Influence of Storage on the Composition
Apple Juice Concentrate
N.E. BABSKY,
J.L. TORIBIO,
The effect of storage on apple juice concentrate was determined by
following changes in composition during a period of 111 days at 37°C.
Results showed that storage caused an 87% loss in the total free amino
acids, which was mostly due to decreases in glutamic acid, asparagine
and aspartic acid. The form01 titration method was inadequate for
determining the amino compounds involved in Maillard-type reactions. Sucrose was hydrolyzed under these conditions at a rate corresponding to a fist order process. The reducing sugars increased at a
rate determined by the inversion of sucrose; no consumption attributable to browning reaction was detected. Reduction of organic acids
was 9% while apparent phenolic compounds increased from 0.149 to
0.215 g/lOOg. A maximum accumulation of HMF was observed after
100 days of storage.
INTRODUCTION
analysis
Total phenolics were determined using the Folin-Ciocalteau reagent
(Singleton and Rossi, 1965). Total acids (titrable acids) were determined by direct titration in accordance with the method reported by
the International Federation of Fruit Juice Producers (IFFJP, 1974).
Hydroxymethylfurfural
(HMF) was also quantitatively determined following the procedure described by the IFFJP (IFFJP, 1974) which is
based on the calorimetric reaction between barbituric acid, p-toluidine
and HMF.
564JOURNAL
and Lozano
1842, 8000
are with Plapiqui
(LJNS Bahia Blanca, Argentina.
OF FOOD SCIENCE-Volume
Acidity
g/lOOg
Formol
index
meq/lOOg
0
7
14
21
28
38
45
49
60
70
84
104
111
2.64
2.58
2.47
2.52
2.52
2.47
2.47
2.41
2.41
2.41
2.41
2.35
2.41
2.85
2.84
2.64
2.68
2.57
2.57
2.48
2.35
2.34
2.27
2.62
2.21
2.35
Phenolic
compounds
g/l oog
0.149
0.153
0.157
0.159
0.164
0.164
0.169
0.174
0.178
0.186
0.196
0.211
0.215
Reducing
sugars
moles/lOOg
Sucrose
moles/lOOg
0.286
0.291
0.290
0.299
0.306
0.313
0.325
0.336
0.332
0.329
0.0390
0.0376
0.0319
0.0291
0.0261
0.0260
0.0267
0.0233
0.0222
0.0208
0.0180
0.0163
0.0150
Total amino acids were determined according to the formol index
(AOAC, 1980). Individual free amino acids were quantified by INTI
Laboratories (Miguelete, Buenos Aires, Argentina) with a Beckman
amino acid analyzer. (Method: As described in Beckman Manual (118/
119 BLiCL IM2, 1977). Analysis of physiological fluids).
Soluble sugars were quantified using a Waters model ALC 244
(Waters Associated Inc., Milford, MA) liquid chromatograph equipped
with a differential refractometer R401 Unit, Model U6K injector and
Model 6000A solvent delivery system under the following conditions:
ambient temperature, refractive index detector, mobile phase acetonitrilejwater (80:20) at 1.5 mUmin and chart speed 0.25 cm/min. Samples were diluted (1 .OOOgconcentrate to 12.OOOg)with double distilled
water, filtered through Sep-pak C ia and Millipore 0.45 pm, injected
in 20 PL aliquots and quantified by the external standard method.
Since the chromatographic system used in this work was unable to
separate glucose from sorbitol, the values of glucose reported here
should be interpreted as including sorbitol.
RESULTS & DISCUSSION
Acidity
AND METHODS
APPLE JUICE CONCENTRATE (AJC) (72” Brix, pH = 3.51) was
obtained from Ind. Cipoletti S.A. (Cipolletti, Rio Negro, Argentina).
The juice was stored in aseptic glass vials without head space in the
dark at 37 2 O.YC and 40-50 mL samples were extracted each week
and stored at - 20°C until analyzed.
Authors
Babsky,
Toribio,
CONICET),
12 de Octubre
Storage
time
days
total phenolics
a Units per 1OOgapple juice concentrate. Average of two determinations.
DURING STORAGE apple juice is exposed to temperatures
which have an adverse influence on quality due to the so-called
nonenzymatic browning reaction. Intensive studies of the problem have helped to clarify some of the involved chemical
mechanisms and allowed Hodge (1953) to present a lucid,
integrated scheme of some of the reactions known to play a
role in Maillard-type browning. As knowledge has unfolded,
it has become apparent that nonenzymatic browning during
storage of apple juice concentrate at relatively high temperatures (Toribio and Lozano, 1984) is highly significant and worthy of study to gain the knowledge necessary to predict and
control the color deteriorative process.
