CN110940639B - Identification method of dead mutton - Google Patents
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Abstract
The method simultaneously determines the concentration C of the peroxidase in a sample to be detected by carrying out double-label detection on the hemoglobin residue and the peroxidase activity 1 And the concentration C of hemoglobin 2 By the formula R = C 2 /C 1 The method obtains the R value, and establishes the standard threshold value of the R value by collecting a large amount of data of the dead mutton with different causes and different parts, thereby completing the absolute standard of the judgment of the dead mutton. By adopting the detection method, whether the sample to be detected is the slaughtered mutton or the slaughtered mutton can be determined only by carrying out a single experiment on the sample to be detected without being limited by the part of the detected sample.
Description
Technical Field
The invention relates to the field of meat detection, in particular to an identification method of dead mutton.
Background
With the improvement of modern material culture level, the quality requirement of people for meat consumption is higher and higher. However, many trade companies sell the dead mutton serving as high-quality mutton, which seriously threatens the physical health of people. At present, the detection of mutton is still a difficult problem.
The dead mutton refers to raw sheep suffering from infectious diseases, parasitic diseases and toxic diseases harmful to human bodies, non-slaughtered dead raw sheep, mutton which is unqualified by quarantine inspection and cannot be eaten and meat products thereof, wherein the raw sheep are regulated by laws and regulations. For the dead mutton and products thereof with complex sources in the market, the detection and identification of the existing dead mutton 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 current method for inspecting the dead mutton by the market quarantine center mainly comprises sensory inspection, physicochemical inspection and bacteriological inspection. The sensory test mainly comprises visual test, olfactory test and touch test, and mainly comprises the steps of detecting the mouth killing state, the bloodletting 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 mostly judged by experience and has strong subjectivity, meat infected with recessive infection and clinical symptoms are difficult to detect, and the meat detection qualified rate is low. The bacteriological examination mainly inspects the types and the quantity of pathogenic bacteria contained in meat, the bacteriological examination method has strict requirements on experimental conditions and certain requirements on the technology of experimenters, and the aim of rapidly or on-site examining the livestock and poultry meat died of diseases is difficult to achieve. The physical and chemical detection methods mainly comprise a pH value determination method, a peroxidase method, a copper sulfate protein precipitation method, a bacterial endotoxin oxidation and color generation method and the like.
In the traditional method for detecting and identifying dead mutton and slaughtered mutton, pH detection and Peroxidase (POD) activity detection have certain objectivity and become common auxiliary means for identifying the dead mutton at present. In the prior art, the dead pork is identified by detecting peroxidase adopted in the Chinese patent application CN201511001061, but in the identification diagnosis, a healthy pork sample is required to be adopted as a reference at the same time, and the identification diagnosis is carried out by comparison. If the control sample is problematic, the entire identified reference standard is wrong. CN201710320285 adopts a MALDI-TOF-MS-based dead meat identification method, and the MALDI-TOF-MS is applied to determine a sample set to obtain a corresponding mass spectrum; 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 is not capable of being accurately implemented by common technicians. In addition, in both of the above patents, the differential diagnosis using pork as a sample is not known, and it can be accurately applied to the discrimination of mutton.
In addition, in clinical slaughter, the dead sheep 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 phenomena that discrete intervals of the killed mutton and the slaughtered mutton are overlapped to cause indistinguishable overlapping intervals exist in both pH detection and POD detection.
Disclosure of Invention
Aiming at the defects of the prior detection technology, the invention establishes a novel method for identifying the dead mutton 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 the concentration C of hemoglobin 2 Through C 2 /C 1 And acquiring an R value, and establishing a standard threshold value of the R value by collecting a large amount of data of the dead mutton with different causes and different parts, thereby finishing the absolute standard of the judgment of the dead mutton.
