J . Sci. Food Agric.

1985,36,561-566

Analysis of Sugars and Organic Acids in Ripening

Mango Fruits (Mangifera indica L. var Keitt)
By High Performance Liquid Chromatography
Andrew P. Medlicott and Anthony K. Thompson
Tropical Development and Research Institute, 56-62 Gray’s Inn Road, London WClX 8LU
(Manuscript received 28 November 1984)

The sugars and organic acids present in the pulp of Keitt mangoes at various stages of
ripeness were analysed by h.p.1.c. Ripening was associated with a loss in firmness, peel
chlorophyll and pulp acidity, with increasing soluble solids and total sugars. The major
sugars were identified as glucose, fructose and sucrose. All three increased during
ripening; sucrose was found to be in the greatest concentration throughout, with
fructose the predominant reducing sugar. Acidity loss was shown by decreasing
titratable acidity and increasing pH values. Citric and malic acids were found to be the
major organic acids. A large decrease in citric acid and a small reduction in malic acid
were responsible for the loss of acidity. Tartaric, ascorbic, oxalic and a-ketoglutaric
acids were also shown to be present at low concentrations.
Keywords: Sugars; organic acids; mango; h.p.1.c.

1. Introduction

Mangoes are of increasing commercial importance in the UK and are a valued source of income for
the producer countries. However, international trade in mangoes is currently restricted because of
unpredictable quality and often high market losses, as only limited information is available concern-
ing their postharvest physiology and biochemistry. More research is required to determine mango
fruit quality characteristics, and to investigate closely the effects of various storage and ripening
techniques on the development of these parameters. This account is concerned with analysing the
sugar and acid contents, which are directly related to flavour and acceptability.
Fruit flavour is based mainly on the balance between sugars and organic acids, and of numerous
aroma compounds. Previous literature has shown that during ripening, mangoes show a decrease in
acidity and an increase in sugars.’-‘ Acidity has generally been assessed by pH or titratable acidity,
and little information is available on individual organic acid metabolism. The predominant acids
have been shown to be citric acid with lesser amounts of succinic and malic acids,’ and tartaric with
smaller amounts of citric.6 The sugar content of mangoes has usually been assessed in terms of
soluble solids content, or non-reducing and reducing sugars. There are conflicting data regarding the
relative contribution of these to the quality of the ripe fruit. Reducing sugars have been found to
increase,’ or remain constant8 during ripening. Only limited information is available regarding the
individual sugar content of mangoes. Glucose, fructose and sucrose have been reported to be in
similar concentrations in ripe m a n g ~ e sand
, ~ sucrose has been shown to be predominant throughout
ripening.*
High performance liquid chromatography (h.p.1.c.) has been successfully used to determine
sugars and organic acids in a number of fruit and ~egetables,”’~ providing accurate and reliable
results. Determination of sugars and organic acids during mango fruit ripening by h.p.1.c. has not
previously been reported. This paper aims to provide a comprehensive and detailed study of these
changes.
561
562 A. P. Medlicott and A. K. Thompson

2. Materials and methods

2.1. Plant material
Air-freighted shipments of mature firm mangoes of the cultivar Keitt, produced in Mexico, were
obtained from a commercial source in Britain. Only undamaged fruit free from infection were
chosen for study. Fruit were ripened at 22°C and 85-90% relative humidity. Analyses were carried
out in duplicate on five fruits at various stages during ripening.

2.2. Sample preparation

2.2.1. Sugars
Fruit pulp (10 g) was homogenised in 25 ml distilled water and the sample clarified by centrifuging at
15000xg for 20 min. The sediment was washed with 25 ml distilled water, centrifuged, and the
supernatant collected and pooled. Samples were filtered through Sep-pak C18 (Waters Associates)
and 0.45 pm Millipore filters prior to h.p.1.c. analysis.
2.2.2. Acids
The clarified extract was filtered through a Sep-pak C18 cartridge and 10 ml was chromatographed
through a cation exchange column (4 g Dowex 50W-X8,50-100 mesh, H + form) and washed with
water to a total volume of 50 ml. Solutions were filtered through a 0.45 pm Millipore filter prior to
h.p.1. c. analysis.
2.3. H.p.1.c. analysis
2.3.1. Sugars
A Waters liquid chromatograph was employed consisting of a 6000 A pump, a Rheodyne 7125
injector fitted with a 20 pl sample loop, a RCM-100 radial compression module, and a R400
differential refractometer connected to a Hewlett Packard 3390A recording integrator. A Waters
Radial-Pak pBondapak NH, (100x8 mm i.d.) cartridge was used. The mobile phase consisted of
acetonitrile: water (750 ml litre-]) which was degassed and filtered through a 0.45pm Millipore filter
at a flow-rate of 2.5 ml min-'.
2.3.2. Acids
The same system used for sugar analysis was employed, except a Pye Unicam LC3 U.V. detector set at
214nm was used for detection. The column was a Waters Radial-Pak C18 cartridge (100X8mm
i.d.); the mobile phase consisted of 0.2 M KH2P04 adjusted to pH2.4 with H3P04, degassed and
filtered, at a flow rate of 2.0ml min-'.
2.3.3. Standards
Individual sugars and acids were identified by comparison with reference compounds; standard
curves relating integrator response to concentration were used to quantify the results. Analyses were
carried out in triplicate for each sample and standard; the coefficients of variation were generally less
than 7.0%.

