Food Research International 32 (1999) 609619

www.elsevier.com/locate/foodres

Studies on the occurrence of non-enzymatic browning during storage of citrus juice

M.G. Roiga, J.F. Bellob, Z.S. Riverac, J.F. Kennedyc,*
a Departamento de Qumica Fsica, Facultad de Qumica, Universidad de Salamanca, Plaza de la Merced s/n, 37008 Salamanca, Spain Departamento de Qumica Fsica, Facultad de Farmacia, Universidad de Salamanca, Avda. Campo Charro s/n, 37007 Salamanca, Spain c Research Laboratory for the Chemistry of Bioactive Carbohydrates and Proteins, School of Chemistry, Birmingham University, Birmingham B15 2TT, UK b

Received 11 March 1999; accepted 22 September 1999

Abstract In freshly produced commercial citrus juice, aseptically lled in TetraBrik cartons, it has been demonstrated that nonenzymatic browning was mainly due to carbonyl compounds formed from l -ascorbic acid degradation. Contribution from sugaramine reactions is negligible as evident from the constant total sugar content of degraded samples. The presence of amino acids and possibly other amino compounds, however, enhance browning. Although formation of 5-hydroxymethyl-furaldehyde (5-HMF) has been detected in degraded juice samples, its presence could not be used as an index of browning. 5-HMF has been found to be unreactive in the browning process in citrus juices and its contribution to browning in this type of products is insignicant, if not negligible. Increasing the l -ascorbic acid concentration extends the nutritional value of the product but also increases the severity of browning. Hence where l -ascorbic acid is added as an antioxidant in foods, care should be taken so that the amount added is just proportional to the level of oxygen present or to the amount of oxidizable substances. # 2000 Canadian Institute of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved.
Keywords: l-Ascorbic acid; Non-enzymatic browning; Citrus juice

1. Introduction Colour, avour, texture and nutritive value are generally regarded as the acceptability criteria of food (Jen, 1989). Interest in oxidative changes particularly occurring in citrus products is increasing continuously due to the nonenzymatic browning reactions which give undesirable o-taste and o-colour on storage. This problem has been addressed in a number of reviews (Handwerk & Coleman, 1988; Hodge, 1953; Wedzicha, 1984). One of the more important food constituents which has drawn attention to these oxidation reactions is l-ascorbic acid. Theories have been proposed which implicate l-ascorbic acid, based on the observations that browning formation follows l-ascorbic acid loss (Clegg, 1964, Clegg & Morton, 1965). Investigators (Shaw, Tatum & Berry, 1977; Tatum, Shaw & Berry, 1969) reported the isolation of furan-type compounds,
* Corresponding author. Tel.: +44-121-414-4385; fax: +44-121414-4384. E-mail addresses: jfkennedy@chemistry.bham.ac.uk (J.F. Kennedy), mgr@gugu.usal.es (M.G. Roig).

lactones, acids and 3-hydroxy-2-pyrone as degradation products of l-ascorbic acid. A number of these compounds were identied as nonenzymatic browning products found in dehydrated orange and grapefruit powders (Shaw et al., 1977). Enzymatic browning and its inhibition has been the subject of numerous studies and review articles (Flurkey & Ingebrigsten, 1989; Hsu, Sheih, Bills & White, 1988; Sapers & Hicks, 1989). So far, among the most eective and safest inhibitors of enzymatic browning are l-ascorbic acid and its isomers and derivatives (Hsu et al., 1988; Sapers & Hicks, 1989). Sulphite is also considered very eective (Sapers & Miller, 1995). Enzymatic browning occurs in bruised or cut fruit or vegetables, such as apples, bananas and potatoes, possibly due to low levels in l -ascorbic acid (Sapers & Miller, 1995), although the acid of citrus fruits may prevent their browning as much as their higher levels of l-ascorbic acid. For this type of browning to occur, three components are essential: enzyme, substrate and oxygen (Bauernfeind, 1982). In the presence of oxygen, highly active phenolases oxidize orthophenolic substrates to form the coloured quinone compounds (Sciancalepore &

0963-9969/00/$20.00 # 2000 Canadian Institute of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved. PII: S0963-9969(99)00128-3

610

M.G. Roig et al. / Food Research International 32 (1999) 609619

Longone, 1984). Addition of l-ascorbic acid reduces these quinone structures. In the absence of l-ascorbic acid, further oxidation and polymerization continues to form the irreversibly oxidized brown compounds (Henshall, 1981; Sapers & Miller, 1995). Reduction of quinone compounds by l-ascorbic acid is accompanied by a decrease of enzyme activity (Sapers & Miller, 1995). It could be argued that discolouration would not occur until all enzyme had been deactivated by an adequate amount of l-ascorbic acid. l-Ascorbic acid and its isomer d -isoascorbic acid have been used interchangeably as inhibitors of enzymatic browning (Santerre, Cash & Vannorman, 1988). However, some investigators suggest that l -ascorbic acid is more eective (Hsu et al., 1988; Sapers & Ziolkowski, 1987). In some cases, l-ascorbic acid, d -isoascorbic acid or its derivatives have been used in combination with sodium benzoate, sulphur dioxide, citric acid, and sodium or calcium chloride (Langdon, 1987; Santerre et al., 1988). The eect of l-ascorbic acid degradation on browning has been reported to be accelerated by amino acids (Clegg, 1964, 1966; Kacem, Cornell, Marshall & Shiremean, 1987; Yu, Wu, Wang & Salunkhe, 1974). Whether the amino acids react with l-ascorbic acid or its degradation products to form the brown pigments is still unknown. Another major component of citrus fruit is citric acid. It has been suggested that it contributes signicantly to the browning of acidic products that contain l -ascorbic acid (Clegg, 1966). As to its exact role in browning is another point of investigation. Studies showed that l-ascorbic acid decomposition resulted in furaldehyde and 5-hydroxymethyl-furaldehyde (5-HMF) formation (Huelin, 1953; Huelin, Coggiola, Sidhu & Kennet, 1971; Kanner, Harel, Fishbein & Shalom, 1981; Robertson & Samaniego, 1986). Since accumulation of these compounds parallels quality loss in citrus products, furaldehyde and/or 5-HMF indexing has been recommended as a basis for quality control (Berry & Tatum, 1965; Kanner et al., 1981; Laing, Schulueter & Labuza, 1978; Meydav & Berk, 1978; Nagy & Randall, 1973; Robertson & Samaniego, 1986). This study was undertaken to check on the reason for nonenzymatic browning and whether sugar amine complexes contribute to the browning of cartoned commercial citrus fruit juices. 2. Materials and methods 2.1. Determination of browning 2.1.1. Spectrophotometric assay of browning Browning indices of the samples were measured by their absorbance at 280 and/or 420 nm on a Shimadzu Model UV 240 UVVis spectrophotometer using a 1 cm

