Method for indicating fish freshness change by adopting cell apoptosis signal molecules and application

Abstract

The invention discloses a method for indicating fish freshness change by adopting cell apoptosis signal molecules and application, and belongs to the technical field of grass carp freshness detection. The invention discloses a method for indicating fish freshness change by adopting cell apoptosis signal molecules, which comprises the following steps: according to the detected contents of cell apoptosis signal molecules Caspase-3, Bax, Bcl-2 and Cyt-c, establishing a corresponding relation model between the content of the cell apoptosis signal molecules and the freshness of grass carp. According to the method, the freshness of the grass carp can be quickly and accurately judged, and a scientific basis is provided for quality control and market supervision of the grass carp. The method can be popularized and applied in actual production and market supervision.

Description

Technical Field

[0001] The present invention relates to the technical field of grass carp freshness detection, and more specifically, to a method and application for indicating the freshness change of grass carp by using apoptosis signaling molecules. Background Art

[0002] Grass carp is one of the important protein sources in people's daily diet, and its freshness directly affects the quality, taste and safety of fish meat. At present, the methods for judging the freshness of grass carp mainly include sensory evaluation, microbiological detection, chemical analysis and new detection technologies, etc. Sensory evaluation mainly relies on the experience and subjective judgment of testers, with poor accuracy and repeatability; although microbiological detection can reflect the growth of microorganisms in fish meat, the detection period is relatively long and cannot meet the requirements of rapid detection; for chemical analysis such as the detection of indicators such as total volatile basic nitrogen (TVB-N) and K value, although it has a certain degree of accuracy, the operation is relatively complex, the detection accuracy for ice-stored fish is poor, and the detection of some indicators requires expensive instrument equipment. New detection technologies such as near-infrared spectroscopy technology are expensive and require professional personnel for operation and maintenance, which limits their popularization in grass-roots inspection and quarantine institutions and consumer markets. At present, some of the existing detection methods in the market have different adaptabilities to different varieties of grass carp and lack universality, and there is a lack of an economic, efficient and rapid grass carp freshness detection method in the market.

[0003] Apoptosis is a programmed cell death process that occurs in cells under normal physiological or pathological conditions. After grass carp dies, a series of physiological changes will occur in its cells, including apoptosis. Apoptosis signaling molecules play a key regulatory role in the process of apoptosis, and the change of their content is closely related to the process of apoptosis. When the fish body begins to deteriorate at an early stage, the apoptosis signaling factors will change accordingly, while some sensory and physical-chemical indicators have no significant differences; therefore, detecting the content of apoptosis signaling molecules to indicate the freshness change of grass carp has potential application value, especially for the freshness change of the fish body at an early stage. Combining this indication method with molecular biology technology may be able to develop a portable device suitable for on-site detection, with higher practicality. However, there is currently no mature method for accurately indicating the freshness change of grass carp by using apoptosis signaling molecules.

[0004] Therefore, it is an urgent problem for those skilled in the art to provide a method and application for indicating the freshness change of grass carp by using apoptosis signaling molecules. Summary of the Invention

[0005] In view of this, the present invention provides a method and application for indicating the freshness change of grass carp by using apoptosis signaling molecules. This method can quickly and accurately judge the freshness of grass carp, providing a scientific basis for the quality control and market supervision of grass carp. The present invention can be popularized and applied in actual production and market supervision.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for indicating the freshness change of grass carp using apoptosis signaling molecules, comprising the following steps:

[0008] (1) Sample collection: Select fresh grass carp. At different time points after the fish dies (such as 0h, 24h, 48h, 72h, 96h, 120h), collect muscle tissue samples of the fish. After quickly freezing the samples in liquid nitrogen, store them in a -80°C refrigerator for later use (freezing preservation has no significant effect on the freshness of fish meat).

[0009] (2) Extraction of apoptosis signaling molecules: After thawing the collected muscle tissue samples, add cell lysate (the mass-volume ratio of muscle tissue sample to cell lysate is 1g:1mL), perform homogenization treatment on ice, and then centrifuge at 12,000 rpm for 15 minutes at 4°C. Take the supernatant to obtain the extraction solution of apoptosis signaling molecules.

