Polyphenol Oxidase Inhibition by Glucose Oxidase in Pink Shrimp ( Pandalus borialis )

The glucose oxidase and catalase (GOX/CAT} enzyme system was studied as an inhibitor of polyphenol oxidase (PPO} in extracts of whole pink shrimp (f~~~~l~2 borealis}. PPO was isolated from shrimp heads and purified by acetone extraction, ultrafiltration, and gel filtration chroma to~ rap h y . A p u r i f i cat i on of 7 • 7 4 f o 1 d w a s ob ta i n e d with a recovery of 54.3% activity. The shrimp PPO activity was compared to that of mushroom PPO activity. The shrimp PPO was found to have an optimum pH of 6 . 0-6.5 and to be 0 stable at 25-35 C. Below freezing, the shrimp PPO was damaged completely and no activity was obtained. Several phenolic compounds were tested as the substrate of shrimp PPO. L-OOPA and OL-OOPA yielded the highest activity. The Km of shrimp and mushroom PPO were 4.54 and 0.5 mM, respectively. Varying the concentration of 0-glucose, a substrate of GOX/CAT; over the range of 0.5 to 4% had no significant effect on the activity of both mushroom and shrimp PPO. GOX/CAT at a concentration of 2 units/ml inhibited 95.2% and 97% of shrimp and mushroom PPO, respectively, with 0.5% 0-glucose. The PPO was completely inhibited by 6 units/ml of GOX/CAT.


THESIS ABSTRACT
The glucose oxidase and catalase (GOX/CAT) enzyme system was studied as an inhibitor of polyphenol oxidase (PPO) in extracts of whole pink shrimp (Pandalus borealis).
PPO was isolated from shrimp heads and purified by acetone extraction, ultrafiltration, and gel filtration chromatography. A purification of 7.74-fold was obtained with a recovery of 54.3% activity. The shrimp PPO activity ' was compared to that of mushroom PPO activity. The shrimp PPO was found to have an optimum pH of 6.0-6.5 and to be s t a b l e a t 2 5 -3 5 °C . Be l o w f r e e z i n g , t h e s h r i m p P P 0 w a s damaged completely and no activity was obtained. The substrates and end products of GOX/CAT were studied for their potential to inhibit mushroom and shrimp PPO. Mushroom and shrimp PPO activity were inhibited completely by the GOX/CAT end products hydrogen peroxide and gluconic acid at concentrations of 2.0 mg/ml, while glucono-lactone showed a weak inhibition of less than 7.6% and 9.0%, respectively.
Oxygen consumption by mushroom and shrimp PPO was secondly, sodium tripolyphosphate (STPP) at a concentration of 9% alone and at concentrations of 3%, 6%, and 9% in c om bi n at i on with G 0 XI CAT at a con c en tr at i on of 6 u n i ts Im 1 . ACKNOWLEDGEMENT I would like to take this opportunity to express my appreciation to my major advisor Dr. Arthur G. Rand . . . . . . . . . . . . . . . . . • . . . . . . • . . . • . . . ii PPO was isolated from shrimp heads and purified by abdominal shell segments and tailfin (Finne and Migget, 1985). This undesirable blackening phenomenon on shrimp causes serious problems to the seafood industry. Although melanosis formation is not related to the eating quality, safety or decomposition of shrimp, it is visually objectionable to most consumers and therefore less acceptable.
Many investigators have tried to prevent black spot in shrimp.
One of the first observations on the prevention and delay of black spot in shrimp involved removal of the shrimp head immediately after the catch was brought on board (Alford and Fieger, 1952). Other methods of inhibiting melanosis include the use of chemicals which interfere with black spot formation, such as ascorbic acid , and cysteine (Mason, 1957;Loomis and Battaile, 1966 (Lindsay, 1976). The effectiveness of these compounds is known to vary with pH. Sulfiting agents have been found to be the most powerful substance demostrated to strongly inhibit shrimp melanosis, especially sodium bisulfite or metabisulfite. Sodium bisulfite is a strong reducing agent and competes with tyrosine , for molecular oxygen. It can take up the oxygen and prevent the formation of black spot Nickelson and Cox, 1986).
The safety of sulfite has been questioned.
Strong asthmatic reactions to bisulfite and other sulfite agents have been reported (Allen and Collett, 1981;Stevenson and Simon, 1981;Werth, 1982). Thus, work must be initiated to find alternatives to replace or reduce the amount of sulfites required to inhibit shrimp melanosis.
The enzyme system of glucose oxidase and catalase (GOX/CAT) has been used in food as a food additive and is considered Generally Recognized As Safe (GRAS) (Searle .
GOX/CAT has been used to remove oxygen and prevent oxidation in food products and also to remove glucose . Therefore, it is possible that GOX/CAT might inhibit PPO activity by removing oxygen, which is 3 essential for PPO to produce melanosis . Shrimp PPO extraction procedure The procedure was a modification of that of Farias (1982 (Worthington, 1982). The activity of GOX/CAT was measured by an assay using 2,2'-Azino-di(3ethylbenzthiazoline)-6-sulphonate (ABTS) and peroxidase (POD). The ABTS/POD assay was developed from a glucose oxidase assay using 0-dianisidine and POD enzyme . ABTS was substituted for 0-dianisidine because of the carcinogenic effect of 0-dianisidine, in addition, ABTS has greater sensitivity, is very soluble in water, and virtually insensitive to light .