In these fruit juices the major constituents believed to be
involved in browning are the reducing sugars, amino acids,
polyphenols and organic acids (Joslyn, 1956; Cornwell and
Wrolstad, 1981). The purpose of the present work was to evaluate changes in the composition of clarified apple juice concentrate during prolonged storage at 37°C. Accumulation of
hydroxymethyl-furtural
(HMF) was also studied to determine
the mode and extent of build-up of this product of hexose
degradation under the above conditions of storage.
Compositional
and J.E. LOZANO
Table l-Changes
in total acidity, amino-N compounds,
and sugars during storage of apple juice concentratea
ABSTRACT
MATERIALS
of Clarified
51, No. 3, 1986
Results presented in Table 1 show that total acidity as malic
acid did not significantly change during storage. The role of
organic acids appears to be essentially catalytic (Reynolds,
1965). The slight decrease in acidity might be partly due to
copolymerization of organic acids with products of the browning reactions. Lewis et al. (1949) also suggested that organic
acids can react with reducing sugars to produce brown pigments.
Total phenolic compounds
Phenolic compounds present in fruit products may react to
form brown polymeric compounds (Abers and Wrolstad, 1979).
If this reaction plays any role in the color development of apple
juice, total phenolics content should not increase during storage as Table 1 shows. Cornwell and Wrolstad (1981) proposed
that reductone compounds present in the juices interfere with
the Folin-Ciocalteau reagent increasing the apparent phenolics
contents.
Table 2-Free
amino
ation during storage
Amino acid
mg/lOOg cone
acid composition
of apple juice concentrate.
Vari-
Storage time, days
0
14
Aspartic acid
40.7
40.1
2.1
Threonine
3.1
Serine
8.8
9.6
Asparagine
296.8 259.5
Glutamine
2.3
6.7
Proline
3.9
3.6
37.1
Glutamic acid
57.6
0.6
Glycine
0.7
Alanine
4.8
4.7
2.0
Valine
2.2
Methionine
1.5
1.6
lsoleucine
3.8
3.5
Leucine
0.7
0.6
Amino Butyric acid
4.3
3.8
Orn,Tyr,Phe,Lys,His,Arg,.
ilLILL
TOTAL
438.5 371.2
% Retention
84.5
100
28
45
60
84
111
35.5
24.2
23.7
22.1 14.8
1.84
2.1
1.4
1.0
0.6
8.4
7.6
6.4
6.2
4.0
233.1 180.3
142.8 140.6 28.7
. . . . . . . . . . . . . . . . . trace.
2.9
2.9
2.8
1.5
2.9
26.5
16.8
6.2
2.9
8.2
0.6
0.6
0.5
0.5
0.3
4.6
4.4
4.0
2.5
4.3
2.0
1.9
. . . trace..
1.3
0.9
0.7
0.6
0.4
2.9
2.7
1.8
1.8
1.0
0.5
0.4
0.4
0.3
0.3
0.8
3.3
3.3
2.6
1.6
. .
. . . . . . . . . . . . . trace.
323.6
73.8
247.8
56.4
195.8
44.6
187.9
42.8
57.9
13.2
I
1
I
I
I
0
,
I
1
I
loo
50
time,days
Amino acids
The amino acids composition of apple juice concentrate is
shown in Table 2. The major constituents were asparagine
(asn), aspartic acid (asp) and glutamic acid (glu). These values
are similar to those found by Burroughs (1957) and Czapski
(1975). However, Bielig and Hofsommer (1982), working with
about 90 samples of apples, apple juices and concentrates found
that every apple juice has a characteristic amino acids spectrum
and no mean value can be specified. The other individual amino
acids amounted to less than 10%. The concentration changes
of total amino acids during storage were very large (Table 2).
Asp and glu decreased more markedly than all the other amino
acids.
Joslyn (1956) indicated that of the many amino acids present
in orange juice lysine (1~s) and glu were more likely to be
involved in browning. Several studies (Warmbier et al., 1976;
Spark, 1969; Eichner and Karel, 1972; Reyes et al., 1982)
reported Maillard browning of reducing sugars with only one
or two amino acids other than asp, asn and glu. Wolfrom et
al. (1974) and Ashoor and Zent (1984) studied the influence
of different amino acids in model systems. None of these studies have shown glu and asn to be very high browning producing
compounds.
The experimental data were fitted to an exponential equation:
AA = 493,23 exp( - 0,0162 t)
Fig. l-Extent
of total amino acids (AA) disappearance and nonenzymatic browning (NEBJ (Toribio and Lozano, 1984) development in apple juice concentrate at 3PC.
01
1
0
(1)
where: AA = total amino acids content, mg/lOOg concentrate;
? = 0.986.
