Method for rapidly distinguishing black pork from white pork

Abstract

The invention relates to a method for rapidly distinguishing black pork from white pork, in particular to a method for rapidly distinguishing black pork from white pork. The method solves the problems that an existing method for distinguishing black pork from white pork is high in operation difficulty, complex in operation, long in time and incapable of achieving rapid and effective distinguishing. The method comprises the following steps: 1, preparing homogenate; ultrasonically crushing or repeatedly freezing and thawing the homogenate, and centrifuging to take supernate; and 2, measuring the content of DHA in the supernatant. According to the method, the content of DHA is rapidly and effectively measured, so that black pork and white pork are distinguished. The method disclosed by the invention is simple to operate, does not need to use expensive instruments and equipment, is short in detection period, and greatly reduces the distinguishing cost of black pork and white pork.

Description

Technical Field

[0001] The invention relates to a method for distinguishing black pork from white pork. Background Art

[0002] Pork is the main meat variety consumed by Chinese people. White pork is the main variety of pork in the domestic market. Although the sales volume of local black pork is small, it is famous for its delicious taste, flavor and high quality. Since the breeding cycle of black pigs is longer than that of white pigs, and the taste and nutritional accumulation are better than those of white pigs, the price of black pork in the market is higher than that of white pork. The public's evaluation method of black pork and white pork is mostly based on superficial observation, such as meat color, but the evaluation method is not scientific enough. Therefore, illegal vendors disguise white pork as black pork to increase profits.

[0003] Therefore, a large number of researchers have conducted relevant research on the evaluation methods of black pigs and white pork. For example, Li Ying et al. evaluated the meat quality of Hainan black pork, Sanjiang white pork, and Hubei white pork from three different breeds of pigs based on the texture parameters of pork, such as adhesion, elasticity, cohesion, colloid, resilience, chewiness, hardness, and shear force, but were unable to measure the main differences in the nutritional chemistry of their meat. Zhao Mengyao et al. studied the hind legs of white pigs (large Yorkshire pigs, 5 to 6 months old) and black pigs (Southern Henan black pigs, 1 month old) and compared the flavor substances of the two meat flavors by using SAFE extraction, combined with gas chromatography-mass spectrometry (GC-MS) online and AEDA-GC-O. However, the instruments used were expensive, difficult to operate, complicated, and time-consuming, and could not be distinguished quickly and effectively. Therefore, it is crucial to establish a method that is fast, simple, efficient, and suitable for distinguishing black pigs from white pork. Summary of the invention

[0004] The present invention provides a method for quickly distinguishing black pork from white pork in order to solve the problems that the existing method for distinguishing black pork from white pork is difficult to operate, complicated, time-consuming and cannot be distinguished quickly and effectively.

[0005] The method for quickly distinguishing black pork from white pork of the present invention is carried out according to the following steps:

[0006] 1. Remove the blood or impurities remaining on the surface of the pork tissue to be distinguished, then mince to obtain tissue fragments, and then use pre-cooled 0.01mol / L, pH7.4 PBS buffer to make the tissue fragments into a homogenate; ultrasonically disrupt the homogenate or repeatedly freeze-thaw, and then centrifuge to obtain the supernatant;

[0007] 2. The DHA content of the supernatant in step 1 is determined by using a DHA ELISA detection kit, and black pork and white pork are distinguished based on the DHA content; among them, black pork has a DHA content range of 480-540 μmol / L, and white pork has a DHA content of only 390-440 μmol / L.

[0008] The present invention distinguishes black pork from white pork by quickly and effectively measuring the DHA content, that is, black pork is when the DHA content ranges from 480 to 540 μmol / L, and white pork is when the DHA content is only 390 to 440 μmol / L. The method of the present invention is simple to operate, does not require the use of expensive instruments and equipment, has a short detection cycle, and greatly reduces the cost of distinguishing black pork from white pork. The existing distinction method solves the problems of high difficulty in operation, complexity, long time, and inability to distinguish quickly and effectively. DETAILED DESCRIPTION

[0009] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments 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.