path length cell. The wavelength used was based on the wavelength maxima obtained from spectral scans of aged samples and on previous reports (Meydav, Saguy & Kopelman, 1977). Juice samples obtained from British and Swiss supermarkets or directly from a lling station and from F. Homann La Roche, Basel (Switzerland) were centrifuged at 10000g RCF at 20 C for 45 min, then passed through a 0.45 mm cellulose nitrate membrane lter (Millipore). Dilution with water was performed when necessary. Dry powder samples were dissolved in sodium dihydrogenphosphate solution (0.04 M, pH 2.40) to produce a solution of 2 mg ml1 concentration. 2.1.2. Comparisons of ltration and ethanol addition as clarication steps prior to spectrophotometric assay of browning Portions of clear supernatants of fresh and degraded juice samples after centrifugation were added with an equal volume of ethanol (95%), shaken then passed through a 0.45 mm membrane lter (Millipore). Other portions were just ltered without ethyl alcohol addition. Dilution of the samples with a solution of ethyl alcohol in water [50% (v/v), a further 12.5 times] or water (25 times) was also performed prior to ltration. The samples were then subjected to spectrophotometric assay of browning (see below). 2.1.3. Visual assessment Visual assessment of the degree of browning in undiluted model solutions was a useful initial guide in terms of what might be acceptable to a retail purchaser. However, optical densities at 420 nm were designated for the visual grading of browning to provide the numerical background required to complement the visual data (Table 1). Optical densities at 420 nm were obtained from freshly squeezed orange juice which was centrifuged at high speed, then incubated at l00 C. Aliquots were taken after 0, 2, 11, 17 and 22 h storage. Samples were passed through a 0.45 mm Millipore microlter immediately prior to measurement of optical density. 2.2. Determination of l-ascorbic acid and some of its precursors and decomposition products An HPLC method based on ion-suppression reverse phase mode was developed by our group (Lloyd, Warner, Kennedy & White, 1988). This method avoids the use of laborious sample derivatization techniques thus eliminating problems of sample loss and long analysis times. Specicity and sensitivity of the method are excellent allowing simultaneous determination of l ascorbic acid and some of its precursors and oxidation products. The technique is straightforward thus its use for routine laboratory analysis is feasible.

M.G. Roig et al. / Food Research International 32 (1999) 609619

611

The HPLC protocol was performed on two macroreticular reverse phase PLRP-S columns (Polymer , 5 mm, 1504.6 mm i.d., conLaboratories Ltd), 100 A nected in series (Lloyd et al., 1988). The eluent used was an aqueous solution of sodium dihydrogenphosphate (0.2 M), pH adjusted to 2.14 with hydrochloric acid (12 M) and pumped at a ow rate of 0.5 ml min1. Analysis was carried out at ambient temperature. The liquid chromatograph consisted of a Knauer Model 64 pump, a Rheodyne Model 7125 sample injector, an LDC Spectro Monitor III UV detector and a Cecil Model 2272 linear UV spectrophotometer set at 220, 242 and/or 268 nm and a Tekman TE220 chart recorder. Calibration of the system with freshly prepared solutions of l -ascorbic acid with concentration levels in the range of 0 to 0.2228 mg ml1 using 15 ml injection volume (equivalent to 0 to 3.342 mg l-ascorbic acid content) generated a linear response (regression coecient=0.9992). Separation of the compounds was straightforward without the need for pre-column or post-column derivatization reactions. The choice of detection wavelength was based on the wavelength maxima of these compounds in the eluting buer (Table 2) obtained from spectral analysis (see below). Detection of l -ascorbic acid was performed at 268 nm in some cases to eliminate possible interference from compounds eluting at the lower wavelengths.
Table 1 Visual assessment of browning formation in juice samples in Tetrabrik cartons from F. Homann-LaRochea Sample Storage temperature ( C) Storage time (months) 0 1 3 6 11 12

From the elution positions of the degradation products identied in the studies that follow and from previous studies (Kagawa, 1962), it could be noted that excellent resolution of l -ascorbic acid from the oxidation products was achieved and their simultaneous determination could be performed (Table 3). 2.3. HPLC analysis of carbohydrates Chromatographic separation of carbohydrates was accomplished on a stainless steel column (2008 mm i.d.) packed with irregular silica, 5 mm (H.S. Chromatography Packings). The eluent used was a solution of acetonitrile-water [70/30% (v/v)] containing 0.01% 1,4diaminobutane as amine modier (Kaanane, Kane & Labuza, 1978) pumped at a ow rate of 1.0 ml min1. A presaturation column (505 mm i.d.) packed with similar silica was connected between the pump and the injector to prevent the dissolution of the packing material in the analytical column. The same HPLC equipment as before was used but a Knauer Model 98 dierential refractometer was used for detection. Freshly prepared aqueous solutions of d-glucose, d-fructose and sucrose were used to calibrate the system. Most of the values obtained for the sugar content varies only to a very small extent (variation coecient=57%).

