CN110763815B - Identification method of dead beef - Google Patents
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Abstract
The invention simultaneously determines the concentration C of peroxidase in a sample to be detected by carrying out double-standard detection on the hemoglobin residue and the peroxidase activity 1 And concentration C of hemoglobin 2 By the formula R = C 2 /C 1 *100 percent, obtaining the R value, and establishing a standard threshold value of the R value by collecting a large amount of data of the dead beef due to different causes and different parts, thereby finishing the absolute standard of the judgment of the dead beef. By adopting the detection method, whether the sample to be detected is the slaughtered beef or the slaughtered beef can be determined only by carrying out a single experiment on the sample to be detected without being limited by the position of the detected sample.
Description
Technical Field
The invention relates to the field of meat detection, in particular to an identification method of dead beef.
Background
With the improvement of modern material culture level, the quality requirement of people for meat consumption is higher and higher. However, the dead beef is sold by many merchants as high-quality beef, and the health of people is seriously threatened. At present, the detection of beef is still a difficult problem.
The dead beef refers to raw cattle suffering from infectious diseases, parasitic diseases and toxic diseases harmful to human bodies, non-slaughtered dead raw cattle, and beef and meat products thereof which are unqualified for quarantine inspection and can not be eaten, which are specified by laws and regulations. For the dead beef and products thereof with complex sources in the market, the detection and identification of the current dead beef in China are quite lacking. The meat hygiene test is a relatively complex process, and mainly comprises meat freshness test and meat health test. The existing market quarantine center inspection method for dead beef mainly comprises sensory inspection, physicochemical inspection and bacteriological inspection. The sensory examination mainly comprises visual examination, sniffing examination and touch examination, and mainly comprises the examination of the mouth killing state, the bleeding degree, the blood weighing condition, the pathological changes of tissues and lymph nodes, the abnormal smell of meat and the like. Although the detection method is simple, the detection method is judged by experience mostly and has strong subjectivity, some recessive infection and infected meat without clinical symptoms are difficult to detect, and the meat detection qualified rate is low. The bacteriological inspection is mainly used for inspecting the types and the quantity of pathogenic bacteria contained in meat, the experimental condition of the bacteriological inspection method is strict, certain requirements are provided for the technology of experimenters, and the aim of rapidly or on-site inspecting the livestock and poultry meat died of diseases is difficult to achieve. The physical and chemical detection method mainly comprises a pH value determination method, a peroxidase method, a copper sulfate protein precipitation method, a bacterial endotoxin oxidation color generation method and the like.
In the traditional method for detecting and identifying the dead beef and the slaughtered beef, pH detection and Peroxidase (POD) activity detection have certain objectivity and become a common auxiliary means for identifying the dead beef at present. In the prior art, the dead pork is identified by detecting peroxidase adopted in the Chinese patent application CN201511001061, but the identification diagnosis needs to be carried out by comparing healthy pork samples used as a control in the identification diagnosis. If the control sample is problematic, the entire identified reference standard is wrong. CN201710320285 adopts a dead meat identification method based on MALDI-TOF-MS, and MALDI-TOF-MS is applied to determine a sample set to obtain a corresponding mass spectrogram; establishing a neural network classifier based on the mass spectrogram of the training sample set and the testing sample set; and classifying the samples in the sample set to be detected by using a neural network classifier to obtain an identification result. The method has the advantages of expensive equipment, complex operation and high technical difficulty, and can not be accurately implemented by common technicians. In addition, in both of the above patents, it is unknown that the diagnostic method can be accurately applied to beef identification by using pork as a sample.
In addition, in clinical slaughter, the dead cattle often have incomplete bleeding, resulting in residual hemoglobin. Therefore, hemoglobin can also be used as an index for evaluating health conditions of meat.
However, in actual work, the meat sample processing method and the detection conditions are inconsistent, and the detection result is difficult to standardize and measure. In addition, the pH detection and the POD detection both have the phenomenon that discrete intervals of the dead beef and the slaughtered beef are overlapped, so that the overlapped intervals cannot be distinguished.
