Food Bioprocess Technol (2010) 3:105110

DOI 10.1007/s11947-008-0053-2

ORIGINAL PAPER

Dextransucrase Stability in Cashew Apple Juice

Talita L. Honorato & Sueli Rodrigues

Received: 8 November 2007 / Accepted: 3 January 2008 / Published online: 29 January 2008
# Springer Science + Business Media, LLC 2008

Abstract The use of agriculture waste as substrate for

biotechnological process became an interesting alternative
to reduce production costs and the negative impact of
agricultural wastes disposal in the environment. In Brazil,
cashew tree cultivation is done aiming the cashew nut
production, and tons of cashew apples are wasted in the
field after the nut removal. Thus, cashew apple juice can be
used as low cost substrate for biotechnological process. In
this study, the stability of dextransucrase produced in a
fermentation medium using cashew apple juice as substrate
was evaluated. The crude enzyme was stable at 30 C for
30 h at pH ranging from 4.5 to 5.5. The partially purified
enzyme was stable in non-fermented cashew apple juice at
pH 5.0 for 96 h at 30 C. The high stability of the enzyme
in cashew apple juice at room temperature allows its
industrial use without needing the enzyme purification,
consequently reducing process costs.
Keywords Agriculture substrates . Enzyme stability .
Glycosyltransferase . Fermentation

T. L. Honorato
Departamento de Processos Qumicos,
Universidade Estadual de Campinas,
Faculdade de Engenharia Qumica,
Baro Geraldo, Caixa Postal 6066,
CEP 13083-970 Campinas, SP, Brazil
S. Rodrigues (*)
Departamento de Tecnologia de Alimentos,
Universidade Federal do Ceara,
Av. Mister Hull, 2977, Bloco 858, Campus do Pici,
CEP 60356-000 Fortaleza, CE, Brazil
e-mail: sueli@ufc.br

Introduction
Alternative substrates have been studied as low-cost substrates
for microbial fermentation to produce several products for
chemical, petrochemical, pharmaceutical, and food industry
(Lacaze et al. 2007; Vazquez et al. 2006; Angumeenal
and Venkappaya 2005; Tony et al. 2004; Adham 2002;
Kuerbanoglu 2004; Kumasr et al. 2003; Hang and Woodams
2000; El-Samragy et al. 1996; Stredanska et al. 1993).
Agriculture residues and wastes are the most suitable lowcost substrates for microbial cultivation.
Cashew apple is the peduncle of the cashew fruit, which
is rich in reducing sugars (fructose and glucose), vitamins,
minerals, and some amino acids (Campos et al. 2004;
Oliveira et al. 2002). The cashew tree grows even on poor
soils with low rainfall and is cultivated in 32 countries
around the world, with Brazil, India, Vietnam, and Nigeria
as the main producers. Although cashew apples can be
consumed as juice, as ice cream, and as other foodstuffs,
the cashew tree cultivation in Brazil is an agriculture
activity that aims mainly to produce cashew nuts. The nuts
represent only 10% of the total fruit weight, and large
amounts of cashew apples are left in the field after the
removal of the nut (Honorato et al. 2007).
According to official data, the Brazilian Northeast presents
an annual production of approximately 2 millions of tons of
cashew apples, and 90% of this production is lost or underutilized. As such, the cashew apple is considered an agriculture
waste, and its nutritive juice can be a suitable low-cost
substrate for oligosaccharide synthesis by the acceptor reaction, as this juice is rich in glucose and fructose (acceptors).
Glycosyltransferases are enzymes that catalyze the
transfer of glycosyl residues from a donor molecule to a
particular acceptor. Lactic acid bacteria produce a wide
variety of a particular group of glycosyltransferases, which

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can synthesize oligosaccharides and polymers. Leuconostoc

