Identification and Differentiation of Species in Cooked Meat by Vertical Plate Gel Electrophoresis

Since quality control for adulteration in meat is very important, the vertical plate polyacrylamide gel electrophoretic technique was studied for the detection and identification of cook e d and canned species extracts. Meat samples from beef, pork, lamb and horse were heated for one hour from 65-75°C at intervals of about 5°C. Be' cause most of the research on the effect of cooking meats on the denatured serum proteins and the resultant electrophoretic patterns were done on beef, beef cooked at 70°C was selected as an initial trial for protein solubilization in order to choose an extraction and applying procedure for cooked meats. The beef was extracted with a variety of potential protein solubilizing agents such as sodium dodecyl sulfate (SOS), urea, Triton X-100 and BOI reagent in an attempt to solubilize the proteins. A single gel containing urea or SOS in conjunction with a discontinuous buffer system was studied as the electrophoresis system. Amido black lOB and Coomassie brilliant blue R-250 were compared as staining dyes. Constant power and constant voltage were compared as power supplies. Polyacrylamide gel electrofocusing was tried. Raw meat extracts were utilized as reference patterns.

' cause most of the research on the effect of cooking meats on the denatured serum proteins and the resultant electrophoretic patterns were done on beef, beef cooked at 70°C was selected as an initial trial for protein solubilization in order to choose an extraction and applying procedure for cooked meats. The beef was extracted with a variety of potential protein solubilizing agents such as sodium dodecyl sulfate (SOS), urea, Triton X-100 and BOI reagent in an attempt to solubilize the proteins. A single gel containing urea or SOS in conjunction with a discontinuous buffer system was studied as the electrophoresis system. Amido black lOB and Coomassie brilliant blue R-250 were compared as staining dyes. Constant power and constant voltage were compared as power supplies. Polyacrylamide gel electrofocusing was tried. Raw meat extracts were utilized as reference patterns. i i A system of tris-chloride buffer at pH 6.7 containing 2M urea and 10% glucose gave the best reslts as an extraction procedure. A single 7% cyanogum gel containing 4M urea in conjunction with a discontinuous buffer system was utilized as the electrophoresis system. Amido black was found to be more sensitive so it was selected as staining procedure. Also, additions of mercaptoethanol to the gel and sample improved the electrophoretic patterns. It was found that with constant power, the front solvent was a very sharp straight line and the bands were sharper than with constant voltage.
Characteristic differences were discernible between beef and lamb versus pork and horse meat heated at 65°C, but the distinction was decreased with increasing temperature.
Canned beef (corned beef) and mixture of canned pork and ham (SPAM) gave some bands but did not give proper separation of the bands. So with more study on solubility and gel electrophoretic patterns heat denatured proteins could be improved and achieved. Use of a densitometer might also improve the utility of the procedure.
Polyacrylamide gel electrofocusing did not work and that may be because the thickness of the gel which generates heat, or other causes, as discussed in Appendix C.

INTRODUCTION
With increases in world population and the limited ability of growing animals to produce meat, meat prices continue to increase, especially some of the kinds which many people prefer (i.e., beef and lamb).
Since quality control for adulteration in meat is very important, electrophoresis with polyacrylamide gel has been studied and successfully applied by Payne (1963) to raw pro-' ducts. This method, the disc polyacrylamide gel electrophoretic technique, developed by Ornstein (1964) and Davis {1964) , has proven to be successful for separation and identification of animal serum proteins. Distinct electrophoretic patterns were obtained between meat and fish species using this technique (Payne, 1963;Mancusa, 1964).
The vertical plate polyacrylamide gel electropho~etic (VPE) technique, which was developed by Raymond (1964) has also been successfully used for the detection and identification of raw meat and fish species Rand, 1972a, 1972b).
The effect of cooking beef on the denatured serum proteins and on the resultant electrophoretic patterns has been studied. Laakkonen ~ ~· (1970) found that changes in the electrophoretic patterns of bovine muscle took place during low temperature and long-time heating . Fogg and Harrison (1975) have studied the effect of two end points, 25 oc and 45*C in semitendinous beef muscle on the electrophoretic patterns of the sarcoplasmic proteins. They concluded that heating to 25°C decreased the number and intensity of the slowest migration protein components of the sarcoplasmic fraction and the effects were more pronounced at 45°C. Lee et al. (1974)  11. lOg sample + lOM urea (ultra pure urea) was prepared according to Krzynowek and Wiggin (1979).

