A method for determining the storage time of semi-fermented stinky tofu
The volatile substances in stinky tofu were analyzed through headspace solid phase microextraction technology (HS-SPME-GC-MS), which solved the problem of judging the storage time of semi-fermented stinky tofu, achieved rapid and accurate detection, and reduced food safety risks.
Patent Information
- Application Number
- CN202210936393.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-08-05
- Publication Date
- 2025-03-21
- Estimated Expiration
- 2042-08-05
AI Technical Summary
The prior art is difficult to quickly and conveniently judge the storage time of semi-fermented stinky tofu, resulting in an increase in food safety risks.
Headspace solid phase microextraction technology (HS-SPME-GC-MS) was used to conduct qualitative and quantitative analysis of volatile substances in stinky tofu, and the storage time was determined by detecting the concentration of volatile and semi-volatile esters.
It realizes the rapid and accurate judgment of the storage time of semi-fermented stinky tofu, reduces food safety risks, is convenient to operate, has small sample volume and no solvent is required.
Smart Images
Figure CN115290793B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of food detection, and in particular to a method for determining the storage time of semi-fermented stinky tofu. Background Art
[0002] Stinky tofu is a traditional fermented food with local characteristics. It is made from soybeans, which are rich in protein. After fermentation, the protein is broken down into a variety of amino acids, many of which are essential for human survival. Stinky tofu has various advantages such as a long history, a broad market and high nutritional value. According to different production processes, it can be divided into fermented stinky tofu and semi-fermented stinky tofu.
[0003] Health and safety are the first priorities in food production, and deterioration is an important factor affecting food safety. Food that is stored for more than a certain period of time will become spoiled and deteriorate, and eating spoiled food will cause harm to the human body. The production process of semi-fermented stinky tofu is relatively rough, and most of the production environments are open to the open air, making it difficult to eliminate the infestation of bacteria, which leads to fluctuations in the edible time of each batch of products and increases food safety risks. In general, the freshness of food can be judged by its appearance and smell, but because stinky tofu has a strong and special smell, and its appearance changes after fermentation in stinky brine, it is difficult to judge its freshness by sight and smell alone. The stinky brine contains a large number of complex bacteria, and complex fermentation reactions will occur during storage, resulting in the production of a large number of ester compounds in the later stages of storage.
[0004] Therefore, developing a fast and convenient method to detect the storage time of semi-fermented stinky tofu is of great significance to ensure the food safety of semi-fermented stinky tofu. Summary of the invention
[0005] In order to overcome the above problems, the present invention adopts headspace solid phase microextraction technology (HS-SPME-GC-MS) to quickly and conveniently perform qualitative and quantitative analysis on volatile substances in stinky tofu, thereby achieving the effect of accurately determining the storage time of semi-fermented stinky tofu.
[0006] Specifically, the first aspect of the present invention provides a method for determining the storage time of semi-fermented stinky tofu based on HS-SPME / GC-MS, comprising the following steps: (1) sample pretreatment; (2) headspace solid phase microextraction; (3) GC-MS analysis; and (4) result analysis.
[0007] In certain embodiments, the (1) sample pretreatment: stinky tofu is sampled, and the sample and internal standard are placed in a headspace bottle.
[0008] In certain embodiments, the internal standard is a standard working solution prepared in 2-octanol.
[0009] In certain embodiments, the (1) sample pretreatment includes sampling the stinky tofu, and after sampling the stinky tofu, the sample is fully crushed in a mortar, weighed, and then added to a headspace bottle.
[0010] In certain embodiments, in the sample pretreatment method of step (1), the semi-fermented stinky tofu marinade and the portion outside the marinade should be evenly taken, the sample weight should be preferably 3-5 g, crushed with a mortar until broken and homogenized, and then put into a 10 mL screw-capped headspace bottle.
[0011] In certain embodiments, the (2) headspace solid phase microextraction: a headspace bottle containing a sample and an internal standard is placed in a 25-100° C. hot water bath for equilibrium, and then an aged extraction head is inserted into the headspace portion of the headspace bottle for adsorption.
