Kinetic Analysis of the Thermal Inactivation Behavior of AMP Deaminase and IMPase in Each Muscle Type of Yellowtail Seriola quinqueradiata

Abstract

In this study, a kinetic analysis was conducted to clarify the thermal inactivation behavior of AMP deaminase and IMPase, enzymes involved in the generation and degradation of inosine 5′-monophosphate (IMP) in the dorsal ordinary muscle (OM) and dark muscle (DM) of yellowtail Seriola quinqueradiata. Both enzymes were extracted from each part of the fish muscle, heated in the range of 50–60 °C, and then measured for residual enzyme activity. Based on these data, kinetic analysis was performed. When comparing fish muscle types, the thermal stability at 50 °C and 55 °C and the temperature dependence of both AMP deaminase and IMPase tended to be higher in the DM. When comparing the two enzymes, the thermal stability of IMPase was higher than that of AMP deaminase at 50 °C in both muscle types. These results suggest that to prepare heated yellowtail muscle with a high IMP content, it is important to consider the thermal inactivation behavior of enzymes and use slow heating to maintain AMP deaminase activity and produce sufficient IMP in OM. For DM, rapidly increasing the product temperature to ≥60 °C to inactivate IMPase is required to preserve the IMP content.

1. Introduction

Yellowtail Seriola quinqueradiata is one of the most important cultured fish species in Japan. The muscles of this fish consist of the dorsal ordinary muscle (OM) and dark muscle (DM), which have different taste characteristics.

Taste-active components, such as nucleotide-related compounds, are the principal taste compounds in fish muscle [1] and can be altered by endogenous enzymes during heating [2,3]. In particular, inosine 5′-monophosphate (IMP) plays a very important role as an umami component in fish flesh [4], and its quantity determines the overall umami taste. IMP is generated from adenosine 5′-monophosphate (AMP) through a deamidation reaction catalyzed by AMP deaminase and then degraded by IMPase through a dephosphorylation reaction to produce inosine (HxR) [4,5]. Additionally, HxR is converted to hypoxanthine (Hx) by purine nucleosidase or purine nucleoside phosphorylase [6]. HxR is tasteless [5], while Hx has a bitter taste [7]; therefore, it is preferable for IMP to be produced and retained or to accumulate without being degraded in fish muscle.

Fish muscle should be heated under conditions that are compatible with both palatability and microbiological safety. However, no study had previously investigated the effects of heating conditions designed to prevent food poisoning on taste-active components of fish. Thus, we previously analyzed the taste-active components of each part of fish muscle heated under different heating conditions [8]. Our results, particularly focusing on the behavior of IMP during heating, showed that IMP levels in DM tended to decrease with heating. Additionally, IMP levels in DM tended to be lower in low-temperature/long-time pasteurization (LTLT, chamber temperature of 63 °C for 30 min) than in high-temperature/short-time heating (HTST, internal temperature reaches 85 °C and is maintained for 90 s). In OM, IMP levels increased slightly after heating. However, it is not clear to what extent the thermal stability of AMP deaminase and IMPase contribute to the formation and degradation of IMP during heating, and whether the type of muscle and heating temperature affect them.

The inactivation reaction of microorganisms follows a first-order reaction and is interpreted based on chemical reactions or thermal sterilization technologies [9]. A similar interpretation can be made for the thermal inactivation of enzymes [10]. The inactivation rate constant (k) or the decimal reduction time (D value), which is the time required for a 1-log cycle reduction under a given condition, is used as an indicator of the thermal stability of the enzyme. In thermal sterilization, the activation energy (Ea) calculated using the Arrhenius equation, or the Z value, which represents the temperature increment required for a 1-log cycle reduction in the D value based on thermal tolerance curves, is used as an indicator of the temperature dependence of enzyme inactivation. Sterilization and enzyme inactivation are important for maintaining food quality. Therefore, a kinetic analysis of the thermal deactivation of key enzymes in various foods has been conducted to determine optimal heat treatment conditions [11,12,13,14,15,16]. Additionally, in cod, kinetic analysis of protein denaturation has been performed to prevent cooking loss and loss of water holding capacity during heating [17]. However, to the best of our knowledge, no studies have been conducted on the enzymes involved in taste-active compounds in fish muscle. Hence, we believe that the relationship between the thermal stability and temperature dependence of AMP deaminase and IMPase and the IMP content of heated fish muscle could be explored by clarifying their thermal inactivation behavior based on the above kinetic analysis. Furthermore, it is highly important to analyze for each edible muscle part (OM and DM), as they exhibit significantly different behaviors in nucleotide-related compounds during heat treatment [18]. Understanding of enzyme thermal stability based on reaction kinetics is useful for setting heat processing conditions and controlling final seafood product quality, thus enabling the preparation of more palatable products for consumers. Therefore, in this study, we conducted a kinetic analysis to clarify the thermal inactivation behavior of AMP deaminase and IMPase, which are enzymes involved in the generation and degradation of IMP, a major umami compound in fish muscle.

