Browning and Decomposed Products of Model Orange Juice

Browning and Decomposed Products of Model Orange Juice

Yuki SHINODA,1 Masatsune MURATA,1;y Seiichi HOMMA,1 and Hajime KOMURA2

1Department of Nutrition and Food Science, Ochanomizu University,

2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan2Institute for Fundamental Research, Suntory Ltd.,

5-2-5 Yamazaki, Shimamoto-cho, Osaka 618-0001, Japan

Received July 22, 2003; Accepted November 29, 2003

A model solution of orange juice containing sugars,

ascorbic acid, and citric acid was prepared and its

browning during storage was examined. The solution

gradually turned brown. Ascorbic acid (AsA) most

contributed to the browning. Citric acid and such amino

acids as Arg and Pro promoted the browning. DTPA, a

strong chelator, inhibited the browning. 3-Hydroxy-2-

pyrone (3OH2P), 5-hydroxymethylfurfural (HMF), fur-

fural, 5-hydroxymaltol, and 2-furoic acid were identified

as decomposed products in the stored solution. When

3OH2P was stored, the solution turned slightly brown.

Furfural solution added with amino acids turned yellow.

3OH2P showed a positive relation with the browning of

retail orange juice during storage.

Key words: orange juice; browning; ascorbic acid; 3-

hydroxy-2-pyrone; furfural

Orange juice is one of the most popular beverages inthe world. When orange juice is stored, it gradually turnsbrown. It is well known that orange juice is rich inascorbic acid (AsA). The nutritional value of orangejuice is related primarily to the content of AsA. Two ofthe major changes during storage of orange juice aredevelopment of off-flavor and browning.1) AsA is anantioxidant and represses browning reaction. However,AsA is also known to contribute to browning of foodsbecause it is easily oxidized and decomposed.1–3) Thedecomposition of AsA together with non-enzymaticbrowning is the main deteriorative reaction that occursduring storage of orange juice.3) Tatum et al. showedseveral degradation products of AsA during the storageof orange juice.4,5) The factors affecting AsA degrada-tion are pH, oxygen, AsA concentration, temperature,light, metal, citric acid, and so on. In the presence ofoxygen, AsA is degraded primarily to dehydroascorbicacid via monoanion. The lactone of dehydroascorbicacid is hydolyzed to form 2,3-diketogulonic acid, whichdoes not show vitamin C activity. Decarboxylation of

2,3-diketoguloninc acid leads to xylosone, which isfurther degraded to reductones and furan compounds.The contribution of AsA to the browning of citrus juicehas been reported.3,6–8) However, the detailed pathwayof browning and the interaction between AsA or AsAdegradation products and other components in the juiceare still not clear. Kanner et al. reported the relationshipbetween the browning of orange juice and the concen-trations of 5-hydoxymethylfurfral (HMF) and furfural.9)

Kacem et al.10) suggested that furfral and HMF formedby the amino-carbonyl reaction contributed to thebrowning of orange juice. Thus, furfural and HMF areconsidered important intermediates and indicators of thebrowning of orange juice.11–13) However, Roig et al. saidthat HMF could not be used as an index of browning ofcitrus juice.8) Thus, the usefulness of these indicators isunclear.The purpose of this study is to clarify the factors

affecting the browning of orange juice and the inter-action between AsA and other components and to find auseful indicator of the browning. The model solution isuseful to analyze the browning and interaction betweencomponents in juice. Here we prepared a model solutionof orange juice containing sugars, AsA, amino acids,and citric acid and stored it to examine the browningduring storage and the decomposed products. We hereshowed the importance of degradation of AsA for thebrowning of orange juice that was stimulated by Arg,Pro, citric acid, and metals and was repressed by radicalscavengers and a chelator, and further suggested thepossibility of 3-hydroxy-2-pyrone (3OH2P) as an in-dicator of the browning of orange juice.

Materials and Methods

Model solution of orange juice. A model solution oforange juice consisting of sugars, AsA, citric acid, andamino acids was prepared according to the compositionof Satsuma mandarins.14) Fundamental composition is

y To whom correspondence should be addressed. Tel: +81-3-5978-5753; Fax: +81-3-5978-5899; E-mail: [email protected]

Abbreviations: AsA, ascorbic acid; HMF, 5-hydroxymethylfurfural; 3OH2P, 3-hydroxy-2-pyrone; DPTA, diethylenetriamine-N,N,N,N,N-

pentaacetic acid; NTA, nitriloacetic acid

Biosci. Biotechnol. Biochem., 68 (3), 529–536, 2004

shown in Table 1, that is the standard model solution. Atthe same time, another solution lacking in a componentof the standard model solution was prepared. Whensugars, citric acid, or amino acids were added to AsAsolution, the concentration of each component followedTable 1. Each solution (15ml) was put in a vial (30ml)screwed with a plastic cap and then stored at 50�C for 2months. Two vials were analyzed at each storage time(0–60 days). Each experiment was repeated at leasttwice. The browning of these solutions was estimated byOD420. The changes in the components were analyzedby 3D-HPLC as described below.