Table 2 Wavelength maximaa of l-ascorbic acid and some of its oxidation products Sample Oxalic acid l -Threonic acid 2,3-Diketo-l-gulonic acid l -Dehydroascorbic acid l -Ascorbic acid Furaldehyde 5-Hydroxymethyl furaldehyde
a

lmx (nm) 220 210 220 225 242 280 280

Degree of browningb Orange juice 16-6-01 5 16-6-02 5 16-6-03 5 16-6-01 16-6-02 16-6-03 RTc RT RT 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0 1 1 1 0 0 0 3 3 3 1 1 1 1 1 1 1 1 1 4 4 4 1 1 1 2 2 2 2 2 2 4 4 4 2 2 2 3 3 3 2 2 2 4 4 4

Substances were dissolved in 0.2 M sodium dihydrogen phosphate solution (pH 2.14). Table 3 Retention times of l -ascorbic acid and some of its oxidation products and/or precursors in its production Sample Oxalic acid l-Threonic acid 2,3-Diketo-l-gulonic acid d -Gulonic acid-1,4-lactone l-Dehydroascorbic acid l-Ascorbic acid 5,6-O-Isopropylidene-l -ascorbic acid 5-Hydroxymethyl furaldehyde Furaldehyde Acetic acid Retention time (min) 6.00 6.30 6.60 6.60 7.40 8.70 8.70 9.25 12.1 12.5

Grapefruit juice 17-6-G1 5 17-6-G2 5 17-6-G3 5 17-6-G1 17-6-G2 17-6-G3 RT RT RT

a Designated O.D. range at 420 nm: 40.15; 0.1550.40; 0.4051.00; 1.0050.5010; 50.5010. b Visual assessment: 0, no browning; 1, yellow colour; 2, slight brown colour; 3, brown colour; 4, intense browning with precipitate. c RT, room temperature.

612

M.G. Roig et al. / Food Research International 32 (1999) 609619

2.4. Amino acid analysis Amino acids were determined using a Jeol Model JLC-6AH fully automated amino acid analyzer equipped with an automated post-column ninhydrin detection system. A mixed amino acid standard solution (all in the l conguration) was used as calibrant. An aliquot (10 ml) of a solution obtained by accurately mixing equal volumes of model solutions which contained l-ascorbic acid and/or carbohydrate additives but dierent amino acids were injected into the amino acid analyzer. 2.5. Polarographic determination of dissolved oxygen A YSI Model 57 polarographic oxygen sensor calibrated with water vapour-saturated air (one-point calibration) was utilized to measure the level of dissolved oxygen in juice samples in TetraBrik cartons. A small opening was made in the carton just enough to t the oxygen probe, which was immediately inserted until the membrane was immersed in the liquid. The sample was continuously shaken at a reproducible rate to ensure a constant oxygen supply to the sensor. The readings were taken when a steady value was registered (after 5 min). 2.6. Spectral analysis Spectral analysis of representative samples were performed on a Shimadzu Model UV 240 UVVis spectrophotometer at wavelengths 700190 nm. 2.7. Determination of sample pH All pH measurements were performed on a Philips Model PW 9418 pH meter which was calibrated with pH 4 and pH 7 buers. 2.8. Statistical analysis Regression analysis (curve tting) and calculation of decomposition rate constants were performed using the SIMFIT package, kindly supplied by Dr. W.G. Bardsley (University of Manchester), run on IBM 486 computer. The goodness of the t was estimated by the coecient of determination r2 which is equal to the square of the correlation coecient. 3. Results and discussion 3.1. Correlation between l -ascorbic acid and nonenzymatic browning 3.1.1. l -ascorbic acid retention The instability of l-ascorbic acid as a function of time in real life samples was observed in dierent single-strength

reconstituted grape-fruit and orange juice samples processed and packaged in TetraBrik cartons stored at various temperatures (Fig. 1A and B). From the slopes of the curves, it could be noted that the rate of loss was enhanced by increase in storage temperature. Under the chromatographic conditions used, however, the presence of l-ascorbic acid in apple juice was not detected. This is not surprising since apples contain very low levels of l-ascorbic acid. l -Ascorbic acid content monitored after specic time intervals for grapefruit and orange juice samples from F. Homann-La Roche, however, demonstrated the patterns shown in Fig. 1. For samples stored at 5 C, the increase in ascorbic acid concentration after 10 months seems due to experimental error. Furthermore, from 2 months up to 12 months within experimental error samples stored at 5 C look to achieve a plateau level of l -ascorbic acid (equilibrium). Regression of l -ascorbic acid retained (in terms of concentration, ln concentration and concentration1) against time for these two samples stored at 5 C and at room temperature gave poor ts as shown by the low r2 values (Table 4). From the UV spectra of samples stored for 12 months, the formation of compounds with wavelength maxima at 280 nm is apparent (Fig. 2A and B). Accumulation of these compounds could account for the interference in the chromatographic peak of l -ascorbic acid which was monitored at 268 nm. Two main compounds which have been isolated in degraded samples of l-ascorbic acid were furaldehyde

Fig. 1. Kinetic evolution of l-ascorbic acid of single-strength reconstituted grapefruit (A) and orange (B) juices processed and packaged in TetraBrik cartons during storage at various temperatures.