2. Materials and Methods

2.1. Sample Preparation

2.1.1. Fish

Samples were prepared according to a previous study [8]. Cultured yellowtail Seriola quinqueradiata were purchased from a local market in Hiroshima, Japan (4.7 ± 0.1 kg, n = 3). Fish were transported to the laboratory on ice within 8 h of death. The fish were fileted, skinned, and sliced. Fish samples were wrapped in polyethylene film and stored at −80 °C until use. In this study, OM and DM were used as fish muscle samples.

2.1.2. Extraction of Crude Enzymes from Fish Muscles

AMP deaminase was extracted from each part of the fish muscle according to the method described by Terauchi et al. (1992) [19], with slight modifications. Fish muscle (approximately 3.0 g) was homogenized with 9.0 mL of buffer A (0.1 mM potassium chloride, 1 mM 2-mercaptoethanol, and 20 mM potassium phosphate buffer [pH 7.0]) using a homogenizer (T18 digital, IKA-Werke GmbH and Co. KG, Staufen, Germany) at 16,000 rpm for 30 s on ice. The enzyme solution was filtered through a polyethylene mesh, and the final volume was adjusted to 18 mL using buffer A. This extract was used as the crude AMP deaminase extract.

IMPase from each part of the fish muscle was extracted as described by Yoshioka et al. (2019) [20], with slight modifications. Fish muscle (approximately 3.0 g) was homogenized with 9.0 mL of buffer B (0.1 M NaCl and 20 mM Tris-HCl [pH 7.5]) using a homogenizer at 16,000 rpm for 30 s on ice. The extracts were dialyzed against buffer B by ultrafiltration (cutoff molecular weight, 10,000 Da, UF-10PS, Tosoh Corporation, Tokyo, Japan) at 4 °C for 48 h. The extract was filtered through a polyethylene mesh, and the final volume was adjusted to 18 mL using buffer B. This extract was used as the crude IMPase extract.

At the time of use, the extracted crude enzyme solution was heated as soon as possible to prevent inactivation of the enzyme prior to heat treatment. Specifically, AMP deaminase was heated on the day of extraction, and IMPase was heated 48 h after dialysis. Each crude enzyme extract was stored at 0–4 °C, except for during heating and substrate reactions.

2.1.3. Heating the Crude Enzyme Extract

Each crude enzyme extract was heated according to the procedure by Tanimoto et al. (2004) [13], with slight modifications. Briefly, 1 mL of crude enzyme extract was enclosed in a glass tube (φ5 mm, AGC Techno Glass Co., Ltd., Shizuoka, Japan) and heated in a hot water bath at 50 °C, 55 °C, and 60 °C for an arbitrary time. The heating temperature and time were set after confirming that the D and Z values were within the range that could be calculated in preliminary experiments by predicting the thermal inactivation temperature and heating time based on previous studies [13,14,15,21,22]. After heating, the glass tubes were immediately cooled in an ice bath and subsequently used to measure the enzyme activity.

2.2. Measurement of Enzyme Activity

AMP deaminase activity was measured as previously described by Terauchi et al. (1992) and Matsumoto et al. (1994) [19,23], with slight modifications. Briefly, a reaction mixture (200 µL of 10 mM AMP solution [pH 7.0], 50 µL of 50 mM potassium chloride solution [pH 7.0], and 230 µL of buffer A) was preincubated for 10 min at 30 °C. Then, 20 µL of crude AMP deaminase extract was added and incubated for a specific period at 30 °C. The reaction was stopped by adding 125 µL of 20% perchloric acid solution (PCA). Then, 90 µL of 50 mM potassium carbonate solution was added to neutralize the solution and filtered through a 0.45 µm hydrophilic PTFE membrane (Shimadzu, Kyoto, Japan). The ammonia produced by the deamination reaction of AMP deaminase in the filtrate was determined using the indophenol method [24], and the amount of ammonia was determined from a calibration curve obtained from ammonium sulfate.