Disclosure of Invention
Aiming at the defects of the prior detection technology, the invention establishes a novel method for identifying the dead beef through research, carries out double-standard detection on the hemoglobin residue and the peroxidase activity, and simultaneously determines the concentration C of the peroxidase in a sample to be detected 1 And concentration C of hemoglobin 2 Through C 2 /C 1 * And obtaining an R value 100%, and establishing a standard threshold value of the R value by collecting a large amount of data of the dead beef due to different causes and different parts, thereby finishing the absolute standard of the determination of the dead beef.
The invention provides an identification method of dead beef, which comprises the following steps:
(1) Preparing a sample treatment solution: taking beef to be detected as a detection sample, adding a buffer solution, stirring and crushing, soaking to obtain a soaking solution, centrifuging the soaking solution, and taking supernatant to obtain a sample treatment solution;
(2) Detecting the concentration of peroxidase in the sample processing solution in the step (1), wherein the concentration of the peroxidase is C 1 ；
(3) Detecting the sample processing liquid in the step (1)The concentration of hemoglobin of (2), the concentration of hemoglobin being C 2 ；
(4) According to the formula R = C 2 /C 1 * Calculating the R value by 100 percent;
(5) And (3) identifying the dead beef according to the R value:
if R is less than or equal to 25%, judging that the detection sample is slaughtered beef;
and if the R is more than or equal to 35%, judging that the detected sample is the dead beef.
Further, the detection sample in the step (1) is the muscle tissue of the beef to be detected; the beef to be detected is fresh beef or frozen beef.
Further, the mass of the buffer solution added in the step (1) is 9-12 times of that of the detection sample;
further, dipping for 8-15 minutes in the step (1) to obtain a dipping solution;
further, the detection method of the hemoglobin concentration in the step (3) is an enzyme-linked immunosorbent assay;
further, in the step (1), the conditions for the agitation pulverization are: the rotating speed is 16000r/min, and the time is 30 seconds. According to the invention, researches show that the mechanical stirring vortex method is superior to a grinder oscillation method in the aspects of the completeness of muscle treatment, the precision and the stability of detection results.
Further, in the step (1), the time for immersion is 8 to 10 minutes. According to the invention, through detecting samples with different soaking times, the fact that the peroxidase activity, the hemoglobin concentration and the R value detection result are not obviously influenced by prolonging the soaking time of the soaking solution is discovered. Therefore, in order to shorten the detection time, the detection requirement of the present invention can be satisfied by setting the immersion time to 8 to 10 minutes.
Further, in the step (1), the conditions of centrifugation are: the rotating speed is 3000-4000 r/min, and the time is 2-4 minutes.
Further, in the step (2), tetramethylbenzidine and carbamide peroxide are used as substrates, and the concentration of the peroxidase is measured by a colorimetric method.
The specific determination method comprises the following steps: sequentially adding sodium dihydrogen phosphate buffer solution, carbamide peroxide solution, sample treatment solution and tetramethyl benzidine solution, reacting for 1-2 min at room temperature, adding sulfuric acid solution to terminate the reaction, measuring the absorbance OD value of the sample at the wavelength of 450nm, and obtaining the concentration of peroxidase in the sample treatment solution through a standard curve.
The standard curve is prepared by diluting horseradish peroxidase solution (10 mg/L) 1000 times, and then diluting by times.
Further, in step (3), the enzyme-linked immunosorbent assay at least comprises the following steps: preparing a standard substance, a reagent and a sample to be detected before an experiment; respectively adding a standard substance and a sample into the micropores, incubating for 2 hours at 37 ℃, then adding a hemoglobin antibody, incubating for 1 hour at 37 ℃, washing the micropore plate for 3 times, adding an HRP (horse radish peroxidase) label after drying, incubating for 1 hour at 37 ℃, thoroughly washing the micropore plate for 5 times, adding a TMB substrate, developing for 15-25min, and adding a stop solution. The absorbance (o.d. value) was measured at a wavelength of 450nm with a microplate reader, and the sample concentration was calculated.
Further, in the step (4), the concentration C 1 With concentration C 2 The units are the same and are all ng/mL.
