Oxygen- and Metal-Ion-Dependent Nonenzymatic Browning of

Chapter 5

Oxygen- and Metal-Ion-Dependent Nonenzymatic Browning of Grapefruit Juice Joseph Kanner and Nitsa Shapira

Downloaded by IOWA STATE UNIV on February 19, 2017 | http://pubs.acs.org Publication Date: September 7, 1989 | doi: 10.1021/bk-1989-0405.ch005

Department of Food Science, ARO, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel

Grapefruit j u i c e was packaged i n glass bottles with various headspaces, calculated to provide several d i f f e r e n t concentrations of oxygen/ml j u i c e . The oxygen played the most important r o l e i n degradation of ascorbic acid (ASA) and non-enzymatic browning of grapefruit j u i c e stored at 23°C. A linear c o r r e l a t i o n was found among the amount of oxygen, the degradation rate of ASA and the browning rate. The lag period of browning of grapefruit j u i c e decreased i n length with increasing oxygen concentration. Ascorbic acid oxidase, which oxidizes ascorbic acid to dehydroascorbic acid, was found capable of eliminating the lag period of the browning i n grapefruit j u i c e . Oxygen also enhanced the browning affected by dehydroascorbic a c i d . As the electronic structure of oxygen i s i n the t r i p l e t state, i t s reaction with molecules of singlet m u l t i p l i c i t y , such as ascorbic acid, i s spin-forbidden. This phenomenon led us to determine the involvement of metal ions i n the process of non-enzymatic browning affected by ascorbic acid oxidation. The oxidative degradation of ASA and the browning of grapefruit j u i c e were i n h i b i t e d by the addition of chelating agents (EDTA). The r e s u l t s demonstrated the involvement of metal ions i n the process of non-enzymatic browning.

Non-enzymatic browning of c i t r u s juices and concentrates has been reported by many researchers as a major cause of quality deterioration i n c i t r u s products (1-6). I t was suggested that sugar-sugar and sugar-amino reactions were unlikely to be the only contributors to the formation of brown pigments during storage of c i t r u s products (7), and especially of those packaged i n p l a s t i c metallized laminate bags 0097-6156/89/0405-0055$06.00/0 o 1989 American Chemical Society

Jen; Quality Factors of Fruits and Vegetables ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


56

QUALITY FACTORS OF FRUITS AND VEGETABLES

or cartons (8). The involvement of ascorbic acid i n non-enzymatic browning of c i t r u s products was studied by many researchers (9-12). Degradation of ascorbic acid provided carboxyl compounds (4,7,13,14) that subsequently reacted v i a aldole condensation or with amino acids groups and polymerized to give brown pigments (10,11,12). Ascorbic acid degradation i n heat-treated c i t r u s products i s due to aerobic and anaerobic reactions of a non-enzymatic nature. The aerobic pathway of non-enzymatic browning i n c i t r u s products required oxygen, which may enter i n a s e p t i c a l l y packed products by dissolved from the headspace or penetrate through packaging material (8). Ascorbic acid destruction rates were d i r e c t l y proportional to the i n i t i a l concentration of dissolved oxygen i n several model sys tems (8,15,16). Storage studies of the loss of ascorbic acid potency i n canned orange juices have shown an i n i t i a l period of rapid loss of vitamin C that was caused by the presence of free oxygen (4,5) . However, the electronic structure of oxygen has two unpaired electrons at the energy l e v e l of π antibonding, i n t r i p l e t state. The reaction of oxygen, therefore, i s spin-forbidden with ground state molecules of singlet m u l t i p l i c i t y such as ascorbic acid (17) (Fig. 1, reaction a ) . Transition metals, e.g. i r o n and copper, with their l a b i l e d-electrons system, are well suited to catalyze redox reaction. Stable paramagnetic states, r e s u l t i n g from the presence of unpaired electrons, are common for t r a n s i t i o n metals and f a c i l i t a t e their reaction with free radicals or t r i p l e t molecules, such as oxygen, which i s b i - r a d i a l i n the t r i p l e t state (17) ( F i g . 1, reaction b ) . Thus, they are able to remove this spin r e s t r i c t i o n between oxygen and ascorbic acid and thereby promote the oxidation of the s i n g l e t molecule ( F i g . 1, reaction b ) . Oxygen could interact with other molecules i n the t r i p l e t state ( F i g . 1, reaction c ) . Activated mole cules of oxygen i n the singlet state (singlet oxygen) could oxidize d i r e c t l y with s i n g l e t molecules ( F i g . 1, reaction d). This study was conducted i n an attempt to understand better the e f f e c t of oxygen on non-enzymatic browning of c i t r u s juices and the involvement of metals i n t h i s process. Materials and Methods L-ascorbic acid, EDTA-Na2, metaphosphoric acid, dichlorophenolindophenol, sodium benzoate and potassium hydroxide were purchased from BDH Chemicals Ltd. (Poole, England), and hydrochloric acid 37% and c i t r i c acid from Merck (Darmstadt, FRG); ascorbate oxidase was obtained from Sigma Chemical Co. (St. Louis, MO). Single-strength grapefruit juice was reconstituted from a fresh APV evaporator concentrate of 58° B r i x with d i s t i l l e d water to 11.2° Brix and poured into 200-ml glass b o t t l e s . The bottles were f i l l e d with j u i c e to a headspace which by calculation was found to provide d i f f e r e n t amounts of oxygen per ml of j u i c e . The j u i c e was preserved with 600 ppm sodium benzoate. Ascorbic acid was determined by t i t r a t i o n with 2,6-dichlorophenol-indophenol (18). The color of the j u i c e was determined d i r e c t l y with a Gardner Tristimulus Colorimeter, model KL 10. The instrument was calibrated against a white plate, L=91.6, a=1.8, b=H-1.8.