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The solutions for the assay were prepared as follows:

In order
to convert the raw data to moles of product formed per minute, each absorbance/min value is divided by 3600.
Assay of GOX/CAT as inhibitor for mushroom and shrimp PPO activity The GOX/CAT enzyme system was examined for its effect as an inhibitor of mushroom and shrimp PPO in vitro.
The concentrations of 0-glucose, a substrate for GOX/CAT, and of GOX/CAT (in units/ml) were varied, in order to find the best conditions for inhibiting mushroom and shrimp P PO.

Protein determination
The method of Lowry et al. (1951) was used to determine the protein concentration in each step of the shrimp PPO purification. A standared curve was prepared with bovine serum albumin, and the concentrations of unknown protein solutions were determined from the graph.

Extraction of shrimp PPO
Most of the shrimp PPO is located in the head area ; therefore, only shrimp heads were extracted. The first supernatant was turbid due to the presence of proteins and other insoluble materials.

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Therefore, further separation was needed to purify the shrimp PPO. Different treatments were evaluated for further purification of the shrimp PPO: ammonium sulfate, polyethylene glycol, and acetone. Among these treatments, acetone produced the highest enzyme activity. This result agreed with the reports of Fairs, 1982; Thomas and Jnave, 1973;Chubey and Danell, 1972;and Pefferi et al, 1974. A summary of the purification procedure developed is given in In the fractionation of the sample by Sephacryl S-200 gel filtration chromtography, there were three protein peaks ( Figure 1). The shrimp PPO activity was found in the first peak (fraction numbers 5, 6, 7, and 8) . · These fractions were pooled and used for the enzyme kinetics study.
Various phenolic compounds were tested as a substrate for shrimp PPO activity. These substrates were L-DOPA, DL-DOPA, catechol, dopamine, and L-tyrosine.   " ''c, and was completely inactivated at 70 C Table 3 shows the activity of GOX/CAT at different concentrations and the absorbance/min for each of these ' concentrations by using the ABTS/POD assay. As the GOX/CAT concentration was increased there was an increase in the absorbance/min and in color development. Therefore, the GOX/CAT enzyme system was demostrated to be effective and was as an inhibitor fro mushroom and shrimp PPO activity.
Lineweaver-Burk plot of shrimp PPO activity ( Figure   4 and  When GOX/CAT was added to the PPO assay without substrate (0-glucose), PPO activity did not decrease. Therefore, the 0-glucose was required as a substrate for GOX/CAT enzyme system to give the inhibitory 18 effect on shrimp and mushroom PPO activity.
The effect of different concentrations of the GOX/CAT enzyme system on shrimp and mushroom PPO is shown in Figure 6; the D-glucose concentration was constant at 0.5%. At the lowest concentration of GOX/CAT, 0 . 2 units/ml, the inhibition was more than 40% and 50% for shrimp and mushroom PPO, respectively. At 2.0 units/ml, the inhibition increased to more than 90% and complete inhibition was reached at a concentration of 6.0 units/ml.    0 Total volume of 3.0 ml, at 25 C and 475 nm. Table 3 The activity of GOX/CAT at different concentrations vs.    Finne, G., and Migget, R. 1985  Recently, however, the safety of sulfites has been questioned. Strong asthmatic reactions to bisulfite and other sulfite agents have been reported Werth, 1982). Therefore, alternatives must be found to replace or reduce the amount of sulfites required to inhibit shrimp melanosis.
The enzyme system of glucose oxidase and catalase (GOX/CAT) has been used as a food additive and is considered Generally Recognized As Safe (GRAS) . GOX/CAT has been used to remove oxygen and prevent oxidation in food products, such as beer , apple wine (Yang, 1955), and canned soda ; to protect animal fat from oxidation ; and to protect water and oil emulsions such as mayonnaise . GOX/CAT, has also been used to remove glucose from egg albumin and whole egg prior to drying (Baldwin et al., 1953;. GOX/CAT has been found to extended the shelf life of fresh fish by inhibiting spoilage  '