Advantageous effects
The meat of healthy livestock and poultry contains peroxidase, and the peroxidase in the meat is obviously reduced or even completely disappeared when the livestock and poultry are in a pathological state or an dying state. Peroxidases have the property of cleaving oxygen from peroxide, which oxidizes amines to form colored compounds, the degree of color change of which is proportional to the amount of peroxidase in the meat. In clinical slaughtering, the phenomenon that the bleeding of a dead cattle is incomplete, so that hemoglobin is remained is common. Therefore, meat-like hemoglobin residues are potential targets for identification of dead beef. However, when the two parameters are independently applied, the phenomenon that discrete detection intervals of the dead beef and the slaughtered beef are overlapped, so that the overlapped intervals cannot be distinguished exists. The single detection of the method can not establish the absolute standard of the dead beef identification.
The method carries out double-standard detection on the hemoglobin residue and the peroxidase activity through sharp research, and simultaneously determines the concentration C of the peroxidase in a sample to be detected 1 And concentration C of hemoglobin 2 Through C 2 /C 1 *100 percent, obtaining the R value, and establishing a standard threshold value of the R value by collecting a large amount of data of the dead beef due to different causes and different parts, thereby finishing the absolute standard of the judgment of the dead beef. By adopting the detection method, the sample to be detected can be determined to be the dead beef or the slaughtered beef only by carrying out a single experiment on the sample to be detected without the limitation of the position of the detected sample.
The invention detects the hemoglobin concentration of the sample treatment solution by an enzyme-linked immunosorbent assay, and overcomes the defects of interference of myoglobin by a colorimetric method and inaccurate determination.
The detection method simplifies the judgment steps, shortens the detection time, does not need to set negative and positive detection control groups, improves the detection efficiency, reduces the workload of experimenters, improves the accuracy of the detection result and ensures the authority of the detection result.
In the invention, the beef to be detected can be not only fresh beef, but also frozen beef with the freezing storage time within 20 days.
Drawings
FIG. 1 is a graph showing the distribution of the total concentration of peroxidase detected in samples of dead beef and slaughtered beef after different immersion times.
FIG. 2 is a graph showing the hemoglobin concentration distributions of the dead beef and the slaughtered beef samples measured after different immersion times.
FIG. 3 is a graph showing the hemoglobin concentration distribution measured after immersing samples of slaughtered beef and slaughtered beef for 10 minutes.
FIG. 4 is a graph showing the hemoglobin concentration distribution measured after 30-minute immersion of the dead beef and the slaughtered beef samples.
FIG. 5 is a graph showing the hemoglobin concentration distribution measured after the samples of the dead beef and the slaughtered beef are immersed for 60 minutes.
FIG. 6 is a graph showing the hemoglobin concentration distribution measured after the samples of the dead beef and the slaughtered beef are immersed for 90 minutes.
FIG. 7 is a graph showing the hemoglobin concentration distribution measured after the samples of the dead beef and the slaughtered beef are immersed for 120 minutes.
FIG. 8 is a graph showing the distribution of R values detected after different immersion times for samples of slaughtered beef and slaughtered beef.
FIG. 9 is the distribution of the R values detected after 10 minutes of immersion of the dead beef and the slaughtered beef samples.
FIG. 10 is a graph showing the distribution of R values detected after 30 minutes of immersion of the dead beef and the samples of the slaughtered beef.
FIG. 11 is a graph showing the distribution of R values detected after the samples of the dead beef and the slaughtered beef have been immersed for 60 minutes.
FIG. 12 is a graph showing the distribution of R values detected after 90 minutes of immersion of the dead beef and the samples of the slaughtered beef.
FIG. 13 is the distribution of the R values detected after the samples of the dead beef and the slaughtered beef have been immersed for 120 minutes.
FIG. 14 is a graph showing the differential significance analysis of the R values detected after different immersion times for the dead and slaughtered beef samples.