[0010] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0011] Specific implementation method 1: The method for quickly distinguishing black pork from white pork in this implementation method is carried out according to the following steps:

[0012] 1. Remove the blood or impurities remaining on the surface of the pork tissue to be differentiated, then mince to obtain tissue fragments, and then use pre-cooled PBS buffer to make the tissue fragments into a homogenate; ultrasonically disrupt the homogenate or repeatedly freeze-thaw, and then centrifuge to obtain the supernatant;

[0013] 2. The DHA content of the supernatant in step 1 is determined by using a DHA ELISA detection kit, and black pork and white pork are distinguished based on the DHA content; among them, black pork has a DHA content range of 480-540 μmol / L, and white pork has a DHA content of only 390-440 μmol / L.

[0014] The PBS buffer solution is prepared in step 1 of the present embodiment as follows: 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 are weighed and dissolved in 800 mL distilled water, HCl is used to adjust the solution to pH 7.4, and finally distilled water is added to make the volume to 1 L to obtain 0.01 M (mol / L) PBS buffer solution.

[0015] In the present embodiment, the cutting into pieces in step 1 is as small as possible so as to allow for sufficient homogenization.

[0016] In this embodiment, the purpose of ultrasonically disrupting or repeatedly freezing and thawing the homogenate in step 1 is to further lyse tissue cells.

[0017] In this embodiment, the supernatant after centrifugation in step 1 can be stored at -20°C or -80°C to avoid repeated freezing and thawing.

[0018] In the present embodiment, the ELISA detection kit for DHA in step 2 is: a porcine docosahexaenoic acid (DHA) ELISA detection kit is used.

[0019] Specific embodiment 2: This embodiment differs from specific embodiment 1 in that: in step 1, the blood or impurities remaining on the surface of the pork tissue to be distinguished are removed by washing with pre-cooled 0.01 mol / L, pH 7.4 PBS buffer. Other steps and parameters are the same as those of specific embodiment 1.

[0020] Specific embodiment 3: This embodiment differs from specific embodiment 1 in that: in the homogenate prepared in step 1, the weight volume ratio of tissue fragments to PBS buffer is 1:7-10. Other steps and parameters are the same as those in specific embodiment 1.

[0021] Specific embodiment 4: This embodiment differs from specific embodiment 1 in that: in step 1, a homogenate is prepared by homogenizing on ice or in an ice bath using a glass homogenizer. Other steps and parameters are the same as those of specific embodiment 1.

[0022] Specific embodiment 5: This embodiment differs from specific embodiment 1 in that: in step 1, the centrifugation conditions of the homogenate are: centrifugation at 3000-10000×g for 5 minutes at 2-8° C. Other steps and parameters are the same as those of specific embodiment 1.

[0023] Specific embodiment 6: This embodiment differs from the specific embodiment 1 in that: the method for determining the DHA content by the DHA ELISA detection kit in step 2 is:

[0024] 1. Take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips in a ziplock bag and return them to 4°C;

[0025] 2. Set up standard wells and sample wells, and add 50 μL of standard of different concentrations to each standard well;

[0026] 3. First add 10μL of the sample to be tested to the sample well, then add 40μL of sample diluent; do not add to the blank well;

[0027] 4. Add 50-200 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard and sample wells except the blank wells, seal the reaction wells with a sealing film, and incubate in a 35-40℃ water bath or incubator for 20-90 minutes;

[0028] 5. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 1 to 10 minutes, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 3 to 6 times;

[0029] 6. Add 30-60 μL of substrate A and B to each well and incubate at 37°C in the dark for 10-50 min.

[0030] 7. Add 30-60 μL of stop solution to each well and incubate for 0-20 min. Measure the OD value of each well at a wavelength of 450 nm to further calculate the DHA content.

[0031] In step five of this embodiment, a plate washer can be used to wash the plate.

[0032] Example 1 The method of the present invention is used to distinguish black pork from white pork. The samples selected in this example are taken from Jishen black pig, Lei pig, Songliao black pig, Tibetan Xiang pig and Duchangda Sanyuan white pig, with 6 pigs of each type.