PPO assay with GOX/CAT
The GOX/CAT enzyme system was examined in vitro for its effect as an inhibitor of mushroom and shrimp PPO.  The probe or sensor, was then plugged into the instrument allowed to polarize for 10 to 15 min.The oxygen calibration switch was adjusted to the local altitude).
A beaker filled with distilled water was mixed with a magnetic stirrer so that the water was continually agitated but without breaking the liquid surface, for about 10 min to attain complete aeration. The stirring was continued and the sensor was immersed in the water so that the stainless steel portion containing the temperature-

RESULTS AND DISCUSSION
Hydrogen peroxide The effect of hydrogen peroxide on mushroom and shrimp PPO activity is shown in Figures 1, 2, (Table 3). Figures 3 and 4, al so show a comparison between the mushroom and shrimp PPO activity with and without the GOX/CAT as inhibitor and 0.5% 0glucose as a substrate, respectively.
The GOX/CAT enzyme system was added to solutions at a concentration of 2 units/ml with 0.5% 0-glucose as a substrate (Figures 3 and 4). GOX/CAT was added before the 53 PPO enzyme was added. The oxygen concentration decreased rapidly in the first min of the reaction. The affinity of GOX/CAT for oxygen was very high; after 20 sec 69% and 46% of the oxygen was removed by GOX/CAT (Table 4), while at the same time only 3.85% and 1% of the oxygen was removed by thePO enzyme of mushroom or shrimp, respectively.
After 60 sec the oxygen removal by GOX/CAT reached about 90% in both PPO enzyme systems. Mushroom PPO had a higher affinit~ for oxygen than shrimp PPO did, but when GOX/CAT was added, the oxygen consumption removal was almost the same for both mushroom and shrimp PPO. Therefore, it appears that the affinity of GOX/CAT for oxygen was very high and that mushroom and shrimp PPO could not compete with GOX/CAT. Finally, it seems that the oxygen removed by GOX/CAT is the main cause of inhibition to the mushroom and shrimp PPO activity.
Glucono-lactone Table 5 shows the effect of different concentrations of glucono-lactone on mushroom and shrimp PPO activity.
Before the glucono-lactone was added, the activity was 56.6 and 6.2 uM/min, respectively. When glucono-lactone was added at a concentration of 2.0 mg/ml, the PPO activity was reduced slightly to 56.10 and 6.12 uM/min with only 0.9% and 1.3% inhibition, respectively. At gl ucono-l actone concentrations of 5.0 mg/ml, the PPO activity was reduced further to 52.3 and 5.64 uM/min, or 7.6% and 9.0% inhibition, respectively. The pH was measured at all concentrations of glucono-lactone before and after the enzyme assay and it was constant at 6. 2. This was expected, since there were only 2 min between preparation and assay of a solution for any inhibition of PPO activity. Therefore, it seems that glucono-lactone was a weak inhibitor of mushroom and shrimp PPO activity, even when it was used in a relativly high concentration (5.0 ' mg/ml). A similar result was reported by . He found glucono-lactone to be a weak inhibitor for glucose oxidation in the GOX assay, and only 30% inhibition was obtained.

