L.S. Mazzaferro, J.D. Breccia / Food Chemistry 134 (2012) 2338–2344
2343
4
3
2
1
0
0
0
0
of a microplate reader. It can be applied within the citrus fruit
industry as well as biochemical research.
3
2.4
3
1.1
2
6.5
Acknowledgements
This work was supported by Consejo Nacional de Investigaci-
ones Científicas y Técnicas (CONICET), Universidad Nacional de
La Pampa (UNLPam), and Agencia Nacional de Promoción Científica
y Tecnológica (ANPCyT) of Argentina. The authors gratefully thank
Dr. Guido Mastrantonio for his help with HLPC system.
1
5.9
1
4.6
12.7
0
Orange juice 1
Orange juice 2
References
Fig. 6. Hesperidin quantification in two orange juices. Enzymatic method using (h)
raw samples and ( ) solvent extracted samples, ( ) HPLC method using solvent
extracted samples.
Barbagallo, R. N., Spagna, G., Palmeri, R., Restuccia, C., & Giudici, P. (2004). Selection,
characterization and comparison of b-glucosidase from mould and yeasts
employable for enological applications. Enzyme and Microbial Technology, 35,
58–66.
Bampidis, V. A., & Robinson, P. H. (2006). Citrus by-products as rumiant feeds: A
review. Animal Feed Science and Technology, 128, 175–217.
Biesaga, M. (2011). Influence of extraction methods on stability of flavonoids.
Journal of Chromatography A, 1218, 2505–2512.
Cano, A., Medina, A., & Bermejo, A. (2008). Bioactive compounds in different citrus
varieties. Discrimination among cultivars. Journal of Food Composition and
Analysis, 21, 377–381.
Doostdar, H., Burke, M. D., & Mayer, R. T. (2000). Bioflavonoids: Selective substrates
and inhibitors for cytochrome P450 CYP1A and CYP1B1. Toxicology, 144, 31–38.
Draper, N. R., & Smith, H. (1981). Applied regression analysis (2nd ed.). New York:
Wiley.
(
Sarry & Gunata, 2004). First, an enzyme recognising the linkage
between the two sugar moieties ( -rhamnosidase, EC 3.2.1.40)
splits-off a rhamnose moiety (Manzanares, van den Broeck, de Gra-
aff, & Visser, 2001; Orrillo, Ledesma, Delgado, Spagna, & Breccia,
2
idic linkage between the glucose moiety and the aglycone (Barba-
gallo, Spagna, Palmeri, Restuccia, & Giudici, 2004). The enzymatic
cocktails containing both activities are commercialised under the
terms ‘‘naringinase’’ and ‘‘hesperidinase’’. The use of these en-
zymes, which are rather promiscuous, does not provide the re-
quired selectivity for hesperidin deglycosylation in presence of
a
007). Then, a b-glucosidase (EC 3.2.1.21) hydrolyses the heteros-
He, D., Shan, Y., Wu, Y., Liu, G., Chen, B.,
& Yao, S. (2011). Simultaneous
determination of flavanones, hydroxycinnamic acids and alkaloids in citrus
fruits by HPLC–DAD–ESI/MS. Food Chemistry, 127, 880–885.
Hilmer, J. -M., Ley, J., & Gatfield, I. (2008). Verfahren zum freisetzen bestimmter
flavanone und dihydrochalkine durch saure hydrolyse (A method of releasing
certain flavanone and dihydrochalcone by acid hydrolysis). European patent
EP2017272A2.
the several glycosides present in fruits. The enzymatic activity
rhamnosyl-b-glucosidase recently described for the first time by
a-
our group was demonstrated to be highly specific for flavonoid
Ishikawa, E., Sakai, T., Ikemura, H., Matsumoto, K., & Abe, H. (2005). Identification,
cloning, and characterization of a Sporobolomyces singularis b-galactosidase-like
enzyme involved in galacto-oligosaccharide production. Journal of Bioscience
and Bioengineering, 99, 331–339.
7-O-rutinosides deglycosylation in one step (Mazzaferro et al.,
010). Sporobolomyces singularis glucosidase-like BglA protein
2
was also shown to hydrolyse hesperidin to hesperetin (Li et al.,
008). However, the enzyme was previously shown to be promis-
cuous regarding the sugar moiety, and it was able to hydrolyse also
monoglycoconjugates: b-galactosidase and b-glucosidase activities
were detected. Because of its broad sugar specificity it was named
as ‘‘b-hexosidase’’ (Ishikawa, Sakai, Ikemura, Matsumoto, & Abe,
Kanaze, F. I., Gabrieli, C., Kokkalou, E., Georgarakis, M., & Niopas, I. (2003).