M.G. Roig et al. / Food Research International 32 (1999) 609619

613

Table 4 Regression equations of level of l-ascorbic acid retained (y) against time (x) for orange and grapefruit juice samples in Tetrabrik cartons stored at 5 C and room temperature Storage temperature ( C) 5a RTb 5 RT
a b

Juice sample Grapefruit Grapefruit Orange Orange

y vs x y=3452.38x, r2=10.3% y=3469.05x, r2=64.0% y=291.815.4x, r2=68.3% y=257.517.31x, r2=75.5%

lny vs x y=5.846.58e3x, r2=9.43% y=5.840.027x, r2=66.0% y=5.670.061x, r2=68.7% y=5.540.081x, r2=81.8%

y1 vs x y=1.82e5x+3.0e3, y=8.36e5x+2.9e3, y=2.47e4x+3.4e3, y=3.93e4x+3.9e3, r2=8.55% r2=68.2% r2=68.9% r2=87.1%

Data obtained after 11 and 12 months storage were excluded. RT, room temperature (2025 C).

Fig. 2. UVVisible spectra of single-strength reconstituted grapefruit (A) and orange (B) juices processed and packaged in TetraBrik cartons during storage at room temperature for 0.5 months (3), 2 months (2) and 12 months (1).

and 5-(hydroxymethyl)furaldehyde (5-HMF) (Huelin, 1953; Huelin et al., 1971; Kanner et al., 1981; Robertson & Samaniego, 1986). Their presence has been proposed as index of browning (Berry & Tatum, 1965; Kanner et al., 1981; Laing et al., 1978; Meydav & Berk, 1978; Nagy & Randall, 1973; Robertson & Samaniego, 1986). Although these compounds absorb at 280 nm (Table 2), interference due to them could not be considered because they are well resolved from the l -ascorbic acid peak using the developed method (Table 3). However, it should also be pointed out that variations in composi-

tion of the juice samples could have contributed to this interference. A close inspection on the l -ascorbic degradation patterns samples stored at 5 C (Fig. 1) shows: (i) a greater rate of loss of ascorbic acid at room temperature than at 5 C; (ii) a plateau prole for samples at 5 C after 23 months storage. This is correlatable to the degree of browning observed at the particular storage temperature. At room temperature, where a greater intensity of browning was observed (Table 1), polymerization or other nonenzymatic browning reactions occurred more rapidly thus possibly depleting the concentration of carbonyl compounds. However, at 5 C it appears that the predominant reaction taking place is the decomposition of l-ascorbic acid forming carbonyl compounds. The nonenzymatic browning process is at the early stage at 5 C and is proceeding at a very slow rate, hence the great accumulation of carbonyl compounds. Considering only the rst 6 months of storage, it appears that the loss of l-ascorbic acid was relatively greater in orange juice than in grapefruit juice. However, since transport and supermarket conditions can vary, another set of juice samples was investigated under more strictly controlled storage conditions. The expected instability of l -ascorbic acid as a function of time and temperature was well demonstrated in Fig. 3. The percentages of l-ascorbic acid retained after storage at various storage temperatures were: 60.4% after 64 days at 4 C, 48.6% after 64 days at 20 C, 11.9% after 64 days at 37 C, 2.0% after 6 days at 76 C, and 3.6% after 3 days at 105 C. Results of l -ascorbic acid retention were tted into zero order (concentration against time), rst order (ln concentration against time) and second order (concentration1 against time) equations (Table 5). r2 values for each storage temperature of 4 and 20 C do not show great variations. For the order of the reaction at this level of degradation at these storage temperatures, the pattern of r2 points to zero order in the range 437 C, rst order at 76 C and second order at 105 C. However, other workers (Kanner, Fishbein, Shalom, Harel & Ben-Gara, 1982) reported that the loss of l ascorbic acid in 58 Brix orange concentrates followed

614

M.G. Roig et al. / Food Research International 32 (1999) 609619

rst order reaction kinetics at a temperature of 25 C and below. At 36 C the degradation did not follow a rst order reaction. A dramatic fall of the initial l -ascorbic acid level for samples stored at 20, 37, 76 and 105 C was noted. This seems to coincide with the initial drop of the dissolved oxygen level (Fig. 4). Supplementation of the original juice sample with l-ascorbic acid which had been previously frozen (20 C) for 23 days but still contained approximately the same level of dissolved oxygen produced a greater initial loss of l -ascorbic acid than the control, i.e. 18.3 mg l1 day1 for the control and 25.5 mg l1 day1 for supplemented samples. Samples which have been previously incubated at 37 C (dissolved oxygen was greatly reduced) exhibited a marked decrease in the rate of loss of l-ascorbic acid even after supplementation to the original l-ascorbic acid level.

3.1.2. Nonenzymatic browning The rate of browning of the samples was directly related to temperature. Measurement of the change in optical density at 420 nm (see above) showed that at zero time, there was already an initial reading registered (Fig. 5). This could not be attributed to brown materials that might have been produced during processing but instead to some other inherent components of the juice such as carotenoids (Lee & Nagy, 1988). The eect of their presence on the change in optical density at 420 nm, however, was not investigated. Correlation of the change in optical density at 420 nm with the loss of l ascorbic acid showed an inverse relationship (Fig. 6).

Fig. 4. Kinetics of dissolved oxygen retention in single-strength reconstituted orange juice processed and packaged in TetraBrik cartons during storage at 4 C (*), 20 C (&), 37 C (~), 76 C (!) and 105 C (*). Fig. 3. Kinetics of l-ascorbic acid retention in single-strength reconstituted orange juice processed and packaged in TetraBrik cartons during storage at 4 C (*), 20 C (&), 37 C (~), 76 C (!) and 105 C (*).