Detailed Description
The technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings, and it is to be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
Example one
1. Sample collection
The method comprises the steps of respectively collecting 4 slaughtered cattle, and collecting 6 different parts (A. Tom-bone-free meat, B. Streaky meat, C. Front hip tip, D. Front leg meat, E. Rear hip tip and F. Rear leg meat) to obtain 24 parts of slaughtered beef from a certain slaughterhouse in Baoding city. The method comprises the following steps of respectively collecting 5 dead cattle, taking 6 different parts (A. Tongqian meat, B. Streaky pork, C. Front hip tip, D. Front leg meat, E. Rear hip tip and F. Rear leg meat) to obtain 30 parts of dead beef from a certain innocent treatment factory in Chengde City. About 50g of each meat sample is collected and frozen at the temperature of minus 20 ℃ for standby.
2. Preparation of sample treatment solution
And (3) freezing a sample to be detected, taking 10g of muscle tissue, adding 100mL of PBS into a stirrer, stirring for 30 seconds at the maximum rotating speed until the stirring is complete, then transferring the muscle tissue into a 200mL beaker, uniformly stirring by using a glass rod, soaking for 10 minutes, taking 4mL of soaking solution, centrifuging for 3 minutes at 3000r/min, and taking 2mL of supernatant for detection.
3. Peroxidase detection
2mL of sodium dihydrogen phosphate buffer (1.56 g/100mL, pH 4.5), 0.2mL of urea peroxide solution (0.94 g/100 mL) (Shanghai ' an Ji chemical Co., ltd.), 0.2mL of sample meat extract, 0.2mL of 3,3', 5' -tetramethylbenzidine solution (TMB) (0.065 g/100 mL) (BIOTOPPED) were placed in the cuvette in this order, reacted at room temperature for 1min, the reaction was terminated by adding a sulfuric acid solution, and the absorbance (OD) of the sample was measured at a wavelength of 450 nm. A standard curve was prepared by diluting horseradish peroxidase (SIGMA) solution (10 mg/L) 1000 times, and then diluting the diluted solution by a multiple ratio. The POD value (denoted as C1) of each beef sample was obtained by calculation, and the experimental data and the comparison results are shown in fig. 1.
As can be seen from FIG. 1, the POD value of slaughtered beef and the parts A, C, D and F of slaughtered beef are distributed in the same interval and cannot be distinguished. It follows that the detection with peroxide alone does not allow to distinguish between slaughtered and dead beef.
4. Bovine hemoglobin ELISA detection
And (3) taking the sample treatment solution in the step (2), and detecting the content (marked as C2) of the bovine hemoglobin. Bovine hemoglobin ELISA detection method reference kit (cat # DL-HB-b, DEVELOP Co., ltd.) instructions. The method comprises the following specific steps:
the enzyme-linked immunosorbent assay method comprises the following steps: preparing a standard substance, a reagent and a sample to be detected before an experiment; respectively adding a standard substance and a sample into the micropores, incubating for 2 hours at 37 ℃, then adding a hemoglobin antibody, incubating for 1 hour at 37 ℃, washing the micropore plate for 3 times, adding an HRP (horse radish peroxidase) label after drying, incubating for 1 hour at 37 ℃, thoroughly washing the micropore plate for 5 times, adding a TMB substrate, developing for 15-25min, and adding a stop solution. And (3) measuring the absorbance O.D. value under the wavelength of 450nm of a microplate reader, and calculating the concentration of the sample.
The experimental data and the comparative results of the bovine hemoglobin content of each sample are shown in fig. 2.
As can be seen from FIG. 2, the hemoglobin values of the A and B parts of the slaughtered beef are the same as the distribution intervals of the A, B and D parts of the slaughtered beef, and cannot be distinguished. Therefore, the detection by adopting the hemoglobin alone cannot distinguish the slaughtered beef from the dead beef.
The schematic diagrams of detailed comparison of hemoglobin detection results under various time conditions are shown in fig. 3 to 7.
As can be seen from FIGS. 3 to 7, the hemoglobin content in different beef samples changes obviously, and serious cross phenomena exist, which are difficult to distinguish.
5. The R value was calculated by substituting the detected peroxidase concentration C1 and the hemoglobin concentration C2 in each sample into the following equation:
calculating the R value according to formula I;
r = C2/C1 100% (formula I);
the experimental data and the comparative results obtained are shown in fig. 9.