2.4. Soluble solids, pH and titratable acidity

Soluble solids of the pulp were determined with a refractometer, and pH with a pH meter. Titratable
acidity of the aqueous extract of the fruit pulp was determined by titration against 0 . 1 NaOH
~ and
expressed as mEq 100 g-' fresh weight.

2.5. Pulp rupture force and chlorophyll contents

Each mango was tested on opposite sides of the fruit. A compression force was applied to the pulp
(with the peel removed) at a speed of 1 cm min-I using a bench top pressure tester, a 6 mm diameter
cylindrical probe, and a 0-10 kg force gauge. The value recorded for the pulp rupture force was the
maximum force required for the pulp to yield to the tip of the penetrometer head.I4Peel chlorophylls
Sugar and organic acid analysis in ripening mangoes 563

Table 1. Compositional changes during ripening of Keitt mangoes at 22°C

Total
sugar: Titratable
Storage Soluble Glucose: soluble acidity Citric: Sugar:
tirne solids Total fructose solids (mEq malic acid
(days) (%I sugar (%j ratio ratio 100 g-'j PH ratio ratio
0 4.9k0.3 3.5150.5 0.59 0.71 20.224.2 3.1k0.1 4.03 0.17
2 9.520.4 8.29k0.6 0.56 0.87 11.9k3.0 3.7k0.2 1.56 0.70
4 11.2k1.2 10.127t1.2 0.64 0.90 8.5k0.6 3.91t0.3 1.I5 1.19
7 11.0k0.7 10.2950.8 0.54 0.94 5.320.5 4.1k0.1 6.56 1.93
9 10.67tl.l 8.40k0.7 0.57 0.79 4.0k0.7 4.3k0.2 5.80 2.09
Values are the mean of five fruitskstandard error.

were extracted from peel discs in 80% acetone and the absorbance determined at 663 and 645 nm;
total chlorophyll contents were calculated according to Arnon,I5and are expressed as pg m r 2 .

3. Results and discussions

The main ripening trends in mangoes are similar to those reported for most other climacteric fruit.
During ripening sugars increased and acidity decreased (Table 1). Soluble solids and total sugars
increased two to three-fold after 7 days storage while titratable acidity decreased five-fold by day 9.
These trends are emphasised by the changes in the sugar :acid ratio, which increased throughout
ripening. The total sugars:soluble solids ratio increased up to day 7 , indicating the increasing
contribution of sugars to the soluble solids content. These general sugar and acidity parameters
showed changes similar to those reported by previous workers. '--) Titratable acidity values in ripe
fruit of 1.9, 4.0, 1.6 and 2.8 mEq 100 g-' have been reported in D ~ s e h r iCarabao,*
,~ Pico3 and
Gaurjeet' mangoes, respectively. The increased total sugars :soluble solids ratio was also found by
Krishnamurthy et a1.* Their initial values of 0.17, 0.13 and 0.33 for Badami, Raspuri and Neelum,
respectively were considerably lower than the 0.71 obtained in this investigation. The ratio of 0.94
obtained here after 7 days storage is similar to the 0.90 shown for ripe fruit by Agnihortri et aL4with
Dusehri, but substantially higher than 0.76 reported by Tripathi' with ripe Gaurjeet mangoes. Both
sugars and acids contribute to the soluble solids content of the pulp; increasing sugars and decreasing
acidity should therefore produce an increasing ratio. However between 7 and 9 days the soluble
solids, total sugars and acidity were all found to decrease. It would be expected that the contribution
of total sugars to the soluble solids would increase slightly or remain constant. However, the ioss in
total sugars shown after 9 days storage (Table 1) probably resulted from respiration. Sugar is
constantly being metabolised and initially this would be replenished by starch hydrolysis. Starch
depletion during ripening could at some point result in the amount of sugar metabolised being
greater than the amount of sugar being produced from starch hydrolysis, causing a net loss in sugar
content. Morga et al.* showed that starch levels in unripe Carabao mangoes were 11.38 g 100 g-'
fresh weight, dropping to zero in ripe fruit. As the fruit ripens the rate of loss of acidity decreases,
therefore making its relative contribution to soluble solids greater.
H.p.1.c. gave rapid, accurate and reproducible results for determination of individual sugars and
organic acids. Typical chromatographic separations are shown in Figure 1. The changes in sugar
content during ripening are shown in Figure 2. Sucrose predominated throughout, with fructose the
major reducing sugar. All three sugars increased during ripening: sucrose by 3.5-fold, and fructose
and glucose by 2.5 and 2.7-fold, respectively. Sucrose contributed 57% to the total sugar content of
the ripe fruit, with fructose and glucose making up 28 and 15% respectively. The data are in
agreement with Cancel and de Perez,16 who found that non-reducing sugar predominated during
ripening of Edward mangoes, and with Krishnamurthy et al.* who showed that sucrose predominated
in three of four varieties studied. Similar trends were obtained by Shashirekha and Patwardhad for
Badami mangoes, but they showed that preclimacteric sucrose levels were lower than those of
glucose and fructose, and postclimacteric levels of all three sugars were similar.
564 A. P. Medlicott and A. K. Thompson

