Characterization and meat freshness application of a serial three-enzyme reactor system measuring ATP-degradative compounds

Analytica Chimica Acta 404 (2000) 75–81

Characterization and meat freshness application of a serial three-enzyme reactor system measuring ATP-degradative compounds In-Seon Park, Yong-Jin Cho, Namsoo Kim ∗ Korea Food Research Institute, San 46-1, Baekhyun-dong, Bundang-ku, Songnam-si, Kyungki-do 463-420, South Korea Received 20 May 1999; received in revised form 1 September 1999; accepted 5 September 1999

Abstract A serial three-enzyme reactor system which measures hypoxanthine (Hx ), inosine (Hx R) and inosine 50 -monophosphate (IMP) simultaneously was prepared, characterized for its properties and applied to freshness evaluation of meats. The biosensor system was operated as a flow injection analysis mode and the reactor length combination of 15, 12 and 7 cm for Hx , Hx R and IMP, respectively, was selected for a complete reaction between substrate and enzyme. The sequential enzymatic reactions by the system were performed best at 0.05 M phosphate buffer, pH 7.5 and 35◦ C. The effects of various possible interferants such as amino acids and sodium chloride on the system were investigated. When applied to meat freshness evaluation, the Ki - and H-values obtained by the system agreed well with those obtained by a conventional method (liquid chromatography (LC)). ©2000 Elsevier Science B.V. All rights reserved. Keywords: Characterization; Serial three-enzyme reactors; ATP-degradative compounds; Meat freshness

1. Introduction It is very important to maintain meat quality during storage and marketing for improvement of the sanitary status of processed foods. Freshness-related compounds and their contents in meats are dependent on postmortem aging, conditions prior to death, sexual maturity, storage temperature and other factors related to handling and storage. Hence, various methods assessing meat freshness have been developed based on the measurement of postmortem deteriorative changes associated with sensory quality, chemical changes and microbial growth [1]. However, sensory ∗ Corresponding author. Tel.: +82-342-780-9131; fax: +82-342-709-9876/77 E-mail address: [email protected] (N. Kim)

evaluation is, in most cases, subjective and costly [1]. Microbial methods estimating bacterial spoilage [1,2] and chemical methods measuring total volatile amines [3,4], trimethylamine [5–7] or pH change [8] encounter problems in measuring early postmortem deterioration. Therefore, rapid and simple methods for meat freshness estimation have been required urgently for the determination of autolytic deterioration shortly following death. The utilization of chemical freshness indices for evaluating fish or meat freshness is appealing because they are quantitative in nature, objective and easily measured by an automatic device. Adenosine triphosphate (ATP) alone cannot be used as a freshness index because it is so rapidly converted into its degradative compounds comprising inosine 50 -monophosphate (IMP), inosine (Hx R) and hypoxanthine (Hx ). The

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concentrations of these compounds also rise and fall during the early postmortem period. Accordingly, attention has focused on freshness prediction by the determination of relative ratios of ATP-degradative compounds [9–12]. Ki -value, (Hx R + Hx )/(IMP + Hx R + Hx ) × 100, % and H-value, Hx /(IMP + Hx R + Hx ) × 100, % [12,13] which were originally proposed to evaluate fish freshness can be applied to the measurement of meat freshness. Shahidi et al. [14] analyzed the contents of nucleotides and their degradative products in harp seal meat under postmortem storage and derived K-value, (Hx R + Hx )/(ATP + ADP + AMP + IMP + Hx R + Hx ) × 100, % and Ki -value for freshness assessment. Bergann and Kleeman [15] calculated K-values from a chart showing the contents of ATP-degradative compounds and applied their system to beef and pork. Other reports dealing with the freshness evaluation in beef, rabbit and quail meat by the measurement of ATP-degradative products have also been found [16,17]. Biosensor systems for convenient measurement of chemical freshness indices such as Ki - and H-values have been developed by making use of the enzymes involved in ATP-degradative pathway shown below and oxygen electrodes [18–20]: IMP

50 -nucleotidase (50 -NT)

+Pi

→ Hx R nucleoside phosphorylase (NP)

+O2

→

Hx

needed for calculating Ki - and H-values separately and simultaneously can be measured, facilitating an effective and accurate estimation of meat freshness. In this work, the optimum conditions for operating this system were determined and the effects of possible interferants on biosensor performance were evaluated. Furthermore, the contents of Hx , Hx R and IMP in beef loins and chickens were measured after the postmortem storage for 7 days at 4◦ C, and the resulting Ki - and H-values were also obtained.