[0012] In certain embodiments, in the headspace solid phase microextraction method of step (2), an SPME extraction head coated with a divinylbenzene / carboxyl / polydimethylsiloxane fused quartz (75 μm) coating fiber is used to perform a volatile compound extraction experiment.
[0013] In certain embodiments, the (3) GC-MS analysis: inserting the extraction head adsorbed with the volatile compounds in the semi-fermented stinky tofu into the GC-MS injection port for desorption, and then performing GC-MS analysis.
[0014] In certain embodiments, the GC-MS operating conditions in step (3) are as follows: the initial column oven temperature of the HP-Innowax column is set to 40°C and maintained for 4 minutes; the temperature is increased to 130°C at a rate of 4°C / min and maintained for 5 minutes; the temperature is increased to 200°C at a rate of 4°C / min and maintained for 5 minutes; carrier gas: helium, flow rate 1 mL / min, mass spectra are recorded in electron impact mode (70 eV) within the scanning range of 35-350 m / z, the ion source temperature is maintained at 250°C, and the solvent delay time is set to 3 minutes.
[0015] In certain embodiments, the (4) result analysis: analyzing the GC-MS characteristic spectrum results of the semi-fermented stinky tofu to be tested, when the volatile compound detection results contain ethyl 2-phenylacetate or ethyl 2-phenylpropionate, or when the total concentration of volatile and semi-volatile ester substances in the sample is above 0.5 mg / kg, it is determined that the storage time of the sample stinky tofu has exceeded 10 days.
[0016] In certain embodiments, the volatile and semi-volatile ester substances include: ethyl acetate, ethyl propionate, butyl acetate, pentyl acetate, hexyl acetate, hexyl propionate, hexyl formate, heptyl acetate, octyl formate, bornyl acetate, butyrolactone, ethyl 2-phenylacetate, ethyl 2-phenylpropionate, etc.
[0017] In certain embodiments, the equilibration time in step (2) is 5-15 min, the adsorption time in step (2) is 45-90 min, and the desorption time in step (3) is 3-5 min.
[0018] In certain embodiments, the present invention as a whole is quantified by the internal standard method. The specific method of the internal standard method is: the internal standard is a standard working solution prepared with 2-octanol. Weigh 3-5g of semi-fermented stinky tofu and crush it, then add 5-20μL of a series of standard working solutions, quickly cover the bottle cap, and measure it with HS-SPME-GC-MS / MS to obtain the integrated peak area of the test compound and the internal standard; when quantifying, the concentration of each test compound in the sample can be inferred based on the known internal standard concentration and peak area.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] The present invention detects volatile and semi-volatile compounds in semi-fermented stinky tofu by HS-SPME / GC-MS, and can quickly and accurately judge the storage time of semi-fermented stinky tofu and determine its deterioration. The detection method has the advantages of convenient operation, small sample amount, no need for solvent, etc., is very easy to promote, and has high application value for quality evaluation of semi-fermented stinky tofu. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:
[0022] Figure 1 This is the HS-SPME / GC-MS characteristic spectrum of the volatile components of fresh semi-fermented stinky tofu stored for 3 days.
[0023] Figure 2 This is the HS-SPME / GC-MS characteristic spectrum of the volatile components of semi-fermented stinky tofu that has been deteriorated after storage for 10 days.
[0024] Figure 3 This is the HS-SPME / GC-MS characteristic spectrum of the volatile components of semi-fermented stinky tofu that has been deteriorated after storage for 13 days. DETAILED DESCRIPTION
[0025] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0026] Unless otherwise defined, technical or scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.
[0027] The following describes a method for determining the storage time of semi-fermented stinky tofu according to the present invention in conjunction with specific embodiments.
