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Sodium chloride as a preferred protein extractant for pork lean meat
MEAT SCIENCE Meat Science 67 (2004) 697–703 www.elsevier.com/locate/meatsci
Sodium chloride as a preferred protein extractant for pork lean meat D.M.S. Munasinghe a, Tadashi Sakai b
b,*
a United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima-shi, Kagoshima 890-0065, Japan Department of Biochemistry and Applied Biosciences, Faulty of Agriculture, Miyazaki University, Miyazaki-shi, Miyazaki 889-2192, Japan
Received 10 October 2003; received in revised form 3 February 2004; accepted 3 February 2004
Abstract The protein extractability of sodium chloride (NaCl), potassium chloride (KCl) and lithium chloride (LiCl) under a range of molarity with in the physiological pH range (pH 6.0–8.0) was assessed to determine the best protein extractant for pork lean meat. The individual proteins in the extracts were identified by electrophoresis. The highest protein extractabilities at pH 7.0 for NaCl, KCl and LiCl were observed at 1.2, 1.1, and 1.1 M, respectively. There was no significant difference in protein extractability for KCl and LiCl within physiological pH range. NaCl had a significant increase of its protein extractability as pH increased from 6.0 to 6.5 followed by a relatively constant extractability. The NaCl had the highest protein extractability followed by LiCl and KCl. The maximum number of proteins (26 bands) was found at the optimum concentration of each salt. However, resolution and clarity of bands were better in NaCl extracts. The pH variation does not affect the number and the intensity of the bands. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: NaCl; KCl; LiCl; Pork; Protein extractability; Salt concentration
1. Introduction The protein extractability of high concentration salt solutions is widely used as a better index of pig lean meat quality compared with color or reflectance measurements (Lopez-Bote, Warriss, & Brown, 1989). In addition, high ionic strength salt solutions are employed to extract proteins for meat batter preparations (Gordon & Barbut, 1992). Apart from those, such extractant are also used for the detection of meat substitutions (Toorop, Murch, & Ball, 1997). In all these instances, it is important to extract maximum amount of individual proteins representing cytoplasmic and myofibrillar fractions. The protein extractability of salt solutions is mainly depends on the ionic strength, pH, and type of the salt (Franks, 1993). Of the different salts, sodium chloride (NaCl), and potassium chloride (KCl) are widely used for protein extraction. Recently, Kelleher and Hultin (1991) reported lithium chloride (LiCl) as a better protein extractant over KCl and NaCl in fish meat. In *
Corresponding author. Tel./fax: +81-985-58-7230. E-mail address: [email protected] (T. Sakai).
0309-1740/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2004.02.001
addition, protein extractability depends on extracting procedure that includes volume of extraction solution, duration of homogenization, centrifugal force and time, etc. Lan, Novakofski, Carr, and McKeith (1993) have investigated the optimum protein extracting procedure for pork. However, to the best of our knowledge, no investigations have been done to establish the best salt for this purpose with its optimum concentration. Moreover, different authors have used different salts under different salt concentrations making it difficult to compare data from one study to another. In this context, we investigated the protein extractability of NaCl, KCl, and LiCl, keep in line with the Lan et al.Õs (1993) extraction procedure, in order to identify the best protein extractant for pork within the physiological pH range (pH 6.0–8.0).
2. Materials and methods 2.1. Materials Sodium chloride (NaCl), potassium chloride (KCl), lithium chloride (LiCl), sodium bicarbonate (NaHCO3 ),
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D.M.S. Munasinghe, T. Sakai / Meat Science 67 (2004) 697–703
potassium bicarbonate (KHCO3 ), and lithium carbonate (LiCO3 ) were of the purest grade available and purchased from Wako Pure Chemical Industries Ltd., Tyuo-ku, Osaka, Japan. Coomassie brilliant blue R-250 was obtained from ICN Biochemicals Inc, Aurora, Ohio, USA. Mark 12e wide range protein standard was purchased from NOVEX, San Diego, California, USA. All other reagents were of electrophoretic grade and purchased from Wako Pure Chemical Industries Ltd., Tyuo-ku, Osaka, Japan. 2.2. Methods 2.2.1. Sample preparation A slice of sirloin was obtained form a pig carcass at the end of 2-day aging period during which carcass was kept at 0 °C, and brought to the laboratory under refrigeration. The visible fatty tissue and epimysial connective tissue were removed, cut into small pieces and minced in a food processor for 1 min to prepare samples. Samples were subjected to analysis immediately. 2.2.2. Preparation of salt solutions The relevant molar concentrations of the salt solutions were prepared by dissolving the appropriate amount of NaCl, KCl or LiCl in distilled water while adjusting the pH with 0.02 M NaHCO3 , KHCO3 , and LiCO3 , respectively. 