[0033] The specific method is as follows:

[0034] 1. Sample collection, processing and storage methods

[0035] Tissue homogenate:

[0036] 1) Rinse the pork tissues to be differentiated (samples from: Jishen Black Pig, Lei Pig, Songliao Black Pig, Tibetan Xiang Pig and Duchangda Sanyuan White Pig, 6 each) with pre-cooled 0.01M (mol / L), pH 7.4 PBS to remove residual blood or impurities on the surface (lysed red blood cells in the homogenate will affect the measurement results);

[0037] 2) Weigh the tissue block and then cut it into as small pieces as possible to obtain tissue fragments for full homogenization;

[0038] 3) adding pre-cooled 0.01 M (mol / L), pH 7.4 PBS buffer to the tissue fragments, and fully homogenizing with a glass homogenizer on ice or in an ice bath. In order to further lyse the tissue cells, the homogenate is ultrasonically disrupted or repeatedly frozen and thawed. The weight-to-volume ratio of the tissue fragments to the PBS buffer is 1:7-10;

[0039] 4) The homogenate was aspirated into a centrifuge tube, centrifuged at 5000 × g for 5 minutes at 2-8°C, the supernatant was collected and stored at -80°C;

[0040] 2. The DHA content of the supernatant in step 1 was determined using a DHA ELISA test kit.

[0041] 1) Take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips in a ziplock bag and return them to 4°C;

[0042] 2) Set up standard wells and sample wells, and add 50 μL of standards of different concentrations to each standard well;

[0043] 3) First add 10 μL of the sample to be tested to the sample well, and then add 40 μL of sample diluent; do not add to the blank well;

[0044] 4) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard well and sample well, seal the reaction wells with a sealing film, and incubate at 37°C in a water bath or incubator for 60 min;

[0045] 5) Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 1 min, shake off the washing solution, pat dry on absorbent paper, and repeat this process 5 times (you can also use a plate washer to wash the plate);

[0046] 6) Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min;

[0047] 7) Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0048] 3. Experimental results

[0049] Table 1 shows the content of DHA in different varieties of pork. The results of the determination of DHA content in different varieties of pork in Table 1 show that there are significant differences in the content of DHA in different black pigs and white pork. The content of DHA in black pork is significantly higher than that in white pigs, ranging from 480 to 540 μmol / L, while the content of DHA in white pork is only about 420 μmol / L. Therefore, the present invention can effectively distinguish black pork from white pork by using DHA content.

[0050] Table 1 DHA content of different pork varieties

[0051]

[0052] 4. Detection of meat quality traits of black pigs and white pork

[0053] In this example, the properties of meat (including pH value 24h, meat color, shear force, and intramuscular fat) of Jishen Black Pig, Lei Pig, Songliao Black Pig and Duchangda Sanyuan White Pig were evaluated, and compared and analyzed with the above-mentioned result trends.

[0054] The detection method is as follows:

[0055] (1) pH value: NY / T 821-2019

[0056] 1) Measurement time: Store at 0-4℃ until 24h±10min after slaughter, recorded as PH24;

[0057] 2) Measurement site: Longissimus dorsi muscle, extending from the front part of the third to last thoracic vertebra to the back until the sample length and weight required by the measurement item are met;

[0058] 3) Determination method:

[0059] Remove the peripheral muscle membrane, cut into strips and mince, pack into 2 containers (the height is about 2 / 3 of the container), and compact until there is no visible gap; calibrate pH to at least two numbers such as pH = 4.00 and pH = 7.00; calibrate pH with standard buffer; measure the temperature of the meat sample and perform temperature compensation according to the instrument requirements;

[0060] a) Insert the electrode into any measuring point of the sample, wait for about 30 seconds, read and record the displayed value;

[0061] b) Pull out the electrode, rinse it with water and then dry it, insert it into another measuring point, wait for about 30 seconds, read and record the displayed value;

[0062] c) Repeat the above two steps

[0063] d. Two specimens were measured for each sample, and two different points were measured for each specimen. The average value was used to express the measurement results.

[0064] eThe relative deviation of the measurement results of the same sample should be less than 5%.

[0065] The results are judged as follows;

[0066] a) pH 15.9-6.5 or pH 245.6-6.0. Normal meat pH;

[0067] b) pH1<5.9 or pH24<5.6, PSE meat pH;

[0068] c) pH1>6.5 or pH24>6.0, DFD meat pH

[0069] (2) Flesh color

[0070] 1) Sampling site: Longissimus dorsi muscle, extending backward from the anterior segment of the third to last thoracic vertebra until the sample length and weight required for the measurement item are met.

[0071] 2) The meat color is measured using the scoring method NY / T 821-2019: the measurement time is 45-60 minutes after slaughter, and the meat color is indicated as 1. After the meat color 1 is measured, it is stored at 0-4℃ until 24h±15min after slaughter. Two pieces of meat are measured for each sample, and three different points are measured for each piece of meat. The measurement results are expressed as the average value of the L value. The relative deviation of the measurement results of the same sample should be less than 5%.