2. Experimental 2.1. Enzymes and reagents XOD (EC 1.1.3.22, from buttermilk, 50 units/mg solid) and NP (EC 2.4.2.1, bacterial, 500 units/mg solid) were obtained from Sigma (MO, USA). 50 -NT (EC 3.1.3.5, from Crotalus adamanteous venom, 1000 units/mg solid) was purchased from Fluka (Germany). ATP and its degradative compounds (IMP, Hx R and Hx ), and 50% glutaraldehyde were also obtained from Sigma. Chitosan porous beads (Chitopearl, diameter 0.1 mm) were purchased from Wako (Japan). All other chemicals were of analytical reagent grade. 2.2. Preparation of enzyme reactors

xanthine oxidase (XOD)

→

uric acid + 2H2 O2

The relative amounts of Hx , Hx R and IMP in fish or meat samples under postmortem storage quite differ according to the species of sample. Moreover, the response factors for Hx R and IMP are different from that of Hx . As expected from the above pathway, a biosensor which measures consumed oxygen concentration cannot selectively discriminate Hx R or IMP against Hx [21,22]. Thus, formulas for calculating the concentration of each ATP-degradative compound were devised for compensation of the signal on the basis of Hx calibration curve [23,24]. Recently, a biosensor system which is composed of serially connected three-enzyme reactors packed with enzyme-immobilized chitosan porous beads (Chitopearl) was developed [25]. The main advantage of this system is that three components (Hx , Hx R and IMP)

A slurry of Chitopearl 3001 (2.5 ml) was immersed into 2.5% glutaraldehyde solution (0.05 M phosphate buffer, pH 7.5) for 2 h at 30◦ C. After activation with glutaraldehyde, the beads were washed with distilled water, followed by washing with 0.05 M phosphate buffer (pH 7.5). To 4.6 ml of the enzyme solution (0.05 M phosphate buffer, pH 7.5) containing 5 units of XOD, 2.5 ml of the glutaraldehyde-treated beads were added. The mixture was stirred for 2 h at 30◦ C and then for 24 h at 4◦ C with an orbital shaker. A simultaneous immobilization of NP (30 units) and XOD (5 units) or 50 -NT (125 units), NP (30 units) and XOD (5 units) was done by the same method as described above. The beads immobilized with the above enzyme(s) were preserved in 0.05 M phosphate buffer (pH 7.5) at 4◦ C. The XOD-bonded beads were packed into a glass column (3 mm i.d.), previously plugged

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Fig. 1. Schematic diagram of the biosensor system with three-enzyme reactors for the determination of Hx , Hx R and IMP. (A) water bath, (B) circulator, (C) buffer solution, (D) peristaltic pump, (E) injector, (F) reactor I, (G) reactor II, (H) reactor III, (I) oxygen electrode, (J) oxygen electrode adaptor, (K) waste solution, (L) multirecorder.