2.2.3. Protein extraction One gram of minced meat was homogenized with 19 ml of ice-cooled buffered salt solution in a homogenizing tube placed in ice for 2 min. The pH of the homogenates (pH 5.4–5.8) was adjusted to the expected pH using a 1 M-hydroxide solution of the relevant salt while stirring. Thus prepared homogenates were centrifuged at 2600g for 20 min at 4 °C. The resultant supernatants obtained were used for the determination of protein and electrophoresis. 2.2.4. Protein measurement Protein concentrations were determined by Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, California, USA) based on coomassie blue dye binding method (Bradford, 1976), measuring absorbance at 595 nm using a UV-1200 UV–Vis spectrophotometer (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan) according to the manufactureÕs instruction. The bovine serum albumin was used as the standard. 2.2.5. Samples for electrophoresis Equal volumes from each replicate of every sample were pooled. The protein contents of the pooled samples were adjusted to unity using their respective salt buffer. Samples were prepared by mixing equal volumes of protein extract with LaemmliÕs (1970) sample buffer and
heating them at 100 °C for 5 min. The samples were stored at )20 °C until electrophoresis. 2.2.6. Electrophoresis Sodium dodecyl sulfate–poly acrylamide gel electrophoresis (SDS–PAGE) was carried out as outlined by Laemmli (1970) using a large gel electrophoresis unit (ATTO Corporation, Bunkyo-ku, Tokyo, Japan). Samples were loaded at 45 lg of protein per well on a 4% stacking gel and proteins were resolved in a 7.5–20% gradient gel at room temperature under an 8 mA constant current. The gels were stained with coomassie blue dye solution (0.1% coomassie brilliant blue, 50% methanol, 10% acetic acid in distilled water) and de-stained with the same solution without coomassie brilliant blue. 2.2.7. Experimental design and statistical analysis Protein extractability of NaCl, KCl, and LiCl was first tested between 0.4 and 1.8 M in 0.2 M increments to obtain the salt concentration that gives the highest protein extractability for each salt at pH 7.0. The protein extractability of each salt was re-assessed between the adjacent lower and upper values of the salt concentration that gave the highest protein extractability for each salt in previous experiment in 0.1 M increments to obtain more accurate concentration for each salt. The effect of pH on protein extractability of each salt was tested between pH 6.0 and 8.0 in 0.5 increments using the concentrations that gave the highest protein extractability for each salt. Each test was done using a separate sample with three replicates. Finally protein extractability of the three salts were compared using their respective concentrations that gave the highest protein extractability at pH 7.0 using a sample prepared from a single slice of sirloin in order to exclude effect of individual variation of pigs. In this instance number of replicates used was four. All data were expressed as the means SEM. The results were analyzed by the analysis of variance (ANOVA) and the significant differences among the means were determined by DuncanÕs multiple range test (Duncan, 1955).
3. Results and discussion 3.1. Effect of ion concentration on protein extractability The protein extractabilities of NaCl, KCl, and LiCl were shown in Figs. 1–3, respectively. All three salts tested showed an initial significant increased of their protein extractability at each increment. Furthermore, these initial significant increments of protein extractability of NaCl, KCl, and LiCl were extended up to the concentrations of 1.2, 1.0, and 0.8 M, respectively. Thereafter both LiCl and KCl extractants showed a plateau followed by gradual decline of their protein
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160
Extractable protein content (mg/ g tissue)
140
c
120
e
e
e
1.2
1.4
1.6
d
e
b
100
a
80 60 40 20 0 0.4
0.6
0.8
1.0
1.8
Molarity
Fig. 1. Effect of NaCl concentration on protein extractability at pH 7.0. Results are means SEM of three replicates. The columns bearing different letters are significantly different from each other (P < 0:05).
140
d
d
Extractable protein content (mg/g tissue)
d
c
120
cd
c
b 100
a 80
60
40
20
0 0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Molarity
Fig. 2. Effect of KCl concentration on protein extractability at pH 7.0. Results are means SEM of three replicates. The columns bearing different letters are significantly different from each other (P < 0:05).
content while a continuous plateau was observed for the NaCl extractants. In order to obtain more accurate concentration for each salt, the protein extractability of each salt was reassessed between the adjacent lower and upper value of the highest observed value of the respective salt in 0.1 M increments. The highest protein
extractabilities for NaCl, KCl, and LiCl were observed at 1.2, 1.1 and 1.1 M, respectively (Table 1). However, Toorop et al. (1997) and Murray and Johnson (1994), respectively reported that 0.4 and 1 M NaCl as suitable NaCl concentrations for porcine meat quality assessment. This may be due to the differences in respective
700
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c
c
c
d
b
120 Extractable protein content (mg/g tissue)
c
100
b
a
80
60
40
20
0 0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Molarity
Fig. 3. Effects of LiCl concentration on protein extractability at pH 7.0. Results are means SEM of three replicates. The column bearing different letters are significantly different form each other (P < 0:05).