mesenteroides NRRL B-512F is a lactic acid bacterium that
produces dextransucrase, dextran, fructose, and prebiotic
carbohydrates (Seo et al. 2007; Eom et al. 2007; Rabelo et
al. 2006; Rodrigues et al. 2003, 2005). Dextransucrase (EC
2.4.1.5) is the extracellular glycosyltransferase produced by
this strain. The same enzyme is also responsible for the
synthesis of prebiotic oligosaccharides through the acceptor
reaction (Pereira et al. 1998; Rabelo et al. 2006; Rodrigues
et al. 2006; Fernandes and Rodrigues 2006; Seo et al. 2007)
L. mesenteroides NRRL B-512F is the main strain used in
industrial fermentations to produce dextransucrase and
dextran. The enzyme expression is inducible and, nowadays, is industrially obtained by fermentation of L.
mesenteroides NRRL B-512F in a synthetic medium
containing only sucrose as carbon source.
Dextransucrase has been included in glycoside hydrolases based on their sequence homologies. Glycoside
hydrolases have been grouped to date into 108 families.
The enzyme dextransucrase belongs to family 70 of
glycoside hydrolases (Majumber et al. 2007). Dextransucrase is inactivated by ethylenediaminetetraacetic acid and
can be reactivated with Ca+2. Dextransucrase is strongly
inhibited by metal ions such as Cu+2, Fe+3, and Mn+2
(Kobayashi and Matsuda 1976). Diethyl pyrocarbonate
(DEP) was shown to inhibit the enzyme activity, which
indicated the presence of essential histidine residues at the
active site (Fu and Robyt 1988). Statistical and kinetic
analyses of the inactivation of enzyme by DEP showed that
two histidine residues are essential for the enzymatic
activity (Majumber et al. 2007).
Usually, free enzymes in crude fermented broth are
unstable and must be stabilized just after the fermentation
process ends. Costly purifications protocols are often
employed, making enzymatic process expensive for largescale application. To reduce the process cost, several
authors have studied enzyme immobilization techniques
because immobilized enzymes can be reused several times
in a process, reducing the production costs. Dextransucrase
is considered an enzyme difficult to immobilize because of
its natural association with native dextran. Some time ago,
the only available technique for dextransucrase immobilization was the entrapment in sodium alginate. However,
this difficulty has been overcome by using dextranases and
new immobilization strategies and supports (Gomez de
Segura et al. 2004; Kubik et al. 2004; Petronijevic et al.
2007; Erhardt and Jordening 2007).
Cashew apple juice is rich in fructose and glucose. These
sugars can act as acceptors to produce oligosaccharides
using tranferases such as dextransucrase (Heincke et al.
1999). Chagas et al. (2007) studied the effect of yeast
extract and phosphate addition to cashew apple juice to
produce dextranscucrase from L. mesenteroides NRRL B-

Food Bioprocess Technol (2010) 3:105110

512F. Good enzyme production was obtained in shake flask

fermentation. The authors reported that the crude enzyme,
free of cells, produced in cashew apple juice was stable for
16 h at 30 C at a pH of 5.0. The enzyme stability study in
the cashew apple crude fermented broth is important
because this enzyme in the synthetic crude fermented broth
is easily denaturized. Enzyme precipitation and stabilization
must be carried out quickly after the fermentation when the
synthetic medium is employed as substrate (Rodrigues et al.
2003). Chagas et al. (2007) studied the use of cashew apple
juice as substrate to produce dextransucrase; their study
was addressed to the enzyme production using cashew
apple juice as substrate, and the enzyme stability at room
temperature (30 C) was only verified and reported.
Enzyme stability assays were not carried out. In this work,
the stability of dextransucrase in fermented and in nonfermented cashew apple juice was investigated.

Materials and Methods

Raw Material
Cashew apple (Anacardium occidentale L.) juice was
obtained through mechanical process. The juice, which
contains high levels of tannins, was clarified by adding
gelatin to remove tannins and suspended solids (Honorato
et al. 2007). The clarified cashew apple juice was
physicochemically characterized, centrifuged at 11,806g
for 10 min, and stored frozen (20 C) before use.
Cashew Apple Juice Characterization
The pH of the juice was determined by direct measurement
in a Marconi PA 200 potentiometer. Sugar content was
determined by high-performance liquid chromatography in
a Varian Pro Star system equipped with two high-pressure
pumps model Polaris, refraction index detector model 335
RI, and a MetaTherm column oven. Separation was
achieved using a Metacarb 87P (3007.8 mm) column at
60 C. Ultrapure water at 0.6 ml/min was used as eluent,
and the detector temperature was 35 C. The software
ProStar WS 5.5 was used to acquire and handle the data.
Nitrogen content of the clarified cashew apple juice was
determined according to the Kjedahl method (AOAC
1990). Results were expressed as total protein using the
conversion coefficient of 5.75.
Strain Activation
A strain of L. mesenteroides NRRL B-512F obtained from
ARS Culture Bacterial Collection (NRRL Culture collection, United States Department of Agriculture, Peoria, IL,