Figure 1 shows the AA value reduction, expressed as percentage of the initial total amino acids content, compared with
the nonenzymatic browning (NEB) development, also expressed in a relative way for the same juice at the same conditions (Toribio and Lozano, 1984).
Total amino acids reduction rate (AA,) can be obtained through
the first derivative of Eq (1). Figure 2 shows the AA, values
relative to the initial rate (t = 0) compared with the nonenzymatic browning rate (NEB,) from previous work (Toribio et
al., 1984). Both figures clearly show a significant correspondence between total amino acids content and color development during the storage of apple juice concentrate.
Form01 value showed no amino acids consumption during
storage. This behavior was also observed by Beveridge and
Harrison (1984). While individual amino acids quantification
showed that these compounds noticeably diminished, the formol index suggested that only a slight reduction in amino-N
occurred during storage (Table 1). It was inferred that formaldehyde reacted with intermediate Maillard compounds
masking the actual level of free amino acids.
I
I
I
I
t
I
time,
I
I
I
I
I
100
50
days
Fig. 2-Nonenzymatic
browning rate (NEBd and rate of reduction of total amino acids (AAd as a function of time of storage.
Carbohydrates
Figure 3 shows the hydrolysis of sucrose after 111 days at
pH 3.55 and 37°C. It is well known (Glasstone, 1946) that the
rate of hydrolysis is a function of the concentration of reactants, temperature and acid-catalyst concentration. However,
if excess water is present the rate of disappearance of sucrose
can be represented by a pseudo-first order reaction rate equation:
S = S, exp (-Kt)
(2)
where: S, = initial sucrose concentration, moles/lOOg concentrate; S = sucrose concentration at time t; K = rate constant; t = time, min. The experimental data were fitted to this
equation, resulting in a value of K = 0.00822 day-‘. Hydrolysis, also called inversion because it is accompanied by an
inversion of the angle of polarization, yields the two simpIe
Volume
51, No. 3, 79864OURNAL
OF FOOD SCIENCE-565
COMPOSITION
CLARIFIED
APPLE
JUICE
CONC.
. .
lo-
‘I
N
0
-
l.O.
Y
100
50
time, days
I
Fig. d-Extent
of sucrose hydrolysis in apple juice concentrate
as a function of time of storage at 3PC: 0 Experimental data;
(--I Full line represents Eq. (7).
sugars, D-glucose and D-fructose. The rate of appearance of
total reducing sugars is described by Eq. (3):
R = 2S,(l-exp(-Kt))
+ R,
HMF
Formation of 5-(hydroxymethyl)-2-furaldehyde
(HMF) from
amino acids and hexoses or from the acidic degration of hexoses has been widely recognized (Scallet and Gardner, 1945;
Reynolds, 1965; Schallenberger and Mattick, 1983). The HMF
increase during storage of foods containing hexoses was posOF FOOD SCIENCE-Volume
2
I
3
\
-I
4
PH
Fig. 4-Dependence
of first order rate constant on pH for sucrose hydrolysis at 3PC: 0 Value of K, this study; (-) Full line
represents Schoebel et al. (1969J correlated data.
(3)
where: R = reducing sugars (glucose + fructose) concentration at time t moles/lOOg concentrate; R, = reducing sugar
concentration at t = t,; and t = time, min.
Experimental data obtained by Schoebel et al. (1969) on the
dependence of the first order reaction rate on pH, ranged from
1.7-2.76, were fitted to the exponential curve K = a brH (r2
= 0,996) and extrapolated to pH = 4. Figure 4 compares this
information with the K value obtained in this work.
Figure 5 shows the development of total reducing sugars
during storage, which increased in concentration in accordance
with the predicted kinetics (Eq. 3). Hence, looking at Fig. 3
to 5, hydrolysis appeared to be the major cause of sucrose
reduction (and reducing sugars increase) at a rate determined
by pH and temperature.
Akhavan and Wrolstad (1980) verified that slight losses (6%)
in total sugars occur after 112 days of storage at 37°C of pear
concentrate. Stadtman (1948) considered the possibility that
relatively small chemical changes are required to produce brown
pigment of intense color. If this is the case, the changes in
reducing sugars necessary to produce large changes in color
might be hard to be detectable. Beveridge and Harrison (1984)
detected no loss of reducing sugar after heating a 72.5” Brix
pear juice at temperatures up to 80°C for 2 hr. Reyes et al.
(1982) found that glucose undergoes more browning than fructose with glycine at 60°C and pH 3.5. Any detectable variation
in the fructose/glucose ratio may indicate unbalanced consumption of these reducing sugars due to nonenzymatic browning reaction. No signticant variation was recorded in this study.
