M. SEKIKAWA, K. SENO, K. SHIMADA, M. FUKUSHIMA and M. MIKAMI

Department of Bioresource Science,

Obihiro University of Agriculture and Veterinary Medicine,

Inada, Obihiro 080, Japan

Abstract

Electrical stimulation (ES) of a beef carcass increases the content of free amino acids, which affects meat flavor. Levels of glutamic acid (Glu)-aminopeptidase activity were similar in both control and ES meat but decreased during storage to about 65% at 14 days. When 2-ketoglutaric acid (a substrate of transaminase) was added to the sarcoplasm, the level of Glu increased. Transaminase activity was unaffected by storage and was about 20% greater in ES than in the control. Transaminase activity in beef was confirmed when radioactive 2-ketoglutaric acid was added to the sarcoplasm. These results suggest that transaminase contributes to the accumulation of some amino acids such as Glu in beef during conditioning.

Key words: electrical stimulation, beef conditioning, aminopeptidase, transaminase, glutamic acid

Electrical stimulation (ES) of a beef carcass increases the rates at which pH drops and glycogen levels decline. Because ES and hot boning may markedly affect processing, economics, and tenderness of meat, both treatments have been the subject of several studies (Cross and Seideman 1985). ES has the advantages of color stability and improving the tenderness of meat (Devine et al., 1984; Chrystall and Devine, 1985; Uytterhaegen et al., 1992; Ho et al., 1996). It also increases the content of free amino acids during storage, which affects meat flavor (Morris et al., 1997; Mikami et al., 1994). Nishimura et al. (1994) considered the primary reason for this accumulation to be the action of protease, peptidase, or both. However, Sekikawa et al. (1996) found that the increase in each amino acid is not uniform: alanine (Ala) increased only slightly compared with glutamic acid (Glu). The aims of this study were to compare the effect of ES on the content of amino acids and to investigate another mechanism; contributing of transaminase for amino acid accumulation in beef during storage.

Four Holstein steers (21?23 months old) were conventionally slaughtered and assigned to 1 of 2 groups: control (not stimulated) and electrically stimulated for 60 s at 40 V, 13.8 Hz, within 10 min of slaughter. Thirty-gram samples of ground quadriceps femoris muscle were taken 24 hr after slaughter and stored at 4℃ in a refrigerator. Samples were then homogenized with 90 g of 0.1 M phosphate buffer (pH 7.2) at 1, 7, and 14 days. The homogenate was centrifuged (11000× g, 20 min at 1℃), and the supernatant was filtered (0.45 μｍ) to produce the sarcoplasm. Free amino acids in the sarcoplasm were analyzed in a 2% trichloroacetic acid?soluble fraction with an amino acid analyzer (Hitachi 835, Tokyo).

Glutamic acid?aminopeptidase was measured by using glutamic acid?beta-naphthylamide as a substrate (Nakai et al., 1995). The sarcoplasm obtained (0.1 ml) was incubated with 0.4 mL of 1.2 mM glutamyl-β-naphthylamide in a 0.1 M phosphate buffer (pH 7.2) containing 1 mM threo-1,4-dimercapto-2,3-butanediol at 37℃ for 10 min. The released β-naphthylamine was measured at 540 nm by using p-dimethylamino-cinnamaldehyde.

Transaminase (glutamic-pyruvic transaminase [GPT], E.C. 2.61.2) activity was measured with a GPT clinical test kit (Wako Co., Tokyo). The sarcoplasm obtained (0.2 mL) was incubated with a 0.08 M phosphate buffer (pH 7.4) containing 80 mM Ala, 18 mM 2-ketoglutaric acid, 0.12 mM β-NADH, and 1400 u/mL LDH. The activity was measured as the decrease in absorbancy at 340 nm for 2 or 10 min. Results were recorded as change in absorption per min per 1 g meat.

Samples of sarcoplasm were treated at day 1 with 10 μmol radioactive 2-ketoglutaric acid (α-[¹⁴CU]-; Dupont, France) and incubated for 48 hr at 4℃. Radioactive amino acids or other substrates were identified by thin-layer chromatography (TLC) with coated silica gels (Wako, Tokyo) and 2 solvents: (1): chloroform/methanol/ammonia (17%) in the ratio 40:40:20 and (2): phenol/water in the ratio 75:25. The radioactivity was recorded on an imaging plate with a Bio-Imaging Analyzer (Fuji Co., Tokyo), and the amino acids were detected for ninhydrin reaction.