m
I
u)

0
a
e
L
0
+
%
c

L-
I I I I I I I I I I I j
I 2 3 4 5 6 7 2 4 6 8 1 0

Retention time (rnins)

Figure 1. H.p.1.c. separation of sugars and organic acids from unripe mango fruits. (a) Acids: 1, tartaric acid; 2, oxalic acid; 3,
malic acid; 4, ascorbic acid; 5, a-ketoglutaric acid: 6, citric acid. (b) Sugars: 1, fructose; 2, glucose; 3, sucrose.

.o

D
2
Figure 2. Changes in the
4

.,
sugar and acid content of Keitt
mangoes during storage at
22°C. 0, sucrose; 0, fructose;
A , glucose; citric acid; 0 ,
" 0 malic acid.
0 3 6 9
Storage time (days)

The glucose :fructose ratio remained constant throughout ripening (Table 1). These results
contradict those of Krishnamurthy et a1.,8 who found the ratio generally increased and then
decreased during ripening of four varieties and Shashirekha and Patwardhan5 who found the ratio
increased from 0.75 to 0.98 during ripening of Badami mangoes.
The major organic acids were citric acid and malic acid but tartaric, oxalic, ascorbic and
a-ketoglutaric acids were present in concentrations less than 2.0 mEq 100 g-I. The decrease in
acidity was due initially to the high rate of loss of citric acid with only a small loss of malic acid. The
increased citric acid :malic acid ratio shown after 7 days storage (Table 1) was due largely to a
reduction of malic acid from 2.7 mEq 100 g-' on day 4 to 0.55 on day 7. The data agree with
Shashirekha and Patwardhan5 who found citric acid was the major organic acid in Badami mangoes
and that its level changed from 22.5 to 2.4 mEq 100 g-' during ripening. However they also found
malic acid increased slightly, being present in lower concentrations than succinic acid in preclimac-
Sugar and organic acid analysis in ripening mangoes 565

8 r o

Figure 3. Changes in peel 5

chlorophyll content (0) and
pulp firmness (0) of Keitt
mangoes during storage at
22°C. 0

Storage time (days)

teric and climacteric fruits. Succinic acid was not found to be present in this investigation. In
disagreement with the current findings, Modi and Reddy' showed that the initial decrease in acidity
in Alphonso mangoes was due to the loss of malic and other acids while citric acid was found to
increase from unripe to partly ripe fruit and to decrease thereafter.
The major loss in pulp rupture force (Figure 3 ) occurred in parallel to the decrease in citric acid
and increase in sucrose. However, the substantial losses in peel chlorophyll content occurred after
the fruit had begun to soften. In mangoes degreening is difficult to correlate with ripening as many
varieties show differing degrees of green peel in the ripe condition.
There are several possible explanations for the discrepancies between some of the present data
and those of previous workers. Analysis of several varieties has shown that considerable variation
exists in the extent of sugar development and acidity losses (data not shown). Total sugar contents of
ripe fruits have been found to vary from 11.1to 17.1% in Tommy Atkins and Alphonso, respec-
tively; with titratable acidity ranging from 0.82 to 5.65 mEq 100 g-' in Julie and Ngowe, respec-
tively. Similar findings have been presented by Palaniswamy et al. l7 who analysed 29 varieties grown
in India.
In addition to the varietal influence on the sugar and acid balance, the ripening conditions may
also affect flavour development. Preliminary experiments with ripening over a range of tempera-
tures have shown that citric acid loss is reduced at high temperatures. Thus. direct comparisons
between varieties appear feasible only if ripening conditions are similar.