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Each mixture was transferred to a 50 ml plastic centri- Urea-polyacrylamide Gel Electrophoresis: The vertical plate polyacrylamide gel electrophoretic technique described by  for the differentiation of meat speces was used with some modification.
Formulation for all reagents used in this method are shown in Table 1. The gel solution was prepared prior to each run, according to the formulation shown in Table 1. The tris, Cyanogum, urea and TEMED were dissolved in 90 ml of water and adjusted to pH 8.9 with 3N HCl, followed by filtration through Whatman #1 filter paper. The volume of the gel solution was adjusted to 150 ml, and 0.2 ml of 10% Tween 80, 0. lml mercaptoethanol and o. 13g of ammonium persulfate were added with gentle agitation on a magnetic stirrer. The gel solution was poured at room temperature into an EC Apparatus Co. EC-470 vertical electrophoresis cell which was assembled horizoinally. A slot form for 10 samples was inserted, and the gel solution allowed to solidify for 40-50 min. The cell was then connected to a circulating refrigerated water bath, and coolant at 10°C was introduced into the cooling plates . The temperature in the refrigerated waterbath was reduced to 0°C over a 1 hour period and maintained at that temperature for the balance of the run. The excess gel above the slot form was removed and the cell was placed in a vertical position. The tris-glycine electrode buffer, precooled overnight to 2°C was added to the cell and allowed to overflow into the lowest chamber. The slot form was then carefully removed, and bromophenol blue was added to the buffer in the upper chamber to mark the position of the glycine-chloride front.
fonditions for Electrophoresis 1 -Constant Voltage -Was carried out as described by .

Staining and Destaining
After completion of the run, the power was turned off and the cell was disconnected. The electrode buffer was drained off and the cell was disassembled. The gel was removed and stained for 15 minutes in the Amido black lOB stain shown in Table 1  (1) Triton X-100 with ME (TM), (2) Triton X-100 with SDS (TS), (3) Triton X-100 with urea (TU), (6) lOM urea (U) , (7) BDI reagents, (9) tris-HCl buffer containing 2M urea and 10% glucose (BGU) . · ' Figure 3. Effect of urea concentration on cooked beef meat extracts, in presence of mercaptoethanol.
• mercaptoethanol which provided a slightly better pattern.
The addition of mercaptoethanol to the running gel was also studied . Figure 4a shows e 1ectrophoretic patterns obtained from cooked meats without addition of mercaptoethanol to the running gel . Figure 4b shows The effect of Amido black 108 and Coomassie brilliant blue R-250 on the electrophoresis of meat protein as a staining procedure was compared. Cooked beef meat extracted with extraction buffer containing lM, 2M and 4M urea containing mercaptoethanol and 2M urea without mercaptoethanol were applied to a gel in duplicate. After the electrophore t ic run was accomplished, the gel was divided down the middle.
One side of it was sta i ne d i n . Amido black 108 accor di ng to   (Ogita and Markert, 1979). As seen in Figure 5, Amido black was sensitive and gave sharper ' Figure 4a. Electrophoretic patterns obtained from cooked meats without addition of ME to the running gel.
I resolution -better than Coomassie blue. Wilson (1979) also found the same results. Therefore, Amido black 108 was selected as the stai.n of preference, following the procedure of coduri and Rand (1972) Samples 1,2,3 and 4 stained with Amido black lOB; 7,8,9, and 10 stained with Coomassie Blue.

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The possibility that the increased volume resulted in dilution of the so~uble material and there may be less soluble material present in horse meat were considered as probable causes for the weaker staining of extraction containing horse meat .  The increased sharpness and appearance of more bands after heating may be due to a low molecular weight fragment derived from one of the proteins initially present (Cohen, 1966).
These figures show the electrophoretic patterns obtained with VPE method using only one gel. Characteristic differences were discernible between beef and lamb versus pork and horse at 65°C, but the distinction decreased with an increase of the heat process temperature. Therefore, this study did  With the vast increase in world population and the limited ability of growing animals to produce meat, meat prices are getting higher day after day, especially some of the kinds which most of t:l)e .people prefer (i.e., beef and lamb)·. Fennema (1976) predicts that the industrialists will look more and more to areas of substituted, fabricated or synthetic foods as a possible solution to world food problems. Various electrophoretic techniques such as: moving boundary electrophoresis, zone electrophore-sis, polyacrylamide gel electrophoresis and thin layer isoelectric focusing have been used for the separation and identification of proteins.
Since the quality control from any adulteration in meat is very important, electrophoresis with polyacrylamioe gel has been successfully applied by Payne (1963) as a method for separation and identification of animal serum proteins using the disc polyacrylamide gel electrophoretic technique. As developed by Ornstein (1964) and Davis (1964), the method has prov~n to be a good method for the detection and identification of mixtures of animal proteins. Distinct electrophoretic patterns were obtained between meat and fish species using this technique (Payne, 1963;Mancuso, 1964 '1964). Nakanichi and Raymond (1962) reported that VEP could be used in the routine process of blood proteins. Laakkonen et al. (1970) found this technique was useful for meat an a 1 y s i s .  were the first to establish that distinct electrophoretic pattern could be obtained from the sarcoplasmic extracts of different meats --beef, lamb, pork, and horse, using vertical plate technique. Also, they applied this technique for the differentiation on fish and shellfish species {1972b).