The invention provides an identification method of dead mutton, which comprises the following steps:
(1) Preparing a sample treatment solution: taking mutton 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 concentration of hemoglobin in the sample processing liquid in the step (1), wherein the concentration of hemoglobin is C 2 ；
(4) According to the formula R = C 2 /C 1 Calculating the R value;
(5) And (4) identifying the dead mutton according to the R value:
if R is less than or equal to 15, judging the detection sample to be slaughtered mutton;
and if the R is more than or equal to 25, judging that the detected sample is the dead mutton.
Further, the detection sample in the step (1) is muscle tissue of mutton to be detected; the mutton to be detected is fresh mutton or frozen mutton.
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 stirring and pulverizing are as follows: 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 of detection results and the stability.
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: and 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 the sample at a 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 at 37 ℃ for 2 hours, then adding a hemoglobin antibody, incubating at 37 ℃ for 1 hour, washing the micropore plate for 3 times, drying, adding an HRP label, incubating at 37 ℃ for 1 hour, 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 And concentration C 2 The units of (A) 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 peroxides, 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 slaughter, the dead sheep often have incomplete bleeding, resulting in residual hemoglobin. Therefore, meat-like hemoglobin residues are potential targets for identification of dead mutton. However, the phenomenon that the detection discrete intervals of the dead mutton and the slaughtered mutton are overlapped when the two parameters are independently applied, so that the overlapped intervals cannot be distinguished exists. The single detection of the method can not establish the absolute standard for identifying the dead mutton.
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 the concentration C of hemoglobin 2 Through C 2 /C 1 Obtaining R value, and establishing standard threshold value of R value by collecting a large amount of data of the dead mutton with different causes and different parts, thereby completing the judgment of the dead muttonAbsolute standards. By adopting the detection method, whether the sample to be detected is the dead mutton or the slaughtered mutton can be determined 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 and inaccurate determination by adopting a colorimetric method.
The detection method provided by the invention simplifies the determination 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 mutton to be detected can be not only fresh mutton, but also mutton frozen within 20 days.
Drawings
Figure 1 is the total concentration profile of peroxidase detected after different immersion times for the dead and slaughtered mutton samples.
Figure 2 is a graph of the hemoglobin concentration profiles measured for the dead mutton and the slaughtered mutton samples after different immersion times.
Fig. 3 is a graph showing hemoglobin concentration distributions measured after the samples of the killed mutton and the slaughtered mutton are soaked for 10 minutes.
Fig. 4 is a graph showing hemoglobin concentration distributions detected after the samples of the killed mutton and the slaughtered mutton are soaked for 30 minutes.
Figure 5 is the hemoglobin concentration profile measured after immersion of dead and slaughtered mutton samples for 60 minutes.
Fig. 6 is a hemoglobin concentration profile measured after the immersion of the dead mutton and the slaughtered mutton samples for 90 minutes.
Fig. 7 is a hemoglobin concentration profile measured after immersion of dead mutton and slaughtered mutton samples for 120 minutes.
Figure 8 is the distribution of R-values detected after different immersion times for the dead mutton and the slaughtered mutton samples.
Figure 9 is the distribution of R-values detected after 10 minutes of immersion of dead and slaughtered lamb samples.
Figure 10 is the distribution of R-values detected after 30 minutes of immersion of dead and slaughtered lamb samples.
Figure 11 is the distribution of R values detected after immersion of dead and slaughtered mutton samples for 60 minutes.
Figure 12 is the distribution of R values detected after 90 minutes immersion of dead and slaughtered mutton samples.
Figure 13 is the distribution of R-values detected after 120 minutes of immersion of dead and slaughtered lamb samples.
Figure 14 is a differential significance analysis of the R values detected after different immersion times for the dead and slaughtered lamb 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 collecting 4 slaughtered sheep, and collecting 6 different parts (A. Tommy meat, B. Streaky pork, C. Front hip tip, D. Front leg meat, E. Rear hip tip and F. Rear leg meat) to obtain 24 parts of slaughtered mutton from a certain slaughterhouse in Shijiazhuang city. Respectively collecting 5 dead sheep, and 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 mutton died by illness, wherein the mutton is from a certain innocent treatment factory of Shijiazhuang. 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 calibration curve was prepared by diluting horseradish peroxidase (SIGMA) solution (10 mg/L) 1000-fold and then diluting it by a double ratio. POD values (marked as C1) of the mutton samples are obtained through calculation, and experimental data and comparison results are shown in figure 1.