As shown in FIG. 9, the R value interval of the slaughtered beef and the dead beef samples is obviously distinguished, and the R value of the dead beef is obviously higher than that of the slaughtered beef.
The oxidase concentration, hemoglobin concentration and R value measured at the time of immersion for 10 minutes in the different parts of slaughtered cattle and dead cattle are shown in Table I.
TABLE oxidases concentration, hemoglobin concentration and R-value of each sample at 10 minutes immersion
6. Determination of the criteria for the identification of dead beef
By further analysis of the results in fig. 9, R values between 20% -40% are valid isolation intervals to distinguish slaughtered beef from slaughtered beef. More precisely, the R value of the slaughtered beef is more than 35%, and the R value of the slaughtered beef is less than 25%. Therefore, according to the above data, the identification standard of the dead beef can be obtained. Namely:
and (3) identifying the dead beef according to the R value obtained by calculation:
r is less than or equal to 25 percent, and the detection sample is judged to be slaughtered beef;
and R is more than or equal to 35 percent, and the detected sample is judged to be dead beef.
Example two
In this example, the immersion time was 30 minutes in preparing the sample treatment solution, and the other steps were the same as those in the first embodiment.
The oxidase concentration, hemoglobin concentration and R value measured at different parts of slaughtered cattle and dead cattle under 30-minute immersion are shown in Table two. Statistical analysis is carried out on the beef, as shown in figure 10, R value intervals of samples of slaughtered beef and dead beef are obviously distinguished, and the R value of the dead beef is obviously higher than that of the slaughtered beef.
TABLE II oxidase concentration, hemoglobin concentration and R value of each sample at 30 minutes immersion
EXAMPLE III
In this example, the immersion time was 60 minutes in preparing the sample treatment solution, and the other steps were the same as those in the first embodiment.
The oxidase concentration, hemoglobin concentration and R value measured at different parts of slaughtered cattle and dead cattle under 60-minute immersion are shown in Table three. Statistical analysis is carried out on the samples, as shown in figure 11, the R value intervals of the samples of the slaughtered beef and the dead beef are obviously distinguished, and the R value of the dead beef is obviously higher than that of the slaughtered beef.
TABLE III oxidase concentration, hemoglobin concentration and R value of each sample after 60 minutes immersion
Example four
In this example, the immersion time was 90 minutes in preparing the sample treatment solution, and the other steps were the same as those in the first embodiment.
The oxidase concentration, hemoglobin concentration and R value measured at different parts of slaughtered cattle and dead cattle under 90-minute immersion are shown in Table four. Statistical analysis is carried out on the beef, as shown in figure 12, R value intervals of samples of slaughtered beef and dead beef are obviously distinguished, and the R value of the dead beef is obviously higher than that of the slaughtered beef.
TABLE IV oxidase concentration, hemoglobin concentration and R value of each sample immersed for 90 minutes
EXAMPLE five
In this example, the immersion time was 120 minutes in preparing the sample treatment solution, and the other steps were the same as those in the first embodiment.
The oxidase concentration, hemoglobin concentration and R value measured at 120 minutes of immersion in different parts of slaughtered cattle and dead cattle are shown in Table five. Statistical analysis is carried out on the beef, as shown in fig. 13, R value intervals of samples of slaughtered beef and dead beef are obviously distinguished, and the R value of the dead beef is obviously higher than that of the slaughtered beef.
TABLE five oxidase concentration, hemoglobin concentration and R value of each sample at 120 minutes immersion
FIG. 8 is the distribution of the R values detected after different immersion times for the dead beef and the slaughtered beef samples, i.e., the results of examples one to five are summarized. By further analysis of the results in fig. 8, R values between 20% and 40% are valid isolation intervals to distinguish slaughtered beef from slaughtered beef.
More precisely, the R value of the slaughtered beef is more than 35%, and the R value of the slaughtered beef is less than 25%. Therefore, according to the above data, the identification standard of the dead beef can be obtained. Namely:
r is less than or equal to 25 percent, and the detection sample is judged to be slaughtered beef;
and R is more than or equal to 35 percent, and the detected sample is judged to be the dead beef.