D-glucose
The effect of D-glcuose as an inhibitor of mushroom and shrimp PPO activity is shown in Table 6. At concentrations of 1.0, 2.0, 3.0, 4.0, and 5.0 mg/ml, no inhibition occurred. Therefore, D-glucose had no effect on the activity of mushroom and shrimp PPO at the concentrations studied.

CONCLUSIONS
The substrates (0-glucose and oxygen) and the end products (hydrogen peroxide, glucono-lactone, and gluconic acid) of the GOX/CAT enzyme system were studied for their potential inhibitory effect on mushroom and shrimp PPO  The pH was held contant at 6.2.
The mushroom and shrimp PPO a~tivity were increased at 0.2 mg/ml to 38.22% and 32.22%, respectively, with respect to the control activity.        Wiseman, A., ed. 1975  Melan'n formation is not related to eating quality, safety, or decomposition of shrimp, but it is visually objectionable to most consumers and, therefore, ~akes the product less acceptable.
The enzyme which causes melanosis is polyphenol oxidase (PPO), and was found to be located in head area, gills, liver, stomach, and blood of shrimp .
To control "black spot" on shrimp shells, freshly harvested shrimp have been dipped in dilute solutions of sodium bisulfite immediately after harvest for many year~.
The U.S. Food and Drug Administration permits a one-minute dip in a 1.25% solution of sodium bisulfite as Current Good Manufacturing Practice (CGMP) (Finne and Miget, 1985). The FDA has determined an acceptable residual s u 1 f i t e l eve 1 i n sh r imp of 1 O O ppm as S 0 2 • Thus , sh r imp containing residual sulfite greater than 100 ppm would be 77 considered adulterated because of an unsafe amount of a food additive (Finne and Migget, 1985 Therefore, alternatives must be found to inhibit melanosis in shrimp. The enzyme system of glucose oxidase and catalase (GOX/CAT) has been used in foods as a additive and is considered as Generally Recognized As Safe (GRAS) . The GOX/CAT enzyme system has been used to remove oxygen and prevent oxidation in food products, and also to r .emove glucose. GOX/CAT can i n h i b i t P P O a c t i v i t y by removing oxygen (Al-Jassir,1987) and might also prevent melanosis. Judokusumo (1985), found that GOX/CAT improved the strongly inhibit oxidation (Shimp, 1985). Since melanosis development is due to activity of shrimp PPO in the presence of phenolic compounds and oxygen, STPP could also help to delay the melanosis.
The purpose of this study was to investigate the potential of the GOX/CAT enzyme system for reducing melanosis development when used as a dip for whole shrimp.

Dip solutions
The solutions into which whole pink shrimp were dipped included: 1) different concentrations of the GOX/CAT enzyme system at 0.5% D-glucose as substrate 80 (Table 3), 2) a combination of sodium tripolyphosphate (STPP) and the GOX/CAT enzyme system at a concentration of 6 units/ml (Table 4), and 3) a combination of sodium metabisulfite and the GOX/CAT at a concentration of 6 units/ml (Table 5).
The GOX/CAT dip was prepared as follows: 0 . 5% Dglucose, the substrate of the GOX/CAT enzyme system, was Dip solutions with STPP concentrations of 3%, 6%, and 9% were used to allow STPP to penetrate the meat under the shell prior to shelling (Shimp, 1985).
In the study of sodium metabisulfite on the rate of melanosis, the concentrations of sodium metabisulfite were

Melanosis evaluation
The evaluation of melanosis was performed with a scale similar to one developed by the National Marine Fisheries Service (Otwell, 1986  Unless otherwise indicated, "significant" means significant at the 5% level ( e><; = 0.05).

Melanosis development in untreated shrimp
Melanosis development was studied as a function of time in untreated pink shrimp (Pandalus borealis) with heads on. The degree of melanosis gradually increased in these control shrimp, which had been dipped in only ' tap water. Table 2 shows the average of melanosis scores on the control shrimp given by the panelists. The shrimp had no melanosis on day 1, but black spots were noticeable in most shrimp by day 3. By day 5, melanosis due to the f u 11 act i v i t y of sh r imp PP 0 , had became a severe defect with an average score of 7.8. This score would be considered unacceptable, according to National Marine Fisheries Service (Otwell, 1986). The statistical study showed that there was a significant difference between control shrimp up to day 7, from day 7 on there was no significant difference between the untreated shrimp samples.