extraction conditions. For an example Toorop et al. (1997) used homogenization time of 15 s, which is quite inadequate for maximum protein extraction when considered the observation of Lan et al. (1993) who found a significant increase of protein extractability in 30 s homogenizing intervals up to 90 s. 3.2. Effect of pH on protein extractability The effect of pH on protein extractabilities of 1.2 M NaCl, 1.1 M KCl, and 1.1 M LiCl was tested
within the physiological pH range. The highest protein extractability for NaCl was observed at pH 6.5 (Table 2). However, KCl and LiCl did not show a significant variation of their protein extractability within the physiological pH range. Toorop et al. (1997) found significant variation of protein extractability for NaCl between pH 6.0 and 7.0. Similarly, we also observed the highest protein extractability for NaCl at pH 6.5. In contrast, Lan et al. (1993) found similar protein extractability for pork at pH 6.0 and 6.5.
Table 1 The effect of molar concentration on protein extractability (mg/g tissue) of NaCl, KCl and LiCl at pH 7.00 Salt
Molar concentration 0.8
0.9
1.0
1.1
1.2
1.3
1.4
NaCl KCl LiCl
– 117.49 1.03a 110.26 1.23a
– 122.04 0.97ab 110.54 2.23a
131.67 0.80a 124.29 0.28bc 111.26 0.51a
133.08 2. 36ab 124.78 1.59b 116.27 2.76a
139.06 2.01b 118.63 3.3ac 112.83 1.89a
133.76 1.05ab – –
131.37 3. 13a – –
Values (means SEM, n ¼ 3) within row with no common superscript differ significantly (P < 0:05).
Table 2 Effect of pH on protein extractability (mg/g tissue) of 1.2 M NaCl, 1.1 M KCl and 1.1 M LiCl Salt
pH 6.0
6.5
7.0
7.5
8.0
NaCl KCl LiCl
118.03 1.71a 116.47 0.88a 117.09 1.2a
123.48 1.70b 118.87 1.29a 117.76 2.42a
120.79 1.51ab 122.57 4.07a 117.48 2.36a
122.80 0.89ab 118.86 0.42a 116.80 0.52a
122.02 1.29ab 119.15 1.56a 121.71 0.60a
Values (means SEM, n ¼ 3) within row with no common superscript differ significantly (P < 0:05).
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3.3. Comparison of protein extractability The protein extractabilities of 1.2 M NaCl, 1.1 M KCl, and 1.1 M LiCl at pH 7.0 were compared to select the best protein extractant for pork. The highest protein extractability was observed for the NaCl followed by LiCl and KCl (Table 3). Furthermore, the extractable protein content of each salt was found significantly different from each other. Gordon and Barbut (1992) also observed the same order of protein extractability of these three salts in comminuted meat mixes. The difference of the protein extractability of the three salts might due to the inherent properties of the three ions. 3.4. Electrophoretic banding pattern of protein extracts The three salts reflected the salt concentrations (0.4– 1.8) dependant total protein extractability in their electrophoretic banding patterns. The buffered NaCl showed a substantial extraction of myosin heavy chain (band a; 200 kDa) only at 0.6 M and continued to increase up to 1.0 M (Fig. 4). The same tendency was observed for the LiCl and KCl extractants (results were not shown). Furthermore, optimum intensity for myosin heavy chain of NaCl extract was observed at 1.2 M and gradual reduction of intensity was observed thereafter. Toorop et al. (1997) have observed salting out effect for myofibrillar proteins of pork at 0.6 M NaCl (pH 7.0) under their extraction condition. However, we did not observe such effects indicating superiority of our extraction condition. Apart from Myosin heavy chain, bands c (26 kDa), d (21 kDa), and e (19 kDa) showed an increase of their intensities with the increase of salt concentration and approached maximum intensity at 1.2 M followed by a decline. The identities of these proteins were not known at present. However, this indicates the importance of optimal high salt concentration for the completeness of individual protein extractability. Though there were differences such as the intensities of the bands, the ionic strength at which particular band first appeared etc., among the electrophoretic profiles of the salts, no differences were observed among NaCl, KCl, and LiCl in relation to the number of bands present at their respective concentrations of 1.2, 1.1, and 1.1 M (results were not shown). Few bands (e.g. band b, 58 kDa) showed an initial reduction of their intensities. This caused by the higher proportion of the particular
Table 3 Protein extractability (mg/g tissue) of 1.2 M NaCl, 1.1 M KCl, and 1.1 M LiCl at pH 7.0 pH 7.0
NaCl
KCl a
146.98 1.27
LiCl b
125.34 1.19
133.76 0.63c
Value (mean SEM, n ¼ 4) within row with no common superscript differ significantly (p < 0:05).
