A Method for the Immobilization of Xanthine Oxidase in CA-Alginate Membranes to Measure Hypoxanthine Concentration in Order to Assess Flesh Food Quality

Clark electrode, oxidase probe, oxidase meter, and batch reactor with free enzymes, an analysis procedure to measure hypoxanthine concentration was developed. An immobilized xanthine oxidase membrane was developed for measuring hypoxanthine concentration in flesh food. Oifferenl types of algin were compared to choose the best agent for membrane manufacture. The effects of CaCl2 concentration on the shape of the membrane and the thinning of the gel membrane were tested. The desired concentration of xanthine oxidase enzyme to immobilize in the membrtane was obtained. The membrane was attached to the oxidase probe, and tried with both a batch reactor and a continuous flow reactor. The influence of glutaraldehyde concentration on the stability of immobilized xanthine oxidase was studied. The effects of assay conditions on the response of the enzyme sensor were studied and evaluated. A system of continuous flow reactor, Clark electrode, oxidase meter and immobilized enzyme membrane of 2% Ca alginate and 1.0 u/ml of xanthine oxidase, incubated in 0.05 M Tris-HCl buffer, pH 8.4, containing 0.0028% glutaraldehyde gave the best results. The optimum


THESIS ABSTRACT
The consumer's acceptance of food quality is an important factor, especially in Third World Countries which import much of their food. Since flesh food (meat, fish) is shipped to those countries frozen or in cold storage~ it is susceptible to temperature fluctuations during shipment, resulting in deterioration of the meat and unacceptable quality. Therefore an acceptable procedure to assess flesh food quality is necessary to protect the consumer and trader and to aid in the manufacture of high quality food products.
Hypoxanthine concentration in flesh food has been a useful index for the assessment of freshness. Therefore, it was selected as the quality control indicator for .the development of a rapid and effective method for quality analysis.
An immobilized enzyme analysis system was developed, showed a high degree of correlation between the two methods.