Effect of GBX/CAT levels on shrimp melanosis
The effect of the GOX/CAT enzyme concentrations on the delay in melanosis formation was studied (Table 3). The effect of sodium metabisulfite concentrations in combination w i th G 0 XI CAT at con cent rations of 6 units Im l and 0.5% D-glucose was studied (Table 5) ' Bailey, M.E., Fieger, E.A., and Novak, A.F. 1960. Phenoloxidase in shrimp and crab. Food Res. ~:565.
seafood leader, Spring. pp 114-119. Simon, R.A., Green, L. and Stevenson, 0.0. 1982 quinone [Duckworth and Coleman, 1970]. For a long time it was not confirmed whether this action was accomplished by two different enzymes or by a dual-function enzyme.
Recently, most results suggested that one enzyme performs two functions [Lerner, 1953;Nelson and Dawson, 1944 ;. The first function, the conversion of monophenol to di phenol, is referred to as the cresolase activity and the second function, the transformation of the diphenol to quinone, is refered to as the catecholase 99 activity. The two activites seem to be independent of one another, as indicated by reports of "high-cresolase" and "high-catechol ase" enzyme preparations [Nelson and Dawson, 1944 ;Smith and Krugger, 1962]. Figure 1 shows the proposed pathway for the enzymatic oxidation of tyrosine to the dark-colored melanin (Liner and Fitzpatrick,1950).
The term melanin is applied to the polymer derived from oxidized phenols; no definition has been established' regarding the size or precise structure of the polymer [Swan, 1963]. The initial step in the reaction is the oxidation of tyrosine to 3,4-dihydroxyphenylalanine (Dopa]. This reaction is quite slow. The next step in the reaction sequence, the conversion of Dopa to Dopa quinone, is a much faster reaction than the hydroxylation of tyrosine [Nelson and Dawson, 1944]. The remaining steps in the reaction sequence may proceed spontaneously, although the reaction rate is increased in the presence of the enzyme [Mathew and Parpia, 1971]. One of the intermediate reaction products in this sequence is 2carboxy-2,3-dihydroindole-5,6 quinone [Dopa chrome]. In a cell-free system, this product accumulates in solution and is commonly used to measure enzyme activity [Fling et al, 1963]. In plants this reaction is thought to function as a protective mechanism for areas exposed by tissue damage result of a wound are highly reactive with nucleophilic groups (hydroxyl, amino, thiol, or activated methylene groups [Butt, 1980]) tend to polymerize readily, and react with amino acids and thiol functional groups or proteins [Van Sumere et al, 1975;Butt, 1980]. In plant metabolism, PPO plays several important roles: 1. Hydroxylation reactions of phenolic biosynthesis . Mason [1957] has proposed that PPO acts as a hydroxylating enzyme in vivo, for instance, during the biosynthesis of O-diphenolit compounds. 2. Respiration, Mapson and Burton [1962] suggested that the oxidation function of the enzyme is involved in the terminal respiratory sequence of the potato tuber . 3. Resistance of plants to various microorganisms. [Oeverall, 1961] concluded that quinone produced by the intensive oxidative action of PPO on injured tissues is toxic to invading pathogens.