HPLC analysis for decomposed products. The HPLCsystems were as follows: pump, L-6000 (Hitachi, Tokyo,Japan); column, YMC pack R-ODS (Yamamura, Kyoto,Japan; 4:6 i.d.� 250mm; for analysis) and YMC packD-ODS (20 i.d.� 250mm; for preparation); detector,photodiode array detector (Hitachi, Tokyo; for analysis),L-4200 UV-VIS detector (Hitachi; for preparation);wavelength, 250–370 nm (for analysis), 283 nm (fordetermination of HMF, furfural, 3OH2P and 5-hydroxy-maltol, and for preparation of 3OH2P and 5-hydroxy-maltol), 265 nm (for determination of AsA), 250 nm (fordetermination of 2-furoic acid), and 210 nm (for oxalicacid); eluent, CH3CN and 5% aqueous acetic acid (2:98)for furfural, 3OH2P, and 5-hydroxymaltol, 50mM

potassium phosphate buffer (pH 6.0) containing 1mM

EDTA and 2.5mM tetrabutyl ammonium sulfate forAsA15) and 2-furonic acid, and 50mM potassiumphosphate buffer (pH 7.7) containing 5mM tetrabutylammonium sulfate for oxalic acid; flow rate, 1.0ml/minfor analysis and 9.99ml/min for preparation.

Preparation and identification of decomposed prod-ucts. HMF, furfral, and furoic acid were identified by thecomparison with authentic samples (Wako Chemicals,Osaka). 3OH2P and 5-hydroxymaltol were preparedfrom AsA solution and identified with spectroscopicdata. AsA solution (0.15%) was incubated for 3 days at

50�C, before 3OH2P was extracted with ethyl ether andisolated with a preparative HPLC as described above.The compound was obtained as white needles and wasdissolved in D2O (for 1H-NMR), CD3OD (for 13C-NMR), or MeOH (for MS). 1H-NMR �H (ppm); 5.95(1H, t, J ¼ 6:0), 6.41 (1H, d, J ¼ 6:0), 6.89 (1H, d,J ¼ 6:0). 13C-NMR �C (ppm), 108.9, 119.5, 143.4,144.5, 163.6. EI-MS (m=z); 112 (Mþ), 69, 43.

AsA solution (0.03%) containing 5% of fructose wasincubated for 18 days at 50�C, before 3HO2P wasextracted with ethylacetate and isolated with a prepara-tive HPLC. The compound was a slightly yellowishpowder and dissolved in CD3OD (for NMR), or MeOH(for MS). 1H-NMR �H (ppm); 2.20 (3H, s), 7.75 (3H, s).13C-NMR �C (ppm), 14.6, 140.0, 142.5, 145.5, 151.5,170.0. EI-MS (m=z); 142 (Mþ), 113, 69.

Instrumental analyses. Spectroscopic measurementswere done using the following instruments; JEOL JNM-GX270 (NMR) and JEOL JMX-102/JMX-DA 6000(EI-MS).

Storage of retail orange juice. Eleven samples oforange juice were purchased from a local market in2000. Samples were stored at 50�C for 2 weeks. The avalue before and after storage were measured by acolorimeter (Model TC3600, Tokyo Denshoku, Tokyo),and AsA, HMF, 3OH2P, furfural, 5-hydroxymaltol, and2-furanioc acid were analyzed by the HPLC method asthe above described.

Results and Discussion

Effect of each ingredient on the browning of modelsolution

The model solution imitated Satsuma mandarin. Thesolution was stored at 50�C with headspace to promote abrowning reaction. The model solution gradually turnedbrown during storage (Fig. 1-A). This browning curveseems to have two phases. At first, the model solution inwhich one component was only removed was stored,and the degree of browning was compared (Fig. 1-B).When AsA was removed, the solution did not turnbrown at all for 30 days and then gradually turnedbrown. AsA decreased during storage and completelydecomposed after 3 days of storage. Degradationproducts seem to contribute to the browning. Whencitric acid or amino acids were removed, the degree ofbrowning was reduced by 40 to 60%. On the other hand,when sugars were removed, the degree of browning hadnot changed for 2 weeks compared with the control. Inlonger storage such as 1 or 2 months, the removal ofsugars reduced the browning. These results suggest thatAsA most contributes to the browning within 2 weeks ofstorage and that an amino-carbonyl reaction betweenamino acids and degradation products derived of AsAcontributes the browning, while sugars do only after 2weeks of storage. This difference in origin of browning

