Chapter_3_Lose_of_quality_in_functional_energetic_orange_juice_drinks_for_Thesis_Draft

Effect of stimulant ingredients on orange juice quality factors Chapter 3
Effect of stimulant functional ingredients on quality factors of
orange juice during storage
Abstract
Functional energy drinks made by mixing taurine, caffeine and/or citric acid
with orange juice were monitored over 1 month of storage at 4, 20 and 30 °C in
presence and in absence of light. Both functional ingredients showed no
protective effect against browning and loss of cloud stability, except when only
citric acid was added. Samples with taurine showed more significant browning,
although adding citric acid substantially reduced the browning index (BI).
Refrigeration did not offer a significant protection, except for samples with
caffeine and citric acid where 4°C showed a better result on browning index, and
light did not statistically affect the browning index results. Cloud loss only
occurred slightly at the higher storage temperature of 30°C. Using aqueous
model solutions with pH 3.2, it was found that a first order model described well
the evolution of the browning index, and a zero order model the loss of caffeine,
which was less than 5%.
Keywords: functional drink, browning index, caffeine stability, cloud stability,
shelf-life
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1. Introduction
1.1. - Framework
Energy drinks containing stimulant ingredients such as caffeine and taurine
have mushroomed in the market since the success of Red Bull. Research on the
individual constituents of these drinks has suggested an improvement in
cognitive performance (Seidl et al. 2000). These products, called “energy
drinks”, have a wide spectrum of use varying from the drink of choice for
sportspeople to keep fluid levels up and energy levels high, to an aid to
concentration when working long hours or driving long distances (Leatherhead
Food, 2002). Abuse ingestion effects of caffeine and taurine, if any, are however
yet to be fully studied.
The disappointing sensory quality of such drinks has been the subject of
many attempts at improvement, generally trying to reach a consumer profile
closer to that of a fruit juice, away from the rather “medicinal” profile of the
original. Ultimately, the ideal sensory profile would correspond to a natural fruit
juice fortified with the stimulant ingredients. However, the influence of these
new beverage additives in storage and shelf-life aspects has not been addressed.
The objective of this work was to evaluate shelf-life and ingredient stability
during storage in orange juice fortified with caffeine and/or taurine, to ascertain
the implications of using these ingredients in natural fruit juices.
1.2. - Critical shelf life factors
Orange juice is known as an excellent source of vitamin C and is a top choice
for consumers concerned with healthy diets (Lee & Coates, 1999). Its shelf-life
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has been suitably studied and it is known that ascorbic acid losses and browning
are the main critical shelf-life factors (Lee & Coates, 1999; Manso, 2000).
Ascorbic acid (AA) is a natural antioxidant and its loss in juices is closely
related to the availability of oxygen in packages. The loss of this vitamin in
processed citrus juice is due to aerobic and anaerobic reactions of non-enzymatic
nature (Shaw et al., 1993) or catalyzed or un-catalyzed aerobic pathways (Villota
& Hawkes, 1992).
Most studies report L-AA degradation to follow first order kinetics (Lee &
Labuza, 1975; Saguy et al., 1978; Lathrop & Leung, 1980; Robertson &
Samaniego, 1986; Buettner, 1988; Sahbaz & Somer, 1993; Lee & Coates, 1999),
which means that the loss rate is proportional to the concentration. In many cases
the retention of ascorbic acid in the conditions and times tested is relatively high,
which may explain the adequacy of this simple mathematical model to describe
L-AA degradation during shelf life.
The colour of a food is the first quality factor that the consumer appreciates
and has a remarkable influence on its acceptance. Colour is also an indicator of
the natural transformation of a fresh food (ripeness) or of changes that occur
during its storage or processing (Calvo & Duran, 1977). Browning is an
oxidative change occurring in citric juices during manufacture and storage which
has been comprehensively studied. Enzymatic browning occurs easily in bruised
or cut fruit or vegetables, such as apples, bananas and potatoes (Sapers & Miller,
1995). However, because ascorbic acid and its isomers and derivatives are
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among the most effective inhibitors of enzymatic browning (Hsu et al, 1988;
Sapers & Hicks, 1989 ), and given its high level in orange juice, non-enzymatic
browning seems to be the main concern in orange juice storage. The non-
enzymatic browning (NEB) reaction is one of the most prevalent and studied
chemical reactions that occurs in foods during heating and storage. The NEB rate
is known to be affected by several physico-chemical factors, being the most
studied: concentration, ratio and chemical nature of the reactants (type of amine
and carbonyl groups involved); pH; relative humidity; temperature and time of
heating (Labuza & Baisier, 1992).