Disease resistance. Upon infection, the PPO activity in
the plant increases and new PPO appears [Frakas and Kiraly, 1962].
Browning is known to occur in many fruits and may result from either enzymatic or non-enzymatic reactions; in these reaction PPO is of major importance in catalysing the brown reaction after cutting, bruising, injury or Picking. This browning reaction is desirable, for example in the manufacture of black tea [Takeo,1966], in grapes and prunes [Grencarevic and Hawker, 1971]. The browning reaction is undesirable in the handling, processing, or thawing of frozen raw fruits and vegetables and in flours with high levels of PPO [Vamos-Vigyazo, 1981]. The reaction of o-quinones may alter the nutritive value of proteins [Szabo, 1979]. In the case of casein this reaction decreases the available lysine and reduces its digestibility. Gross and Coombs [1975] observed high levels of PPO in the manufacture of sugar from beets ~nd cane, causing discoloration and yields.
lower There are many reports on the substrate specificity of PPO. Walker [1964] suggested that chlorogenic acid is a major substrate for PPO in apple and pear fruit. The browning of apple tissue resulting from the action of PPO is very important; it may be involved in the development of physiological disorders of apples, such as "bitter pit", and affects the quality of processed apple products . Palmer [1963] found that dopamine is the main substrate in the banana browing reaction. Mayer et al. [1964] [Luh and Phithakpol, 1972] catechins, [Walker, 1975], and 3,4dihydroxyphenylalanine [Dopa] and tyrosine [Williams, 1963].
In microorganisms, PPO is normally synthesized under unfavorable conditions [e.g . , starvation], but not during vegetative growth. PPO is responsible for the conversion of L-tyrosine to melanin, the main pigment of perithecia and ascospores [Feldman and Thayer, 1974].  [Bouchilloux et al., 1963;Jolley and Mason, 1965], in potatoes and apples [Constantinides and Bedford, 1967], and in chloropla.sts fr om a p p 1 e s [ Mey e r an d B i e h 1 , 1 9 81 ] . Two e n z y me s w i t h P P 0 activity have been purified from the frog Rana pipiens [Mikkelsen and Triplett, 1975]; both enzymes are isolated in an inactive form that can be activated with trypsin.
The analysis of black spot in shrimp as a post-mortem deteriorative change was first reported by Fieger et al. [1950]. Fir~t it was suggested that melanosis was the result of mold growth [Fieger, 1950] Later investigation dismissed the possibility of microbial action, concluding that the black discoloration on the shells of shrimp packed in ice was caused by an enzyme naturally present in the organism [Alford and Fieger, 1952]. In an effort to locate the area of high activity in shrimp,  found that the most active preparations were obtained by pressing juice from the gill area of shrimp heads. That juice contained not only blood but also cellular fluids from internal organs as liver and stomach.
These tissues appeared to be the principal source of enzymes involved in melanogensis.
There is little information on how enviromental factors can accelerate the onset of black spot or increase its intensity. One factor that has proven to affect the amount of active enzyme present in crustacean fluids is the stage of the molting cycle [Summer, 1967]. In the premolt and molt stages, the activity of phenoloxidase and the levels of phenolic compounds increase [Cobb, 1977].
Thi s results i n harden i n g of the new ex o skeleton . After molting, both enzyme activity and the level of substrate drop back to their normal levels. [Massayoshi and Perdigao [1984] showed that discoloration in female shrimp was stronger than that in males. This study showed that injuries did not always induce discoloration in shrimp stored . ' 1 n 1 c e , but when shrimps were subjected to heavy trauma such as a blow, discoloration was found as . deep as the superficial muscle. On the other hand most of the common, natural black spots remained in the shell after peeling, and those on the superficial muscle could be scraped off the black membranes.
PPO is extracted by a large variety of methods, depending on the source and the desired degree of purity. Most of the extraction procedures include all or some of the following steps : fractionation with organic solvents and buffer extraction, use of detergents to solubil ize the enzyme, such as Triton x-100 [Harel and Mayer, 1968] or Tween-80 [Ben-Shalom et al.;, precipitation with ammonium sulfate [Scopes, 1978], and presence of a reductant such as ascorbic a c i d [Constantinides and Bedford, 1967] and a binding agent to avoid the reaction of the enzyme with quinones [Rhodes, 1977].
The purification of PPO is extremely difficult, because the enzyme is generally present in very low concentrations. Kertesz and Zito, 1965]