Food Bioprocess Technol (2010) 3:105110

USA) was activated in an optimized synthetic medium

composed of sucrose, 50 g/l (food grade); yeast extract,
20 g/l (Himedia); MgSO47H2O, 0.20 g/l; MnSO42H2O,
0.01 g/l; FeSO47H2O, 0.01 g/l; CaCl22H2O, 0.02 g/l;
NaCl, 0.01 g/l; and K2HPO4 (anhydrous), 20 g/l in an
orbital shaker (Tecnal TE 420) at 30 C and 150 rpm
(Rabelo et al. 2006; Rodrigues et al. 2003). The initial pH
of the culture medium was adjusted to 6.5 with H3PO4 and
autoclaved (121 C/15 min). Ten percent of this cell culture
was used as inoculums to the cashew apple juice fermentation medium.
Cashew Apple Juice Fermentation
The fermentation medium was composed by diluted cashew
apple juice, containing 27.351.25 g/l of fructose and
22.470.57 g/l of glucose, 50 g/l of added sucrose (food
grade), 20 g/l of yeast extract, and 20 g/l of K2HPO4. Sucrose
was added to the cashew apple juice to induce the enzyme
production (Chagas et al. 2007; Dols et al. 1998). Because
of the high mineral content of the juice, no mineral addition
was necessary (Honorato et al. 2007; Chagas et al. 2007).
The initial pH of the culture media was adjusted to 6.5 with
H3PO4, and the culture media was autoclaved (121 C/
15 min). Ten percent of the activated cell culture (10 ml),
prepared as described earlier, was inoculated in the culture
media after cooling to room temperature. Fermentations
were carried out for 8 h in a rotary shaker (Tecnal TE 420)
at 30 C and 150 rpm in a 250-ml Erlenmeyer flask closed
with cotton plugs containing 100 ml of culture medium.
Enzyme Activity Assay
After harvesting the cells by centrifugation at 11,806g for
10 min, the supernatant was used to determine enzyme
activity. Enzyme activity was determined by quantification
of the fructose released during the reaction by the
dinitrosalicylic acid method (Miller 1959). The crude
fermented broth (100 l) was mixed with 400 l of a
10% (w/v) sucrose solution in an acetate buffer pH 5.2
(20 mM containing 0.05 g/l of CaCl2) and then incubated
for 1 h at 30 C. Three volumes of ethanol 96% (v/v) were
added to stop the reaction (Chagas et al. 2007; Rodrigues et
al. 2005). The enzyme activity was expressed in IU/ml. One
international unit is defined as the amount of enzyme that
releases 1 mol of fructose per minute under ideal reaction
conditions (30 C and pH 5.2).
Enzyme Stability Assay
Stability assays were carried out with crude cashew apple
fermented broth and with the partially purified enzyme. The
stability of the crude enzyme produced in the cashew apple

107

juice medium was assayed at 301 C using the crude

fermented broth free of cells with pH adjusted with NaOH
to 4.5, 5.0, and 5.5. Sodium azide (0.1% w/v) was added to
the crude fermented broth to avoid microbial contamination. The initial activity for each pH value was determined
as previously described. Samples were taken at regular time
intervals, and the residual activity was determined. The
crude fermented broth, containing the enzyme obtained in
the synthetic medium, was also used as control.
The partially purified dextransucrase was prepared using
the same synthetic medium used to activate the strain
according to the standard protocol used in our laboratory
where sucrose is the sole carbon source (Rabelo et al. 2006;
Rodrigues et al. 2005). After harvesting the cells by
centrifugation, the enzyme was recovered from the culture
broth by adding one volume of 50% (w/v) polyethylene
glycol (PEG 1500) aqueous solution. The enzyme was
precipitated by centrifugation (11,806g for 10 min) at 4 C
(Rodrigues et al. 2005). Then, the pellet was resuspended in
10 mM acetate buffer containing 0.05 g/l of CaCl2. The
enzyme preparation was stored frozen (20 C). The
enzyme is called partially purified because the precipitation
protocol used in this work is not selective and other
enzymes and/or proteins are co-precipitated along with
dextranscurase (Rodrigues et al. 2005; Lopretti et al. 1999).
The enzyme was recovered immediately after harvesting
the cells because the enzyme is not stable in the crude broth
unless frozen at 80 C (Rodrigues et al. 2003).
The partially purified enzyme was diluted in nonfermented cashew apple juice with pH adjusted to 5.2, the
optimum pH for partially purified enzyme assay (Rodrigues
et al. 2003, 2005). The assay was carried out at 301 C,
which is the optimum temperature for dextransucrase
activity. This assay was carried out to verify if the enzyme
stability in the cashew apple crude fermented broth was
caused by some metabolite produced during the fermentation when this substrate is used instead of the synthetic
medium, or if the stability is caused by the cashew apple
juice itself. Of the partially purified enzyme, 1 IU/ml was
added to the non-fermented cashew apple juice containing
0.1% of sodium azide. Samples were taken every 24 h
during 96 h, and enzymatic activity was determined as
previously described. The assay was carried out with and
without addition of sucrose (10 g/l) to the non-fermented
cashew apple juice.
Statistical Analyses
Results are shown as meansSD, and the data were
statistically examined by analysis of variance and Tukey
test at a 95% of confidence level. The software Microcal
Origin Pro 7.5 (OriginLab Corporation, Northhamptom,
MA) was used to process the data.