INTRODUCTION
Hypoxanthine, a metabolic by-product of ATP breakdown during autolysis, accumulates in the fish and meat tissues and increases steadily until a maximum is reached. It then decreases as bacterial degradation occurs. If little 0 r n o h Y-P o x a n t h i n e i s p r e s e n t , t h e f i s h a n d m e a t i s considered to be fresh. Thus hypoxanthine can be used as an index of fish and meat quality.
A Colorimetric enzyme assays for the measurement of hypoxanthine concentration in flesh food (meat and fish) have been established and proven to be useful in the assessment of flesh food freshness in quality control (Pizzocaro, 1978Platz et al., 1978;Burt et al., 1968;Jahns 1975;Beuchat, 1973;. This method however is time consuming and expensive. In recent years, a great deal of interest has been shown in the applications of immobilized enzymes, because they are reusable and open the way to continuous processing (Beeby, 1983). Different immobilization t~chniques and procedures have been used and discussed (Carr et al., 1980;Hultin, 1974;Klibonov, 1983;Lasking 3 et al., 1984;Mosback, 1976;Trevan, 1980;Wingard, 1972;zaborsky, 1973). These immobilization techniques and procedures were classified according to the reactions or processes. 1) covalent attachment of enzymes to solid supports, 2) adsorption of enzymes on solid supports, 3) entrapment of enzymes in polymeric gels, 4) encapsulation of enzymes. Although a number of enzyme immobilization methods have been studied, no one method is ideal, since each meth~d has specific advantages and disadvantages.
Therefore, in practice, it is necessary to find a suitable method and conditions for the immobilization of a particular enzyme in light of the intended application (Chibata, 1978).
Several different materials have been used to immobilize enzymes by entrapment method. Among these are cellulose triacetate, collagen, k-carrageenan, algin, agar, and polyacrylamide.
The reason for the previous researchers choosing alginate immobilization is its simplicity. Futhermore, the immobilization reagents _are of low cost and safe, making the procedure attractive for large scale application; also the immobilization procedure is rapid and mild (Kierstan, 1981;Brodelius, 1984;Klibanov, 1983 A few years ago, the use of biochemical electrodes having bio-specificity attracted considerable attention for the checking or control of the concentration of the metabolites in body fluids. This bio-specificity was based on enzymes, and immobilized enzymes were used in many cases. These electrodes were suitable for the measurement of substrates, coenzymes, and inhibitors of an enzyme, and were called "enzyme electrodes" or "microbial electrodes" when microbial cells were used (Chibata, 1978). Clark and Lyons (1962) proposed the first amperometric enzyme electrode. Since then the literature reviewed shows numerous references to enzyme electrode 5 production and use (Olson and Richardson, 197 4;Hasselberger, 1978;Car and Bowrers, 1980;Wingard et al., 1981;Laskin et al., 1984).
To 0.5 ml of each solution was added 2 ml of a 2,6dichloroindophenal-xanthine oxidase solution (DIP/XOD). in the batch reactor.
The results (as shown in Table 1 and Figure 3) indicated that the resulting current was directly proportional to hypoxanthine presence. The reaction time seemed long, especially with the higher amounts of hypoxanthine, but because in the bulk solution the enzyme has relatively less concentrated, a longer time was required to attain a steady state. Therefore, the enzyme concentration was increased to 0.64 unit/ml and 0.90 u/ml as shown in Table 2 and Table 3 respectively.
The results showed that increasing the enzyme concentration shortened the time required to attain a steady state.
At the conclusion of this step, the approach showed promise using the YSI Clark 2510 electrode and YSI Model 25 oxidase meter as a tool for measuring the hypoxanthine concentration.
14 The second step was the immobilization of the xanthine oxidase. A simple, rapid, low cost and applicable method for immobilization was preferable.