As can be seen from FIG. 1, the POD values of the C, D, E and F parts of the slaughtered mutton and the diseased and dead mutton are distributed in the same interval and cannot be distinguished. It follows that the use of peroxide alone for detection does not allow discrimination between slaughtered and dead mutton.
4. Sheep hemoglobin ELISA detection
And (3) taking the sample treatment solution in the step (2), and detecting the content (marked as C2) of the sheep hemoglobin. The sheep hemoglobin ELISA detection method is described in the specification of a kit (cat # DL-HB-g, DEVELOP Co.). 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 content of the sheep hemoglobin detected by each sample are shown in figure 2.
As can be seen from FIG. 2, the hemoglobin values of the B and C parts of the slaughtered mutton are partially crossed with the hemoglobin value distribution interval of the slaughtered mutton, which cannot be distinguished. Therefore, hemoglobin is independently adopted for detection, and the slaughtered mutton and the dead mutton cannot be distinguished.
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 mutton samples changes obviously, so that serious cross phenomenon exists and the mutton samples are difficult to distinguish.
5. The peroxidase concentration C detected in each sample was measured 1 And its hemoglobin concentration C 2 The R value is calculated by substituting into the following equation:
calculating the R value according to formula I;
R＝C 2 /C 1 (formula I);
the experimental data and the comparative results obtained are shown in fig. 9.
As shown in figure 9, the R value interval of the slaughtered mutton and the slaughtered mutton samples is obviously distinguished, and the R value of the diseased and dead mutton is obviously higher than that of the slaughtered sheep.
The oxidase concentration, hemoglobin concentration and R value measured at different parts of slaughtered sheep and slaughtered sheep under 10-minute immersion are shown in Table I.
TABLE oxidases concentration, hemoglobin concentration and R-value of each sample at 10 minutes immersion
6. Determination of standard of identification of dead mutton
By further analysis of the results in figure 9, R values between 20 and-30 are effectively isolated intervals to distinguish between dead and slaughtered mutton. More precisely, the R value of the slaughtered mutton is more than 25, and the R values of the slaughtered mutton are all below 15. Therefore, according to the above data, the identification standard of the dead mutton can be obtained. Namely:
and (3) identifying the dead mutton according to the R value obtained by calculation:
r is less than or equal to 15, and the detection sample is judged to be slaughtered mutton;
and R is more than or equal to 25, and the detected sample is judged to be dead mutton.
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 30-minute immersion in different parts of slaughtered and dead sheep are shown in Table two. Statistical analysis is carried out on the mutton sample, as shown in figure 10, the R value interval of the slaughtered mutton and the slaughtered mutton sample is obviously distinguished, and the R value of the diseased and dead mutton is obviously higher than that of the slaughtered sheep.
TABLE II oxidase concentration, hemoglobin concentration and R value of each sample at 30 minutes of 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 60 minutes immersion in different parts of slaughtered and dead sheep are shown in Table three. Statistical analysis is carried out on the mutton, as shown in figure 11, the R value intervals of the samples of the slaughtered mutton and the dead mutton are obviously distinguished, and the R value of the ill-killed mutton is obviously higher than that of the slaughtered sheep.
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 when the sample treatment solution was prepared, and the other steps were the same as those in the first embodiment.
The oxidase concentration, hemoglobin concentration and R value measured at 90-minute immersion in different parts of slaughtered and dead sheep are shown in Table four. Statistical analysis is carried out on the mutton sample, as shown in figure 12, the R value interval of the slaughtered mutton and the slaughtered mutton sample is obviously distinguished, and the R value of the diseased and dead mutton is obviously higher than that of the slaughtered sheep.
