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thawed pork
M e a t Science 29 (1991) 177-181
Research Note Simple Test for Differentiation between Fresh Pork and Frozen/Thawed Pork
ABSTRACT Enzyme profile of the exudates from fresh and frozen/thawed pork has been assayed by using a semi-quantitative method ( A P I Z Y M ) . Only three enzymes (esterase-lipase, fl-glucuronidase and ct-glucosidase) showed significant differences (P
INTRODUCTION Different methods of differentiation between fresh and frozen/thawed meat, based on the release of mitochondrial enzymes into the sarcoplasm, have been proposed in the literature (Hamm, 1979; Gottesmann & Hamm, 1983, 1984; Chen et al., 1988). Most ofthem are, however, difficult to assay because they require specific techniques or long processing times. Other methods are based on the assay of the fl-hydroxyacyl-Co A-dehydrogenase (HADH) by both photometric and colour test (Gottesmann & Hamm, 1983). The colour test has been recently improved by stabilising the colour after reaction with fresh meat extracts (Chen et al., 1988). However, these methods are only reliable when the meat is frozen at --12°C or lower (Gottesmann & Hamm, 1983). On the other hand, the photometric method requires scientific instruments and qualified personnel which would reduce the availability for many industries. 177 M e a t Science 0309-1740/90/$03"50 O 1990 Elsevier Science Publishers Ltd, England. Printed
in Great Britain
178
Fidel Toldrd, Yolanda Torrero, Jos~ Flores
Other enzymes may be assayed as indicators of freezing treatment. The 'Apizym' system (Api system, Montalieu-Vercieu, France) consists of a semiquantitative method for the simultaneous measurement of 19 enzyme activities usually found in tissues, cells, microorganisms.... It has already been used for the detection of enzyme activities in both raw and heat processed muscle tissue (Townsend & Blankenship, 1987). This technique is very simple and seems attractive as an easy tool for the control of frozen/thawed meat. In the present work, we have tested the 'Apizym' system as a new and easy method for the differentiation between fresh pork and frozen/thawed pork.
MATERIALS A N D METHODS
M. Longissimus dorsi was obtained from an industrial meat processor within 60 min from slaughter and immediately cut into 100-125 g portions. Each sample was vacuum packed in plastic bags and either stored at - 18°C for various periods of time (11, 22, 35, 43 and 54 days) or stored during 13 days at four different temperatures ( - 10, - 13, -- 18 and -60°C). All samples were done in triplicate. Thawing temperature was always 25°C. Meat juice was obtained by squeezing out. Twenty-five microlitres of the collected exudate was diluted 1:200 with 0-1M phosphate buffer (pH 6-0) and used for the photometric test as described by Gottesmann and Hamm (1983). Seventy five microlitres of the exudate were also diluted 1:20 with distilled water and, then, 60/al added to each of the galleries in the Apizym strip (Api system, Montalieu-Vercieu, France). The strips were incubated at 37°C for 4 h. Color intensity values, ranging from 0 to 5, were determined in accordance with the manufacturer's instructions. Catalysis of N A D H to N A D + by ~-hydroxyacyl-Co A-dehydrogenase (HADH) was continuously monitored at 340 nm for 3 min at 25°C. Activity is expressed in International Units per ml (U/ml). The differences between means were evaluated by t-test. Differences between values with P < 0"01 were considered statistically significant.