[0010] (3) Detection of apoptosis signaling molecules: Use enzyme-linked immunosorbent assay (ELISA) or Western Blot method to quantitatively detect the apoptosis signaling molecules in the extraction solution. Apoptosis signaling molecules include but are not limited to Caspase-3, Bax, Bcl-2, and Cyt-c.

[0011] (4) Freshness judgment: According to the content of apoptosis signaling molecules detected, establish a corresponding relationship model between the content of apoptosis signaling molecules and the freshness of grass carp. Specifically, as the death time of grass carp prolongs, the contents of Caspase-3, Bax, and Cyt-c gradually increase, and the content of Bcl-2 gradually decreases. According to the above change rules, when the expression level of Caspase-3 is less than 4 times the expression level of Caspase-3 in fresh fish (A), the expression level of Bax is less than 1.5 times the expression level of Bax in fresh fish (B), the expression level of Bcl-2 is greater than 0.5 times the expression level of Bcl-2 in fresh fish (C), and the expression level of Cyt-c is less than 1.25 times the expression level of Cyt-c in fresh fish (D), these are used as judgment conditions. When all four conditions A, B, C, and D are met, it can be judged that the grass carp is in the fresh stage; as long as one of the four conditions A, B, C, and D is not met, it is determined that the grass carp is in the non-fresh stage with a significant difference from freshly killed live fish.

[0012] Furthermore, the application of the method for indicating the freshness change of fish using apoptosis signaling molecules in the freshness detection of grass carp.

[0013] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses a method and application for indicating the freshness change of fish by using apoptosis signaling molecules, which has the following beneficial effects:

[0014] The present invention indicates the freshness change by detecting the content of apoptosis signaling molecules in grass carp muscle tissue, and can quickly and accurately judge the freshness of grass carp, overcoming the drawback of traditional detection methods that only stay in judging spoilage or not and lack of evaluation of freshness.

[0015] The detection methods adopted by the present invention, such as ELISA method and Western Blot method, are relatively simple to operate and do not require expensive large-scale instrument equipment, and are suitable for popularization and application in actual production and market supervision.

[0016] The corresponding relationship model established by the present invention between the content of apoptosis signaling molecules and the freshness of grass carp provides a scientific and quantitative basis for the quality control of grass carp, and helps to improve the quality and safety of grass carp. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0018] Figure 1 The drawings are the results of the changes in the contents of Caspase-3, Bax, Bcl-2 and Cyt-c in the present invention over time; wherein, a: Caspase-3; b: Bax; c: Bcl-2; d: Cyt-c. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0020] The Caspase-3 detection kit, Bax detection kit, Bcl-2 detection kit, and Cyt-c detection kit were all purchased from Wuhan Yunclon Technology Co., Ltd.; Caspase-3 mouse monoclonal antibody (66470-2-Ig), Cyt-c rabbit polyclonal antibody (11242-1-AP), and Lamin B1 rabbit monoclonal antibody (66095-1-Ig): Wuhan Sanying Biotechnology Co., Ltd.; Bcl-2 rabbit polyclonal antibody (AF0060), Bax mouse monoclonal antibody (AF0054), RIPA lysis buffer, PMSF: Shanghai Beyotime Biotechnology Co., Ltd.; Tanon enhanced hypersensitive chemiluminescence solution (ECL): Shanghai Tianneng Technology Co., Ltd.

[0021] Example 1

[0022] A method for indicating the freshness change of grass carp using apoptosis signaling molecules, comprising the following steps:

[0023] (1) Sample collection: Select grass carp with similar body weights and good health as the experimental subjects. Collect muscle tissue samples from the back of the fish at 0 hours, 24 hours, 48 hours, 72 hours, 96 hours, and 120 hours after the grass carp is slaughtered. Collect 3 samples at each time point. Quickly freeze the samples in liquid nitrogen, and then transfer them to a -80°C refrigerator for storage for later use.