Our previous studies (Mikami et al., 1994; Sekikawa et al., 1996) and others showed that almost all free amino acids in beef increase during storage (Field et al., 1971; Feidt et al., 1996). The mean values for each storage day indicated that Ala was present at the highest concentrations, and that hydroxyproline and cysteine were present at the lowest. In this study, the mean values for Ala were also the highest overall of the free amino acids analyzed, regardless of treatment. Table 1 shows the mean values for Ala and Glu found during storage. Nishimura et al. (1988) stated that Ala is one of the major free amino acids in beef, pork, and chicken muscle tissue. This accumulation of free amino acids in meat may contribute to an improvement in flavor development during roasting (Morris et al., 1997). However, glutamine (Gln), which was not determined in either our study or that of Nishimura et al.(1988), is the most abundant free amino acid in human plasma and muscle (Perriello et al., 1995). Gln can be synthesized by skeletal muscle, where high concentrations of it are found.

In our previous study (Mikami et al., 1994; Sekikawa et al., 1996), leucine and phenylalanine in the control increased more than Ala, and Ala in ES decreased slightly. We again saw this decrease in this study, but do not yet understand the cause. The increase in amino acid levels during storage has been thought to be due to the action of certain proteolytic enzymes located in cytoplasm or released partly from lysosomes, including aminopeptidase and cathepsins (Etherington et al., 1990; Koohmaraie, 1992; Nishimura et al., 1994; Dransfield, 1994; Sekikawa et al., 1998). The increase in ES meat is probably related to the action of lysosomal enzymes (Dutson et al., 1980) following disruption of the muscle structure (Savell et al., 1978; Will et al., 1980; Takahasi et al., 1987; Chrystall and Devine, 1985). In these studies, it appears that the lysosomal enzymes released by ES acted more rapidly on the meat.

Daily means of Glu-aminopeptidase activity were similar in both treatments but decreased during storage to about 65% at 14 days (Fig. 1). Nakai et al. (1995) also stated that aminopeptidase activity in beef decreased with storage. The levels of most amino acids identified in our previous and present studies increased almost linearly throughout storage, but the increase in individual amino acid levels was not uniform: Ala changed only slightly compared with Glu in ES meat. Therefore, aminopeptidase alone is not the explanation for the increases.

A possible reason for the increasing Glu vs. decreasing Ala might be transaminase, which transfers the alpha-amino group of Ala to 2-ketoglutaric acid to yield Glu. GPT and glutamic-oxaloacetic transaminase are found in the liver, the heart, and skeletal muscles (Garber et al., 1976; Townsend and Davis, 1992). The transaminase activity we measured was unaffected by storage and was about 20% greater in ES than in the control (Fig. 2). Hamm et al. (1969) reported that the activity of transaminase in dead beef and pig muscles decreased only slightly during storage at 4℃ for a week.

Transaminase activity in beef was confirmed when 2-ketoglutaric acid (a substrate of transaminase) was added to the sarcoplasm. The level of Glu increased (Table 2), but remained unaffected upon the addition of maleic acid (an inhibitor of transaminase; Garber et al., 1976) Moreover, when radioactive 2-ketoglutaric acid was added to the sarcoplasm, the hot spot corresponding to Glu was detected on the TLC plate (Fig. 3). However, because there were a few hot spots of other amino acids on the TLC, detailed analyses will be needed.

As well as providing movement and locomotion, skeletal muscle also generates heat, helps to maintain body temperature, and generates amino acids to maintain a homeostatic level. Muscle lacks glucose-6-phosphatase and is therefore unable to manufacture glucose. During fasting it serves as an energy reservoir, and its proteins are degraded to amino acids, many of which are converted to pyruvate, which is then transaminated to alanine. The alanine travels in the bloodstream to the liver, where it is transaminated back to pyruvate, a glucose precursor (Voet and Voet, 1995).

Our data would indicate that transaminase contributes to the accumulation of amino acids such as Glu during beef conditioning. We presume that ES has an effect not only on proteases but also on other enzymes such as transaminase. The mechanism by which amino acids accumulate in meat is important to both meat scientists and biochemists, such as those investigating ischemia of muscle cells. To elucidate the role of various enzymatic systems such as transaminase during meat conditioning, further experiments are therefore needed.

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Acknowledgment

We are indebted to Dr. M. Kroger, Department of Food Science, The Pennsylvania State University for revision of manuscripts. This research was supported by the Grant-in Aid for Science Research from the Ministry of Education, Science and Culture, Japan (MS: #10660253).