RESULTS A N D DISCUSSION H A D H activities of exudates from both fresh and frozen/thawed meat are reflected in Table 1. As stated by Gottesmann and Hamm (1983), H A D H activity should not exceed 6"0 U/ml for unfrozen pork meat. But, according
Differantiation between fresh pork and frozen~thawed pork T A B L E
179
1
Activities of HADH in Exudates from Unfrozen Fresh Pork and Frozen/thawed Pork tin U/ml)* Acticity
Fresh unfrozen pork
3'09
Frozen/thawed pork Temp. storage (°C) 10 - 13 18 60
3-80 37"60 6-85 9-92
Time storage (days) 11 22 35 43 54
6"66 6"85 7-92 8-15 6"03
* Each experimental point represcnts the mean of three samples. to the same authors, the m e t h o d is only reliable for meat frozen at temperatures below - 1 2 ° C . In fact, we observed an anomalous result, (3-80 U/ml), when the meat was frozen at - 10°C (see Table 1). In this case, the meat would be erroneously classified as fresh. In the case o f the other storage conditions, H A D H activities in the exudates were found to be higher than 6-0U/ml (see Table l) and would be correctly classified as frozen/thawed meat. The Apizym system was also assayed as a test for frozen/thawed meat. The same samples as those used for the H A D H method were assayed. Color intensity values for different enzymes in the exudates from fresh unfrozen pork and frozen/thawed pork are shown in Table 2. As may be observed, only three enzymes (esterase-lipase, fl-glucuronidase and ~-glucosidase) showed significant differences between fresh and frozen/thawed pork. No significant differences (P<0-01) were found a m o n g the conditions for freezing temperatures ( - 10 to - 6 0 ° C ) for 13 days and times o f storage (11 to 54 days at - 18°C) (see Table 3). The Apizym system would constitute an easy m e t h o d for differentiation between fresh and frozen/thawed meat for a wide range o f freezing temperatures, - 10 to - 60°C. A n o t h e r advantage is that it does not require special scientific instruments or qualified personnel.
TABLE
2
Colour Intensity Values for Different Enzymes from Exudates of Unfrozen Fresh Pork and Frozen/thawed Pork
Enzymes detected
Fresh pork
Frozen~thawed pork
.2
s
~
s
Control Alkaline phosphatase Esterase C4 Esterase lipase C8 Lipase C14
0 3-1 2"8 4.0 0-2
0 0-3 0-3 0 0-3
0 2-9 2.7 2-8 0"3
0 0-8 0-7 0-5* 0-2
Leucine aminopeptidase Valine aminopeptidase Cystine aminopeptidase Trypsin ~t-Chymotrypsin
4.3 4.2 2'9 1"3 0-6
0-3 0-3 0-3 0-5 0"5
4-4 4.7 2-7 1-4 0"5
0-4 0-4 0-5 0-6 0-4
Acid phosphatase Phosphoamidase ~,-Galactosidase fl-Galactosidase fl-Glucuronidase
5-0 2-5 0.3 0.6 0.6
0 0-5 0.5 0.3 0-3
4-6 1.8 0 0-7 0
0-5 0.7 0 0.4 0*
~t-Glucosidase fl-Glucosidase N-Acetyl fl-glucosaminidase ~t-Mannosidase ~t-Fucosidase
0"6 0 2-0 0 0'2
0'3 0 0 0 0-3
2-0 0 2"0 0-1 0
0"5* 0 0'8 0-2 0
* Significant differences between means (P < 13-01).
TABLE 3 Colour Intensity Values for Esterase Lipase Ca, fl-Glucuronidase and ~t-Glucosidase from Exudates as Affected by Freezing Temperature and Time of Frozen Storage
Enzymes activities Esterase lipase C 8
fl-Glucoronidase
~t-Glucosidase
Temp. storage (°C) - - 10
3-0
0
2-5
- - 13
3-0
0
2-0
- 18
2"5
0
2-5
60
2"5
0
2-5
I1
3-0
0
22 35 43 54
2"5 3-0 3"0 2"5
0 0 0 0
2"0 1-5 2-0 2-5 1-5
-
Time of storage (days)
Differentiation betweenfresh pork and fro:en/thawed pork
181
ACKNOWLEDGEMENTS Financial support from the Comisi6n Tecnologia (CICYT) is acknowledged.
Interministerial de Ciencia y
REFERENCES Chen, M. T., Yang, W. D. & Guo, S. L. (1988). Meat Sci., 24, 223. Gottesmann, P. & Hamm, R. (1983). Fleischwirtsch. 63, 219. Gottesmann, P. & Harem, R. (1984). Fleischwirtsch., 64, 203. Hamm, R. (1979) In Proteins at Low Temperatures, ed. O. Fennema, Amer. Chem. Soc., Washington, DC, pp. 191-204. Townsend, W. E. & Blankenship, L. C. (1987). J. Food Sei., 52, 51 I.