Fig. 4. SDS–PAGE pattern of protein extractants of NaCl at different molar concentrations. MM designates molecular weight markers in kDa. Lanes 1–8 represent salt concentrations of extractants 0.4–1.8 M in 0.2 increments. Arrow heads a–e represent bands with molecular weights 200, 58, 26, 21 and 19 kDa, respectively.
protein due to low extraction of myosin heavy chain when every lane contains same quantity of protein. As shown in Fig. 5, a significant difference was not observed for the number of bands and their intensities throughout physiological pH range. The same phenomena were observed for the KCl and LiCl extracts too (results were not shown). However, there were differences in relation to intensities of the bands among the three slats reflecting the differences of the protein extractability of the three salts. As shown in Fig. 5, salting our or salting in effects were not observed with in the physiological pH range. This gives the possibility of adjusting the pH of buffers suite to the pH of subsequent procedures. The electrophoretic banding pattern of the protein extractants of comparison study showed a similarity in banding pattern of all the three salts tested (Fig. 6). However, differences in individual protein extractability among the salts may surface if more sensitive methods such as two-dimension SDS–PAGE were used. Furthermore, low extractability of myosin was clearly visible for KCl extractant. The inability of KCl to extract enough myosin may be the reason for its very low total protein extractability. Even in LiCl extractants, the intensity of the myosin band was lighter than that of NaCl extractants indicating low myosin extractability. Cheung and Cooke (1971) have shown Li+, Na+, and K+ ions have different effects on myosin confirmation. Such conformational changes might have caused the
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number of individual protein bands (26) than the crude extraction and soluble fractions of pork muscle prepared by Sentandreu and Toldra (2000).
4. Conclusions and implications
Fig. 5. SDS–PAGE pattern of proteins extracted at 1.2 M NaCl with in physiological pH range. MM designates molecular weight markers in kDa. Lanes 1–5 represent pH of extractants 6.0–8.0 in 0.5 increments.
The buffered NaCl had significantly higher protein extractability at its optimum concentration (1.2) compared with those of LiCl and KCl making NaCl as a better protein extractant. The higher protein extractability of NaCl seems to be due to its higher extraction of myosin. The amount of myosin present, being one of the major factors that determine gel forming ability and its quality in surami products, buffered NaCl could be applied as a better protein extractant particularly when comminuted meat are used. Moreover, the utilization of NaCl is more attractive in industrial wise due to its inexpensiveness and known safety as a food additive. The higher total and individual protein extractability of buffered NaCl make it a better protein extractant for lean meat quality assessment of pork than that of KCl or LiCl. In addition, the extractability of clearly separated higher number of individual protein bands may enable it to be used as a protein extractant for the detection of meat substitutes where optimum individual protein representation make a high impact on accuracy and sensitivity of such experiments.
Acknowledgements We thank Mrs. T. Okubo and T. Tagami (Faculty of Agriculture, Miyazaki University) for their technical assistance.
References
Fig. 6. SDS–PAGE pattern of proteins extracted by 1.2 M NaCl, 1.1 M KCl, and 1.1 M LiCl at pH 7.0. MM designates molecular weight markers in kDa. Lanes 1–3 represent protein extractants of NaCl, KCl, and LiCl, respectively. Arrow head indicates the myosin band.
differences in myosin solubility in different salts resulting in differences of total protein extractability among the three salts. The 1.2 M NaCl extractant had a higher
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254. Cheung, H. C., & Cooke, R. (1971). Effect of alkali ions on myosin confirmation. Biopolymers, 10, 523–528. Duncan, D. B. (1955). Multiple range and multiple F tests. Biometrics, 11, 1–42. Franks, F. (1993). Solution properties of protein. In F. Franks (Ed.), Protein biotechnology (pp. 133–190). New Jersey: Humana Press. Gordon, A., & Barbut, S. (1992). Effect of chloride salts on protein extraction and interfacial protein film formation in meat batters. Journal of the Science of Food and Agriculture, 58, 227–238. Kelleher, S. D., & Hultin, H. O. (1991). Lithium chloride as a preferred extractant of fish muscle proteins. Journal of Food Science, 56(2), 315–317. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685. Lan, Y. H., Novakofski, J., Carr, T. R., & McKeith, F. K. (1993). Assay and storage conditions affected yield of salt soluble protein from muscle. Journal of Food Science, 58(5), 963–967.