5.

57

Browning of Grapefruit Juice

KANNER & SHAPIRA

Results are the means of t r i p l i c a t e s , and i n the figures each error bar (I) denotes the standard deviation.


Results and Discussion Non-enzymatic browning of grapefruit j u i c e i s accelerated by increasing oxygen concentration. During the f i r s t days of storage there was a lag period where the browning rate was low. After this period, the browning rate accelerated and decreased once more a f t e r a l l the ascorbic acid had been degradated ( F i g . 2). Figure 3 i l l u s t r a t e s the l i n e a r relationship between browning and the amount of oxygen. In contrast to j u i c e browning, ascorbic acid degradation was rapid during the f i r s t period of storage ( F i g . 4). Figures 5 and 6 i l l u s t r a t e the l i n e a r relationship between ascorbic acid oxidation and oxygen concentration, and the c o r r e l a t i o n between browning and ascorbic a c i d degradation. In order to better understand the e f f e c t of ascorbic acid on non-enzymatic browning of c i t r u s j u i c e s , we oxidized endogenous ascorbic acid i n grapefruit with ascorbic acid oxidase. The enzyme oxidized ascorbic acid to dehydroascorbic acid and H2O without forming H 0 (19). This study has shown that grapefruit j u i c e treated with the enzyme, browned without developing a lag period. The treated j u i c e browned anaerobically, but i n an aerobic environment the browning rate was s i g n i f i c a n t l y higher (Fig. 7). I t was demonstrated that oxygen i s involved i n browning reactions not only to convert ascorbic acid to dehydroascorbic acid, but also to convert some other reductones generated from dehydroascorbic a c i d to by-products which form brown pigments. The brown pigments which were formed through the oxidative pathway are almost twice those formed through the anaerobic pathway. This e f f e c t could be derived from the involvement of H2O2 and oxy- r a d i c a l s i n the aerobic oxidation pathway of ascorbic acid (20,21). The oxy-radicals may generate carbonyls and amino acid free radicals which, by polymerization, could accelerate the formation of more brown pigments. Although the i n h i b i t i o n of ascorbic acid oxidation i n c i t r u s juices (22,23) and other foods by EDTA was reported by several researchers (22-24), the p o s s i b i l i t y that EDTA could i n h i b i t nonenzymatic browning i n grapefruit or other c i t r u s juices has not been explored. The r e s u l t s i l l u s t r a t e d i n Figures 8 and 9 showed that EDTA inhibited non-enzymatic browning and ascorbic acid oxidation i n grapefruit j u i c e . This demonstrates the involvement of metal ions in the non-enzymatic browning of grapefruit j u i c e and possibly of other c i t r u s products. The involvement of metal ions, oxygen, and oxygen species in non-enzymatic browning of grapefruit j u i c e could be described by the following reactions: 2

2

(low pH) AH + Fe+ (high pH) AH" + Fe+ Fe+ + 0 (low pH) AH- + 0 (high pH) AT + 0 0J + HO2

3

2

3

2

slow fast

2

2

7

2

2

2

H+

AH. + Fe+ + H+ J A" + Fe+ H+ Fe+ + O j A + HO2 A + 0J H 0 + 0 3

2

2

2

(1) (2) (3) (4) (5) (6)