IMPase activity was measured as described by Yoshioka et al. (2019) [20] with slight modifications. Briefly, a reaction mixture (200 µL of 10 mM IMP solution [pH 7.5], 50 µL of 50 mM magnesium chloride solution [pH 7.5], and 50 µL of buffer B) was preincubated for 10 min at 25 °C. Subsequently, 200 µL of crude IMPase extract was added and incubated for a specific period at 25 °C. The reaction was stopped by adding 125 µL of PCA and filtered through a 0.45 µm hydrophilic PTFE membrane. The phosphoric acid produced by the dephosphorylation reaction of IMPase in the filtrate was determined using the Fiske–Subbarow method [25], and the amount of inorganic phosphoric acid was calculated from a calibration curve obtained from potassium phosphate.

The activity of each enzyme, both before and after heating for an arbitrary period, was determined by measuring the amount of ammonia or phosphoric acid produced after the enzymatic reaction of each crude enzyme extract. One unit of AMP deaminase and IMPase activities was defined as the amount producing 1 µmol/min of ammonia or phosphoric acid, respectively [19]. The residual activity (%) after heat treatment was expressed as the relative value of enzyme activity after heat treatment to before treatment.

2.3. Data Analysis

For the kinetic analysis of enzyme inactivation, a linear relationship was observed between the logarithm of the residual activity and treatment time for each enzyme (Figure 1).

Thus, the thermal inactivation behavior of each enzyme was assumed to follow a first-order reaction and was calculated using the following equation [13,14]:

$$\mathrm{l}\mathrm{n}\left(\mathrm{A}\right)=-\mathrm{k}\mathrm{t}$$

(1)

where A is the residual activity (%) at any heating time t (s) and k is the reaction rate constant (s⁻¹) under a given condition. The value of k was obtained from the slope of the regression of ln(A) versus the heating time.

The natural logarithm is converted into a common logarithm as follows:

$$\mathrm{L}\mathrm{o}\mathrm{g}\left(\mathrm{A}\right)=-(\mathrm{k}/2.303)\mathrm{t}$$

(2)

The D value is related to the reaction rate constant and Equation (3); thus, Equation (3) was transformed and substituted into Equation (2) to derive Equation (4). The D value was calculated from the slope of Equation (4) using the least squares method.

$$\mathrm{D}=2.303/\mathrm{k}$$

(3)

$$\mathrm{L}\mathrm{o}\mathrm{g}\left(\mathrm{A}\right)=-\mathrm{t}/\mathrm{D}$$

(4)

The Z value was calculated from the negative reciprocal slope of the log (D value) versus the heating temperature:

$$Z=(T_2-T_1)/(\mathrm{L}\mathrm{o}\mathrm{g}{D}_1-\mathrm{L}\mathrm{o}\mathrm{g}{D}_2)$$

(5)

where T₁ and T₂ are the heating temperatures and D₁ and D₂ are the D values at T₁ and T₂, respectively.

2.4. Statistical Analysis

The experiments were conducted independently for each fish (n = 3). Student’s t-test was used to test the differences among the mean values using SPSS Statistics 26 (IBM Japan, Tokyo, Japan). The significance level was set at 5%.

3. Results and Discussion

3.1. Enzymatic Activity of AMP Deaminase and IMPase Before Heat Treatment

Table 1. Enzymatic activities of AMP deaminase and IMPase in raw yellowtail muscles.

	AMP Deaminase		IMPase
	Dorsal Ordinary Muscle	Dark Muscle	Dorsal Ordinary Muscle	Dark Muscle
Enzyme activity (unit/fish muscle g)	240 ± 17	128 ± 14 *	1.6 ± 0.1 †	2.1 ± 0.1 *†

Values indicate the mean ± standard deviation of triplicate measurements (n = 3). Asterisks indicate significant differences in different muscle types at the same heating temperature for each enzyme (p < 0.05). Daggers indicate significant differences between different enzymes in the same muscle type (p < 0.05).

shows the activity of each enzyme before the heat treatment. The AMP deaminase activity was significantly higher (p < 0.05) in OM than in DM. However, IMPase was significantly higher (p < 0.05) in DM than in OM. When comparing the enzymes, AMP deaminase activity was significantly higher (p < 0.05) than IMPase activity in both OM and DM. This result supports the findings of previous studies [5,8,26], which showed that IMP accumulated more in OM than in DM in raw samples. Additionally, the higher enzyme activity of AMP deaminase compared to IMPase in both OM and DM suggests that AMP deaminase causes rapid AMP degradation and IMP generation, whereas IMP degradation by IMPase proceeds more slowly, resulting in the accumulation of IMP in fish muscle.

3.2. Kinetic Analysis of Thermal Inactivation Behaviors of AMP Deaminase and IMPase

Table 2. D and Z values for thermal inactivation of AMP deaminase and IMPase in yellowtail muscles.