108

Food Bioprocess Technol (2010) 3:105110

Results
The experiments were carried out in duplicate, and the
assays were done in triplicate. Results are presented as mean
valueSD. The clarified cashew apple juice used in the
present work had 45.252.34 g/l of fructose and 46.34
1.75 g/l of glucose and 2.580.58 g/l of total nitrogen. The
medium formulated without addition of yeast extract
contained about 1.420.29 g/l of total nitrogen, considering
the juice dilution done to reach about 50 g/l of initial
reducing sugar (27.351.25 g/l of fructose and 22.47
0.57 g/l of glucose). The enzyme activity in the synthetic
crude fermented broth (2.5 IU/ml) was completely lost after
6 h at 30 C for all studied pH values. This result was in
agreement to the results reported by Rodrigues et al. (2003)
for dextransucrase activity in synthetic crude fermented
broth.
Figure 1 illustrates the microbial growth and the enzyme
activity during fermentation. The cashew apple juice crude
fermented broth presented final enzyme activity of 27.95
0.03 IU/ml and pH of 4.940.01. The results obtained herein
were similar to the ones reported by Chagas et al. (2007) for
dextransucrase fermentation at the same condition.
Figure 2 presents the dextransucrase stability for the
cashew apple crude fermented broth free of cells. The enzyme
activity was expressed as relative activity. The enzyme in
cashew apple crude fermented broth at pH 4.5 presented good
stability during 30 h. A slight activity loss occurred in the first
6 h of the assay, but the enzyme activity was recovered after
20 h of assay. No significant difference was observed
comparing the initial activity to final activity (30 h) at
pH 4.5. At pH 5.0, the enzyme activity increased and
decreased with the time, reaching a maximum of about
threefold of the initial activity in 6 h. The relative activities
values presented significant differences from the initial
activity until 24 h of assay. Again, no significant difference

Fig. 1 Biomass, dextransucrase activity, and pH in shake flask

fermentation of L. mesenteroides NRRL B512-F in cashew apple juice

Fig. 2 Dextransucrase stability in crude cashew apple fermented

broth. Values bearing different letters are significantly different at p<
0.05

was observed comparing the initial activity to final activity

(30 h) at pH 5.0. At pH 5.5, the relative enzyme activity only
increased with the time, presenting significant differences
during the whole assay. Comparing the assays carried out at
the three different pH values, it is clear that although the
enzyme presented good stability at pH 4.5 during all the
evaluated period, the assays carried out at pH 5.0 and 5.5
presented higher relative values than the one carried out at
pH 4.5 until the first 18 h of the assay. However, the assay
carried out at pH 5.5 presented the highest enzyme activity
increase, reaching about fivefold the initial activity after 30 h
of the assay.
Because of the presence of residual non-fermented
sucrose (about 12 g/l), dextran was formed during the
enzyme stability assay. According to Willemont et al.

