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Inhibition of Enzymatic and Non-Enzymatic Browningby Use of Gallic AcidDela Cruz, C.; Felongco, G.; Garcia, F.; Gomez, G.; Velmonte, J.Department of Food Science and Nutrition, College of Home Economics

ABSTRACTBrowning reactions can be enzymatic, which involves an enzyme polyphenol oxidase; and non-enzymatic, which involves the reaction of amines and reducing sugars. In this study, gallic acid was used andwas tested for its potential as an inhibitor for both browning reactions. Spectrophotometric methods were employed to detect browning reactions through absorbances. A tissue extract from banana wasused to determine the extent of enzymatic browning and its inhibition, while a model browning system consisting of an inverse sugar and glycine was used for non-enzymatic browning studies. Resultsobtained from the two analyses yielded positive results for the inhibitory ability of gallic acid: at pH 3 and 5, enzymatic browning is inhibited; while at pH 7, non-enzymatic browning is inhibited. Inhibition ofgallic acid for enzymatic browning can be explained by its structure similar to that of phenolic substrates, while its inhibition for Maillard reaction is due to the modifications it induces to the intermediates inthe reaction. Results from this study may be used in the food industry, as Gallic acid is found commonly in nature.

INTRODUCTION

Browning reactions may either be non-enzymatic or enzymatic. Non-enzymatic brown-ing occurs due to t he chemical reaction between reducing sugars and a free amino acid or a freeamino group of an amino acid which is part of a protein chain, resulting in the production ofbrown pigments. Enzymatic browning, on the other hand, involves a group of enzymes calledpolyphenol oxidase that cause the formation of brown colors in foods, especially in fruits andvegetables, in the presence of phenolic compounds that act as substrates. [1]

Gallic acid, or trihydroxybenzoic acid (3,4,5-trihydroxybenzoate), is a kind of phenolicenzyme and organic acid which can be found in gallnuts, sumac, witch hazel, tea leaves, oakbark, and other plants. It is white, yellowish-white or pale f awn-colored[2] and exists as free acids,esters, catechin derivatives, and hydrolysable tannins.[3] It is considered as one of the most-biologically active phenolic compounds from plants.[3]

In the study of Dogan, Turan, Dogan, Alkan, & Arslan[4] which involved the inhibition ofthe activity of purified polyphenol oxidase activity from basil, gallic acid was used as one of theinhibitors while 4-methylcatechol, catechol, and pyrogallol were utilized as substrates. The re-sults showed that gallic acid exhibited competitive inhibition when it used catechol as a substrateand uncompetitive inhibition when it utilized 4-methylcatechol. The study also stated that gallicacid was effective in the inhibition of t he enzyme-catalyzed browning reaction.

Banana is one of the most popular and easily available fruits worldwide. It is normallyeaten raw as a fruit and is used in making desserts. Bananas are good sources of vitamins,

minerals, sugars, and other nutrients. It has four times more protein content compared to anapple and contains a huge amount of easily digestible sugars. Banana readily undergoes brown-ing as it contains phenolic compounds such as 3,4-dihydroxyphenylethylamine (dopamine),leucodelphinidin, and leucocyanidin which can be utilized as substrated for enzymatic browning.[5]

This study aims to analyze the effect of the addition of gallic acid in the inhibition ofenzymatic browning reaction in the banana sample. A model browning system containing bothsugar and protein shall be employed to observe the any inhibitory effect of gallic acid on non-enzymatic browning. Both processes shall utilize the spectrophotometer to detect absorbances.

DISCUSSION

I. Enzymatic Browning Inhibition

Enzyme activity is influenced by many factors,such as pH or temperature that may change the three-dimensional shape of an enzyme and alter its rate of activ-ity. Specific chemicals may also bind to an enzyme andmodify its shape. Chemicals that must bind for the enzymeto be act ive are c alled activators. Co-fact ors are non-protein substances that usually bind to the active site on theenzyme and are essential for the enzyme to work. Organic

co-factors are called co-enzymes, but other co-factors may simply be metal ions. Chemicals that shut off en-zyme activity are called inhibitors and their action can be class ified as competitive, uncompetitive, or non-competitive inhibitors.[6]

Small amounts of catechol occur naturally in fruits and vegetables, along with the en-zyme polyphenol oxidase (also known as catecholase, or catechol oxidase). Upon mixing the enzyme with thesubstrate and exposure to the colorless catechol oxidizes to reddish-brown melanoid pigments, derivatives ofbenzoquinone (Fig. 4).[7] The enzyme is inactivated by adding an acid, such as lemon juice, and slowed withcooling. Excluding oxygen also prevents the browning reaction. Benzoquinone is said to be an antimicrobial,which slows the spoilage of wounded fruits and other plant parts, acting as a defense mechanism of fruits andvegetables when they are stressed.[7] Once the reaction starts, the subsequent reactions occur spontaneously

and no longer depend on oxygen or enzyme. [8]Gallic acid (Fig. 5) was the inhibitor used in the study of enzymatic in Musa acu-

minata (banana). Spectrophotometric techniques are used to measure the concentration ofsolutes in solution by measuring the amount of light that is absorbed by the solution in acuvette placed in the spectrophotometer. In this experiment, the extent of browning will bedetermined by the amount of light which will be absorbed. Thus, a high absorbance readingentails a greater amount of browning in the solutions. It can be observed that at pH 3 ( Fig.1), the rate of inhibition is evident due to the immediate stopping of the browning as depictedby the early constancy of values. It can be inferred that there was little amount of browningthat occurred. The same can be stated for pH 5 (Fig. 2). At pH 7 (Fig. 3), however, the ab-sorbances of the solution with inhibitor exceeded that of the maximum absorbance of thesolution without inhibitor. Based on this, it can be inferred that the activity of gallic acid as inhibitor in the enzy-matic browning in banana is more effec tive under an acidic environment.