with a polypropylene cap covered with a circular piece of nylon net (200 mesh) to yield reactor I (Hx reactor, column length of 15 cm) (Fig. 1). After completion of packing, another polypropylene cap was inserted into the upper end of the packed column. In the same way, two-enzyme (NP and XOD) and three-enzyme (50 -NT, NP and XOD)-bonded beads were packed into glass columns to make reactor II (Hx R reactor, column length of 12 cm) and reactor III (IMP reactor, column length of 7 cm), respectively (Fig. 1). 2.3. Structure of the biosensor system The flow injection analysis system of this study consists of a buffer reservoir, a peristaltic pump (Miniplus 3, Gilson, France), a Rheodyne injector (model 7725i, Cotati, CA, USA), three-enzyme reactors, three oxygen electrodes (model BO-G, ABLE, Japan) and a multirecorder (model PRR-5041, TOA, Japan) (Fig. 1). The buffer solution (0.05 M phosphate buffer, pH 7.5) which was adjusted to 35◦ C with a water bath (model 912474, Cole-Parmer, IL, USA) flowed continuously into the enzyme reactors and flow cells installed with the oxygen electrodes at the flow rate of 0.3 ml/min. The total system was connected with capillary tubing (0.8 mm i.d.) of LKB (England) in a

serial manner. The response signal from each oxygen electrode was recorded with the multirecorder via an oxygen electrode adapter (model FA-1, ABLE). 2.4. Preparation of meat extract Beef loins and chickens were purchased from a local market and treated immediately and after 7 days of storage at 4◦ C. A 5 g sample of meat was added into 25 ml of 0.05 M phosphate buffer (pH 7.5) and crushed for 5 min at room temperature with a homogenizer (model HR-1358, Philips, Austria). The homogenate was filtered through Whatman no. 1 filter paper (Whatman, England) and 5 ml of the filtrate was passed through a 0.45 ␮m PVDF syringe filter (Whatman) to obtain the meat extract for biosensor measurement. For liquid chromatography (LC), a 10 g sample of meat was added into 25 ml of 10% perchloric acid. After homogenization for 5 min at room temperature, the homogenate was filtered through Whatman no. 1 filter paper. The residue was treated in the same way with 20 ml of 10% perchloric acid. The combined filtrate was then adjusted to pH 6.5 with 5 M KOH and the final volume was made up to 50 ml with 10% perchloric acid. This solution was passed through the above syringe filter twice before measurement.

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2.5. Biosensor measurement After the baseline signals from three-enzyme reactors of Fig. 1 had been stabilized, a 20 ␮l aliquot of sample was injected into the flow line and the response signals from these columns were measured separately and converted into the concentrations of Hx , Hx R and IMP using an external standard method. The Ki - and the H-value for each sample were also determined. 2.6. Precision of the biosensor system

IMP were shown within 30 min [25]. The system also demonstrated an acceptable long-term stability. After 2 months of storage in 0.05 M phosphate buffer (pH 7.5) at 4◦ C, three-enzyme reactors in the system have approximately showed 80% of their original responses (data not shown). 3.2. pH and temperature profiles Enzyme activity is generally affected by pH change. Therefore, the effects of pH on the immobilized

The analytical results from the biosensor system were cross-checked with those from LC. For LC analysis, a liquid chromatograph (model PU-980, Jasco, Japan) equipped with an UV detector (model UV-975, Jasco) was used. The column was ␮Bondapak C18 (3.9 mm i.d. × 300 mm, Waters, MA, USA) and the column temperature was adjusted to 40◦ C to shorten the time for analysis. A 10 ␮l aliquot of sample was injected into the liquid chromatograph and an isocratic elution of 1% triethylamine (to pH 4.5 with H3 PO4 ) was done at the flow rate of 2.0 ml/min [26]. The measurement was made at 254 nm with the absorbance range of 0.5 and the chromatograms obtained were handled by Borwin software (Rev. 1.2150).

3. Results and discussion 3.1. Operational merits of the biosensor system As shown in Fig. 1, we constructed a serially connected unique three-enzyme reactor system. As an organic support for enzyme immobilization, Chitopearl 3001, which has the particle size of 0.1 mm and gave a good sensor response, was selected based on its good diffusional property and mechanical strength, and its broad surface area for enzyme immobilization [27]. In this system, Hx and Hx R were completely decomposed at their respective enzyme reactors by adjusting the length combination of three-enzyme reactors to 15 cm–12 cm–7 cm and by having a flow rate 0.3 ml/min, facilitating a separate and simultaneous determination of Hx , Hx R and IMP [25]. When a sample containing Hx , Hx R and IMP was injected into the system, the three peaks representing Hx , Hx R and

Fig. 2. Effects of pH on the response peaks of the reactors measuring ATP-degradative compounds. (A) reactor I (Hx ), (B) reactor II (Hx R), (C) reactor III (IMP) (䊉 0.05 M acetate buffer, 䊊 0.05 M phosphate buffer, N 0.05 M Tris–HCl buffer, 4 0.05 M carbonate buffer).