Mean value was F:G = 1.53 +- 0.05 (w:w) where the range
shown is the standard deviation.
566-JOURNAL
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51, No. 3, 7986
m
0.35 t
i
time,
days
Fig. 54ncrease
of reducing sugars as a function of time of
storage: 0 Experimental data: (-) Full line represents Eq. (2J.
itively identified and quantified (Keeney and Bassette, 1958;
Drilleau and Prioult, 197 1; Resnik and Chirife, 1978).
Figure 6 shows the HMF increase of the concentrated apple
juice at 37°C during 111 days of storage. The rate of accumulation can be divided into three period. First period is characterized as an induction time of approximately 2 wk. During
the second period the rate showed a rapid increase of HMF
with a maximum at 50 days. After that maximum the rate of
formation diminished rapidly, and the HMF production approached a plateau of 44 mg/lOOg concentrate at approximately
100 days. A similar behavior attributable to a second order
autocatalytic reaction (Frost and Pearson, 1961) was also recognized by Schallenberger and Mattick (1983) during the acidic
degradation of hexoses. It would appear that after 50 days of
storage under the present conditions, HMF started to form
brown pigments (melanoidins) at such a rate that 40-50 days
Akhavan, I. and Wrolstad, R.E. 1980. Variation of sugars and acids during
ripening of pears and in the production and storage of pear concentrat<
J. Food Sci. 45: 499.
Ashoor, S.H. and Zent, J.B. 1984. Maillard browning of common amino
acids and sugars. J. Food Sci. 49: 1206.
Beveridge, T. and Harrison, J.E. 1984. Nonenzymatic browning in pear
juice concentrate at elevated temperatures. J. Food Sci. 49: 1335.
Bielig. H.J. and Hofsommer, H.J. 1982. On the importance of the amino
acid spectra in apple juices. Fliissiges Obst. 2: 50.Burrouehs. L.F. 1957. The amino-acids of anole
*. ”iuices and eiders. J. Sci.
Food &g&. 3: 122.
Cornwell, C.J. and Wrolstad, R.E. 1981. Causes of browning in pear juice
concentrate during storage. J. Food Sci. 46: 515.
Czapski, J., 1975. W lw wolnych aminokwas6w na zmiany jak6sci zageszcmnych sokbw job1Kowych podczas przschowuwania. Presem. Ferm. Rolny
R-9.19
Drilleau, J.F. and Prioult, C. 1971. Gout de cuit et presence de, 5-HMF
clly le jus de pommes. Etude en solutions modeles. Indust. Ahm. Agr.
time, days
Fig. GEffect
of storage
time on the accumulation
of HMF.
Eichner, K. and Karel, M. 1972. The influence of water content on the
amino browning reaction in model systems under various conditions. J.
Agr. Food Chem. 20: 218.
Frost, A.A. and Pearson, R.G. 1961. “Kinetics and Mechanisms.” John
Wilev and Sons. New York.
Glass&e. S. 1946. “Textbook of Phvsical Chemistrv.” D. Van Nostrand.
Prince&, N.J.
Hodge, J.E. 1953. Dehydrated foods. Chemistry of browning reactions in
model systems. J. Agric. Food Chem. 1: 928.
IFFJP. 1974. International Federation of Fruit Juice Producers Methods.
Analysen-analyses; Fruit-Union Suisse Assoc. Svissera Frutta, Zug, Suiza,
1962-1974.12:
later the consumption equalled the formation via Amadori rearrangement of hexose degradation.
SUMMARY
THE CHROMATOGRAPHIC
OBSERVATIONS
reported in
this work indicated that the amino acids present in apple juice
were involved in browning reaction. However, results indicated that they reacted at different rates. For example, glutamic
acid and asparagine were reduced 20 times and 10 times, respectively, whereas only half of the initial glycine, leucine and
proline were consumed during the storage.
Carbohydrate analysis indicated inversion of sucrose under
acidic conditions. No loss of reducing sugars due to nonenzymatic browning was detected.
Results of the present study indicate that in an apple juice
concentrate properly produced and stored the HMF content is
considerably lower than 10 mg/lOOg but increases with storage
and more than 40 mg/lOOg were found after 100 days at 37°C.
Rate of formation of HMF resembled that of a second order
autocatalytic reaction which was shown to be characteristic of
sugar decomposition. In this case simultaneous formation and
consumption of HMF by reaction with other compounds seems
to have occurred. It could be inferred that HMF stopped accumulating when the rates of formation and disappearance were
equal.
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MS received 6114185;revised 10/21185; accepted 11/25/85.
Volume 51, No. 3, 19864OURNAL
OF FOOD SCIENCE-567
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