edited by
JAMES0. SCHRECK
filtrates e residues
University of Nonhern Colorado Greeley, CO 80639
lnhibition of Enzymatic Browning Reaction by Sulf ite Hie-Joon ~ i m ' U S . Army Natick Research, Development & Engineering Center, Natick, MA01760 When certain fruits or potatoes are cut, the cut surface turns brown. This change is due to a chemical reaction known as the enzymatic browning reaction (I, 21, in contrast with nonenzymatic browning reaction often seen in the crust of bread. In a typical enzymatic browning reaction shown in the figure, polyphenol oxidase (PPO) with a bound cupric ion catalyzes oxidation of polyphenols, such as catechol, by oxygen in the air. The oxidation product, o-quinone in this case, undergoes a series of polymerization reactions leading to dark brown end products collectively known as melanins. Why such browning reaction takes place is not well understood. It probably represents a protective mechanism equivalent to blood clotting in animals. Sulfur dioxide, produced by burning sulfur, was used in ancient Greece as an antiseptic. Sulfivous acid is formed when sulfur dioxide is dissolved in water. Sulfite, commonly used as sodium or potassium sulfite, bisulfite or metabisulfite, is a collective term for these salt forms of sulfurous acid. Sulfite is used in processed foods as antimicrobial agent, antioxidant, inhibitor of enzymatic and nonenzymatic browningreactions, and bleaching agent (3).Sulfite is also essential in winemaking for controlling contaminating microbes during fermentation. The following classroom activity is designed to demonstrate visually the multiple functions of sulfite as an effective food additive. lnhibition of Enzymatic Browning in Sliced Apple Procedure
Dip sliced apple in water (control), lemon juice (bottled or fresh), 2% ascorbic acid (vitamin C) solution (2 g ascorbic acid i n 100 mL H20), and 2% sodium sulfite solution (2 g NazSOs in 100 mL HzO) for about 5 min. The ascorbic acid and sulfite solutions should be prepared fresh daily. Read the label and make sure that the bottled lemon juice contains no sulfite. Freshly squeezed lemon juice can be used. Remove excess liquid with a paper towel. Leave the apple slices in the open air and let students record the color observed on the surface initially and after about 1h (at the end of the class) and 4-6 h (at the end of the day). Keep the slices in a refrigerator overnight and make another observation the following day. Which slice of apple turns brown most quickly? Which is the best inhibitor of the browning reaction: lemon juice, ascorbic acid, or sulfite? Suggest how lemon juice or sulfite might inhibit the reactions in the figure. Discussion The different inhibitory mechanism by ascorbic acid in lemon juice and sulfite should he noted. Initially all apple slices will look the same. However, the control apple slice will soon start to turn brown while the slices dipped in 'Adjunct Professor, Department of Chemistry, Framingham State College Framingham. MA01701. 242
Journal of Chemical Education
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acid
\ polymerizstion A' melanin PPO : polyphenol axidase
Typical enzymatic browning. lemon juice, ascorbic acid, or sulfite solution will remain lightly colored. At the end of the day, the slices dipped in lemon juice or ascorbic acid solution will turn light brown. The difference i n color among different slices will be prominent after overnight exposure to air. The sulfited slice will stay fresh-looking for several days. If a tristimulus colorirneter is available, the color change on the surface can he monitored by reflectance measurements of the Hunter L, a, and b values. Instructors may want to try different concentrations of ascorbic acid and sulfite with apple slices to be used in order to achieve maximum contrast i n the surface color consistent with the above outline. Also different varieties of apples may be tried to see if there is a varietal difference in browning potential. Acidity in both lemonjuice and the ascorbic acid solution has some denaturing effect on the PPO. More importantly, ascorbic acid that is present a t a high concentration in lemon juice reduces the oxidation products such as o-quinone back to the polyphenols before they undergo polymerization to melanin. Concurrently, ascorbic acid is oxidized to dehydroascorbic acid. This type of inhibition will be effective only a s long a s sufficient reduced ascorbic acid is available. One should remember that ascorbic acid is also oxidized to dehydroascorbic acid by oxygen in the air. The higher effectiveness of sulfite is due to the fact that sulfite not only inhibits the PPO directly but also combines with o-quinone preventing formation of melanin (4). As demonstrated in the second experiment, sulfite also can
bleach the melanin oimnents if anv were formed bv escaDing the inhibitory a&& of sulfite,-which is quite Glikeiy. Sulfite had been used in salad bars to keep the fruits and vegetables fresh. A small percentage of the population, especially asthmatic patients, are hypersensitive to suKte and can be affected adversely. Therefore, the Food and Drug Administration banned the use of sulfite on fresh fruits and vegetables (5).It is easy to see why several sulfite substitute formulations are based on the reducinp proper%' of ascorbic a c ~ dor its s t ~ ~ c t ~imi~logcrythorbic ~ral arid. 'rhriimitation of surh l'ormulntionl; ran be dedured from an nndvsii ofthe experimental results. The use of sulfite in processed foods is permitted, because there is no alternative in certain foods, hcluding dehydrated fruits and potatoes, Maraschino cherries, wine, etc. However, the processors are required to indicate the presence of sulfite on the label if the sulfite level exceeds 10 parts per million (6). Inhibition of Enzymatic Browning in Potato Extract Procedure 1. Dissolve 1 g catechol in 100 mL Hz0 to make a 1% catechol solution. 2. Boil 200-250 mL water in a 500-mL beaker on a hot plate. 3. Blend about 30 g (or about a n ounce) of cut potatoes with about 200 mL (or 1 CUD) of water i n a small food blender or mixer F ~ l t e ar portion of the extract using a filtcr u a w r rExrrsct A!. .4lternativelv c c n ~ r i f-w ethe extract a n d u i e the supernatant. 4. Heat about 1 mL of Extract A in a test tube i n the boiling water bath for about 5 min and let cool (Extract B). 5. Prepare the following mixtures and note color after 5 min and about 1 h. The catechol and sulfite solutions should be prepared fresh daily. Test tube 1:2 mL water + 0.2 mL Extract A. Test tube 2: 2 mL 1%catechol solution + 0.2 mL Extract A. Test tube 3: 2 mL 1%eateehal solution + 0.2 mL Extract B. Test tube 4: 1.8 mL 1%cstechol solution + 0.2 mL 2% sulfite solution + 0.2 mL Extract A. 6. Add 0.2 mL 2% sulfite solution to the mixture that developed brown color and note the color change. What did sulfite do to the melanin? What does i t tell you about the efficacy of sulfite a s a browning reaction inhibitor?
Discussion I n the second experiment, 1%catechol solution as a n added substrate is used to increase the concentration of melanins for easy visual comparison. (The rate of color development also can be monitored by absorbance measuremenis a t 420 nm.) The potato extract contains PPO a t a sufficient concentration to catalyze the oxidation and subs e q u e n t polymerization of catechol, h u t not enough polyphenols (chlorogenic acid is the primary polyphenol in potatoes) to cause substantial browning by itself within a short period of time (control in tube 1). The content i n tube 2 will turn yellow within a few minutes and gradually turn into dark brown by the catalytic action of PPO in the potato extract. Such color development will not take place in tube 3 because the activity of PPO has been lost upon heating. The concept of protein denaturation can be introduced to the class a t this point. One also can discuss why certain vegetables in the frozen foods section of a mocerv store are blanched ~ r i o to r freezing. Another relevant point to make is that, unlike proteins, DNAis extremely stable due to the thousands of hydrogen bonds between the two strands that make up the double helix. Therefore. no b a c k u ~c o ~ vis necessarv and only one copy of DNA isfound in aAcel