By ionic interactions since most proteins and
Phenolics at neutral and acidic pHs have a charge. 108 4. By hydrophobic interactions, due to interaction between the aromatic ring of the polyphenols and the hydrophobic areas of proteins [Loomis, 1974]. These interactions could yield an inactive enzyme, which would change most of its hydrodynamic properties. In recent years this problem has been solved to some degree by binding the phenol compounds to an insoluble polymer.  [Mathew and Parpia, 1971].
Column chromatography has often been used in purification and separation of PPO. While both adsorption chromaotography and ion-exchange chromatography have been employed, the latter has been found to be particularly successful, especially when substituted celluloses such as diethylaminoethyl eel l ul ose [DEAE] and carboxymethyl ' cellulose [CMC] are used [Peterson and Sober, 1962] Celite has been found to be selective as an adsorbent for phenolases, but enzyme adsorption was found to be dependent on copper. When copper was removed from phenolases of Neurospora crassa, no adsorption was noted for the apoenzymes [Fling et al, 1963]. Hydrophobic adsorption chromotography has been used to separate phenoloxidases from peaches [Flurkey and Jen, 1980 [2] the methods are less complicated to perform [Soto,1983].

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Inhibition of polyphenol oxidase can be achieved by inactivating the enzyme by a physical or chemical method.
Physical methods include the exclusion of oxygen, the storage of raw products at freezing temperatures, and heat inactivation of the enzyme [Yankov, 1963]. Chemical methods are usually preferred over physical methods.
Only a few of the substances used to inhibit the activity of PPO in foods are accepted due to restrictive requirements such as non-toxicity, wholesomeness, and effect on taste, flavor, and texture, etc. [Walker, 1975].
Since PPO is a metalloprotein with copper as the prosthetic group, it can be inhibited by metal-chelating agents such as cyanide [Walker, 1975], carbon monoxide diethyldithiocarbamate [Pierpoint, 1966]; sodium [DIECA] [Robb, et a 1 , 1966], mercaptobenzithiazole [Palmer and Roberts, 1967]. Most of these reagents are used by biochemists to avoid the interaction with polyphenols during the isolation of different molecules from plant tissues [Rhodes, 1977]. Benzoic acid and some substituted cinnamic acids were found to be the most competitive inhibitors of the enzyme from sweet cherries [Piffere, et al. 1974], apples [Walker, 1964], pears [Walker, 1976], and apricots [Soler et al. 1965].
Many investigators have tried to prevent black spot in shrimp. One of the first observations on the preventio~ and delay of black spot in shrimp involved removal of the shrimp head immediately after the catch is brought on board [Alford and Fieger, 1952].
Other methods of inhibiting melanosis include the use of chemicals which interfere with black spot formation. One of these compounds is ascorbic acid, which has been proven to be useful in delaying black spot formation in shrimp [Flaukner et al., 1954]. Ascorbic acid delays the reaction by competing for available oxygen. Cysteine and glutathione have been used to reduce PPO activity by combining with quinones to form thioethers [Mason, 1955;Loomis and Bataille, 1968]. Many  prompted by an increasing concern for adverse "allergic" 113 reactions most common among hyper-[sulfite-] sensitive asthmatics [Otwell and Marshall, 1986] (Furia, 1977).
Glucose oxidase shows a high affinity for -D-glucose Adams, et al., 1960). Other hexoses, pentoses, or dissacharides are not oxidized, or are oxidized only at negligible rates. (Pazur and Kleepe, 1966). For all practical purpose purified glucose oxidase reacts only with glucose, and this was made the enzyme availuable tool for analysis, as well as for other purposes.
The usual commercial preparations of glucose oxidase contain catalase, which is advantageous in most of the a pp 1 i cations . The over a 11 re a c ti on s of the co mm er c i a 1 ' glucose oxidase/catalase system (GOX/CAT) are shown in Figure 2.
For the industrial application the GOX/CAT enzyme system is used to remove glucose and to remove oxygen.
The most important application of glucose oxidase for the removal of glucose is from the egg albumen and whole eggs prior to drying Baldwim, et al., 1953).
Oxygen is responsible for a wide types of deterioration of foods. It has been demonstrated that addition of glucose oxidase is very effective in removing residual oxygen from beer and stabilized it Reinke, et al., 1963). Glucose oxidase found to remove oxygen form unpasteurized apple wine, prevented microorganism growth, and off-flavor development (Yang, 1955). Glucose oxidase have been found to be effective in protecting cans of carbonated beverages against oxidative corrosion, such as canned soda ; to