D.M.S. Munasinghe, T. Sakai / Meat Science 67 (2004) 697–703 Lopez-Bote, C., Warriss, P. D., & Brown, S. N. (1989). The use of muscle protein solubility measurements to assess pig lean meat quality. Meat Science, 26, 167–175. Murray, A. C., & Johnson, C. P. (1994). Calpain and cathepsin activities, and protein extractability during ageing of longissimus porcine muscle from normal and PSE meat. Food Chemistry, 63(3), 385–390 (cited by Gill, M., Hortos, M., Sarraga, C., 1998).
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Sentandreu, M. A., & Toldra, F. (2000). Purification and biochemical properties of dipeptidyl peptidase 1 from porcine skeletal muscle. Journal of Agriculture Food Chemistry, 48(10), 5014–5022. Toorop, R., Murch, S. J., & Ball, R. O. (1997). Development of a rapid and accurate method for separation and quantification of myofibrillar proteins in meat. Food Research International, 30(8), 619–627.
Sodium chloride as a preferred protein extractant for pork lean meat D.M.S. Munasinghe a, Tadashi Sakai b
b,*
a United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima-shi, Kagoshima 890-0065, Japan Department of Biochemistry and Applied Biosciences, Faulty of Agriculture, Miyazaki University, Miyazaki-shi, Miyazaki 889-2192, Japan
Received 10 October 2003; received in revised form 3 February 2004; accepted 3 February 2004
Abstract The protein extractability of sodium chloride (NaCl), potassium chloride (KCl) and lithium chloride (LiCl) under a range of molarity with in the physiological pH range (pH 6.0–8.0) was assessed to determine the best protein extractant for pork lean meat. The individual proteins in the extracts were identified by electrophoresis. The highest protein extractabilities at pH 7.0 for NaCl, KCl and LiCl were observed at 1.2, 1.1, and 1.1 M, respectively. There was no significant difference in protein extractability for KCl and LiCl within physiological pH range. NaCl had a significant increase of its protein extractability as pH increased from 6.0 to 6.5 followed by a relatively constant extractability. The NaCl had the highest protein extractability followed by LiCl and KCl. The maximum number of proteins (26 bands) was found at the optimum concentration of each salt. However, resolution and clarity of bands were better in NaCl extracts. The pH variation does not affect the number and the intensity of the bands. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: NaCl; KCl; LiCl; Pork; Protein extractability; Salt concentration
1. Introduction The protein extractability of high concentration salt solutions is widely used as a better index of pig lean meat quality compared with color or reflectance measurements (Lopez-Bote, Warriss, & Brown, 1989). In addition, high ionic strength salt solutions are employed to extract proteins for meat batter preparations (Gordon & Barbut, 1992). Apart from those, such extractant are also used for the detection of meat substitutions (Toorop, Murch, & Ball, 1997). In all these instances, it is important to extract maximum amount of individual proteins representing cytoplasmic and myofibrillar fractions. The protein extractability of salt solutions is mainly depends on the ionic strength, pH, and type of the salt (Franks, 1993). Of the different salts, sodium chloride (NaCl), and potassium chloride (KCl) are widely used for protein extraction. Recently, Kelleher and Hultin (1991) reported lithium chloride (LiCl) as a better protein extractant over KCl and NaCl in fish meat. In *
Corresponding author. Tel./fax: +81-985-58-7230. E-mail address: [email protected] (T. Sakai).
0309-1740/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2004.02.001
addition, protein extractability depends on extracting procedure that includes volume of extraction solution, duration of homogenization, centrifugal force and time, etc. Lan, Novakofski, Carr, and McKeith (1993) have investigated the optimum protein extracting procedure for pork. However, to the best of our knowledge, no investigations have been done to establish the best salt for this purpose with its optimum concentration. Moreover, different authors have used different salts under different salt concentrations making it difficult to compare data from one study to another. In this context, we investigated the protein extractability of NaCl, KCl, and LiCl, keep in line with the Lan et al.Õs (1993) extraction procedure, in order to identify the best protein extractant for pork within the physiological pH range (pH 6.0–8.0).