[0024] (2) Extraction of apoptosis signaling molecules: Take out the stored muscle tissue samples and thaw them on ice. Weigh 0.5 g of the thawed muscle tissue, add 500 μL of cell lysis buffer (RIPA:PMSF volume ratio 87:1), and homogenize it on ice using a tissue homogenizer. Transfer the homogenate to a centrifuge tube, centrifuge it at 12,000 rpm for 15 minutes at 4°C, and take the supernatant, which is the extraction solution of apoptosis signaling molecules. Store the extraction solution in a -80°C refrigerator for later use.

[0025] (3) Detection of apoptosis signaling molecules:

[0026] ELISA method: According to the instructions of the ELISA kit (Caspase-3 detection kit, Bax detection kit, Bcl-2 detection kit, and Cyt-c detection kit), after appropriately diluting the extraction solution, add it to the enzyme-linked immunosorbent assay (ELISA) plate, and perform operations such as incubation, washing, adding enzyme conjugate, and color development. Finally, measure the absorbance value at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the contents of Caspase-3, Bax, Bcl-2, and Cyt-c in the extraction solution according to the standard curve.

[0027] Western Blot method: Take an appropriate amount of the extract, perform SDS-PAGE electrophoresis, transfer the protein to a PVDF membrane, and block it with 5% skim milk for 2 hours. Add the primary antibodies against Caspase-3, Bax, Bcl-2, and Cyt-c (Caspase-3 mouse monoclonal antibody (66470-2-Ig), Bax mouse monoclonal antibody (AF0054), Bcl-2 rabbit polyclonal antibody (AF0060), Cyt-c rabbit polyclonal antibody (11242-1-AP)) respectively, and incubate overnight at 4°C. The next day, wash the membrane 3 times with TBST for 10 minutes each time, add the corresponding secondary antibody (LaminB1 rabbit monoclonal antibody (66095-1-Ig)), and incubate at room temperature for 1 hour. Wash the membrane 3 times again with TBST for 10 minutes each time, develop the color with a chemiluminescent reagent, observe and analyze the intensity of the bands through a gel imaging system, and calculate the contents of Caspase-3, Bax, Bcl-2, and Cyt-c in the extract according to the content of the standard protein.

[0028] (4) Freshness judgment: By detecting the samples collected at different time points, the changing data of the contents of Caspase-3, Bax, Bcl-2, and Cyt-c over time are obtained. The results are shown in Figure 1 .

[0029] Sampling point at 24 h: The multiple relationships of the contents of Caspase-3, Bax, Bcl-2, and Cyt-c to the freshly killed fresh fish are 2.04 times, 1.31 times, 0.65 times, and 1.06 times respectively. It can be determined that the fish meat is fresh fish meat at this time.

[0030] Sampling point at 48 h: The multiple relationships of the contents of Caspase-3, Bax, Bcl-2, and Cyt-c to the freshly killed fresh fish are 3.97 times, 1.17 times, 0.87 times, and 1.05 times respectively. It can be determined that the fish meat is fresh fish meat at this time.

[0031] Sampling point at 72 h: The multiple relationships of the contents of Caspase-3, Bax, Bcl-2, and Cyt-c to the freshly killed fresh fish are 6.31 times, 2.24 times, 0.87 times, and 1.13 times respectively. It can be determined that the fish meat is not fresh fish meat at this time.

[0032] Sampling point at 96 h: The multiple relationships of the contents of Caspase-3, Bax, Bcl-2, and Cyt-c to the freshly killed fresh fish are 6.00 times, 2.37 times, 0.46 times, and 1.18 times respectively. It can be determined that the fish meat is not fresh fish meat at this time.

[0033] 120 h sampling point: The multiple relationships between the contents of Caspase-3, Bax, Bcl-2, and Cyt-c and the freshly killed fish were 4.74 times, 2.46 times, 0.31 times, and 1.37 times, respectively. It can be determined that the fish meat at this time is not fresh fish meat.

