step (4) to 0.1mL, adding 1.5mL of pentane into the 0.1mL of concentrated solution, then concentrating to 0.1mL by nitrogen purging at a micro flow rate, repeatedly adding pentane and concentrating to 0.1mL for three times. The concentrated sample was applied to a silica gel column activated with methylene chloride (see FIG. 5 for operation of fractional analysis), the sample was sequentially eluted with 6mL of pentane and 6mL of methylene chloride, the fractions eluted with pentane and methylene chloride were collected, respectively, and concentrated to 0.1mL with a micro-flow nitrogen purge, and the two concentrates were measured using the same GC-MSD conditions as described in step 5, and total ion spectra of the sample fractionated with pentane and methylene chloride are shown in FIGS. 2 and 3, respectively.

As can be seen from fig. 1, the total ion spectrum of the volatile organic compound concentrate without fractionation contains a large number of high and broad alkane peaks, especially hexadecane and octadecane. These alkanes do not contribute to flavor. After elution with pentane, the peaks of these alkane compounds were all present in the pentane fraction (FIG. 2), while the effect of the large peaks of these alkanes was not seen in the subsequently fractionated dichloromethane fraction (FIG. 3), indicating that the fractionation operation of the present invention facilitates efficient and accurate identification of the flavor compounds.

Through identification, odor substances represented by the main peaks appearing in FIG. 3 are (i) hexanal in sequence; ② E-2-pentenal; ③ octanal; fourthly, 1-octen-3-ol; e, E-2, 4-heptadienal; sixthly, E-2-nonanal; seventhly, Z-2, 6-nonadienal; eighty percent of phenylacetaldehyde; ninthly E, E-2, 4-decadienal; phenol in the R;

4-methylphenol;

(E, Z) -2,4, 7-tridecatrienal;

(Z, Z, Z) -7,10, 13-hexadecatrienal. In particular

Substance No. is covered by the alkane peak in figure 1 and cannot be identified in the total ion spectrum of the unfractionated concentrate. Therefore, the method improves the effectiveness and accuracy of identifying the odor substances, and the simplified flavor fractionation test is feasible.

Example 2 extraction and detection of volatile odor substance from silver carp surimi gel

(1) Cutting the silver carp minced fillet gel into about 1-2 cm³The block of (2) is quickly frozen with liquid nitrogen and then crushed into powder with a crusher.

(4) Adding anhydrous sodium sulfate into the collected volatile organic compound extract until no crystal is formed, standing overnight, concentrating the extract to 5mL at 46 ℃ by using a K-D concentration device connected with a Vickers fractionating column, and further concentrating the sample to 0.1mL by using micro-flow nitrogen flow blowing and concentration.

(5) 1.5mL pentane was added to 0.1mL VOC extract concentrate, then concentrated to 0.1mL by gentle nitrogen purge, and pentane was added repeatedly and concentrated to 0.1mL three times. The concentrated sample was loaded onto a silica gel column activated with dichloromethane, the sample was sequentially eluted with 6mL of pentane and 6mL of dichloromethane, and the dichloromethane-eluted fractions were collected and concentrated to 0.1mL using a micro-flow nitrogen purge. The concentrated samples were analyzed using an Agilent 7890A Gas Chromatography (GC) system with an Agilent 5975 Mass Selective Detector (MSD) and sniffer. The sample introduction amount was 1. mu.L, the GC inlet temperature was 250 ℃, the carrier gas was helium, the flow rate was 2mL/min, and the column was a DB-wax column (60 m.times.0.32 mm.times.0.25 μm). The temperature-raising procedures are respectively as follows: after keeping the temperature at 40 ℃ for 4min, the temperature is increased to 230 ℃ at 4 ℃/min and kept for 5 min. The split ratio of the sniffing instrument to the MSD is 1:1, and the temperature of the sniffing instrument is 250 ℃. The electron impact energy of MSD is 70eV, the ion source temperature is 230 ℃, and the system software control and data management/analysis adopt enhanced ChemStation software. Mass spectra of the compounds were compared to the NIST database. The Retention Index (RI) was calculated according to the modified Kov-ts method. The compounds were identified by comparing the mass spectrum, aroma and RI of the standards, and the results of the identification of the substances are shown in table 1.

As can be seen from table 1, by using the method of the present invention, a total of 62 flavor compounds were successfully identified in surimi gel, which contained 27 aldehydes, 10 alcohols, 10 ketones, 6 acids, 4 phenols, 3 lactones, and 2 nitrogen-containing compounds. It is worth mentioning that polyunsaturated aldehydes with obvious characteristic fish flavor, such as (E, E, E) -2,4, 6-nonatrienoic aldehyde, (E, Z, Z) -2,4, 7-tridecatrienoic aldehyde and (Z, Z, Z) -7,10, 13-hexadecatrienoic aldehyde, are successfully identified, and the polyunsaturated aldehydes are difficult to be found by methods such as solid phase microextraction, and related reports are not found at present. Therefore, the method greatly improves the accuracy and comprehensiveness of the detection of the volatile odor substances of the freshwater fish and the products thereof.

Table 1 identification of odorants of surimi gel

Note: RI indicates that the linear retention index of the material is consistent with that of the standard; RIL indicates that the substance is consistent with the linear retention index reported in the literature; MS shows that the mass spectrum of the substance is consistent with the comparison result of the map; the flavor description of the substance represented by A is consistent with standard products and literature reports.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A sample processing method for detecting volatile substances in minced fillet or products thereof is characterized by comprising the following steps:

quickly freezing a sample by liquid nitrogen, and crushing to obtain sample powder;

oscillating and extracting organic compounds in the sample powder by using an organic solvent, filtering, and collecting a leaching solution; the organic solvent is dichloromethane, and the leaching time is 2-24 h; the mass volume ratio of the sample powder to the organic solvent is (1-100): (2-200);

extracting the leaching solution by solvent-assisted distillation to obtain volatile organic compound extract; the conditions of the solvent-assisted distillation extraction are as follows: the temperature is 30-50 ℃, and the vacuum degree is 1.0 multiplied by 10^-3~1.0×10^-2 Pa；

Concentrating the extracting solution to obtain a volatile organic compound extracting concentrated solution; drying the extracting solution, and then concentrating; the method for concentrating the extractive solution comprises concentrating the extractive solution at 50 deg.C by a K-D concentrating device connected with a Vickers fractionating column; the method also comprises the steps of purging and concentrating by using micro-flow nitrogen;

fractionating the volatile organic compound extraction concentrated solution by using pentane, and eluting by using dichloromethane to collect volatile odor substances; the operation of fractionating the volatile organic compound extraction concentrate by using pentane specifically comprises: adding the volatile organic compound extraction concentrated solution into pentane, and concentrating; adding the concentrated sample to a silica gel column activated by dichloromethane, and eluting the sample by sequentially using pentane and dichloromethane; collecting the fraction eluted by the dichloromethane, and purging and concentrating the fraction by using micro-flow nitrogen to obtain the volatile odor substance concentrated solution of the sample, wherein the volatile odor substance concentrated solution can be used for subsequent detection.

2. The use of the sample treatment method for detecting volatile substances in surimi or products thereof as claimed in claim 1 in detecting volatile substances in surimi or products thereof.

3. Use according to claim 2, characterized in that: the volatile substances include polyunsaturated aldehydes.

4. Use according to claim 3, characterized in that: the polyunsaturated aldehyde substance comprises at least one of (E, E, E) -2,4, 6-nonatrienal, (E, Z, Z) -2,4, 7-tridecatriene aldehyde and (Z, Z, Z) -7,10, 13-hexadecatriene aldehyde.