Table 1. Composition of the Model Solution of Orange Juice

Component Concentration

Sugars

Sucrose 5.0 g/100ml

Glucose 2.5 g/100ml

Fructose 2.5 g/100ml

Acids

Citric acid 1.0 g/100ml

Ascorbic acid 30mg/100ml

Amino acids

L-Ser 7.0mmol/l

L-Asp 5.4mmol/l

L-Ala 1.9mmol/l

L-Arg 0.75mmol/l

L-Glu 0.54mmol/l

L-Pro 0.42mmol/l

pH 3.0

530 Y. SHINODA et al.

seems to correspond to the two phases of browning.To ascertain the contribution of AsA to browning,

AsA concentration was changed. When the AsAconcentration was raised from 0.03%, 0.15%, 0.3% to0.6%, the absorbance at 420 nm of the solution becamemore intensely from 0.03, 0.77, 1.07, to1.14 at 14 daysof storage and from 0.19, 2.29, 3.72, to 4.62 at 60 daysof storage, respectively. This result coincided with thatof Kacem et al.10) As the solution lacking in AsA did notturn brown during 2 weeks of storage, such componentsas sugars, amino acids, and citric acid was then added toan AsA solution to examine the interaction with AsAand other components. All of the amino acids, sugars,and citric acid stimulated the browning of AsA solution(Fig. 2-A). Among them, amino acids stimulated thebrowning most intensively. The degree of browning inthe solution containing AsA and amino acids was morethan 3 times than that of AsA solution. The stimulationof browning by amino acids such as Arg and 4-aminobutyric acid in orange juice was reported by

Wolfrom et al., however, they did not use AsA in themodel solution.16) Therefore, an amino acid was addedto AsA solution to examine the effects of each aminoacid (Fig. 2-B). L-Ser, L-Asp, L-Pro, L-Arg, L-Ala, and L-Glu promoted the browning. Among them, L-Arg and L-Pro was the most effective on the browning. Theseresults suggest that the interaction as the Maillardreaction between AsA degradation products and suchamino acids as Arg and Pro is an important factor of thebrowning.Next, the effects of metals and citric acid on the

browning were examined. It is reported that citric acid3)

and metals17) promote the degradation and browning ofAsA. The model solution contained 0.05 ppm Fe and0.01 ppm Cu, which might be derived from distilledwater or reagents used here. As retail orange juicecontained about 0.5 ppm Fe and 0.2 ppm Cu, 0.5 ppm Fe(FeCl2) or 0.2 ppm Cu (CuCl2) was added to the modelsolution. As a result, the browning was promoted by theaddition of Fe or Cu (Fig. 3), especially in the early

Fig. 1. Browning of Model Orange Juice Solution during Storage (A) and Effect of Each Component on the Browning (B).

The model solution was stored at 50�C in a vial with headspace. Averages and standard deviations (n ¼ 8) were shown (A). One component

(1, AsA; 2, sugars; 3, amino acids; 4, citric acid) was removed from the model solution and then stored at 50�C (B). Degree of browning (A420)

was shown as % of control (no removal).

Fig. 2. Effects of Amino Acids on the Browning of the Model Orange Juice Solution during Storage.

A, sugars (2), amino acids (3), or citric acid (4) was added to 0.03% of AsA solution (1, control) (pH 3.0). B, each amino acid (1.7mM) was

added to 0.03% of AsA solution (1, no addition (control); 2, Ser; 3, Asp; 4, Pro; 5. Arg; 6, Ala; 7, Glu).