58

a i l

-11


o i l

e A H

2

+

11

+

11

*

1

•

f|

• NO REACTION

1 — 1

11

- 11

1—11

11

+

11 11—11

U

+

^ C u ^ 0

2

- ° 2 - ^ H 0 2

_

_

2 +

0

11

2

Figure 1. Reactions among oxygen, ascorbic acid and metal ion i l l u s t r a t e d by the interaction of electrons from the valence orbitals. a, Reaction between t r i p l e t oxygen and a s i n g l e t molecule. b, Reaction between t r i p l e t oxygen and metal ions. c, Reaction between t r i p l e t oxygen and other molecules i n the t r i p l e t state. d, Reaction between singlet oxygen and other singlet molecules. e, Reaction among ascorbic acid, cupric ion and oxygen which generates superoxide (θ|), ascorbyl r a d i c a l (ΑΗ') and, by dismutation and further oxidation, produce hydrogen peroxide ( H 0 ) , oxygen and dehydroascorbic acid. 2

2

Figure 2. The e f f e c t of oxygen concentration on grapefruit j u i c e browning at 23±2°C.


KANNER & SHAPIRA

Browning ofGrapefruit Juice


5.

0

10

20 TIME

30

(days)

Figure 4. Ascorbic acid oxidation i n grapefruit j u i c e as a f f e c ted by oxygen concentration.


59


60


0

2

OXYGEN

4 (pmole

6

8

«0

0 / m l juice) 2

Figure 5. Correlation between oxygen concentration and ascorbic acid oxidation i n grapefruit j u i c e during storage a t 23±2°C.



5.

KANNER & SHAPIRA


CONTROL-N

4Q

J 4

61

2

I— 8

12

16

20

TIME (days) Figure 7. The e f f e c t of ascorbic acid oxidation by ascorbic acid oxidase on grapefruit j u i c e browning under d i f f e r e n t conditions. Δ, the presence of nitrogen; A, control i n the presence of 9.65 ymole oxygen/ml j u i c e ; 0, grapefruit j u i c e treated with ascorbic acid oxidase (1 U/5 ml juice) i n the presence of nitrogen; #, grapefruit j u i c e treated with ascorbic acid oxidase (1 U/5 ml juice) i n the presence of oxygen.


62



60 ι

TIME

(days)

Figure 8. The effect of a chelating agent (EDTA) on non-enzymatic browning of grapefruit j u i c e incubated at 23±2°C. ·, control j u i c e i n the presence of 9.65 umole 02/ml; 0, as control, but with added 100 uM EDTA.

EDTA (100 μΜ)

12

24

36

48

TIME (days) Figure 9. The e f f e c t of a chelating agent (EDTA) on ascorbic acid oxidation i n grapefruit j u i c e . ·, control j u i c e i n the presence of 9.65 limole 02/ml; 0, as control, but with added 100 μΜ EDTA.


4

2

Fe " Carb AA + Free AA +

63


5. KANNER & SHAPIRA

+ 3

+ H 0 Fe + HO- + HO" (7) + HO^ Carb · + HO" (8) HO' AA- + HO" (9) radicals polymerization ^ brown compounds (10) Carbonyls condensation ^ brown compounds (11) 2

2

AH = ascorbic acid; A" = semiquinone of ascorbic acid; 0 = super oxide r a d i c a l ; H02 = perhydroxyl r a d i c a l ; Carb- = carbohydrate r a d i c a l ; A = dehydroascorbic acid; AA = amino acids.


2

2

The e f f e c t of EDTA i n oxidation reaction i s complicated. EDTA is known to accelerate ascorbic acid oxidation by f e r r i c ions at neutral pH (21,25,26). In a medium at the same pH, EDTA also enhanced the formation of hydroxyl r a d i c a l s by f e r r i c ion i n the presence of ascorbic acid (21,25). I t i s known that the redox p o t e n t i a l of the F e / F e pair can vary by complexing ligands (27) . EDTA reduces the redox p o t e n t i a l of F e (28) and this increases the rate constant transfer of the electron from F e to H 0 , which i s formed during autooxidation of ascorbic acid (29), and decomposition of the l a t t e r to H0-. However, at low pH 3-4, EDTA was found to i n h i b i t ascorbic acid oxidation by f e r r i c ions (29) . Thus, the form the metal chelate takes, as a function of pH, plays a key role i n i t s effectiveness as a c a t a l y s t . Cupric ions are known to accelerate ascorbic acid oxidation; however, EDTA i n h i b i t s i t s c a t a l y t i c e f f e c t at both neutral and low pH (24). C i t r i c j u i c e s contain i r o n and copper ions (30,31) at a concen t r a t i o n which could catalyze ascorbic acid oxidation. Contamination of c i t r u s juices by t r a n s i t i o n metals w i l l a f f e c t the rate of oxidative browning, especially i n products exposed to a i r , such as c i t r u s j u i c e s stored i n p l a s t i c metallized laminate bags which are permeable to oxygen. + 3