Table 5 Rate of l-ascorbic acid degradation in single-strength orange juice aseptically processed in Tetrabrik cartons Storage temperature ( C) Zero order First order Second order

k (mgl1 day1) 4 20 37 76 76a 105

a

r2

k (day1) 9.18e-3 1.13e-2 3.49e-2 6.41e-1 7.45e-1 7.89e-1

r2

k (lmg1 day1) 4.09e-5 5.66e-5 4.97e-4 9.54e-2 4.39e-2 2.5e-2

r2

2.116 2.347 3.897 30.52 4.73e2 59.4

0.995 0.975 0.972 0.832 0.945 0.634

0.992 0.975 0.894 0.974 0.982 0.933

0.986 0.947 0.682 0.654 0.901 0.988

Data at 9.5 h was excluded.

Fig. 5. Kinetic evolution of the absorbance at 420 nm of singlestrength reconstituted orange juice processed and packaged in TetraBrik cartons during storage at 4 C (*), 20 C (&), 37 C (~), 76 C (!) and 105 C (*).

M.G. Roig et al. / Food Research International 32 (1999) 609619

615

Fig. 6. Correlation between the absorbance at 420 nm and the l-ascorbic acid content of single-strength reconstituted orange juice processed and packaged in TetraBrik cartons during storage at 4 C (*), 20 C (&), 37 C (~), 76 C (!) and 105 C (*).

As discussed before, 5-HMF and furaldehyde have been used as indices of nonenzymatic browning. Simultaneous measurements of the levels of 5-HMF and furaldehyde (see above) showed the presence of 5-HMF (0.45 mg l1) even at zero time (Kennedy, Rivera, Lloyd, Warner & Jumel, 1990). This amount could have been produced during processing. The presence of furaldehyde, however, was not detected. The rate of formation of 5-HMF is dependent on the loss of l -ascorbic acid and is likewise directly related to storage temperature (Kanner et al., 1982; Kennedy et al., 1990). At 4 and 20 C storage temperatures, the increase in concentration of 5-HMF seems to be linear with time but at a very slow rate. An increase of 0.59 mg l1 of 5-HMF was obtained after storage for 64 days at 4 C and 1.43 mg l1 was obtained for samples stored at 20 C for the same duration. High temperature storage (76 and 105 C) remarkably accelerated the formation of 5-HMF similar to the accelerated decomposition of l-ascorbic acid. However, calculation of the theoretical amount of 5-HMF expected to be formed from l-ascorbic acid that degraded showed that there is a large excess of 5-HMF formed (303 mg l1 l -ascorbic acid will theoretically yield 217 mg l1 5-HMF). There is extensive evidence on the formation of 5-HMF and furaldehyde caused by the acid-catalyzed dehydration of sugars (Considine & Considine, 1982; Roig, Bello, Rivera & Kennedy, 1994; Wedzicha, 1984). Subsequent formation of browning from these solutions was observed and its rate was intensied by the presence of amino compounds. An investigation on the contribution of this type of sugar reaction is particularly relevant in this study since d -fructose and d -glucose are natural components of foods particularly fruit juice or

are products of the hydrolysis of sucrose. Results of the analysis of the sugar content after storage (not shown) indicate that sugars do not contribute to 5-HMF or furaldehyde formation in juice. This was previously shown in the study of orange juice based model system and in other studies on nonenzymatic browning (Clegg & Morton, 1965; Lee & Nagy, 1988; Wedzicha, 1984). However, other investigators reported the loss of d -glucose during storage of dehydrated orange juice at 100 C (Wolfrom, Kashimura & Horton, 1974) and about 9% reduction of total sugars after storage of grapefruit juice at 50 C for 6 weeks (Kanner et al., 1982). Except for sucrose inversion which was observed in samples stored at 37, 76 and 105 C, there was no change detected in the sugar content. The total amount of sugar present remained constant. The stability of d -glucose and d -fructose present in orange juice could be due to the extent to which juice is naturally pH buered (Lee & Nagy, 1988; Shallenberger & Mattick, 1983). Some investigators reported that during the degradation of l-ascorbic acid, a decrease in pH due to the formation of carbonyl compounds was noted. Measurement of sample pH after storage showed a constant pH of 3.65. Moreover, dehydration of sugars are favoured at relatively high hydrogen ion concentrations (Wolfrom et al., 1974). Non-detection of furaldehyde which has been identied as one of the degradation products in orange juice is quite unusual. Standard addition of furaldehyde into the juice sample prior to extraction gave a relatively good percentage recovery, i.e. 81%. Likewise, furaldehyde was well resolved from 5-HMF using the optimized HPLC conditions. Nonetheless, further studies have to be undertaken to investigate its non-detection. A correlation of the rate of browning with the rate of 5-HMF formation (Fig. 7) at high temperature showed that whilst the intensity of browning was increasing, 5-HMF was continuously accumulating although l-ascorbic acid concentration was already at a minimum. This could be explained by the relatively low reactivity of 5-HMF towards browning (Flink, 1983). Other more reactive carbonyls such as the a, b-unsaturated carbonyls which have formed as a result of the oxidation of l -ascorbic acid appear to be the more potent browning agents (Clegg & Morton, 1965; Flink; Wedzicha, 1984). Therefore, the possible reaction taking place during the initial stage of browning is between amino compounds and some other degradation products of l-ascorbic acid (not identied in this study) followed by condensation of the products with each other or with nitrogen-free intermediates (Fig. 8) (Wedzicha, 1984). In systems containing low l -ascorbic acid content such as apple and pear juice, the main reaction contributing to nonenzymatic browning is considered to be the Maillard browning reaction (Babsky, Toribio & Lozano, 1986, O'Beirne, 1986). However, in citrus juice

616

M.G. Roig et al. / Food Research International 32 (1999) 609619

Fig. 7. Correlation between the absorbance at 420 nm and 5-HMF formation in single-strength reconstituted orange juice processed and packaged in TetraBrik cartons during storage at 4 C (*), 20 C (&), 37 C(~), 76 C (!) and 105 C (*).