In order to prepare the membrane much time was spent to obtain a good membrane (thin gel), since the main reaction to form the membrane was between algin and CaC1 2 • ' several articles were reviewed to choose the best concentration of algin and CaCl2 and the effect of their concentrat1on on the diffusion of substrate Ca-alginate gels (Tanaka, 1984;Kierstan, 1981;Kierstan and Bucke, 1977;Cheetham et al., 1979). They found that substrates with a molecular weight (MW) less than 2 X 104 could ·diffuse freely into and from Ca alginate gel beads. Also, neither the Ca-alginate concentration in the beads, nor the Cac1 2 concentration used in the gel preparation, had any effect on the diffusion of the substrates. The efficiency of retention of large molecular-weight compounds increased when 2% (W/V) sodium alginate was used (Kierstan and Bucke, 1977). Tanaka (1984) found that substrates with small molecular weight (such as glucose) could diffuse into 2% Ca-alginate gel beads as freely as water. Therefore, a 2% algin concentration was chosen. So a new attempt was tried: spraying the CaC1 2 by an atomizer connected to an air pressure pump. Also, a small 16 flat dish with edges was found to be important to control the desired volume and shape of the gel membrane, and a Gelman Sciences small flat dish with dimensions of 4.5 cm.
diameter and 0.5 cm. depth was found to be ideal These changes dramatically improved the membrane preparation procedures, and a thin gel could be formed, which was used throughout the study.
The effect of the CaC1 2 concentration on the shape of the membrane and the thinning of the gel membrane was ' tested. Concentrations of 1, 2, 3, 4, 5, and 6% CaC1 2 were prepared. It was found that at low concentrations (1-4%), after spraying and adding extra CaC1 2 , the gel membranes had shrunk to almost half their size and increased in thickness. However, at 5% and 6% Cacl 2 level, there was no decrease in size and a uniform gel membrane formed. Therefore, 5% CaC1 2 was used for preparing the membrane. Also, it was found that spraying with CaCl2 several times over a ten-minute period, and then covering the membrane with CaC1 2 solution for 20 minutes more gave the best results for membrane formation. cut and attached to the electrode as described above. The immobilized enzyme sensor was attached to the reaction chamber as shown in Figure 2. The results, as seen in Figure 4, indicated that the new system was working and was a usable procedure. The desirable enzyme concentration was 1.0 u/ml, since higher levels of XOD produced no further increases in peak height response.
The only weakness of this immobilization technique has been reported that the enzyme often leaked from the support. It was felt that this could be overcome by treatment of the entrapped enzyme (membrane) with a cross ' linking reagent. Glutaraldehyde has been the most recommended and usable compound of this type for immobilization procedures due to the low cost and minimal effects on enzymes (Kl ibonov, 1983;Watanable et al., 1983;Watonable et al., 1984;Messing, 1975;Zaborsky, 1973;Jacober and Rand, 1980;Chibata, 1978).
Therefore, an initial trial was made to see which was the best way to prepare XOD immobilized membrane stabilized with glutaraldehyde. Glutaraldehyde (0.0028%) was mixed with the alginate solution and the enzyme before gelation or by incubating the membrane in the gluraldehyde solution for 24 hours after gelation. A control membrane was prepared (no glutaraldehyde). The results,  The effect of glutaraldehyde incubation time on the xoo immobilized membrane was also studied. Two membranes were prepared and incubated for 12 and 24 hours. As seen in Table 6, incubation of a XOD membrane in 0.05 M Tris-HCl buffer~ pH 8.4, for 12 hours gave the best enzyme sensor response and reduced the preparation time.
The effects of substrate concentration and injection volume on the response of the enzyme sensor were studied.
Hypoxanthine solutions of 0.001, 0.002, and 0.003% were prepared and 25, 50, 100 ul of the hypoxanthine solution were injected. The results, as shown in Figure 6 and As one of the factors to be considered in the design of an enzyme reactor, the relation between the flow rate and the response of the enzyme sensor had to be studied. Figure 10 shows this relationship. Above 0.4 ml min-1, the XOD sensor response decreased due to insufficient time for the reaction.
The effect of pH on the XOD sensor reaction rate was evaluated between pH 7.6-8.5, as seen in Figure 11. The maximum response was at pH 7.85.
The effe~t of circulating buffer concentration on tbe response of the XOD enzyme sensor was studied as shown in Table 7. The results indicated a substantial enhancement using 0.05 M for the circulating buffer and using 0.01 M for substrate preparation.
Standard sample solutions of hypoxanthine were analyzed and compared, using the immobilized enzyme analysis procedure developed here and the standard colorimetric enzyme analysis. As seen in Figure 12 a linear relationship was obtained between hypoxanthine concentration and the peak height responses over the range of 0-10 ug/ml. Comparing this result to the colorimetric analyses in Figure 13 shows a high degree of correlation over the same range. 22