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Table 1 Effects of various amino acids on biosensor response

Fig. 3. Effects of temperature on the response peaks of the reactors measuring ATP-degradative compounds. (䊉) Reactor I (Hx ), (䊊) reactor II (Hx R), (N) reactor III (IMP).

enzymes of three-enzyme reactors were studied by varying buffers and pH (Fig. 2). The maximum response signals for reactors I, II and III were obtained at 0.05 M phosphate buffer (pH 7.5), 0.05 M Tris–HCl buffer (pH 8.0) and 0.05 M carbonate buffer (pH 9.5), respectively. On the other hand, the optimum pH values for free XOD, NP and 50 -NT of this study were 7.5, 7.4 and 9.0, respectively. As shown by previous reports [28,29], alkaline shifts of the pH optimum, due to immobilization, were found in cases of two-enzyme (NP and XOD) and three-enzyme (50 -NT, NP and XOD)-bonded beads. Taking into account that XOD is the final degradative enzyme relevant to signal formation in each reactor and showed a sensitive pH profile, the selected buffer and its pH for further experiments was 0.05 M phosphate buffer (pH 7.5). The optimum temperatures of the individual enzyme reactors were measured (Fig. 3). The response signals for reactors I and II were maximal at 35◦ C, whereas that for reactor III was the highest at 30◦ C. It seemed undesirable to operate the biosensor system at a temperature higher than 35◦ C owing to a conspicuous decrease in long-term stability. Therefore, the system was maintained at 35◦ C.

Treatmenta

Relative response (%)b

Hypoxanthine +Alanine +Aspartic acid +Arginine +Glycine +Glutamic acid +Histidine +Lysine +Proline +Serine +Threonine +Tryptophan +Valine

100 117.8 ± 1.1c 113.9 ± 0.4 114.3 ± 0.4 107.4 ± 0.4 137.6 ± 1.1 110.8 ± 0.4 105.0 ± 0.2 109.7 ± 0.1 119.3 ± 0.7 116.6 ± 0.9 103.6 ± 0.9 119.1 ± 0.8

a Each amino acid was added as 4.0% concentration into 0.05 M phosphate buffer (pH 7.5) containing 2.9 mM Hx . b Biosensor response in the control was arbitrarily taken as relative response 100%. c Mean ± SD.

reactor) (Table 1). A four-fold excess of each amino acid expected to be present in acid-hydrolyzed meat samples was added into 2.9 mM Hx solution and a 20 ␮l aliquot of the resulting solution was injected into reactor I. When glutamic acid was injected with the Hx solution, a relative response of 137.6% was found. Relative responses were, however, less than 120% in cases of the other amino acids. Considering that most portions of nitrogen-containing materials in meats are proteins and four-fold excess of each amino acid possibly present in the acid-hydrolyzed samples was added, interference caused by only free amino acids in raw meats seemed to be insignificant (difference less than 5%). When sodium chloride was added into 2.9 mM Hx solution at 0.20–0.90% (concentrations normally found in biological fluids) and a 20 ␮l aliquot of the resulting solution was injected into reactor I, biosensor response was still maintained at above 95% of the original response. Over this NaCl concentration, however, biosensor response decreased considerably, resulting in a relative response of 86.2% at 2.00% NaCl.