with high l -ascorbic acid, degradation is the major contributor to browning and the browning rate is augmented by amino acids. 3.1.3. Chemical additives as possible preservatives of nonenzymatic browning In addition to proper control of variables such as temperature, oxygen and light to prolong the shelf-life of products containing l-ascorbic acid, some chemical substances have been added to arrest avour and quality deterioration. Of the food additives commonly used, sulphiting agents have the longest history of use. Sulphiting agents are used to control enzymatic and nonenzymatic browning, to arrest microbial spoilage and to act as an antioxidant, as a reducing agent and as a bleaching agent (Taylor, Higley & Bush, 1986). In synthetic systems containing l -ascorbic acid and tryptophan, polyphosphates have been shown to reduce browning (Yu et al., 1974). An assessment of the degree of browning in the presence of additives (Table 6) showed that the addition of sodium metabisulphite tends towards reducing browning, particularly at the higher storage temperatures of 76 and 105 C where a greater accumulation of 5-HMF was observed. Nonetheless, even at the lower storage temperatures where the same level of 5-HMF was formed, in samples stored at the same temperature, the degree of browning was still much lower in samples preserved with sodium metabisulphite. These results could be explained by the two-fold eect of sulphite species on the oxidative browning resulting from l-ascorbic acid decomposition (Wedzicha, 1984). Firstly, the additive inhibits autooxidation of l-ascorbic acid. Secondly, it prevents subsequent

Fig. 8. Possible repeating units of polymeric brown products and their precursors.

browning due to l-dehydroascorbic acid and other carbonylic degradation products. The mechanism involved in the inhibition of autooxidation of l -ascorbic acid is not fully understood but it could involve interactions with the free radical intermediate (semidehydroascorbic acid) (Wedzicha, 1984). Interaction of sulphite with the free radical intermediate interrupts the formation of l-dehydroascorbic acid which has been reported to be very reactive in the nonenzymatic browning process (Kurata & Sakurai, 1967; Namiki, Terao, Ueda & Hayashi, 1986). The interaction of sulphite with the free radical intermediate could be a probable explanation for the arrested reactivity of oxygen. Nonetheless, if the oxidation reaction of l -ascorbic acid proceeded to the formation of l -dehydroascorbic acid, sulphite will reduce its carbonyl reactivity through hydroxysulphonate formation (Wedzicha, 1984). Over the range pH 25, sulphites are in the ionic form HSO 3 (Wedzicha, 1981). This could attack at positions bearing a positive or partial positive charge or take part in nucleophilic displacements. This reaction mechanism explains the preservative action of sulphite species in food. The degradation of l -ascorbic acid causes the formation of carbonyl compounds which can undergo a series of reactions leading to the formation of browning. The attack of sulphites on the carbonyl groups forms hydroxysulphonate compounds which are less reactive towards browning than their precursors.

M.G. Roig et al. / Food Research International 32 (1999) 609619 Table 6 Eect of additives on nonenzymatic browning in single-strength reconstituted orange juice in Tetrabrik cartons Storage Storage Change in optical densitya at 420 nm temperature time ( C) (day) Additiveb Control Na2S2O5 Na5P3O10 CMC Sorbitol 4 0 7 13 27 44 64 6 13 27 44 64 3 9 16 23 35 44 64 2 6 9 1 3 0.019 0.024 0.024 0.025 0.034 0.040 0.026 0.026 0.027 0.040 0.051 0.032 0.036 0.040 0.042 0.046 0.051 0.066 0.075 0.180 0.249 0.210 0.978 0.019 0.019 0.020 0.023 0.033 0.038 0.020 0.025 0.027 0.034 0.039 0.024 0.024 0.027 0.030 0.032 0.041 0.048 0.057 0.150 0.190 0.165 0.906 0.019 0.026 0.029 0.030 0.040 0.042 0.030 0.033 0.037 0.054 0.062 0.030 0.033 0.042 0.043 0.050 0.063 0.072 0.081 0.231 0.318 0.264 1.158 0.019 0.026 0.030 0.032 0.040 0.042 0.027 0.029 0.037 0.045 0.058 0.033 0.038 0.042 0.045 0.058 0.065 0.073 0.019 0.026 0.030 0.032 0.038 0.042 0.030 0.032 0.035 0.040 0.045 0.034 0.038 0.040 0.042 0.049 0.057 0.062

617

20

37

Fig. 9. Storage stability at 20 C of l-ascorbic acid retained (A), dissolved oxygen (B), 5-HMF formation (C) and increase in absorbance at 420 nm of orange juice samples unsupplemented (~) and supplemented (*) with 270 mg l1 l-ascorbic acid.

76

0.072 0.084 0.180 0.240 0.318 0.360 0.297 0.228 1.431 1.110

105

a Samples were diluted with water (10) prior to measurement of absorbance. b Na2S2O5, sodium metabisulphite; Na5P3O10, sodium tripolyphosphate; CMC, carboxymethyl cellulose; Sorbitol, d -sorbitol.

However, the reaction between the carbonyl intermediates formed from the degradation of l-ascorbic acid, and sulphites is reversible. Hence, sulphites only retard the formation of browning pigments (Wedzicha, 1984). Another method of controlling nonenzymatic browning is the inhibition of formation of reactive carbonyl intermediates e.g. the use of antioxidants which can retard oxidation of l -ascorbic acid, the precursor of browning. Studies on replacement of sulphites with cysteine or other mercaptans as reducing agents have been performed (Li, Sawamura & Yano, 1989; Maeda & Mussa, 1986). However, these substitutes produce undesirable organoleptic properties and colour. Hence, addition of l -ascorbic acid, a very potent antioxidant, was investigated. Fig. 9 A showed that a greater initial loss of l -ascorbic acid was incurred in the supplemented sample. However, after the initial stage, the rate of decomposition in the control and supplemented sample appears to be comparable. This supports earlier ndings which demonstrated that supplementation with l -ascorbic acid merely extends the nutritional value of the product.