References
1. Tripathi, J . S . Note on postharvest changes during storage and ripening of Gaurjeet mango. Curr. Agric. 1980,4,227-228.
2. Morga, N. S . ; Lustre, A. 0.;Tunac, M. M . ; Balogot, A. €1.; Soriano, M. R. Physicochemical changes in Philippine
Carabao mangoes during ripening. Food Chem. 1979, 4, 225-234.
3. De Leon, S . Y.; De Lima, L. S.Postharvest changes in some physical and chemical properties of Pico mangoes (Mangifera
indica Linn. var. Pico). Philipp. J. Sci. 1968, 97, 337-347.
4. Agnihortri, B. N.; Kapoor, K. L.; Srivastava, J. C. Physico-chemical changes in Dusehri mango during storage. Punlab
Hort. J. 1963,3,286-291.
5 . Shashirekha, M. S.;Patwardhan, M. V . Changes in amino acids, sugars and non-volatile organic acids in a ripening mango
fruit (Mangifera indica Badami variety). Lebensm.-Win. Techno[. 1976, 9, 369-370.
6. Sarker, S. C.; Muhsi, A. A. A. A study on the content and interconversions of organic acids of mango (Mangifera indica
Lin.) at various stages of fruit development. Bangladesh J. Agric. Sci. 1981, 8, 6%75.
7. Modi, V. V.; Reddy, V. V. R . Carotenogenesis in ripening mango. fndian J. Exp. Biol. 1967, 5, 23S235.
566 A. P. Medlicott and A. K. Thompson

8. Krishnamurthy, G. V.; Jain, N . L . ;Bhatia, 9. S. Changes in the physico-chemical composition of mangoes during ripening
after picking. Food Sci. (Mysore) 1960, 9 , 277-279.
9. Reyes, F. G. R.; Wrolstad, R. E .; Cornwell, C. J . Comparison of enzymic, gas liquid chromatographic and high
performance liquid chromatographic methods for determining sugars and organic acids in strawberries at three stages of
maturity. J. Assoc. O/f. Anal. Chem. 1982, 65, 126131.
10. Iverson, J. L.; Bueno, M. P. Evaluation of Hplc and Glc for quantitative determination of sugars in foods. J. Assoc. Of/.
Anal. Chem. 1981, 64, 139-143.
11. Wade, N . L.; Morris, S. C. Rapid determination of sugars in cantaloupe melon by high performance liquid chromatogra-
phy. J. Chromatogr. 1982. 240, 257-261.
12. Shaw, P. E.; Wilson, C. W. Organic acids in orange, grapefruit, and cherry juices quantified by high performance liquid
chromatography using neutral resin or propylamine columns. J. Sci. Food Agric. 1983,34, 1285-1288.
13. Salvo, F.; Tripodo, M. M.; Dugo, G. Determination of fructose and glucose in Cucurbitaceae by high performance liquid
chromatography. J. Sci. Food Agric. 19R4,35,212-214.
14. Blanpied, G. D.; Bramlage, W.; Dewey, D. H.; Lahelle, R. L.; Massey, L. M.; Mattas, G. E.; Stiles, W. E.; Watada, A . E.
A standardised method for collecting apple pressure test data. N . Y. Food Life Sci. Bull. Plant Sci. 1978, 74, S 8 .
15. Arnon, D . 1. Copper enzymes in isolated chloroplasts, polyphenoloxidase in Beru vulgaris. Plant Physiol. 1949, 24, 1-15.
16. Cancel, H. L.; de Perez, T. A. Effect of fruit size on the ripening of Edward mangoes. J. Agric. Univ. Puerto Rico
1979, 63, 460-464.
17. Palaniswamy, K. P.; Mathukrishan, C. R.; Shanmugavelu, K. G. Physico-chemical characteristics of some varieties of
mango. Indian Food Packer 1975, 28, 12-19.