2. Materials and methods 2.1. Materials Sodium chloride (NaCl), potassium chloride (KCl), lithium chloride (LiCl), sodium bicarbonate (NaHCO3 ),
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D.M.S. Munasinghe, T. Sakai / Meat Science 67 (2004) 697–703
potassium bicarbonate (KHCO3 ), and lithium carbonate (LiCO3 ) were of the purest grade available and purchased from Wako Pure Chemical Industries Ltd., Tyuo-ku, Osaka, Japan. Coomassie brilliant blue R-250 was obtained from ICN Biochemicals Inc, Aurora, Ohio, USA. Mark 12e wide range protein standard was purchased from NOVEX, San Diego, California, USA. All other reagents were of electrophoretic grade and purchased from Wako Pure Chemical Industries Ltd., Tyuo-ku, Osaka, Japan. 2.2. Methods 2.2.1. Sample preparation A slice of sirloin was obtained form a pig carcass at the end of 2-day aging period during which carcass was kept at 0 °C, and brought to the laboratory under refrigeration. The visible fatty tissue and epimysial connective tissue were removed, cut into small pieces and minced in a food processor for 1 min to prepare samples. Samples were subjected to analysis immediately. 2.2.2. Preparation of salt solutions The relevant molar concentrations of the salt solutions were prepared by dissolving the appropriate amount of NaCl, KCl or LiCl in distilled water while adjusting the pH with 0.02 M NaHCO3 , KHCO3 , and LiCO3 , respectively. 2.2.3. Protein extraction One gram of minced meat was homogenized with 19 ml of ice-cooled buffered salt solution in a homogenizing tube placed in ice for 2 min. The pH of the homogenates (pH 5.4–5.8) was adjusted to the expected pH using a 1 M-hydroxide solution of the relevant salt while stirring. Thus prepared homogenates were centrifuged at 2600g for 20 min at 4 °C. The resultant supernatants obtained were used for the determination of protein and electrophoresis. 2.2.4. Protein measurement Protein concentrations were determined by Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, California, USA) based on coomassie blue dye binding method (Bradford, 1976), measuring absorbance at 595 nm using a UV-1200 UV–Vis spectrophotometer (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan) according to the manufactureÕs instruction. The bovine serum albumin was used as the standard. 2.2.5. Samples for electrophoresis Equal volumes from each replicate of every sample were pooled. The protein contents of the pooled samples were adjusted to unity using their respective salt buffer. Samples were prepared by mixing equal volumes of protein extract with LaemmliÕs (1970) sample buffer and
heating them at 100 °C for 5 min. The samples were stored at )20 °C until electrophoresis. 2.2.6. Electrophoresis Sodium dodecyl sulfate–poly acrylamide gel electrophoresis (SDS–PAGE) was carried out as outlined by Laemmli (1970) using a large gel electrophoresis unit (ATTO Corporation, Bunkyo-ku, Tokyo, Japan). Samples were loaded at 45 lg of protein per well on a 4% stacking gel and proteins were resolved in a 7.5–20% gradient gel at room temperature under an 8 mA constant current. The gels were stained with coomassie blue dye solution (0.1% coomassie brilliant blue, 50% methanol, 10% acetic acid in distilled water) and de-stained with the same solution without coomassie brilliant blue. 2.2.7. Experimental design and statistical analysis Protein extractability of NaCl, KCl, and LiCl was first tested between 0.4 and 1.8 M in 0.2 M increments to obtain the salt concentration that gives the highest protein extractability for each salt at pH 7.0. The protein extractability of each salt was re-assessed between the adjacent lower and upper values of the salt concentration that gave the highest protein extractability for each salt in previous experiment in 0.1 M increments to obtain more accurate concentration for each salt. The effect of pH on protein extractability of each salt was tested between pH 6.0 and 8.0 in 0.5 increments using the concentrations that gave the highest protein extractability for each salt. Each test was done using a separate sample with three replicates. Finally protein extractability of the three salts were compared using their respective concentrations that gave the highest protein extractability at pH 7.0 using a sample prepared from a single slice of sirloin in order to exclude effect of individual variation of pigs. In this instance number of replicates used was four. All data were expressed as the means SEM. The results were analyzed by the analysis of variance (ANOVA) and the significant differences among the means were determined by DuncanÕs multiple range test (Duncan, 1955).
3. Results and discussion 3.1. Effect of ion concentration on protein extractability The protein extractabilities of NaCl, KCl, and LiCl were shown in Figs. 1–3, respectively. All three salts tested showed an initial significant increased of their protein extractability at each increment. Furthermore, these initial significant increments of protein extractability of NaCl, KCl, and LiCl were extended up to the concentrations of 1.2, 1.0, and 0.8 M, respectively. Thereafter both LiCl and KCl extractants showed a plateau followed by gradual decline of their protein
D.M.S. Munasinghe, T. Sakai / Meat Science 67 (2004) 697–703
699
160
Extractable protein content (mg/ g tissue)
140
c
120
e
e
e
1.2
1.4
1.6
d
e
b
100
a
80 60 40 20 0 0.4
0.6
0.8
1.0
1.8
Molarity
Fig. 1. Effect of NaCl concentration on protein extractability at pH 7.0. Results are means SEM of three replicates. The columns bearing different letters are significantly different from each other (P < 0:05).