[0028] Example 1 HS-SPME / GC-MS Detection and Analysis of Stinky Tofu Stored for 3 Days
[0029] This embodiment provides a method for identifying the storage time of semi-fermented stinky tofu, which identifies stinky tofu that has been stored for 3 days, including the following:
[0030] Step a: Sample preparation: Take a stinky tofu sample stored for 3 days, sample and mix the parts soaked in brine and the parts not soaked in brine evenly. Accurately weigh 3g of stinky tofu as a sample, fully mash the sample in a mortar, and then add it to a 10ml special screw-mouth headspace bottle.
[0031] Step b: Add internal standard: Add 7 μl of 2-octanol (solvent is water) with a concentration of 220 μg / kg as internal standard into the headspace bottle in step a, then quickly tighten the bottle cap for testing. Refrigerate in a 4°C refrigerator before testing to prevent volatilization of the compound.
[0032] Step c: Aroma enrichment: Place the headspace bottle in step b in a 60°C hot water bath and balance for 15 minutes to ensure that the aroma substances are completely volatilized into the headspace phase in the bottle. Insert the aged divinylbenzene / carboxyl / polydimethylsiloxane fused quartz (75μm) coated fiber SPME extraction head into the headspace area of the headspace bottle to allow the extraction head to fully enrich the volatile compounds in the stinky tofu, and wait for 50 minutes before pulling out the extraction head.
[0033] Step d: GC-MS analysis: The extraction head in step c was analyzed by GC-MS, and the desorption time was 5 min.
[0034] The operating conditions of GC-MS were as follows:
[0035] The initial oven temperature of the HP-Innowax column was set to 40°C and maintained for 4 minutes; then increased to 130°C at a rate of 4°C / min and maintained for 5 minutes; then increased to 200°C at a rate of 4°C / min and maintained for 5 minutes; carrier gas: helium, flow rate 1 mL / min, mass spectra were recorded in electron impact mode (70 eV) in the scan range of 35-350 m / z, the ion source temperature was maintained at 250°C, and the solvent delay time was set to 3 minutes.
[0036] Step e: Result analysis: The results are shown in Table 1.
[0037] Table 1 Types and concentrations of esters in fresh stinky tofu stored for 3 days
[0038]
[0039] As can be seen from Table 1, the total concentration of ester substances is 0.085 mg / kg, which is much lower than 0.5 mg / kg. At the same time, no ethyl 2-phenylacetate or ethyl 2-phenylpropionate was detected, indicating that the sample is fresh stinky tofu suitable for consumption.
[0040] Example 2 HS-SPME / GC-MS Detection and Analysis of Stinky Tofu Stored for 10 Days
[0041] This embodiment provides a rapid detection method for the storage time of semi-fermented stinky tofu based on HS-SPME-GC-MS, and identifies spoiled stinky tofu stored for 10 days. The specific method is the same as that in Example 1.
[0042] The results are shown in Table 2.
[0043] Table 2 Types and concentrations of esters in spoiled stinky tofu stored for 10 days
[0044]
[0045] It can be seen from Table 2 that the total concentration of ester substances is 0.918 mg / kg, which is greater than 0.5 mg / kg. At the same time, ethyl 2-phenylacetate or ethyl 2-phenylpropionate was detected, which shows that the sample is a spoiled stinky tofu that is not suitable for consumption.
[0046] Example 3 HS-SPME / GC-MS Detection and Analysis of Stinky Tofu Stored for 13 Days
[0047] This embodiment provides a rapid detection method for the storage time of semi-fermented stinky tofu based on HS-SPME-GC-MS, and identifies spoiled stinky tofu stored for 13 days. The specific method is the same as that in Example 1.
[0048] The results are shown in Table 3.
[0049] Table 3 Types and concentrations of esters in spoiled stinky tofu stored for 13 days
[0050]
[0051] As can be seen from Table 3, the total concentration of ester substances is 18.602 mg / kg, far exceeding 0.5 mg / kg. At the same time, ethyl 2-phenylacetate or ethyl 2-phenylpropionate was detected, indicating that the sample is a spoiled stinky tofu that is not suitable for consumption.