The main pathways involved in non-enzymatic browning in juice products
are the Maillard reaction, ascorbic acid degradation and the oxidation and
condensation of tannins (Lui, Chang & Wu, 2003). 5-Hydroxymethyl-2-
furfuraldehyde (HMF) is produced during fruit juice processing due to heating
and can cause browning reactions with amino compounds and sugars. The role of
organic acids in non-enzymatic browning reactions is essentially catalytic
(Reynolds, 1965). Ascorbic acid is decomposed to furfural, which is known to
undergo polymerisation and, as an active aldehyde, may combine with amino
acids and contribute to the browning of the juice (Solomon, Svannberg &
Sahlstrom, 1995).
Cloud stability (formation of cloudy haze during storage) is another
important quality problem in the citrus juice industry, which has been directly
related to the activity of pectin methyl esterase (PME) (Wenzel et al, 1951;
Versteeg et al, 1980). PME is released into the juice during the extraction
process, which demethoxylates pectin thereby causing calcium pectate
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precipitates (Rouse & Atkins, 1995). Several methods have been applied to
inactivate PME, including pasteurisation (90°C for 1 min) (Eagerman & Rouse,
1976) and more recently Ultra High Pressure (Takahachi et al, 1993).
2. Materials and Methods
2.1. - Measurement of browning and cloud stability in fortified
orange juice
A commercial brand of orange juice produced by lightly pasteurising fresh
juice (not from concentrate - Dunnes 100% Pure Squeezed Florida Premium
Orange Juice, Dunnes, Ireland) was used as a base for fortification, after fine
sieving. All possible combinations of Caffeine (300 mg/l), Taurine (4000 mg/l)
and Citric Acid (250 mg/l) were produced in duplicate, placed in clear (light
exposed) and dark (light protected) 12 ml test tubes with head space limited to a
fifth of the total volume, and cap screwed. All samples were stored for 30 days at
30°C (in a water bath-Memmert) and 4°C (in a chilled chamber- UMS Cooled
incubator). Browning and cloud stability changes were measured as optical
density (OD) at 420 nm by HACH spectrophotometer (Garza et al, 1999) and
650 nm (Parish, 1998), respectively, (Hach DR/2000 spectrophotometer) against
a reference of distilled water.
The Browning Index (BI) is the difference between the OD 420nm
of a sample
at the end of storage and the original. That of untreated (freshly squeezed) orange
juice has been reported to be 0.15 ± 0.07 (Johnson et al, 1995).
The Cloud Stability Index (CSI) is the percentage loss of light transmission
measurement at 650 nm. According to the Parish scale (Parish, 1998), the effect
97

is negligible when CSI<24%, slight for 25 %< CSI<35%, definitive for 65 %<
CSI<60% and extreme for CSI>61%.
2.2. - Kinetics of browning and caffeine loss in model solutions
Model solutions consisting of fructose (25 g/l) and citric acid (250mg/l) were
used to determine the shelf life kinetics over 6 weeks storage. Caffeine fortified
samples (300 mg/l) were analysed for caffeine loss at room temperature (20°C
average) and 30 °C storage temperature, and taurine fortified samples (4,000
mg/l) were analysed for browning (preliminary experiments showed no
significant losses of taurine) . Samples were placed in 250 ml clear (light
exposed) and dark (light protected) beakers with headspace limited to 20% of
total volume.
The concentrations used in the model solution were those needed to obtain
the same amount of sugar and pH (~ 3.2) as normal orange juice, and the same
level of fortification as commercial energy drinks.
2.3. - Caffeine Quantification
Caffeine concentration was determined with a modified HPLC method
(Phenomenex, 2003), with UV detector at 254 nm, using a mobile phase
containing 1% formic acid in a mixture of (60-40%) water-methanol. The
modification consisted in the use of a longer column (SYNERGYtm
POLAR-RP®
,
150x4.6 mm) than the one recommended in Phenomenex (2003) (SYNERGYtm
POLAR-RP®
, 50x4.6 mm), giving a longer retention time compared with the
method referenced.
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All procedures were carried out isocratically. A number of standard samples
were generated by making some caffeine/water solutions of known
concentrations. A thermo separation TSP “Spectra Series P4000” HPLC was
used with a UV detector. Guard column cartridges were Polar-RP, 4mm in length
and 3mm internal diameter (Phenomenex, Cheshire, England). The mobile phase
solution was degassed by sonication and filtered (Sartorious cellulose acetate
0.2µm filter) before passing through the column at 1ml/min. Standard samples
were used to determine the caffeine elution time which was determined as 8 min,
longer than the 1 min caffeine elution time reported in the Phenomenex (2003)
catalogue. Separation was done at room temperature. Samples were injected
manually using a Hamilton 25µl syringe.