140
d
d
Extractable protein content (mg/g tissue)
d
c
120
cd
c
b 100
a 80
60
40
20
0 0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Molarity
Fig. 2. Effect of KCl concentration on protein extractability at pH 7.0. Results are means SEM of three replicates. The columns bearing different letters are significantly different from each other (P < 0:05).
content while a continuous plateau was observed for the NaCl extractants. In order to obtain more accurate concentration for each salt, the protein extractability of each salt was reassessed between the adjacent lower and upper value of the highest observed value of the respective salt in 0.1 M increments. The highest protein
extractabilities for NaCl, KCl, and LiCl were observed at 1.2, 1.1 and 1.1 M, respectively (Table 1). However, Toorop et al. (1997) and Murray and Johnson (1994), respectively reported that 0.4 and 1 M NaCl as suitable NaCl concentrations for porcine meat quality assessment. This may be due to the differences in respective
700
D.M.S. Munasinghe, T. Sakai / Meat Science 67 (2004) 697–703 140
c
c
c
d
b
120 Extractable protein content (mg/g tissue)
c
100
b
a
80
60
40
20
0 0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Molarity
Fig. 3. Effects of LiCl concentration on protein extractability at pH 7.0. Results are means SEM of three replicates. The column bearing different letters are significantly different form each other (P < 0:05).
extraction conditions. For an example Toorop et al. (1997) used homogenization time of 15 s, which is quite inadequate for maximum protein extraction when considered the observation of Lan et al. (1993) who found a significant increase of protein extractability in 30 s homogenizing intervals up to 90 s. 3.2. Effect of pH on protein extractability The effect of pH on protein extractabilities of 1.2 M NaCl, 1.1 M KCl, and 1.1 M LiCl was tested
within the physiological pH range. The highest protein extractability for NaCl was observed at pH 6.5 (Table 2). However, KCl and LiCl did not show a significant variation of their protein extractability within the physiological pH range. Toorop et al. (1997) found significant variation of protein extractability for NaCl between pH 6.0 and 7.0. Similarly, we also observed the highest protein extractability for NaCl at pH 6.5. In contrast, Lan et al. (1993) found similar protein extractability for pork at pH 6.0 and 6.5.
Table 1 The effect of molar concentration on protein extractability (mg/g tissue) of NaCl, KCl and LiCl at pH 7.00 Salt
Molar concentration 0.8
0.9
1.0
1.1
1.2
1.3
1.4
NaCl KCl LiCl
– 117.49 1.03a 110.26 1.23a
– 122.04 0.97ab 110.54 2.23a
131.67 0.80a 124.29 0.28bc 111.26 0.51a
133.08 2. 36ab 124.78 1.59b 116.27 2.76a
139.06 2.01b 118.63 3.3ac 112.83 1.89a
133.76 1.05ab – –
131.37 3. 13a – –
Values (means SEM, n ¼ 3) within row with no common superscript differ significantly (P < 0:05).
Table 2 Effect of pH on protein extractability (mg/g tissue) of 1.2 M NaCl, 1.1 M KCl and 1.1 M LiCl Salt
pH 6.0
6.5
7.0
7.5
8.0
NaCl KCl LiCl
118.03 1.71a 116.47 0.88a 117.09 1.2a
123.48 1.70b 118.87 1.29a 117.76 2.42a
120.79 1.51ab 122.57 4.07a 117.48 2.36a
122.80 0.89ab 118.86 0.42a 116.80 0.52a
122.02 1.29ab 119.15 1.56a 121.71 0.60a
Values (means SEM, n ¼ 3) within row with no common superscript differ significantly (P < 0:05).