[0052] Example 4 Comparison of the effects of the sensory evaluation method and the method of the present invention
[0053] 4.1 Sensory evaluation method:
[0054] Take 5g each of stinky tofu A (3 days), B (10 days), and C (13 days) and place them in an opaque container with a sniffing port. Select a ventilated place with appropriate lighting, temperature, and humidity to conduct a sensory evaluation experiment on the samples, and then have the sensory evaluators judge the freshness of the samples.
[0055] The 19 sensory evaluators (10 females and 9 males, aged 22-28 years old) who participated in this experiment had all been fully investigated and trained in advance according to international standards (ISO 8589-2007.14).
[0056] 4.2 Comparison of the effects of the sensory evaluation method and the method of the present invention:
[0057] The results of sensory evaluation and the accuracy of the method of the present invention are shown in Table 4. Due to the special smell of stinky tofu itself, it is difficult to distinguish its freshness by smell alone. Compared with the sensory evaluation method, the accuracy of the method of the present invention is obviously higher. In order to ensure the accuracy of the sensory evaluation experiment, a sensory laboratory dedicated to tasting is required, as well as several or even dozens of professional sensory evaluators to participate in the experiment. Compared with gas chromatography, it consumes more manpower and has higher costs.
[0058] Table 4 Determination results of stinky tofu stored for different lengths by sensory evaluation method and the method of the present invention
[0059]
[0060] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It is obvious to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention. Therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the attached claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention. Any figure mark in the claims should not be regarded as limiting the claims involved.
[0061] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This description method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.
Claims
1. A method for determining the storage time of semi-fermented stinky tofu based on HS-SPME / GC-MS, characterized in that: The steps include: (1) Sample pretreatment: Take samples of stinky tofu and put the samples and internal standards into headspace bottles; (2) Headspace solid phase microextraction: Place the headspace bottle containing the sample and internal standard in a hot water bath at 25-100°C for equilibrium, and then insert the aged extraction head into the headspace part of the headspace bottle for adsorption. The internal standard is a standard working solution prepared with 2-octanol. (3) GC-MS analysis: The extraction head adsorbed with volatile compounds in semi-fermented stinky tofu was inserted into the GC-MS injection port for desorption, and then GC-MS analysis was performed; (4) Result analysis: Analyze the GC-MS characteristic spectrum results of the semi-fermented stinky tofu to be tested. When the volatile compound detection results include ethyl 2-phenylacetate and ethyl 2-phenylpropionate, and the total concentration of volatile and semi-volatile ester substances in the sample is above 0.5 mg / kg, it is determined that the storage time of the stinky tofu sample has exceeded 10 days.
2. The method according to claim 1, characterized in that: After the stinky tofu is sampled, the sample is placed in a mortar and fully crushed, then weighed and added to a headspace bottle.
3. The method according to claim 1 or 2, characterized in that: In the sample pretreatment method of step (1), the semi-fermented stinky tofu marinade and the portion outside the marinade should be evenly taken, the sample weight should be preferably 3-5 g, crushed with a mortar until broken and homogenized, and then put into a 10 mL screw-capped headspace bottle.
4. The method according to claim 1, characterized in that: The equilibrium time is 5-15 min, the adsorption time is 45-90 min, and the desorption time is 3-5 min.
5. The method according to claim 1, characterized in that In the headspace solid phase microextraction method of step (2), an SPME extraction head coated with divinylbenzene / carboxyl / polydimethylsiloxane fused silica coating fiber is selected to carry out volatile compound extraction experiments.
6. The method according to claim 1, characterized in that The GC-MS operating conditions in step (3) are as follows: the initial column oven temperature of the HP-Innowax column is set to 40°C and maintained for 4 minutes; the temperature is increased to 130°C at a rate of 4°C / min and maintained for 5 minutes; the temperature is increased to 200°C at a rate of 4°C / min and maintained for 5 minutes; the carrier gas is helium, with a flow rate of 1 mL / min, and the mass spectrum is recorded in the electron impact mode at 70 eV within the scanning range of 35-350 m / z, the ion source temperature is maintained at 250°C, and the solvent delay time is set to 3 minutes.