The rate of consumption of dissolved oxygen showed similar patterns for both control, and supplemented samples (Fig. 9B). There was a drastic initial drop of dissolved oxygen until the level reached equilibrium. The concentration of 5-HMF and degree of browning were both higher in the supplemented samples (Fig. 9C and D). This is not surprising since the initial concentration of l-ascorbic acid was higher thus producing an increased concentration of reactive degradation products. Therefore, in food systems where l-ascorbic acid is used as an antioxidant, it is most useful in inhibiting enzymatic browning. However, in citrus products, addition of l -ascorbic acid is not eective in extending avour and colour qualities. l -Ascorbic acid in its reduced form is of utmost importance in nutrition and in food processing. However, once it has degraded, it results in the production of reactive carbonyl compounds which act as intermediates in nonenzymatic browning in foods. Although these reactive intermediates have not been identied in this study, they have been observed to undergo further reactions leading to the formation of brown pigments. Acid-catalyzed sugar reactions and sugar-amine reactions do not contribute to browning in l -ascorbic acid solutions. This has been evident from the virtually constant total sugar content of degraded samples. The presence of amino acids and possibly other amino compounds, however, enhance browning. Although formation of 5-HMF has been detected in degraded juice samples, its presence could not be used as an index of browning. 5-HMF has been found to be

618

M.G. Roig et al. / Food Research International 32 (1999) 609619 packaged orange drinks: eect of ascorbic acid, amino acids and oxygen. Journal of Food Science, 52(6), 1668. Kagawa, Y. (1962). Enzymatic studies on ascorbic acid catabolism in animals. Journal of Biochemistry, 51, 134. Kanner, J., Fishbein, J., Shalom, P., Harel, S., & Ben-Gara, I. (1982). Storage stability of orange juice concentrate packaged aseptically. Journal of Food Science, 47, 429. Kanner, J., Harel, S., Fishbein, Y., & Shalom, P. (1981). Furfural accumulation in stored orange juice concentrates. Journal of Agricultural and Food Chemistry, 29, 948. Kennedy, J. F., Rivera, Z. S., Lloyd, L. L., Warner, F. P., & Jumel, K. (1990). Studies on nonenzymic browning in orange juice using a model system based on freshly squeezed orange juice. Journal of the Science of Food and Agriculture, 52, 85. Kurata, T., & Sakurai, Y. (1967). Degradation of l -ascorbic acid and mechanism of nonenzymic browning reaction (II) nonoxidative degradation of l -ascorbic acid, including the formation of 3-deoxyl-pentosone. Agricultural and Biological Chemistry, 31(2), 177. Laing, B. M., Schlueter, D. L., & Labuza, T. P. (1978). Degradation kinetics of ascorbic acid at high temperature and water activity. Journal of Food Science, 43(5), 1440. Langdon, T. T. (1987). Preventing of browning in fresh prepared potatoes without the use of sulphate agents. Food Technology, 41(5), 64. Lee, H. S., & Nagy, S. (1988). Relationship of sugar degradation to determination changes in citrus juice quality. Food Technology, 42, 91. Lee, H. S., & Nagy, S. (1988). Quality changes and nonenzymatic browning intermediates in grapefruit juice during storage. Journal of Food Science, 53(1), 168. Li, Z., Sawamura, M., & Yano, H. (1989). Eect of oxygen on the browning and formation of furfural in Yazu juice. Agricultural and Biological Chemistry, 53, 1979. Lloyd, L. L., Warner, F. P., Kennedy, J. F., & White, C. A. (1988). Quantitative analysis of vitamin C (l -ascorbic acid) by ion suppression reversed phase chromatography. Food Chemistry, 28, 257. Maeda, E. E., & Mussa, D. M. D. N. (1986). The stability of vitamin C (l -ascorbic acid) in bottle and canned orange juice. Food Chemistry, 22, 51. Meydav, S., & Berk, Z. (1978). Colorimetric determination of browning precursors in orange juice products. Journal of Agricultural and Food Chemistry, 26(1), 282. Meydav, S., Saguy, I., & Kopelman, J. I. (1977). Browning determination in citrus products. Journal of Agricultural and Food Chemistry, 25, 602. Nagy, S., & Randall, V. (1973). Furfural measurements as an index of temperature abuse in stored grapefruit juice. Journal of Agricultural and Food Chemistry, 21, 272. Namiki, M., Terao, A., Ueda, S., & Hayashi, T., 1986, In M. Fujimaki, M. Namiki, & H. Kato, Amino-carbonyl reactions in food and biological systems (pp. 105114), Tokyo: Kodansha Ltd.; Amsterdam: Elsevier Science Publishers. O'Beirne, D. (1986). Eect of pH on nonenzymic browning during storage in apple juice contrate prepared from Bramley's seedling apples. Journal of Food Science, 51(4), 1073. Robertson, G. L., & Samaniego, C. M. L. (1986). Eect of initial dissolved oxygen levels on the degradation of ascorbic and the browning of lemon juice during storage. Journal of Food Science, 51(1), 184. Roig, M. G., Bello, J. F., Rivera, Z. S., & Kennedy, J. F. (1994). Possible additives for extension of shelf-life of single-strength reconstituted citrus juice aseptically packaged in laminated cartons. International Journal of Food Sciences and Nutrition, 45, 15. Santerre, C. R., Cash, J. N., & Vannorman, D. J. (1988). Ascorbic acid/citric acid combinations in the processing of frozen apple slices. Journal of Food Science, 53(6), 1713. Sapers, G. M., & Hicks, K. B. (1989). Inhibition of enzymic browning in fruits and vegetables. American Chemical Society Symposium Series, 405, 29.

unreactive in the browning process in citrus juices and its contribution to browning in this type of products is insignicant, if not negligible. Increasing the l-ascorbic acid concentration extends the nutritional value of the product but also increases the severity of browning. Hence where l -ascorbic acid is added as an antioxidant in foods, care should be taken so that the amount added is just proportional to the level of oxygen present or to the amount of oxidizable substances.