Browning and Decomposed Products of Model Orange Juice 531

stage of storage in the case of Fe. As the model solutioncontained 1.0% citric acid, a metal chelator, a solutionlacking in citric acid was prepared and stored. Whencitric acid was removed, the browning was repressedeven in the case of Cu or Fe addition. This shows thatmetal ions chelate with citric acid and promote thedecomposition of AsA. Khan and Martell reported thatmetal chelate catalyzed the oxidation of AsA.18)

Effects of radical scavengers and chelating agents onthe browningAsA most contributed to the browning of model

orange juice, which suggests that the degradation ofAsA is the most important process of the browning.Therefore, the effect of air or oxygen on the browningwas examined. When the bottle was degassed bynitrogen or the headspace of bottle was filled with themodel solution, the degree of browning was promoted(Table 2). In these conditions, oxidative decompositionof AsA was repressed because of the decrease inoxygen. AsA existed for 20 days in the case of noheadspace, while AsA disappeared in 3 days of storagewith 15ml of headspace. The reason why the browningwas promoted in this relatively oxygen-less condition

was not clear, however, non-oxidative decompositionproducts might more contribute to browning in anaero-bic condition. For example, furfural is one of the majornon-oxidative degradation products of AsA.19) Asdescribed later, furfural is a reactive compound for thebrowning with amino acids. Another possibility is thatthe oxidative degradation of brown pigment mighthappen. Robertson and Samaniego showed that thebrowning in lemon juice with a high oxygen level wasmore intense than that with a low oxygen level,12) whileKacem et al. reported that an orange drink with high O2

showed more intense browning than that with low O2.10)

The group of Sawamura reported that dehydroascorbicacid solution produced a browner color under non-aerobic conditions than under oxidative conditions.7,20)

Next the effects of radical scavengers and chelatingagents on browning were examined. When 20% (V/V)of ethanol, a radical scavenger, was added to the modelsolution, the browning was repressed (Fig. 4-A). Therepression was more definite in the longer storage time.When 0.57mmole/l of mannitol was added to thesolution as another scavenger, the browning was re-pressed by 72.9% after 14 days of storage and by 65.5%after 60 days of storage. These results suggest thatradicals participate in the browning because ethanol andmannitol are scavengers of hydroxyl radical.21) Next, theeffects of chelators on the browning were examined.When diethylenetriamine-N,N,N,N,N-pentaacetic acid(DTPA) or nitriltriacetic acid (NTA) was added to themodel solution, the degree of browning was repressed orpromoted by the addition of DTPA or NTA, respectively(Fig. 4-B). AsA was more maintained in the solutionadded with DTPA (Fig. 4-C). The different effects ofDTPA and NTA on the browning seem to be due to thedifference in chelating ability between the two com-pounds. DTPA is a much stronger chelator than NTA.22)

It was reported as a similar phenomenon that oxidativestress by active oxygen was repressed by DTPA, whilestimulated by NTA.23) This result shows that metalcomplex with DTPA represses the AsA-degradatingactivity of metals, while a metal complex with NTA orcitric acid promotes the degradation of AsA to lead tobrowning.

On the base of these results, the factors affecting thebrowning of the model solution of orange juice duringstorage was summarized in Table 3. The storage timewas divided into two periods, that is, the early stage (1or 2 weeks of storage) and the later stage (1 or 2 monthsof storage), on this condition that it was stored at 50�Cwith headspace. AsA was essential for the browning inthe early stage of storage. Amino acids and citric acidstimulated the browning of AsA, while sugars had littleeffect on the browning. These results coincided withother reports.3,7) In the later stage, AsA contributed themost to the browning, however, the browning happenedwhen AsA was removed from the solution. Sugars aswell as amino acids and citric acid stimulated thebrowning. These results suggest that in the early stage of

Fig. 3. Effects of Metals and Citric Acid on the Browning of the

Model Orange Juice Solution during Storage.

Cu2þ (0.2 ppm) or Fe2þ (0.5 ppm) was added to the standard

model solution (control). Solutions lacking in citric acid and with

added Cu or Fe were also prepared. , control; , control minus

citric acid; , control plus Cu; , control minus citric acid plus Cu;

, control plus Fe; , control minus citric acid plus Fe.

Table 2. Effect of Headspace of a Vial and Degassing on the

Browning of Model Orange Juice Solution during Storage

Storage time Headspace (ml)Degassing

(days) 0 15 25

7 n.d. 100 n.d. 150a

14 178a 100 96.6 n.d.

60 323a 100 96.3 n.d.

Values show degree of browning expressed by % of control with 15ml of

headspace. n.d., not determined. a, significant difference from control

(p < 0:01).


storage, the AsA degradation products are essential forbrowning and that the Maillard reaction between thedegradation products and amino acids stimulates theformation of brown pigments. In the later stage ofstorage, the AsA degradation product as well as sugardegradation products also contribute to the formation ofbrown pigments, which is also produced by the Maillardreaction. Active oxygen species seems to be importantfor the degradation of AsA and browning, because metalions stimulated the browning and DTPA, a strongchelator, and ethanol and mannitol, radical scavengers,repressed the browning.