2.4. - Statistical analysis
Tukey HSD tests were performed on the data obtained by using
STATISTICA (Release 7, StatSoft Inc., Tulsa OK, USA) to establish statistically
significant differences.
3. Results and Discussion
3.1. - Browning in fortified orange juice
Figure 3.1 shows the browning indexes of all samples. Orange juice with
added citric acid show negative browning index value, indicating a clearer juice
after storage. All other cases monitored, including the orange juice with no
additives, showed significant browning (this is not surprising as the juice used
was only lightly processed and requires refrigeration) with the exception of
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samples of orange juice with caffeine and citric acid added at 4°C storage
temperature. Light did not affect the results (no statistically significant
differences for p<0.05 for all paired comparisons).
-0.8
-0.65
-0.5
-0.35
-0.2
-0.05
0.1
0.25
0.4
0.55
OJ
OJ+Caff
OJ+Caff+CA
OJ+Tau
OJ+Tau+CA
OJ+Tau+Caff
OJ+Tau+Caff+CA
OJ+CA
BI
Figure 3.1: Browning index obtained after 30 days storage. White fills indicate light exposed
samples at 4 °C, diagonal brick striped fills light exposed samples at 30 °C, grey fills light
protected samples at 4 °C and grey wide downward diagonal striped fills light protected samples
at 30 °C. OJ is orange juice, CA citric acid, Tau taurine and Caff caffeine. The dashed line
indicates the BI of untreated orange juice (Johnson et al, 1995).
Figure 3.1 shows that BI was higher when storing at 30 °C and when taurine
was added, hence refrigeration did not actually offer a significant protection with
the exception of sample with caffeine and citric acid how it was referred before.
Caffeine has been investigated for its potential antioxidant activity;
Devasagayam (1996) reported that the antioxidant ability of caffeine was similar
to that of the established biological antioxidant glutathione and significantly
higher than ascorbic acid. However, no significant effect on browning protection
of the orange juice was found in this work (no statistically significant differences
at p<0.05). In fact, while citric acid addition prevented browning in the
unfortified orange juice, it did nothing to improve browning in the caffeine-
fortified samples when samples were storied at 30°C, so the synergistic effect
100

caffeine/citric acid even eliminated the beneficial effect of citric acid on its own
under these conditions. However, it was found the synergy caffeine/citric acid
with a positive effect on preventing browning for samples storied at 4°C (light
did not affect the results)
The samples with taurine showed a more significant browning problem.
Refrigerated storage led to BI values slightly higher (statistically significant
differences in a Tukey HSD test) than those of samples without taurine, and
storage at 30 °C gave almost twice as much BI. Taurine therefore promotes
browning in orange juice. The effect of L-ascorbic acid degradation on browning
has been reported to be accelerated by amino acids (Clegg, 1966, Clegg, 1964 &
Kacem et al, 1987) and hence the addition of taurine might increase non-
enzymatic browning in the orange juice.
Citric acid gave some significantly better results, totally preventing browning
in the fresh orange juice, and significantly reducing the BI in all samples
containing taurine. The combination of L-ascorbic acid (naturally present in the
juice) and citric acid has been used as inhibitor of enzymatic browning, as it is
recognized that citric acid has a double inhibitory effect on phenolase enzymes
by lowering the pH in the media and by chelating the copper portion of certain
phenolases (Landon, 1987). Furthermore, citric acid also has a protective effect
on ascorbic acid loss and tends to slow its auto oxidation. On the other hand, it
has been suggested that citric acid contributes significantly to the non-enzymatic
browning of solutions containing ascorbic acid (Clegg, 1966).
3.2. - Cloud stability in fortified orange juice
Figure 3.2 shows the Cloud Stability Indexes of all samples. Generally, cloud
loss was higher in dark than in light exposed samples, with the main exception of
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the sample containing both caffeine and taurine (but not citric acid). Cloud loss at
refrigerated storage was below the threshold considered significant by Parish
(Parish, 1998); while at the higher storage temperature some samples showed a
slight loss.
0
5
10
15
20
25
30
35
OJ
OJ+CA
OJ+Caff
OJ+Caff+CA
OJ+Tau
OJ+Tau+CA
OJ+Tau+Caff
OJ+Tau+Caff+CA
OJ+G.
OJ+G.+C.A.
OJ+G.+Caff.
OJ+G.+Caff.+
CA
OJ+G.+Tau
OJ+G.+Tau+
CA
OJ+G.+
Tau+Caff
OJ+G.+
Tau+Caff+CA
CSI
(%)
Figure 3.2: Cloud stability index after 30 days storage. White fills indicate light exposed samples
at 4 °C, diagonal brick striped fills light exposed samples at 30 °C, grey fills light protected
samples at 4 °C and grey wide downward diagonal striped fills light protected samples at 30 °C.