Fidel Toldrfi, Yolanda Torrero & Jos~ Flores lnstituto de Agroquimica y Tecnologia de Alimentos (CSIC), Jaime Roig, 11-46010 Valencia, Spain (Received 3 February 1990; accepted 4 March 1990)
Research Note Simple Test for Differentiation between Fresh Pork and Frozen/Thawed Pork
ABSTRACT Enzyme profile of the exudates from fresh and frozen/thawed pork has been assayed by using a semi-quantitative method ( A P I Z Y M ) . Only three enzymes (esterase-lipase, fl-glucuronidase and ct-glucosidase) showed significant differences (P
INTRODUCTION Different methods of differentiation between fresh and frozen/thawed meat, based on the release of mitochondrial enzymes into the sarcoplasm, have been proposed in the literature (Hamm, 1979; Gottesmann & Hamm, 1983, 1984; Chen et al., 1988). Most ofthem are, however, difficult to assay because they require specific techniques or long processing times. Other methods are based on the assay of the fl-hydroxyacyl-Co A-dehydrogenase (HADH) by both photometric and colour test (Gottesmann & Hamm, 1983). The colour test has been recently improved by stabilising the colour after reaction with fresh meat extracts (Chen et al., 1988). However, these methods are only reliable when the meat is frozen at --12°C or lower (Gottesmann & Hamm, 1983). On the other hand, the photometric method requires scientific instruments and qualified personnel which would reduce the availability for many industries. 177 M e a t Science 0309-1740/90/$03"50 O 1990 Elsevier Science Publishers Ltd, England. Printed
in Great Britain
178
Fidel Toldrd, Yolanda Torrero, Jos~ Flores
Other enzymes may be assayed as indicators of freezing treatment. The 'Apizym' system (Api system, Montalieu-Vercieu, France) consists of a semiquantitative method for the simultaneous measurement of 19 enzyme activities usually found in tissues, cells, microorganisms.... It has already been used for the detection of enzyme activities in both raw and heat processed muscle tissue (Townsend & Blankenship, 1987). This technique is very simple and seems attractive as an easy tool for the control of frozen/thawed meat. In the present work, we have tested the 'Apizym' system as a new and easy method for the differentiation between fresh pork and frozen/thawed pork.
MATERIALS A N D METHODS
M. Longissimus dorsi was obtained from an industrial meat processor within 60 min from slaughter and immediately cut into 100-125 g portions. Each sample was vacuum packed in plastic bags and either stored at - 18°C for various periods of time (11, 22, 35, 43 and 54 days) or stored during 13 days at four different temperatures ( - 10, - 13, -- 18 and -60°C). All samples were done in triplicate. Thawing temperature was always 25°C. Meat juice was obtained by squeezing out. Twenty-five microlitres of the collected exudate was diluted 1:200 with 0-1M phosphate buffer (pH 6-0) and used for the photometric test as described by Gottesmann and Hamm (1983). Seventy five microlitres of the exudate were also diluted 1:20 with distilled water and, then, 60/al added to each of the galleries in the Apizym strip (Api system, Montalieu-Vercieu, France). The strips were incubated at 37°C for 4 h. Color intensity values, ranging from 0 to 5, were determined in accordance with the manufacturer's instructions. Catalysis of N A D H to N A D + by ~-hydroxyacyl-Co A-dehydrogenase (HADH) was continuously monitored at 340 nm for 3 min at 25°C. Activity is expressed in International Units per ml (U/ml). The differences between means were evaluated by t-test. Differences between values with P < 0"01 were considered statistically significant.