Conclusion
The emphasis in this research has been on developing a procedure to measure hypoxanthine concentration and on employing an oxidase meter and Clark electrode oxidase probe as an instrument, when combined with a new method for membrane manufacture which is simple and safe, employing algin.
The oxidase probe and oxidase meter combined with immobilized xanthine oxidase on a Ca-alginate membrane is a simple, easy, as well as relatively quick and inexpensive procedure. · The system seems to be a good procedure to study substances that produce or influence        Gui 1bau1 t, G. G. and Hr a b'a n k ova, E.
197 0 a. Deter min at ion of urea in blood and uring with a urea-sensitive electrode.
Guilbault, G.G. and Hrabankova, E. for determination of amino acids.
1970b. An electrode Anal. Chem 42:1779. Guibault, G.G. and Lubrano, G.J. 1972    This analysis procedure was simple, rapid and may prove to be a useful procedure to assess fish freshness and as an alternative or adjunct to the more time consuming and expensive colorimetric analysis, which may also encourage the extension of this work to other food fish.

INTRODUCTION
An acceptable proced~re to assess fish quality is necessary to protect the consumer and trader and to help the seafood industry with the manufacture of high quality products. Several methods and procedures have been developed, tried, and described to measure the quality of fish, such as volatile reducing substances, volatile nitrogen bises, ammonia (Farber and Ferro, 1956;Dugal, 1967), pH, Trimethylamine (Beatty and Gibbons, 1937), hypoxanthin~ and xanthine (Burt et al.;1968, Beuchat, 1973Jahns et al., 1976;Jones et al., 1964;Uchiyma, 1969;.
These methods and procedures, however, require complicated operations, a long time for preparation, or are inaccurate, or costly; therefore, quick and simple methods are required.
Following the death of a fish, adenosine-5'triphosphate (ATP) breaks down to the adenosine-5'diphosphate and other related compounds, such as hypoxanthine and xanthine (Saito et al., 1959). Whereas, hypoxanthine or xanthine is accumulated with an increase of storage time and can be analyzed using xanthine oxidase.
Studies have shown hypoxanthine concentration to increase with increasing storage time and conclude that hypoxanthine concentration is a useful index of the freshness assessment of fish (Spinelli, 1964;Beuchat, 1973;Jahns et al., 1976;Collette, 1983).
An enzyme sensor specific for hypoxanthine in fish tissue was developed by Watanabe et al., 1983 using immobilized xanthine oxidase-membrane and an oxygen probe.
The prepara~ion of the immobilized enzyme membrane in that study was time-consuming and could result in loss of activity. The disadvantage of this method (covalent) were also described by Klibanov (1983) and others. In addition, the critical reagent 1, 8-di-amino-4-animomethyl octane, was not available in the United States and the only source was Japan (A Sahi Kasei Co.). Therefore, a rapid, simple, low cost, and routinely applicable procedure for immobilization would be preferable.
The Ca-alginate criteria (Klibanov, 1984; ' similar to the one described by Watanabe et al. (1983) with some modifications as reported by .

Colorimetric XOO Analysis Procedure
Hypoxanthine in standards and fish extraction solutions was determined by the method of , as modified by . Figure 1 and Table 1  be easy to use the ratio formula method.

RESULTS AND DISCUSSION
As shown in Figure 1 and Table 1 b~th methods gave hypoxanthine concentration values which were close to each other. It was found that using the ratio formula was very quick, simple, and less time consuming, and more applicable for routine work.
A significant finding of this study was that the results of the immobilized enzyme analysis resembled other studies which have been reported to assess the measurement of the freshness of fish using hypoxanthine as index of freshness (Collette and Rand, 1983;Jahns et al., 1976;Beuchat, 1973;Spinelli et al., 1964;Shewan and Jones, 1957). Hypoxanthine accumulated in the fish muscle post-harvest to a peak value (8 days for Whiting), or about the point of incipient spoilage.
Then the hypoxanthine exhibited the usual pattern of decline as the fish entered the spoilage phase and became unacceptable.

CONCLUSION
The immobilized enzyme analysis to measure hypoxanthine in fish presented in this paper was rapid, simple to perform, and less time-consuming, and could be used for routine work. This analysis procedure may prove to be a useful procedure to assess fish freshness and as an altern~tive or adjunct to the more time consuming colorimetric analysis.
76 Rapid measures of iced fish muscle. and of innorine 5-Sci. Food Agric. Kierstan, M. 1981. The use of calcium alginate gel for solids separation adn diffusional chromatography of biological materials.
Science. 29:722. Kobayashi, H., and Uchiyama, H. 1970 increased. This analysis procedure was simple, rapid, and may prove to be a useful procedure to assess meat freshness.