OJ is orange juice, CA citric acid, Tau taurine and Caff caffeine. The dashed line indicates the
limit of significant effect (Parish, 1998).
Citric acid could have a positive effect in cloud stability of the orange juice,
as it helps to inactivate PME, the enzyme associated with cloud stability
problems in fruit juices. This did in fact happen with unfortified orange juice, but
when adding taurine, caffeine or both there was only a slight improvement in
some cases, and in others even a negative effect.
3.3. - Kinetics of browning in model solutions
The kinetics of browning was studied in solutions containing taurine, as this
was the case that showed a greater extension of the problem. The results showed
that even in model solutions taurine enhance browning in beverages. The
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increase in optical density at 420 nm was well approximated by a first order
model (exponential increase of the OD up to an equilibrium value), with the
equilibrium value being 0.030486 (k= 0.0548, R2
= 0.993) for light exposed and
0.036696 (k= 0.1417, R2
= 0.985) for light protected samples. Results are shown
in figure 3.3. The time constant of the first order model (time required for one
log cycle increase of the optical density, that is, to reach 63.2% of the
equilibrium value) expresses the rate of browning. The values obtained were
16.99 days for the light exposed and 6.45 days for the light protected samples.
The effect of light is therefore very significant not only in terms of the maximum
extent of browning but also in terms of its rate, with darkness significantly
accelerating the process.
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0 10 20 30
time (days)
O.D.(420nm)
Figure 3.3: Increased of optical density at 420 nm in Taurine model solution. () light exposed
and () light protected. The lines show the first order model fits.
The effect of light on the often claimed taurine’s anti-oxidant character has
not been fully studied yet. Therefore, information on this topic has been found to
be scarce in the literature where the anti-oxidative effects of taurine have been
often attributed to its ability to stabilize bio-membranes, to scavenge reactive
oxygen species, and to reduce the peroxidation of unsaturated lipids (Nakaya et
al, 2000; Aruoma et at, 1998; Timbrell, Seabra & Waterfield, 1995; Eppler and
103

Dawson, 2001; Franconi et al, 2006). However, a recent study shows enhanced
antioxidant ability of taurine in the retina (Masatoshi et al, 2007) suggesting a
possible synergistic effect of taurine and light which would be in agreement with
this study where the samples exposed to light showed a significantly lower
browning rate.
3.4. - Kinetics of caffeine loss in model solutions
Small losses of caffeine were detected during storage for 6 weeks in a model
solution containing caffeine, fructose and citric acid. This was not in agreement
with results of caffeine stability in other fluids (urine, Ventura et al, 2003, tea,
Sang-Hee et al, 2005), which may be due to the lower pH. The data were well
described by a zero order model (linear decrease of caffeine concentration with
time), and are shown in figure 3.4. The slightly different initial concentrations
help to distinguish between temperatures.
250
260
270
280
290
300
310
320
330
340
0 10 20 30 40 50
time (days)
Caffeinecontent(mg/L)
Figure 3.4: Loss of caffeine during storage in a model solution.  20 C, light protected;  20 C,
light exposed;  30 C, light protected;  30 C, light exposed.
The slope of the caffeine loss model was not significantly affected by
temperature in the 20 - 30 °C range, but significantly higher (over twice higher)
when the samples were exposed to light. Light therefore plays a major role in the
mechanism of caffeine loss at pH 3.1. The slopes were 0.4348 ± 0.01849 and
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0.446 ± 0.032138 at 20 and 30 °C, respectively, in samples exposed to light and
0.1752 ± 0.013475 and 0.204 ± 0.0144 at 20 and 30 °C, respectively, in samples
protected from light.
4. Conclusions
Taurine increases browning problems in orange juice, while caffeine does not
increase them, but does not help them either, in spite of its claimed anti-oxidant
effect. Orange juice fortified with caffeine and/or taurine does not develop
significant cloud stability problems provided refrigeration is maintained. Citric
acid can reduce browning and cloud stability problems somewhat, but is not the
remedy to them when caffeine and/or taurine are present, unlike what happens in
pure orange juice and in samples with caffeine storied at 4°C.
The kinetics of browning in model solutions with taurine followed a first
order model, with the equilibrium value and the rate itself significantly higher in
containers protected from light, especially the latter.
Small losses of caffeine in model solutions with the same pH and sugar as
orange juice were detected during storage (under 5%). The process followed a
zero order model, with light significantly influencing the rate loss.
Acknowledgments
The authors acknowledge financial support from the Food Institutional Research
Measure of the Department of Agriculture, Fisheries and Foods of the Republic
of Ireland, under the National Development Plan 2006-2012
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