+ 2

+ 2

+ 2

2

2

Acknowledgment This work was

supported i n part by the Gan-Shmuel Factory,

Israel.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

L o e f f l e r , H. J . Ind. Eng. Chem. 1941, 33, 1308-14. Moore, E. L.; Wiederhoald, E.; Atkins, C. D. Food Products J . 1944, 23, 270-75. Joslyn, Μ. Α.; March, G. L. Ind. Eng. Chem. 1935, 27, 186-89. Kanner, J . ; Fishbein, J.; Shalom, P.; Harel, S.; Ben-Gera, I . J . Food S c i . 1982, 47, 429-31. Trammell, D. J . ; Dalsis, D. E.; Malone, C. T. J . Food S c i . 1986, 51, 1021-23. Mannheim, C. H.; Avkin, M. J . Food Process. Preser. 1981, 5, 1-6. Clegg, Κ. M.; Morton, A. D. J . S c i . Food Agric. 1965, 16, 191-96. Shapira, Ν. M.Sc. thesis, Faculty of Agric., Univ. of Jerusalem, Rehovot, I s r a e l , 1986. Joslyn, Μ. Α.; Marsh, G. L.; Morgan, A. F. J . B i o l . Chem. 1934, 105, 17-28. Joslyn, M. A. Food Res. 1957, 22, 1-4.


64 11. 12. 13. 14. 15. 16.


17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

30. 31.

QUALITY FACTORS OF FRUITS AND VEGETABLES Clegg, K. M. J . S c i . Food Agric. 1964, 15, 878-85. Amram, T. (1980) M.Sc. thesis, Faculty of Agriculture, Univ. of Jerusalem, Rehovot, I s r a e l , 1980. Kanner, J . ; Harel, S.; Fishbein, Y.; Shalom, Paulette J . Agric. Food Chem. 1981, 29, 948-50. Kurata, T.; Wakabayashi, H.; Sakurai, Y. Agric. B i o l . Chem. 1967, 33, 101-05, 170-84. Kefford, J . E.; McKenzie, Η. Α.; Thompson, P. C. A. J . S c i . Food Agric. 1950, 10, 51-63. Singh, R. P.; Heldman, D. R.; Kirk, J . R. J . Food S c i . 1976, 41, 304-8. Kanner, J . ; German, B.; K i n s e l l a , J . O. Science and Nutrition 1987, 25, 317-64. A.O.A.C. O f f i c i a l Methods of Analysis, American Assoc. of A n a l y t i c a l Chemistry: Washington DC, 1975; 12th ed. Kanner, J . ; Harel, S. J . Food S c i . 1981, 46, 1407-9. Scarpa, M.; Stevanato, R.; V i g l i n o , P.; Rigo, A. J . B i o l . Chem. 1983, 258, 6695Kanner, J . ; Harel, S.; Hazan, B. J . Agric. Food Chem. 1986, 34, 506-10. Furia, T. E. Food Technol. 1964, 18, 50-58. Furia, T. Ε. Sequestrants i n Foods; Handbook of Food Additives; Furia T. Ε., Ed.; CRC Press: Cleveland, 1972; 2nd ed., p.271-94. L i c c i a r d e l l o , W. B.; Esselsen, J . R.; F e l l e r s , C. R. Food Res. 1952, 17, 338-42. Mahoney, J . R.; Graf, E. J . Food S c i . 1986, 51, 1293-96. Beutter, G. R. Free Rad. Res. Comms. 1986, 1, 349-53. B o t a r i , Ε.; Anderegg, G. Helv. Chim. Acta 1967, 50, 2349-52. Koppenol, W. H. Phytochem. Photobiol. 1978, 28, 431-34. Martell, A. E. In Ascorbic Acid Chemistry Metabolism and Uses; Seib, P.Α.; Tolbert, Β. Μ., Eds.; Advances i n Chemistry Series No. 200; American Chemical Society: Washington DC, 1982; p.153. McHard, J . Α.; Winefordner, J . D.; Ting, S. V. J . Agric. Food Chem. 1976, 24, 950-53. Nagy, S. In Citrus Science and Technology; Nagy, S.; Shaw, P.W.; Veldhuis, M. K., Eds.; Avi Publish Company: Westport CT, 1977; Vol. 2, 479-95.

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Oxygen- and Metal-Ion-Dependent Nonenzymatic Browning of

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