INTRODUCTION
Food quality as a fresh, desirable produced with high consumer acceptance is an important factor especially in the Third World countries who import much of their food.
The meat shipped to those countries either frozen or in ' cold storage, is a food product which is particularly susceptible to temperature fluctuations during shipment, resulting in deterioration of the meat and poor consumer acceptance. Therefore, a rapid, inexpensive, and acceptable procedure is necessary to assess imported meat quality, to protect the consumer and trader, and to help ensure that food industries ship only high quality products to these Third World countries. Several methods and procedures have been developed to measure the quality of meat, such as extract-release volume , pH (Shelef and Jay, 1970;Swift et al., 1960); monoamines ; color (Strange et al., 1974), thiabarbituric acid (Witte et al., 1970), total volatile nitrogen and tyrosin (Pearson, 1968a), free fatty acid levels (Vasundhara, 1983), and hypoxanthine, xanthine and inosine monophosphate (Parris et al., 1983;Pizzacaro, 1978;Platz et al., 1978). Most of these and other methods and procedures were reviewed by Pearson (1968b), Gil (1983). Most of these methods and procedures required complicated operations, long preparation time or were inaccurate and/or costly; therefore, quick, simple and reliable methods still must be developed.
Following the death of the animal, adenosine-5'-' triphosphate (ATP) breaks down to the adenosine-5'diphosphate and other related compounds, such as hypoxanthine and xanthine (Tsai et al., 1972, Price and Schweigert, 1970. Because hypoxanthine or xanthine accumulates with an increase of storage time, these compounds can be analyzed using xanthine oxidas (XOD). Studies have shown that hypoxanthine concentration is a useful index of the freshness assessment of meat (Parris et al., 1983;Pizzacaro, 1978;Platz et al., 1978).
Recently, a biological electrode combined with an immobilized membrane has been developed and proven to be a useful method for measuring amines in meat . A biological electrode for analysis of hypoxanthine in fish has recently been developed (Watanabe et al., 1983;. However, at this time there have been no reports on an enzyme sensor specific for 85 measuring hypoxanthine in meat.
The objective of this study was to measure the hypoxanthine in meat using the Ca-alginate-xanthine oxidase immobilized membrane developed by  combined with the YSI Model 25 oxidase meter and YSI Clark 2510 electrode in a continuous flow reactor.

Sensory evaluation
On sampling day, pieces of meat samples were presented in well iced, coded stainless steel trays to a panel of at least 8 graduate students (untrained) for raw sensory evaluation based on odor, color and appearance, and edibility. The raw odor score scale as decribed by Pearson (1968) was used, and the scaling system of preparation of Ca-alginate Immobilized XOD Membrane The Ca-alginate immobilized enzyme membrane was prepared as described by . Briefly, 1 g of Kelco gel LV (Algin) was added gradually to 50 ml distilled deionized water in a Waring blendor jar. The mixture was blended at high speed until a uniform solution was obtained. The blended mixture was stored at 4°C until needed. In' a 0.5 cm depth and 4.5 cm diameter flat dish, 2 ml of the 2% alginate solution and 46 ul of stock xanthine oxidase concentrate (21.74 u/ml) were mixed very well with a spatula. Then the mixture was sprayed with 5% Cacl2 solution several times for about 10 minutes. Extra Cacl 2 was added to fill the dish and cover the whole membrane.
After that, the membrane was washed with distilled water and incubated in 4 ml. 0.05 M Tris-HCl buffer, pH 8.4, which contained 0.14 ml ('0.0028%) 50% glutaraldehyde, for 12 hours. Then the membrane was washed with distilled water and stored in 0.05 M Tris-HCl, pH 7.85, at 4°C until utilized.

Immobilized Enzyme Hypoxanthine Assay
The continuous flow reactor was similar to the one described by Watanabe et al. (1983) with some modification as reported by   Since peak height was used to measure the enzyme sensor response, it was though that method B (formula) would be easy to use. As shown in Table 3 and 4, good agreement was obtained between the values determined by both methods. It was concluded that using method B was very quick, simple, and less time consuming, and more applicable for routine ' work.
These results show that the sensory evaluation results, especially the edibility index, indicated that through day 7 the majority of potential consumers would purchase and cook the given sample of lamb and through day 11, the given sample of beef. Generally, these were meat samples in which the hypoxanthine concentration was below about 14 mg%. After these key points in storage, the acceptability declined for the majority of consumers, and the hypoxanthine concentration increased dramatically.
This relationship between the consumer's sensory evaluation results and hypoxanthine accumulation provided a consistent index of quality. Generally, when consumer acceptability declined, the hypoxanthine concentration exceeded 14 mg%. Therefore, determining hypoxanthine concentration by the procedure presented in this study 95 would be effective method for the assessment of meat quality.
. , Comparison of this study with previous workers (Pizzacaro, 1978; provides further evidence that hypoxanthine could reflect sensory evaluation and can be used as a freshness index. The significance of this study was using immobilized enzyme analysis may prove to be a useful procedure to    were proposed (Chibata, 1978).
Whereas enzymes convert from water soluble to water insoluble molecules, several definitions are necessary for an immobilized enzyme. An immobilized enzyme is an enzyme that has been chemically or physically attached to a water-insoluble gel matrix or water insoluble microcapsule (Zaborsky, 1973). Enzyme immobilization is the imprisonment of an enzyme molecule in a distinct phase that allows exchange with, but is separated from, the bulk phase in which a substrate effector or inhibitor molecules are dispersed and monitored (Trevan, 1980).
Immobilization is the conversion of enzymes from a watersoluble, mobile state to a water-insoluble, immobile state (Klibanov, 1983).