	Temperature (°C)	AMP Deaminase		IMPase
		Dorsal of Ordinary Muscle	Dark Muscle	Dorsal of Ordinary Muscle	Dark Muscle
D value (s)	50	196 ± 21	1009 ± 418	298 ± 23 †	1889 ± 192 *†
	55	91 ± 32	300 ± 106 *	128 ± 20	450 ± 44 *
	60	51 ± 7	139 ± 67	71 ± 14	203 ± 97
Z value (°C)		17 ± 1	12 ± 2 *	16 ± 1	10 ± 2 *

Values indicate the mean ± standard deviation of triplicate measurements (n = 3). Asterisks indicate significant differences in different muscle types at the same heating temperature and of Z value for each enzyme (p < 0.05). Daggers indicate significant differences between different enzymes of the same muscle type at the same heating temperature and for Z value (p < 0.05).

shows the D and Z values for enzyme inactivation in each part of the fish muscle during the heat treatment. Comparing the parts of the fish muscle, with AMP deaminase, the D value showed no significant difference between OM and DM at 60 °C. However, at 55 °C, the values were significantly higher in DM than in OM (p < 0.05), and at 50 °C, they tended to be higher in DM than in OM, although the difference was not significant (p = 0.078). The Z value of AMP deaminase was significantly lower in DM than in OM (p < 0.05). For IMPase, the D value showed no significant difference between OM and DM at 60 °C; however, D values at 50 °C and 55 °C were significantly higher (p < 0.05), and the Z values were significantly lower (p < 0.05) in DM compared to OM. These results indicate that the thermal stability of both AMP deaminase and IMPase tended to be higher in DM than in OM at 50 °C and 55 °C and that the temperature dependence was also higher in DM. It was also suggested that the thermal stability of the enzymes in OM and DM may become equal or even reverse when the temperature exceeds 60 °C. The heat stability of IMPase in OM and DM of saury was significantly higher in DM at 10–50 °C; however, there was almost no difference between the two parts at 60 °C [5]. This is similar to the IMPase behavior observed in the present study, and it was inferred that AMP deaminase in this fish likely exhibited a similar pattern. The findings from the present study, indicating that DM has higher thermal stability and greater temperature dependence than OM for both enzymes, are interesting. ATPase in sardines was also more stable at 0 °C and 30 °C in DM than in OM [27]. The chemical composition, physiological importance, and nutritional value of OM and DM differ. DM uses energy obtained from aerobic metabolism for continuous swimming, whereas OM is used for rapid and sudden movements, relying mainly on anaerobic glycolysis for energy [28]. In particular, DM is exposed to greater oxidative stress than OM and may be more stable to conformational changes in enzyme proteins. Additionally, the stability of enzymes also differs among species and organs [21,29]. In addition, the thermal stability of several enzymes in fish muscles is also correlated with the temperature of the fish habitat [22]. This is thought to be due to the enzyme adaptation to its environment at the molecular level [30]. Therefore, it is assumed that differences in thermal stability also occur in OM and DM depending on their respective physiological environments. On the other hand, the temperature dependence of both enzymes was greater in DM than in OM because the thermal stability of both enzymes in DM tended to be higher at 50 °C and 55 °C, but the inactivation by thermal denaturation of the enzymes occurred rapidly as the heating temperature approached 60 °C [4,5].