[0034] Basis for judgment: Consumers who often eat grass carp were screened and trained in professional sensory experiments. Finally, 20 members of the sensory evaluation group were determined. The freshness of grass carp was judged using Table 1. The fish meat at different times after the death of grass carp (0 h, 24 h, 48 h, 72 h, 96 h, 120 h) was selected for sensory evaluation (Table 2) to determine the fresh and non-fresh intervals of the fish meat, and the above determination results of the expression levels of Caspase-3, Bax, Bcl-2, and Cyt-c were combined for judgment.

[0035] Table 1 Sensory evaluation criteria for the freshness of grass carp

[0036]

[0037] Note: All indicators that meet the "fresh" standard are judged as fresh. As long as one indicator does not meet the "fresh" standard, the fish is judged as "not fresh". Sensory evaluation is used to judge when the fish starts to be not fresh after death, and the content intervals of apoptotic signaling molecules for fresh and non-fresh are statistically analyzed (the human sense is complex and superior to machines, so the sensory evaluation method of evaluators can better judge the fresh interval of grass carp).

[0038] Table 2 Sensory evaluation results for the freshness of grass carp

[0039]

[0040] Based on the sensory evaluation results of the freshness of grass carp and the detection of the expression levels of relevant indicators at each sampling point, the judgment criteria for the freshness of grass carp were obtained through statistical analysis: When the expression level of Caspase-3 is less than 4 times the expression level of Caspase-3 in fresh fish (A), the expression level of Bax is less than 1.5 times the expression level of Bax in fresh fish (B), the expression level of Bcl-2 is greater than 0.5 times the expression level of Bcl-2 in fresh fish (C), and the expression level of Cyt-c is less than 1.25 times the expression level of Cyt-c in fresh fish (D) are used as the judgment conditions. When all four conditions of A, B, C, and D are met, it can be judged that the grass carp is in the fresh stage; as long as one of the four conditions of A, B, C, and D is not met, it is judged that the grass carp is in the non-fresh stage with a significant difference from the freshly killed live fish.

[0041] The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for indicating changes in fish freshness using apoptosis signal molecules, characterized in that: The following steps are involved: (1) Sample collection: Collect muscle tissue samples of grass carp to be tested, freeze them in liquid nitrogen, and store them in a -80°C refrigerator for later use; (2) Extraction of apoptosis signaling molecules: After thawing the collected muscle tissue samples, add cell lysis buffer, homogenize on ice, and then centrifuge at 12,000 rpm for 15 minutes at 4°C. Take the supernatant to obtain an extract of apoptosis signaling molecules; the mass volume ratio of muscle tissue sample to cell lysis buffer is 1 g:1 mL; (3) Detection of apoptosis signaling molecules: Enzyme-linked immunosorbent assay or protein immunoblotting is used to quantitatively detect apoptosis signaling molecules in the extract; the apoptosis signaling molecules include Caspase-3, Bax, Bcl-2, and Cyt-c; (4) Freshness judgment: Based on the detected content of apoptosis signal molecules, a corresponding relationship model between the content of apoptosis signal molecules and the freshness of grass carp was established. The specific freshness judgment was as follows: when the expression level of Caspase-3 was less than 4 times that of Caspase-3 in fresh fish (A), when the expression level of Bax was less than 1.5 times that of Bax in fresh fish (B), when the expression level of Bcl-2 was greater than 0.5 times that of Bcl-2 in fresh fish (C), and when the expression level of Cyt-c was less than 1.25 times that of Cyt-c in fresh fish (D) as judgment conditions; when the four conditions A, B, C, and D were all met, the grass carp could be judged to be in the fresh stage; as long as one of the four conditions A, B, C, and D was not met, the grass carp was judged to be in the stale stage that was significantly different from the freshly killed live fish.

2. Use of the method of using apoptosis signal molecules to indicate changes in fish freshness as described in claim 1 in detecting fish freshness.

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