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3.3. Comparison of protein extractability The protein extractabilities of 1.2 M NaCl, 1.1 M KCl, and 1.1 M LiCl at pH 7.0 were compared to select the best protein extractant for pork. The highest protein extractability was observed for the NaCl followed by LiCl and KCl (Table 3). Furthermore, the extractable protein content of each salt was found significantly different from each other. Gordon and Barbut (1992) also observed the same order of protein extractability of these three salts in comminuted meat mixes. The difference of the protein extractability of the three salts might due to the inherent properties of the three ions. 3.4. Electrophoretic banding pattern of protein extracts The three salts reflected the salt concentrations (0.4– 1.8) dependant total protein extractability in their electrophoretic banding patterns. The buffered NaCl showed a substantial extraction of myosin heavy chain (band a; 200 kDa) only at 0.6 M and continued to increase up to 1.0 M (Fig. 4). The same tendency was observed for the LiCl and KCl extractants (results were not shown). Furthermore, optimum intensity for myosin heavy chain of NaCl extract was observed at 1.2 M and gradual reduction of intensity was observed thereafter. Toorop et al. (1997) have observed salting out effect for myofibrillar proteins of pork at 0.6 M NaCl (pH 7.0) under their extraction condition. However, we did not observe such effects indicating superiority of our extraction condition. Apart from Myosin heavy chain, bands c (26 kDa), d (21 kDa), and e (19 kDa) showed an increase of their intensities with the increase of salt concentration and approached maximum intensity at 1.2 M followed by a decline. The identities of these proteins were not known at present. However, this indicates the importance of optimal high salt concentration for the completeness of individual protein extractability. Though there were differences such as the intensities of the bands, the ionic strength at which particular band first appeared etc., among the electrophoretic profiles of the salts, no differences were observed among NaCl, KCl, and LiCl in relation to the number of bands present at their respective concentrations of 1.2, 1.1, and 1.1 M (results were not shown). Few bands (e.g. band b, 58 kDa) showed an initial reduction of their intensities. This caused by the higher proportion of the particular
Table 3 Protein extractability (mg/g tissue) of 1.2 M NaCl, 1.1 M KCl, and 1.1 M LiCl at pH 7.0 pH 7.0
NaCl
KCl a
146.98 1.27
LiCl b
125.34 1.19
133.76 0.63c
Value (mean SEM, n ¼ 4) within row with no common superscript differ significantly (p < 0:05).
Fig. 4. SDS–PAGE pattern of protein extractants of NaCl at different molar concentrations. MM designates molecular weight markers in kDa. Lanes 1–8 represent salt concentrations of extractants 0.4–1.8 M in 0.2 increments. Arrow heads a–e represent bands with molecular weights 200, 58, 26, 21 and 19 kDa, respectively.
protein due to low extraction of myosin heavy chain when every lane contains same quantity of protein. As shown in Fig. 5, a significant difference was not observed for the number of bands and their intensities throughout physiological pH range. The same phenomena were observed for the KCl and LiCl extracts too (results were not shown). However, there were differences in relation to intensities of the bands among the three slats reflecting the differences of the protein extractability of the three salts. As shown in Fig. 5, salting our or salting in effects were not observed with in the physiological pH range. This gives the possibility of adjusting the pH of buffers suite to the pH of subsequent procedures. The electrophoretic banding pattern of the protein extractants of comparison study showed a similarity in banding pattern of all the three salts tested (Fig. 6). However, differences in individual protein extractability among the salts may surface if more sensitive methods such as two-dimension SDS–PAGE were used. Furthermore, low extractability of myosin was clearly visible for KCl extractant. The inability of KCl to extract enough myosin may be the reason for its very low total protein extractability. Even in LiCl extractants, the intensity of the myosin band was lighter than that of NaCl extractants indicating low myosin extractability. Cheung and Cooke (1971) have shown Li+, Na+, and K+ ions have different effects on myosin confirmation. Such conformational changes might have caused the
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number of individual protein bands (26) than the crude extraction and soluble fractions of pork muscle prepared by Sentandreu and Toldra (2000).
4. Conclusions and implications
Fig. 5. SDS–PAGE pattern of proteins extracted at 1.2 M NaCl with in physiological pH range. MM designates molecular weight markers in kDa. Lanes 1–5 represent pH of extractants 6.0–8.0 in 0.5 increments.
The buffered NaCl had significantly higher protein extractability at its optimum concentration (1.2) compared with those of LiCl and KCl making NaCl as a better protein extractant. The higher protein extractability of NaCl seems to be due to its higher extraction of myosin. The amount of myosin present, being one of the major factors that determine gel forming ability and its quality in surami products, buffered NaCl could be applied as a better protein extractant particularly when comminuted meat are used. Moreover, the utilization of NaCl is more attractive in industrial wise due to its inexpensiveness and known safety as a food additive. The higher total and individual protein extractability of buffered NaCl make it a better protein extractant for lean meat quality assessment of pork than that of KCl or LiCl. In addition, the extractability of clearly separated higher number of individual protein bands may enable it to be used as a protein extractant for the detection of meat substitutes where optimum individual protein representation make a high impact on accuracy and sensitivity of such experiments.
Acknowledgements We thank Mrs. T. Okubo and T. Tagami (Faculty of Agriculture, Miyazaki University) for their technical assistance.
References
Fig. 6. SDS–PAGE pattern of proteins extracted by 1.2 M NaCl, 1.1 M KCl, and 1.1 M LiCl at pH 7.0. MM designates molecular weight markers in kDa. Lanes 1–3 represent protein extractants of NaCl, KCl, and LiCl, respectively. Arrow head indicates the myosin band.
differences in myosin solubility in different salts resulting in differences of total protein extractability among the three salts. The 1.2 M NaCl extractant had a higher
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