Simultaneous reversed-phase high-performance liquid chromatographic
method for the determination of diosmin, hesperidin and naringin in different
citrus fruit juices and pharmaceutical formulations. Journal of Pharmaceutical
and Biomedical Analysis, 33, 243–249.
2
Kelebek, H., Silli, S., Cnabas, A., & Cabaroglu, T. (2009). HPLC determination of
organic acids, sugars, phenolic compositions and antioxidant capacity of orange
juice and orange wine made from a Turkish cv. Kozan. Microchemical Journal, 91,
187–192.
2
005).
Under the appropriate
Lai, L. B., Gopalan, V., & Glew, R. H. (1992). Continuous spectrophotometric assays
a
-rhamnosyl-b-glucosidase concentra-
for b-glucosidases acting on the plant glucosides
Analytical Biochemistry, 2, 365–369.
L-picein and prunasin.
tion, temperature and pH, stoichiometric hydrolysis of hesperidin
to hesperetin was performed in only 5–10 min and without gener-
ation of by-products. The traditional method for flavonoid determi-
nation in food material is HPLC based in UV–visible detection.
Limits of quantitation (LOQ) are usually around 0.25 mg/L (Kanaze,
Gabrieli, Kokkalou, Georgarakis, & Niopas, 2003), which are compa-
rable with the LOQ obtained by the enzymatic–spectrophotometric
method developed in the present work. The use of detectors such
as diode array and electrospray ionisation mass spectrometry
detection resulted in an increment of the resolution capacity for
different compounds rather than an increase in the sensitivity. As
an example, He et al. (2011) recently described the simultaneous
determination of alkaloids, flavanones and hydroxycinnamic acids
in citrus fruits using HPLC with a LOQ for hesperidin of 0.26 mg/L.
On the other hand, the AdSV method was proven to lower the limit
of quantitation of flavonoids to the nanomolar range (Sims et al.,
Li, Y.-M., Li, X.-M., Li, G.-M., Du, W.-C., Zhang, J., Li, W.-X., et al. (2008). In vivo
pharmacokinetics of hesperidin are affected by treatment with glucosidase-like
BglA protein isolated from yeasts. Journal of Agricultural and Food Chemistry, 23,
5550–5557.
Manthey, J. A., & Grohmann, K. (1996). Concentrations of hesperidin and other
orange peel flavonoids in citrus processing byproducts. Journal of Agricultural
and Food Chemistry, 44, 811–814.
Marín, F. R., Soler-Rivas, C., Benavente-García, O., Castillo, J., & Pérez-Álvarez, J. A.
(
2007). By-products from different citrus processes as a source of customized
functional fibres. Food Chemistry, 100, 734–741.
Manzanares, P., van den Broeck, H. C., de Graaff, L. H., & Visser, J. (2001). Purification
and characterization of two different a-L-rhamnosidases, RhaA and RhaB, from
Aspergillus aculeatus. Applied and Environmental Microbiology, 67, 2230–2234.
Massart, D. L., Vandeginste, B. G. M., Buydens, L. M. C., De Jong, S., Lewi, P. J., &
Smeyers-Verbeke, J. (1997). Handbook of chemometrics and qualimetrics: Part A
(
1st ed.). Amsterdam: Elsevier.
Mauludin, R., & Müller, R. H. (2008). Hesperidin smart crystals: Redispersibility and
Mazzaferro, L., Piñuel, L., Minig, M.,
& Breccia, J. D. (2010). Extracellular
2
009; Temerk, Ibrahim, & Kotb, 2011). The number of recent re-
monoenzyme deglycosylation system of 7-O-linked flavonoid b-rutinosides
and its disaccharide transglycosylation activity from Stilbella fimetaria. Archives
of Microbiology 192, 383–393. Erratum (2011). Archives of Microbiology, 193, 461.
Orrillo, A. G., Ledesma, P., Delgado, O. D., Spagna, G., & Breccia, J. D. (2007). Cold-
ports dealing with hesperidin and related flavonoids detection
and quantification gives account on the current interest in the
development of analytical techniques for such compounds. Fea-
tures such as sensitivity, robustness or simplicity make one or an-
other superior for the application to a particular purpose.
active
a-L-rhamnosidase from psychrotolerant bacteria isolated from a sub-
Antarctic ecosystem. Enzyme and Microbial Technology, 40, 236–241.
Peterson, J. J., Beecher, G. R., Bhagwat, S. A., Dwyer, J. T., Gebhardt, S. E., Haytowitz,
D. B., et al. (2006). Flavanones in grapefruit, lemons, and limes: A compilation
and review of the data from the analytical literature. Journal of Food Composition
and Analysis, 19, S74–S80.
The method developed in this work is fast, easy to perform and
reliable, and can be adapted for high-throughput assays by means