-Entrapment of enzymes in polymeric gel
4 -Cross-linking of enzymes with bifunctional reagents.

-Encapsulation of enzymes.
From Immobilized Enzymes and Cells as Practical Catalysts, by A.M. Klibanov, in Science, vol. 219, pp. 722-727, 1983. 121 Each one of these methods has its advantages and no o n e i s b e s t for a l l a-pp l i c at i on s • Klibanov (1983) reported that comparison of the enzymes immobilization methods listed in Table 2 leads to some important conclusions. The advantage of covalent methods 1 and 4 is that they result in stronger chemical bonds between the enzyme and the support. The disadvantages are that covalent binding is relatively laborious and expensive and ' often leads to significant inactivation of enzymes due to attachment through their active centers. The latter problem, however, can be alleviated in many cases if immobilization is carried out in the presence of substrates or other ligands (inhibitors, cofactors, and so on) that selectively protect the active center from the attachment. Methods of immobilization such as adsorption and gel entrapment are very simple and efficient, but since such methods create no strong bonds between the enzyme and the matrix, enzymes often leak from the supports. This problem can be overcome by the treatment of adsorbed or entrapped enzymes with a cross-linking reagent such as glutaraldehyde.
Preparation methods and characteristics of immobilized enzymes are summarized broadly in Table 3, though there are many exceptions. Although a number of enzyme immobilization methods have been studied, no ideal 122 general methods applicable for the immobilization of many enzymes have yet been developed. Each method has specific disadvantages. Therefore, in practice, it is necessary to find a suitable method and conditions for the immobilization of a particular enzyme in light of the intended application (Chibata, 1978 After studying all the methods of immobilization presented thus far, the entrapment method was chosen.
To entrap an enzyme in polymeric gel, the enzyme is added to a solution of monomers before the gel is formed.
Then gel formation is initiated by either changing the temperature or adding a gel-inducing chemical.
The matrices which have been most employed in entrapment methods are polyacrylamide, collagen, cellulose triacetate, agar and alginate. The first polymeric matrix used to immobilize enzyme was polyacrylamide (Bernfield and Wan, 1973). Polyacrylamide, Kappa-carrageena~ and alginate have been used industrially (Klibanov, 1983).
Since the YSI-Clark 2510 electrode and YSI Model 25 oxidase meter (Yellow Springs, Ohio), which were developed by this company, were used for this study, the principles and operation of the system as described by this company are summarized as follows: The proportional to the H202 present.

Reaction l
The circuit is completed by a silver cathode at which oxygen is reduced to water, reaction II.

Reaction II
The membrane covering the probe is porous, serving both to protect the electrodes and to define a diffusion path to them. Various methods can be used to introduce the enzyme(s) necessary to the system. They can be placed behind the membrane, in the bulk solution being measured, or both.
The level of the final, stable current reading and the time required to attain it depends upon whether the enzyme is placed in the solution or isolated behind the membrane.
In the bulk solution the enzyme is relatively 128 less concentrated and a longer time is required to attain a steady state. Also the final current is higher because the conversion is essentially total. Behind the membrane the enzyme is highly concentrated and converts only the substrate which has diffused through the membrane. In this case the steady state is reached more quickly but the final current is lower.
The system is a research tool (not for diagnostic ' use) specifically designed to study substances that produce or consume, or influence production or consumption In operation the probe produces an el·ectric current proportional to the H 2 o 2 in its immediate vicinity. When an enzyme is used to produce H 2 o 2 , the probe current is a measure either of enzyme activity or substrate concentration, depending upon the experiment design, reaction III.