Decomposed products in the model solutionDecomposed products in the model solution were

analyzed by ODS-HPLC. Figure 5 shows a typicalchromatogram monitored by absorbance at 283 nm.Several peaks were detected and four decomposedproducts (A, 3OH2P;24,25) B, HMF; C, furfural; D, 5-hydroxymaltol that is 2-methyl-3,5-dihydoxy-4H-pyr-ane-4-one) were identified with the comparison ofstandards and instrumental analyses. 2-Furoic acid19)

Fig. 4. Effects of Ethanol (A), DTPA (B), and NTA (B) on the Browning (A and B) and Residual AsA (C) of the Model Orange Juice Solution

during Storage.

Ethanol (20%v/v), DTPA (1mM; 2) or NTA (1mM; 3) was added to the standard model solution (control, 1) and then stored for 60 days.

Table 3. Factors Affecting the Browning of Model Orange Juice

Solution

FactorEffect on browning

7�14 days of storage 40�60 days of storage

AsA essential essential

Sugars no effect essential/stimulation

Amino acids stimulation stimulation

Citric acid stimulation stimulation

Metals stimulation stimulation

Chelators inhibition (DTPA) no effect (DTPA)

/stimulation (NTA) /stimulation (NTA)

Oxygen inhibition inhibition

Ethanol and inhibition inhibition

mannitol

Fig. 5. Typical Chromatogram of the Stored Model Orange Juice

Solution on HPLC.

The model orange juice solution stored for 7 days at 50�C was put

on ODS-HPLC (column, Mightysil RP-18 (4:6 i.d.� 250mm);

eluent, CH3CN:5%AcOH = 2:98; flow rate, 1.0ml/min).


was also detected by another HPLC condition. Oxalicacid, which is one of decomposed products of AsAthrough 2,3-diketogulonic acid in physiological condi-tions,26) was not detected in this stored solution. 3OH2P,furfural and 2-furoic acid were derived of AsA throughdehydroascorbic acid,27) while HMF and 5-hydroxymal-tol were derived of fructose (data not shown). 5-Hydroxymaltol is sometimes identified as a flavorcomponent of heated foods,28–30) and formed by theMillard reaction.31) However, there is no report that 5-hydroxymaltol is formed during storage of orange juice.When retail orange juice was stored, these five com-pounds were all detected (data not shown). 3OH2Pincreased till 3 days and then decreased, while HMF,furfural, 5-hydroxymaltol and 2-furoic acid graduallyincreased during storage (Fig. 6). This suggests that3OH2P might be used for browning reaction. Next, therelationship between these decomposed compounds and

browning was examined. When 3OH2P was stored, thesolution gradually turned brown (Fig. 7-A). When aminoacids were added to 3OH2P solution, the browning wasa little promoted. When furfural solution was stored, thebrowning was not observed. However, when furfuraladded with amino acids turned yellow and the browningduring storage was observed (Fig. 7-B). Hoffmanreported a yellow compound formed by the Maillard-type reaction between furfural and proline.32) Thesolution of HMF, 5-hydroxymaltol, and 2-furoic aciddid not turn brown in the absence or presence of aminoacids. These results suggest that 3OH2P and furfuralderived from AsA and the Maillard reaction contributeto the browning of orange juice.

Browning and decomposed products in retail orangejuice

Retail orange juice was stored at 50�C, before thebrowning and decomposed products were measured. Thebrowning was estimated by the difference of a value (�a

value) before and after storage. The relationship be-tween the �a value after 6 days of storage and 3OH2Pafter 14 days of storage was observed (Fig. 8), while therelation between the �a value and HMF or furfural was

Fig. 6. Changes in the Decomposed Products of the Model Orange

Juice Solution during Storage.

, 3OH2P; , 2-furoic acid; , furfural; , HMF; , 5-

hydroxymaltol.

Fig. 7. Browning Potential of 3OH2P (A) and Furfural (B).

3OH2P (5.0mM) or furfural (5.7mM) solution was stored with ( ) or without ( ) amino acids at pH 3.0 for 60 days. Degree of browning and

residual 3OH2P or furfural was measured.

Fig. 8. Relationship between �a Value after 2 Weeks Storage and

3OH2P Concentration after 7 Days of Storage.


not observed. This result suggests that 3OH2P becomesan indicator for the browning of orange juice instead ofHMF and furfural.

Conclusion

A model solution of orange juice gradually turnedbrown during storage. AsA contributed most to thebrowning. Amino acids such as Arg and Pro, citric acid,and metals promoted the browning. Among five decom-posed products detected here, 3OH2P and furfuralcontributed to the browning. 3OH2P seems to becomea browning indicator of orange juice.

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