Comparing the enzymes, IMPase had significantly higher D values at 50 °C than AMP deaminase for both OM and DM (p < 0.05), suggesting greater thermal stability of IMPase compared to AMP deaminase at this temperature, regardless of muscle type. However, no significant differences were observed in the D values at 55 °C and 60 °C, indicating that the thermal stabilities of the two enzymes at these temperatures were equivalent. Z values were also not significantly different between the two enzymes. The purified 5′-nucleotidase (IMPase) from carp skeletal muscle retained approximately 90% of its activity at 50 °C and approximately 15% at 60 °C after a 5 min heat treatment [31]. The purified 5′-nucleotidase from snapper muscle was almost completely inactivated at 60 °C after 5 min, while about 60% of its activity was retained at 50 °C for 5 min and 90% at 40 °C for 30 min [32]. The partially purified acid phosphomonoesterase (IMPase) from mackerel DM was stable at 40 °C, 50% inactivated at 50 °C for 10 min, and fully inactivated at 60 °C for 5 min [33]. In contrast, purified AMP deaminase from the white muscle of common carp was 80% inactivated when treated in pure water at 50 °C for 5 min but was stabilized in the presence of low concentrations of KCl [34]. AMP deaminase from snapper muscle was inactivated at 50 °C for 5 min and 40 °C for 20 min, with approximately 30% of its activity remaining after 30 min at 30 °C [19]. Crude AMP deaminase from mackerel muscle was inactivated at 55 °C after 30 min of treatment, with approximately 30% and 50% of its activity remaining at 50 °C and 40 °C, respectively [35]. Although these studies on carp, mackerel, and snapper used purified enzymes without kinetic analysis, the enzyme inactivation behavior in OM showed a similar trend to the present study. In addition, previous studies have confirmed interspecific differences in the thermal stability of enzymes in beef [36] and sake [13]. At temperatures above 55 °C, the treatment temperature approached approximately 60 °C, which is the typical inactivation temperature for enzymes [4,5] and is assumed to cause rapid inactivation of enzymes [4], resulting in no observable differences. Furthermore, the results of the present study, which showed that IMPase tended to exhibit greater thermal stability at 50 °C in both muscle parts, support the findings that HxR and Hx levels in OM and DM of yellowtail were significantly higher under LTLT [8]. The temperature dependence of both enzymes did not differ when they were present in the same muscle type. This suggests that, similarly to the results from the heat resistance (D value) of the enzyme at each temperature, temperature dependence is more influenced by the muscle type than the enzyme type. Therefore, it was inferred that the inactivation behavior of enzymes in the muscle is affected by the type of muscle.

Based on the above results, the regression curves of D values obtained in the 50–60 °C range and the calculated Z values in this study can be utilized when considering the application to actual heat treatment, for example, in determining the heating time required for the inactivation of each enzyme at 70 °C. In future studies, measuring the D value over a wider range of temperatures and calculating the Z value will enable the development of mathematical models to establish more accurate sterilization and heating conditions. In a previous study [8], yellowtail muscle was heated based on food hygiene guidelines and regulations [37,38,39], and the core temperature of 85 °C at HTST and 63 °C at LTLT were reached in approximately 11 min, respectively. As a result, when OM and DM were heated with LTLT, the equivalent umami concentrations were higher than those for HTST. Therefore, the kinetic findings of this study strongly support this result. The kinetic parameters of the thermal inactivation behavior of AMP deaminase and IMPase have the potential to provide accurate recommendations or guidelines for commercial cooking and processing.

The limitations of this study stand in the fact that it is unclear whether our results on enzyme inactivation are fully consistent with their inactivation behavior in fish muscle because crude enzymes were used to evaluate the inactivation behavior of enzymes in yellowtail muscle. It is necessary to confirm that there are no sample variations in the kinetics of enzyme inactivation between fish species or between wild and farmed species. Therefore, it is necessary to purify the enzyme and clarify the relationship between structural changes and thermal stability in the future. In addition, since no sensory evaluation was conducted, it is necessary to examine in detail whether the results in the present study are consistent with the actual taste changes in fish muscle. However, we hope that the findings obtained in this study will be applied to the production of seafood products that are more palatable.

4. Conclusions

The thermal stability of AMP deaminase and IMPase was higher in DM than in OM at 50 °C and 55 °C but was equivalent at 60 °C. The temperature dependence of the inactivation of these enzymes was greater in DM than in OM. This indicates that enzyme thermal stability at 50 °C and 55 °C, as well as temperature dependence, rely on the muscle where the enzymes are present. A comparison between the enzymes showed that the thermal stability of IMPase was higher than that of AMP deaminase at 50 °C, but they were equivalent at 55 °C and 60 °C. Additionally, the temperature dependence of the inactivation of these enzymes was similar. This indicates that thermal stability at 50 °C may depend not only on the muscle part where the enzymes are present but also on the enzyme type. Therefore, based on the thermal inactivation behavior of both enzymes, since adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), and AMP remain in OM even before heating [8] and the AMP deaminase activity before heat treatment is higher than IMPase activity (Table 1), to prepare heated yellowtail muscle with a high IMP content, that is, with a high umami flavor, slow heating is required to maintain AMP deaminase activity in OM. However, in DM, almost no ATP, ADP, and AMP remained before heating [8]. IMPase activity was higher than that in OM, and thermal stability below 55 °C was also greater (Table 1). Therefore, it is important to rapidly inactivate IMPase at a temperature of ≥60 °C to preserve as much IMP as possible. In other words, this suggests that HTST is advantageous for retaining the umami flavor in DM. In the future, the relationship between the structure and heat stability of both proteins in different muscle parts should be clarified using purified enzymes. Additionally, our results need to be further validated in wild fish and other fish species.