3.3. Effects of amino acids and NaCl on biosensor response

3.4. Calibration curves

The effects of various amino acids on biosensor response were determined making use of reactor I (Hx

Standard solutions containing different concentrations of Hx , Hx R and IMP were injected into the

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Table 2 Determination of Hx , Hx R and IMP in the beef loins and chickens, and the resulting Ki -and H-values Samplea

After 7 days of storage at 4◦ C

Controlb Hx (␮mol/g)

Hx R (␮mol/g)

IMP (␮mol/g)

Ki -value (%)

H-value (%)

Hx (␮mol/g)

Hx R (␮mol/g)

IMP (␮mol/g)

Ki -value (%)

H-value (%)

Loin Loin Loin Loin

1.149 1.258 1.536 1.265

0.198 0.194 0.290 0.182

0.994 1.158 1.306 0.573

57.5 55.6 58.3 71.6

49.1 48.2 49.0 62.6

2.156 2.033 1.203 2.005

0.222 0.210 0.134 0.225

1.248 1.050 0.382 0.328

65.6 68.1 77.8 87.2

59.5 61.7 70.0 78.4

Chicken Chicken Chicken Chicken

1.139 0.718 0.637 0.890

0.473 0.335 0.349 0.368

1.884 1.655 1.882 1.632

46.1 38.9 34.4 43.5

32.6 26.5 22.2 30.8

1.308 1.938 3.164 1.980

0.741 0.332 0.480 0.259

1.185 0.570 0.806 0.206

63.4 79.9 81.9 91.6

40.4 68.2 71.1 81.0

a b

Treated from different samples. Delivered from a market and treated immediately.

3.5. Freshness measurement of meat samples

Fig. 4. Calibration curves for Hx (䊉), Hx R (䊊) and IMP (N).

biosensor system composed of the enzyme reactors having the length combination of 15 cm–12 cm–7 cm and the corresponding calibration curves for these components were drawn by measuring the response signals from reactors I, II and III (Fig. 4). Normally, the time required for obtaining three peaks was maximally 30 min (data not shown). Considering the injection procedure and the time for baseline stabilization, the total time needed for a single measurement might be estimated at 30–35 min. Linear relationships were found between substrate concentration and peak height up to 4 mM individual substrate. Above that concentration, the ratio of increase in peak height versus increase in substrate concentration decreased gradually (data omitted).

The meat extracts prepared from the beef loins and chickens were determined for the concentrations of Hx , Hx R and IMP with the biosensor system. Just after preparation of the meat extracts, Hx contents in the beef loins were much higher than those in the chickens. On the other hand, Hx R and IMP contents in the beef loins were by far lower than those in the chickens (Table 2). The Ki - and H-values of the beef loins were much higher than those in the chickens, which meant that the beef loins were less fresh than the chickens. In a specific circumstance, this difference might reflect a species-specific nature of the meats tested [17,30]. After storage for 7 days at 4◦ C, Hx contents increased sharply, whereas IMP contents decreased abruptly, in the beef loins and chickens. As a result, the Ki - and H-values increased conspicuously. The Ki - and H-values of the beef loins were in the ranges of 65.6–87.2 and 59.5–78.4%, whereas those of the chickens were 63.4–91.6 and 40.4–81.0%. This fact clearly demonstrated that the meat samples of this study deteriorated after cold-storage for 7 days at 4◦ C. 3.6. Comparison of the analytical results The contents of ATP-degradative compounds and the corresponding Ki - and H-values for the meat samples were also measured by LC (data not shown). Fig. 5 illustrates the correlation between the biosensor method and LC with respect to the Ki - and H-values.

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Fig. 5. Correlation between the biosensor method and LC with respect to meat freshness indices. (䊉) Ki -value, (䊊) H-value.

A good agreement was found with a correlation coefficient (r) of 0.956. This correlation obviously proves the precision and reliability of the data in Table 2.

4. Conclusions The accuracy of a biosensor system for separate and simultaneous determination of Hx , Hx R and IMP in meats was proved by a comparison with LC. By employing the present system, freshness of a meat sample can be estimated by a continuous mode within 35 min. As sample pre-treatment is simple, this system seems to be an easy and economical substitute for conventional methods such as LC for routine measurement of meat freshness.

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