A schematic diagram showing the detailed comparison of the detection results of the R values under the respective time conditions is shown in fig. 9 to 13, and the data of all the slaughtered beef and the slaughtered beef are processed and analyzed to obtain fig. 14.
As can be seen from fig. 9 to 13: compared with single hemoglobin target differentiation, the R values of the slaughtered beef and the slaughtered beef are obviously differentiated under each soaking time, and the difference is extremely obvious.
Under each time condition, the identification accuracy is over 90 percent, and at 60 minutes, the distinguishing area of the dead beef and the healthy beef is most obvious.
As can be seen from fig. 14, the analysis result of the significance of the difference between the R values detected after the samples of the dead beef and the slaughtered beef have been subjected to different dipping times is extremely significant, and therefore, it is reliable to use the R value as an index for distinguishing the dead beef from the healthy beef.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered as the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (7)
1. The identification method of the dead beef is characterized by comprising the following steps:
(1) Preparing a sample treatment solution: taking beef to be detected as a detection sample, adding a buffer solution, stirring and crushing, adding the buffer solution with the mass 9-12 times that of the detection sample, soaking for 8-15 minutes to obtain a soaking solution, centrifuging the soaking solution, and taking supernatant to obtain the sample treatment solution;
(2) Detecting the concentration of peroxidase in the sample treatment solution in the step (1), wherein the concentration of the peroxidase is C 1 ；
(3) Detecting the hemoglobin concentration of the sample treatment solution in the step (1) by adopting an enzyme-linked immunosorbent assay, wherein the concentration of the hemoglobin is C 2 ；
(4) According to the formula R = C 2 /C 1 * Calculating the R value by 100 percent;
(5) And (3) identifying the dead beef according to the R value:
if R is less than or equal to 25%, judging that the detection sample is slaughtered beef;
if R is more than or equal to 35%, judging the detection sample as dead beef;
by adopting the identification method, only a single experiment is carried out on the detection sample, and whether the detection sample is the dead beef or the slaughtered beef can be determined without the limitation of the position of the detection sample.
2. The method for identifying dead beef as claimed in claim 1, wherein the test sample in step (1) is the muscle tissue of the beef to be tested, and the beef to be tested is fresh beef or frozen beef.
3. The method for identifying dead beef as claimed in claim 1, wherein in the step (1), the conditions for stirring and grinding are as follows: the rotating speed is 16000r/min, and the time is 30 seconds.
4. The method for identifying dead beef as claimed in claim 1, wherein in step (1), the centrifugation conditions are as follows: the rotating speed is 3000-4000 r/min, and the time is 2-4 minutes.
5. The method for identifying dead beef as claimed in claim 1, wherein in step (2), tetramethylbenzidine and carbamide peroxide are used as substrates, and the peroxidase concentration is determined colorimetrically.
6. The method for identifying dead beef as claimed in claim 5, wherein the specific determination method of peroxidase concentration is: sequentially adding a sodium dihydrogen phosphate buffer solution, a urea peroxide solution, a sample treatment solution and a tetramethylbenzidine solution, reacting for 1-2 min at room temperature, adding a sulfuric acid solution to terminate the reaction, measuring the absorbance OD value of a sample at a wavelength of 450nm, and obtaining the concentration of peroxidase in the sample treatment solution through a standard curve; wherein, the standard curve is prepared by diluting the horseradish peroxidase solution with the concentration of 10mg/L by 1000 times, and then diluting by times.
7. The method for identifying dead beef as claimed in claim 1, wherein the enzyme-linked immunosorbent assay comprises the following steps: preparing a standard substance, a reagent and a sample to be detected before an experiment; respectively adding a standard substance and a sample into the micropores, incubating for 2 hours at 37 ℃, then adding a hemoglobin antibody, incubating for 1 hour at 37 ℃, washing the microporous plate for 3 times, spin-drying, adding an HRP (horse radish peroxidase) label, incubating for 1 hour at 37 ℃, thoroughly washing the microporous plate for 5 times, adding a TMB substrate, developing for 15-25min, and adding a stop solution; and (4) measuring the OD value of the absorbance under the wavelength of 450nm of a microplate reader, and calculating the concentration of the sample.
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