RESULTS A N D DISCUSSION H A D H activities of exudates from both fresh and frozen/thawed meat are reflected in Table 1. As stated by Gottesmann and Hamm (1983), H A D H activity should not exceed 6"0 U/ml for unfrozen pork meat. But, according
Differantiation between fresh pork and frozen~thawed pork T A B L E
179
1
Activities of HADH in Exudates from Unfrozen Fresh Pork and Frozen/thawed Pork tin U/ml)* Acticity
Fresh unfrozen pork
3'09
Frozen/thawed pork Temp. storage (°C) 10 - 13 18 60
3-80 37"60 6-85 9-92
Time storage (days) 11 22 35 43 54
6"66 6"85 7-92 8-15 6"03
* Each experimental point represcnts the mean of three samples. to the same authors, the m e t h o d is only reliable for meat frozen at temperatures below - 1 2 ° C . In fact, we observed an anomalous result, (3-80 U/ml), when the meat was frozen at - 10°C (see Table 1). In this case, the meat would be erroneously classified as fresh. In the case o f the other storage conditions, H A D H activities in the exudates were found to be higher than 6-0U/ml (see Table l) and would be correctly classified as frozen/thawed meat. The Apizym system was also assayed as a test for frozen/thawed meat. The same samples as those used for the H A D H method were assayed. Color intensity values for different enzymes in the exudates from fresh unfrozen pork and frozen/thawed pork are shown in Table 2. As may be observed, only three enzymes (esterase-lipase, fl-glucuronidase and ~-glucosidase) showed significant differences between fresh and frozen/thawed pork. No significant differences (P<0-01) were found a m o n g the conditions for freezing temperatures ( - 10 to - 6 0 ° C ) for 13 days and times o f storage (11 to 54 days at - 18°C) (see Table 3). The Apizym system would constitute an easy m e t h o d for differentiation between fresh and frozen/thawed meat for a wide range o f freezing temperatures, - 10 to - 60°C. A n o t h e r advantage is that it does not require special scientific instruments or qualified personnel.
TABLE
2
Colour Intensity Values for Different Enzymes from Exudates of Unfrozen Fresh Pork and Frozen/thawed Pork
Enzymes detected
Fresh pork
Frozen~thawed pork
.2
s
~
s
Control Alkaline phosphatase Esterase C4 Esterase lipase C8 Lipase C14
0 3-1 2"8 4.0 0-2
0 0-3 0-3 0 0-3
0 2-9 2.7 2-8 0"3
0 0-8 0-7 0-5* 0-2
Leucine aminopeptidase Valine aminopeptidase Cystine aminopeptidase Trypsin ~t-Chymotrypsin
4.3 4.2 2'9 1"3 0-6
0-3 0-3 0-3 0-5 0"5
4-4 4.7 2-7 1-4 0"5
0-4 0-4 0-5 0-6 0-4
Acid phosphatase Phosphoamidase ~,-Galactosidase fl-Galactosidase fl-Glucuronidase
5-0 2-5 0.3 0.6 0.6
0 0-5 0.5 0.3 0-3
4-6 1.8 0 0-7 0
0-5 0.7 0 0.4 0*
~t-Glucosidase fl-Glucosidase N-Acetyl fl-glucosaminidase ~t-Mannosidase ~t-Fucosidase
0"6 0 2-0 0 0'2
0'3 0 0 0 0-3
2-0 0 2"0 0-1 0
0"5* 0 0'8 0-2 0
* Significant differences between means (P < 13-01).
TABLE 3 Colour Intensity Values for Esterase Lipase Ca, fl-Glucuronidase and ~t-Glucosidase from Exudates as Affected by Freezing Temperature and Time of Frozen Storage
Enzymes activities Esterase lipase C 8
fl-Glucoronidase
~t-Glucosidase
Temp. storage (°C) - - 10
3-0
0
2-5
- - 13
3-0
0
2-0
- 18
2"5
0
2-5
60
2"5
0
2-5
I1
3-0
0
22 35 43 54
2"5 3-0 3"0 2"5
0 0 0 0
2"0 1-5 2-0 2-5 1-5
-
Time of storage (days)
Differentiation betweenfresh pork and fro:en/thawed pork
181
ACKNOWLEDGEMENTS Financial support from the Comisi6n Tecnologia (CICYT) is acknowledged.
Interministerial de Ciencia y
REFERENCES Chen, M. T., Yang, W. D. & Guo, S. L. (1988). Meat Sci., 24, 223. Gottesmann, P. & Hamm, R. (1983). Fleischwirtsch. 63, 219. Gottesmann, P. & Harem, R. (1984). Fleischwirtsch., 64, 203. Hamm, R. (1979) In Proteins at Low Temperatures, ed. O. Fennema, Amer. Chem. Soc., Washington, DC, pp. 191-204. Townsend, W. E. & Blankenship, L. C. (1987). J. Food Sei., 52, 51 I.
Fidel Toldrfi, Yolanda Torrero & Jos~ Flores lnstituto de Agroquimica y Tecnologia de Alimentos (CSIC), Jaime Roig, 11-46010 Valencia, Spain (Received 3 February 1990; accepted 4 March 1990)