Possible sources of error include the preparation of solutions and the materials used for measure-ments. Since minute amounts are required, there might have been excesses in drops from the pipette, resultingto increased absorbances. Other possible source is the re-using of plastic cuvettes, which may bring about ahigher absorbance due to scratches and haziness introduced by its previous users. The early preparation ofsolutions, wherein a week had passed before each was used, might have had aff ected the readings too.

II. Non-enzymatic Browning Inhibition

Various factors affect the rate of Maillard reaction such as type of reagent, temperature, wate activ-ity, and pH. In systems with intermediate moisture content, Maillard reactions are favoured at temperatures>50C and pH 4-7. Amadori products undergo degradation to give 1- and 3-deoxydicarbonyl compounds

(deoxyosones).[6]Based on Table 1, at pH 3, the absorbance of the sample with inhibitor was greater than the sample

with no inhibitor. This indicates that at this pH level, the addition of gallic acid actually increased the rate ofMaillard browning instead of inhibiting it. The same phenomenon was also observed at pH 5. However, theabsorbance at pH 5 with no inhibitor was greater than the absorbance at pH 3 with no inhibitor.

On the other hand, the effect of gallic acid at pH 7 exhibited a lower degree of absorbance than thesample with no inhibitor. This indicates that there is a lower rate of Maillard reaction with the addition of theinhibitor at pH 7. The absorbance at pH 7 with no inhibitor was the greatest among the other samples with noinhibitor. Based from the results, it can be said that at higher levels of pH, the rate of Maillard reaction in-creases in the samples containing no inhibitor; and in the group of samples where the inhibitor was added, thedegree of inhibition can be said to increase with increasing pH. It exhibited the greatest degree of inhibition atpH 7.

Based on the study conducted by Elisei and Leonte [9], it was confirmed that naturally occurring phe-nolic compounds such as gallic acid, ferulic acid, quercetin, and four types of tannins, prevents Maillard reac-tion. These phenolic compounds interact in the Maillard reaction to modify the formation of alkylpyrazines anddegree of browning produced. The phenolic compounds act as antioxidants and scavenge these pyrazine freeradicals in the Maillard reaction, and retard their activity. Among the phenolic compounds tested in their study,gallic acid has been found to be the most efficient inhibitory agent on the alkylpyrazines In addition, gallic acidwas shown to decrease the quantities of methylpyrazine, 2,5-dimethylpyrazine, and 2,35-trimethylpyrazine.

Aside from preventing the browning of foods, the results of the study may be beneficial to preventthe effects of Maillard reaction on the development of diabetic complications and aging, by preventing the accu-mulation of end-stage products of the Maillard reaction, also called advanced glycation end products (AGEs).

REFERENCES

[1] Fennema, O. (1996). Food Chemistry(3rd ed.). New York: Marcel Dekker, Inc.

[2] Gallic Acid. (n.d.). Retrieved March 21, 2012, from Chemical Land Website: http://chemicalland21.com/specialtychem/finechem/GALLIC%20ACID.htm

[3] Karamac, M., Kosinska, A., & Pegg, R. (2006). Content of Gallic Acid in Selected Plant Extracts. Pol-ish Journal of Food and Nutrition Sciences, 55-58.

[4] Dogan, S., Turan, P., Dogan, M., Alkan, M., & Arslan, O. (2007). Inhibition Kinetics of Ocimum basili-cum L. Polyphenol Oxidase. International Journal of Chemical Reactor Engineering, 1-13.

[5] Wageningen University. (n.d.). Enzymatic Browning. Retrieved March 24, 2012, from Food-Info Web-site: http://www.food-info.net/uk/colour/enzymaticbrowning.htm

[6] deMann, J. M. (1990). Principles of Food Chemistry. (2nd Ed. ed.). USA: Van NostrandReinhold Intl. Company Ltd.

[7] n.a. (n.d.). Cathecol oxidase- A Study of Inhibition. Retrieved 27 March 2012 http://www.science-projects.com/Tyrosinase.htm

[8] Prontes, Isabel (n.d.). About Cathecol in Bananas. Retrieved 27 March 2012 http://www.ehow.com/about_4619323_catechol-in-bananas.html

[9] Elisei, A. &Leonte, M. (2004).The Evaluation of the Scavenging Activities of Some Phenolic Com-pounds in the Maillard Reaction.The Annals of the University Dunera de Jos. Retrievedfromwww.ann.ugal.ro/tpa/Annals%2004%20papers/03%20Full%20paper%20Elisei.pdf&ei=JoxxT-e2GsLmrAeD88TIDQ&usg=AFQjCNGOkcVO8btidhbxfIrTnpXJIc3DOA

RESULTS