Fig. 3 Dextransucrase stability in non-fermented cashew apple juice

with and without sucrose addition. Values bearing different letters are
significantly different at p<0.05

Food Bioprocess Technol (2010) 3:105110

(1989), dextransucrase activity can be increased because of

the presence of dextran. Majumber et al. (2007) reported
that the purified enzyme is stable and active only in the
presence of dextran. The absence of dextran causes the
enzyme denaturation. According to the authors, adding
dextran to the enzyme preparation could prevent dextransucrase activity loss.
Thus, the observed increase in enzyme activity could
be attributed to the formation of dextran from the
residual sucrose in the crude fermented broth. To verify
this effect on the enzyme activity, the assay with the
partially purified enzyme was carried out with and
without sucrose addition to evaluate the effect of the
dextran on the enzyme activity in cashew apple juice
because sucrose addition to the non-fermented cashew
apple juice promotes the dextran formation in cashew
apple juice containing dextransucrase.
Figure 3 presents the stability assay for the partially
purified enzyme in non-fermented cashew apple juice with
pH adjusted to 5.2 (optimum stability pH value for the
partially purified enzyme). The enzyme activity increased up
to 48 h of assay and then decreased until the end of the assay
for cashew apple juice with and without addition of sucrose
(10 g/l). Significant differences were observed in both cases
during the assay period. According to the results presented in
Fig. 3, cashew apple juice is able to increase dextransucrase
activity at 30 C and pH 5.2 until 48 h. On the other hand,
comparing the initial with the final activity value (96 h), no
significant differences were observed. Thus, the crude
enzyme can be considered stable at the assay condition
for 96 h. The assay without addition of sucrose presented
higher relative activity after 48 h compared to the assay
carried out with sucrose addition. On the other hand, at 24 h
of assay, the juice containing sucrose presented higher
enzyme activity than the one without sucrose addition.
Sucrose had a negative effect on the enzyme relative.
The increase and decrease of the relative enzyme activity
(presented in Figs. 2 and 3) was also reported by Girard and
Legoy (1999) for lyophilized dextransucrase from L.
mesenteroides NRRL B-512F in several solventwater
systems at 30 C such as: dimethyl sulfoxidewater 5, 25,
30, and 40% (v/v) and ethanolwater 5, 10, and 20% (v/v).
According to the authors, the increase followed by the
decreased of the enzyme activity was caused by changes on
the enzyme conformation in such solutions. On the other
hand, the crude enzyme obtained using the standard
synthetic medium (Rabelo et al. 2006; Rodrigues et al.
2005) was very unstable, and a significant activity loss
occurred even when the crude broth is frozen at 20 C
(Rodrigues et al. 2003).
As the activity of dextransucrase is related to its stability,
crude dextransucrase produced using cashew apple juice
can be considered very stable at 30 C according to the

109

results presented. The results suggest that at least one

component of cashew apple juice has a positive impact on
dextransucrase structure, preserving its stability for a long
time at temperatures that can be considered high for
enzyme storage (30 C) in crude fermented broth. Good
enzyme stability was found even when non-fermented
cashew apple juice was used, suggesting that the stability
is caused by the cashew apple juice itself and not caused by
a metabolite produced when this substrate is used.
Considering industrial applications, these results are very
interesting because enzyme purification and stabilization
are expensive steps.
As an agriculture substrate, cashew apple juice presents
complex composition, and besides glucose, fructose, and
mineral salts, the juice also presents other compounds such
as organic acids (mainly ascorbic and citric), carotenoids,
and phenolics. These compounds might interfere in the
enzyme structure, affecting and stabilizing its activity. The
enzyme stabilization mechanism in cashew apple juice is
still not known and may be caused by a specific juice
component or the combination of several components of the
juice acting synergistically.

Conclusion
In this work, the production and stability of dextransucrase
in cashew apple juice was investigated. The results showed
that this substrate did not inhibit microbial growth.
Dextransucrase presented high stability in cashew apple
juice at 30 C, which makes this substrate suitable for
industrial application. The high stability of the enzyme as
well as the enzyme activity changes during time is still
unknown because of the complex composition of the
substrate. The use of dextransucrase in cashew apple juice
to produce oligosaccharides through the acceptor reaction
without needing the enzyme purification and/or stabilization, which are costly steps in biotechnological process, is
interesting and viable because of the high enzyme stability
at room temperature. The use of crude cashew apple juice
fermented broth for oligosaccharide synthesis will be
subject of future works.
Acknowledgments The authors thank Conselho Nacional de Pesquisa
de Desenvolvimento Cientifico e Tecnolgico (CNPq) for the financial
support of this work and the ARS culture collection for providing the
microbial strain.

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