References
Babsky, N. E., Toribio, J. L., & Lozano, J. E. (1986). Inuence of storage on the composition of claried apple juice concentrate. Journal of Food Science, 51(3), 564. Bauernfeind, J. C., 1982, In: P.A. Seib & B. M. Tolbert, Ascorbic acid: Chemistry, metabolism and uses (pp. 395476). Adv. Chem. Ser. 200. Washington: American Chemical Society. Berry, R. E., & Tatum, J. H. (1965). Comparison of orange concentrate and foammat powders by chromatographic procedures. Journal of Agricultural and Food Chemistry, 13(6), 588. Clegg, K. M. (1964). Nonenzymic browning of lemon suice. Journal of the Science of Food and Agriculture, 15(12), 878. Clegg, K. M. (1966). Citric acid and the browning of solutions containing ascorbic acid. Journal of the Science of Food and Agriculture, 17(12), 546. Clegg, K. M., & Morton, A. D. (1965). Carbonyl compounds and the nonenzymic browning of lemon juice. Journal of the Science of Food and Agriculture, 16, 191. Considine, D. M. & Considine, G. D., 1982. In Foods and food production encyclopedia (pp. 13791391). New York:Van Nostrand inhold Co. Re Flink, J. M. (1983). Nonenzymatic browning of freeze-dried sucrose. Journal of Food Science, 48, 539. Flurkey, W. H., & Ingebrigsten, J. (1989). Polyphenol oxidase activity and enzyme browning in mushroom. American Chemical Society Symposium Series, 405, 44. Handwerk, R. L., & Coleman, R. L. (1988). Approaches to the citrus browning problem. Journal of Agricultural and Food Chemistry, 36, 231. Henshall J. D., 1981, In Gounsell, J. N. & D. H. Hornig, Vitamin C (ascorbic acid) (pp. 123137). London: Applied Science Publishers Ltd. Hodge, J. E. (1953). Dehydrated foods. Chemistry of browning reaction in model systems. Journal of Agricultural and Food Chemistry, 1(15), 928. Hsu, A. F., Shieh, J. J., Bills, D. D., & White, K. (1988). Inhibition of mushroom polyphenoloxidase by ascorbic acid derivatives. Journal of Food Science, 53(3), 765. Huelin, F. E. (1953). Volatile products of apples (III) identication of aldehydes and ketones. Food Research, 18, 633. Huelin, F. E., Coggiola, I. M., Sidhu, G. S., & Kennett, B. H. (1971). Anaerobic decomposition of ascorbic acid in the pH range of foods and in more acid solutions. Journal of the Science of Food Agriculture, 22(10), 540. Jen, J. J. (1989). Quality factors of fruits and vegetables. American Chemical Society Symposium Series, 405, 1. Kaanane, A., Kane, D., & Labuza, T. P. (1978). Time and temperature eect on stability of moroccan processed orange juice during storage. Journal of Food Science, 53(5), 1470. Kacem, B., Cornell, J. A., Marshall, M. R., Shiremean, R. B., & Matthews, R. F. (1987). Nonenzymatic browning in aseptically

M.G. Roig et al. / Food Research International 32 (1999) 609619 Sapers, G. M., & Miller, G. F. (1995). Heated ascorbic/citric acidsolution as browning inhibitor for prepeeled potatoes. Journal of Food Science, 60, 262. Sapers, G. M., & Ziolkowski, M. A. (1987). Comparison of erythorbic and ascorbic acids as inhibitors of enzymatic browning in apple. Journal of Food Science, 52, 1732. Sciancalepore, V., & Longone, V. (1984). Polyphenol oxidase activity and browning in green olives. Journal of Agricultural and Food Chemistry, 32(2), 320. Shallenberger, R. S., & Mattick, L. R. (1983). Relative stability of glucose and fructose at dierent acid pH. Food Chemistry, 12, 159. Shaw, P. E., Tatum, J. H. and Berry, R. E., 1977, In G.G. Birch & R.S. Shallenberger, Developments in food carbohydrates (Vol. 1, pp. 91107). London: Applied Science Publishers Ltd. Tatum, J. H., Shaw, P. E., & Berry, R. E. (1969). Degradation products from ascorbic acid. Journal of Agricultural and Food Chemistry, 17, 38.

619

Taylor, S. L., Higley, N. A., & Bush, R. K. (1986). Sulfates in foods: uses, analytical methods, residues, fate, exposure, assessment, metabolism, toxicity and hypersensitivity. Advances in Food Research, 30, 1. Wedzicha, B. L. (1981). Sulfur dioxide: the reaction of sulte species with food components. Nutrition and Food Science, 11/12, 14. Wedzicha, B. L., In Chemistry of sulphur dioxide in foods (pp. 183 229). Essex, UK: Elsevier Applied Science Publishers Ltd. Wolfrom, M. L., Kashimura, N., & Horton, D. (1974). Factors aecting the Maillard browning reaction between sugars and amino acids. Studies on the nonenzymatic browning of dehydrated orange juice. Journal of Agricultural and Food Chemistry, 22(5), 796. Yu, M. H., Wu, M. T., Wang, D. J., & Salunkhe, D. K. (1974). Nonenzymic browning in synthetic systems containing ascorbic acid, amino acids, organic acids, and inorganic salts. Journal du Institut Canadien de Science et Technologie Alimentaire, 7(4), 279.