TABLE IV oxidase concentration, hemoglobin concentration and R value of each sample at 90 min immersion
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 different parts of slaughtered sheep and slaughtered sheep under 120-minute immersion are shown in Table five. Statistical analysis is carried out on the mutton sample, as shown in figure 13, the R value interval of the slaughtered mutton and the slaughtered mutton sample is obviously distinguished, and the R value of the diseased and dead mutton is obviously higher than that of the slaughtered sheep.
TABLE five oxidase concentration, hemoglobin concentration and R value of each sample at 120 minutes immersion
Fig. 8 is a graph showing the distribution of R-values detected after different immersion times for the samples of the killed and slaughtered mutton, i.e., the results of examples one to five were summarized. By further analysis of the results in fig. 8, R values between 20-30 are effectively isolated intervals to distinguish between slaughtered mutton and slaughtered mutton. The effective identification criteria for the dead mutton according to the calculated R value are as follows:
r is less than or equal to 15, and the detection sample is judged to be slaughtered mutton;
and R is more than or equal to 25, and the detected sample is judged to be dead mutton.
More precisely, the R value of the slaughtered mutton is more than 25, and the R values of the slaughtered mutton are all below 15. Therefore, according to the above data, a more stringent identification standard of the dead mutton can be obtained. Namely:
r is less than or equal to 15, and the detection sample is judged to be slaughtered mutton;
and R is more than or equal to 25, and the detected sample is judged to be dead mutton.
A schematic diagram showing the detailed comparison of the R value detection results under each time condition is shown in fig. 9 to 13, and the data of all the slaughtered mutton and the slaughtered mutton 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 mutton died of illness and the mutton slaughtered 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 10 minutes, the distinguishing area of the dead mutton and the healthy mutton is most obvious.
As can be seen from fig. 14, the significant difference analysis results of the R values detected after the samples of the dead mutton and the slaughtered mutton are subjected to different immersion times are very significant, and therefore, the R value is reliable as an index for distinguishing the dead mutton from the healthy mutton.
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 to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
1. The identification method of the dead mutton is characterized by comprising the following steps:
(1) Preparing a sample treatment solution: taking mutton 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 concentration of hemoglobin in the sample treatment solution in the step (1) by adopting an enzyme-linked immunosorbent assay, wherein the concentration of hemoglobin is C 2 ；
(4) According toFormula R = C 2 /C 1 Calculating an R value;
(5) And (3) identifying the dead mutton according to the R value:
if R is less than or equal to 15, judging the detection sample to be slaughtered mutton;
if R is more than or equal to 25, judging the detection sample as dead mutton;
by adopting the identification method, only a single experiment is carried out on the detection sample, and whether the detection sample is the slaughtered mutton or the slaughtered mutton can be determined without the limitation of the position of the detection sample.
2. The method for identifying dead mutton according to claim 1, wherein the sample to be detected in the step (1) is muscle tissue of mutton to be detected, and the mutton to be detected is fresh mutton or frozen mutton.
3. The method for identifying dead mutton according to claim 1, wherein in the step (1), the conditions of stirring and crushing are as follows: the rotating speed is 16000r/min, and the time is 30 seconds.
4. The method for identifying dead mutton according to claim 1, wherein in the 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 mutton according to claim 1, wherein in the step (2), tetramethylbenzidine and carbamide peroxide are used as substrates, and the concentration of peroxidase is measured by a colorimetric method.
6. The method for identifying dead mutton according to claim 5, wherein the specific determination method of peroxidase concentration comprises: sequentially adding sodium dihydrogen phosphate buffer solution, carbamide peroxide solution, sample treatment solution and tetramethylbenzidine 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; 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 killed mutton as claimed in claim 1, wherein the detection method of hemoglobin concentration in step (3) is enzyme-linked immunosorbent assay, and 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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