[0072] 3) Calculation result: keep two decimal places

[0073] MC=(∑ni / 3+∑nj / 3) / 2

[0074] Where:

[0075] MC-meat color L value determination results;

[0076] ∑ni-L value of meat slice 1, i = 1 to 3

[0077] ∑nj - L value measured for slice 2, j = 1 to 3.

[0078] 2. Determination method:

[0079] MC=(n1+n2) / 2(the result is rounded to one decimal place)

[0080] Where: MC-assessment result

[0081] n1-meat slice 1 score

[0082] n2-Slice 2 score

[0083] Result evaluation: L value ≥ 60, corresponding to a meat color score of 1 point, PSE meat;

[0084] The L value is 53-59, corresponding to a meat color score of 2 points, which is close to PSE meat;

[0085] The L value is 37-52, corresponding to a meat color score of 3 to 4 points, which is normal meat color;

[0086] The L value is 31-36, corresponding to a meat color score of 5 points, which is close to DFD meat;

[0087] L value ≤30, corresponding to meat color score of 6 points, DFD meat.

[0088] The experimental results are shown in Table 2.

[0089] Table 2 Test results of relevant indicators of black pig and white pig meat

[0090]

[0091] As can be seen from Table 2, the difference between black pork and white pork cannot be effectively determined by testing the meat quality related indicators. However, the present invention can quickly and effectively determine the difference between black pork and white pork by measuring the DHA content in black pork and white pork.

Claims

1. A method for quickly distinguishing black pork from white pork, characterized in that The method to quickly distinguish black pork from white pork is as follows:

1. Remove the blood or impurities remaining on the surface of the pork tissue to be distinguished, then mince to obtain tissue fragments, and then use pre-cooled 0.01mol / L, pH7.4 PBS buffer to make the tissue fragments into a homogenate; ultrasonically disrupt the homogenate or repeatedly freeze-thaw, and then centrifuge to obtain the supernatant; 2. The DHA content of the supernatant in step 1 is determined by an ELISA detection kit, and black pork and white pork are distinguished according to the DHA content; among them, black pork has a DHA content range of 480-540 μmol / L, and white pork has a DHA content of only 390-440 μmol / L.

2. The method for quickly distinguishing black pork from white pork according to claim 1, characterized in that In step 1, the blood or impurities remaining on the surface of the pork tissue to be differentiated are removed by washing with pre-cooled 0.01 mol / L, pH 7.4 PBS buffer.

3. The method for quickly distinguishing black pork from white pork according to claim 1, characterized in that In the homogenate prepared in step 1, the weight volume ratio of tissue fragments to PBS buffer is 1:7-10.

4. The method for quickly distinguishing black pork from white pork according to claim 1, characterized in that Prepare a homogenate in step 1: homogenize using a glass homogenizer on ice or in an ice bath.

5. The method for quickly distinguishing black pork from white pork according to claim 1, characterized in that The centrifugation conditions of the homogenate in step 1 are: centrifugation at 3000-10000×g for 5 minutes at 2-8°C.

6. The method for quickly distinguishing black pork from white pork according to claim 1, characterized in that Method for determining DHA content using the ELISA test kit in step 2:

1. Take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips in a ziplock bag and return them to 4°C; 2. Set up standard wells and sample wells, and add 50 μL of standard of different concentrations to each standard well; 3. First add 10μL of the sample to be tested to the sample well, then add 40μL of sample diluent; do not add to the blank well; 4. Except for the blank wells, add 50-200 μL of the detection antibody labeled with horseradish peroxidase (HRP) to each well of the standard well and sample well, seal the reaction wells with a sealing film, and incubate in a water bath or incubator at 35-40°C for 20-90 minutes; 5. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 1 to 10 minutes, shake off the washing solution, pat dry on absorbent paper, and repeat this washing process 3 to 6 times; 6. Add 30-60 μL of substrate A and B to each well and incubate at 37°C in the dark for 10-50 min.

7. Add 30-60 μL of stop solution to each well and incubate for 0-20 min. Measure the OD value of each well at a wavelength of 450 nm to further calculate the DHA content.

Images