Coduri, Bonatti and Simpson (1979) applied a vertical
Plate gel electrophoresis to the separation of pigmented and nonpigmented trout and salmon species.
In order to import meat from many countries of the world the mea _ must be cooked to 69°C to insu~e destruction of the foot and mouth disease virus if present (Heidelbaugh and Graves, 1968), a number of methods therefore have been tried to determine the cooking temperature such as extractability, coagulation test, determination .of acid phosphatase activity and direct spectroscopy of extracted meat pigments. Cohen (1966) studied the protein changes related to ham processing temperatures and found that the rate of ·heatng as well 24 as the temperature reduced the amount of extractable proteins.
In 1969 he studied the determination of acid phosphatase activity in canned hams as an indicator of temperatures attained during cooking. He found the method lacked accuracy when appli~d to hams processed to higher internal temperatures. Ooesburg and Papendrof (1969) stated that the degree of heating of some fish muscle could be calculated from the coagulation test. Helmke and Froning (1971) studied the effect of endpoint cooking temperature on the color of turkey meat. They found that the extracted pigment from turkey meat, which was cooked to 82°C, had a spectra quite different than that observed at lower temperatures.
None of these methods, however, has proved to be acceptable because they were only applicable to narrow ranges of cooking temperature or other experimental conditions.
Since cooked meat will be denatured and protein solubility will decrease, solubilizing agents such as urea and sodium dodecyl sulphate (SOS) are necessary to solubilize the protein in cooked meat. Since SOS and urea are two of the most commonly used reagents in protein chemistry, their applications in protein denaturation, solubilization, dissociation and purification are much too numerous to summarize here.
An advantage of these agents is the ability to obtain constant electrophoretic mobilities of proteins independent of isoelectric point and amino acid composition, which can be slightly modified during cooking (Lee~~., 1974).  (1980) reported that when beef muscles were cooked at temperatures between 60-80°C, protein bands gradually disappeared and could not be detected above 80°C. Deschrreider and Meaux (1974) (Lee _tl ~·, 1974;Mafinen _tl ~·, 1979 andCalderoni andBazan, 1980), and it appeared to be a good procedure to try for analysis of cooked meats. Therefore SDS-polyacrylamide gel electrophoresis was performed according to Ogita and Markert (1979) with some modifications as shown in Table 2.   Table 2. The proportions of 3g of the IA solution to water were varied to give different gel concentrations up to 20% acrylamide gel (Table 3), then ID solution was added.
Preparation of the stacking gel . -The stacking gel mixture was prepared by mixing the solution as given in Table   3. A 4% acrylamide stacking gel was optional for nearly all experiments.
Preparation of the sample. -Cooked beef, lamb, pork and horse meats were prepared as described by Ogita and Markert (1979)

RESULTS AND DISCUSSION
Polyacrylamide gel electrofocusing procedure as recommended by E-C Apparatus Corporation was tried with some modification. Initially the heat generation proved excessive after short time of the run at the recommended 20 watts (w).
So, the run was repeuted using lOW but heat generation proved excessive after a period of time and therefore, the power was cut to 2.5W. Even so, there was still a small heat ef-' feet on the gel. Figure 9 shows polyacrylamide gel electrofocusing of raw, cooked (beef, pork, lamb and horse meats) and canned meat (corned beef and mixture of pork and ham).
It shows no migration or bands, and that may be because of the thickness of the gel which generates heat or because the sample contained salt, which contributed to generaLed heat, or both. Also it could be a problem caused by oxygen generated at the anode. This is partially dissolved in the medium, and may oxidize the SH-groups of protein _ (Goal~~·, 1980).
An overview or the times for running, fixing, staining, and destaining of electrophoresis and isoelectric focu_ing in polyacrylamide gel is shown in Table 4. These results indicated that electrophoresis is less consuming of time and more applicable for routine work.