Journal of Food Biochemistry ISSN 1745-4514

ENYMOLOGIC CHARACTERIZATION OF
GARLIC FRUCTAN EXOHYDROLASE jfbc_532 1..6

KIT LEONG CHEONG, FENGCHAO YAN and XUESONG HUANG1

Department of Food Science and Engineering, Jinan University, 601 HuangpuWest, Guangzhou 510632, China

1
Corresponding author. TEL: 86-13535046421;
FAX: 86-020-85226630; EMAIL:
thxs@jnu.edu.cn
Accepted for Publication October 1, 2010
doi:10.1111/j.1745-4514.2010.00532.x
ABSTRACT
Fructans represent over 75% of the dry mass of garlic (Allium sativum L.). Under-
standing the enzymologic characteristics of garlic fructan exohydrolase (FEH) can
provide theoretical basis on garlic germination inhibition and mass production of
high-quality garlic fructans. In this study, we isolated and localized FEH by ammo-
nium sulfate fractional precipitation, analyzed the fructan enzymolysis products by
thin-layer chromatography (TLC) and ion chromatography, and determined FEH
activity by measuring changes of reducing sugars via 3,5-dinitrosalicylic acid assay.
We further characterized the hydrolysis site of FEH and effects of temperature, pH
level, ionic strength and substrate concentration on its activity. Results showed that
FEH was present in the 0–20% ammonium sulfate fraction. Analysis of the hydroly-
sis product by both TLC and ion chromatography suggested that FEH was an exci-
sion enzyme. The optimum conditions for FEH activity were 45C, pH 5.5, ionic
strength of 1.5 M (sodium chloride) and substrate concentration of 4 mg/mL.

PRACTICAL APPLICATIONS
During garlic storage, quality degradation such as shrinkage, softening, drying and
decreased crispiness often occurs. These deteriorations might be related to garlic
fructan exohydrolase (FEH), because FEH could hydrolyze storage carbohydrates
(fructan) to produce monosaccharide for energy metabolism. Thus, to keep the high
quality of garlic during storage, the key is to control the activity of garlic FEH. We
studied the activity of the enzyme. Results obtained from this study will provide
foundation for FEH research and insights for manufacturing homogeneous-quality
garlic fructan products.

enough to cause severe degradation of garlic fructans

INTRODUCTION
Over 75% of the garlic (Allium sativum L.) dry mass is made
up of polysaccharides (Wang et al. 2004) Numerous studies
demonstrated that the main polysaccharide of garlic is
fructan (Baumgartner et al. 2000; Huang 2005; Shi et al.
2008), which possesses antioxidative (Yang et al. 1992;
Li et al. 2007; Zheng et al. 2008), antiviral (Cai et al. 2003;
Zheng et al. 2005), probiotic (Gibson et al. 2008), and many
other medical effects and health benefits. Experience from
laboratory experiments suggested that garlic fructans pro-
cessed from the same starting material could have signifi-
cantly different molecular weights (Kong 2006; Liu 2006).
Although garlic fructans are prone to degradation under
acidic conditions, the pH of the garlic juice is not low
(Huang and Yang 2007). Instead, we believe this phenom-
enon was probably caused by the endogenous fructan exo-
hydrolase (FEH) activity of garlic. However, there are no
reports available in the literature on the modes of action
of garlic fructan hydrolase and synthase. Because fructan
hydrolase could play an important role in harvesting garlic
fructans of uniform molecular weight and in improving
the quality and freshness of garlic after periods of storage,
we were interested in whether this enzyme is present. The
objective of this work therefore was to determine whether
fructan hydrolase is present in garlic and, if so, to provide
basic information on its hydrolytic site(s) and characteristic
enzymology and to facilitate future in-depth study of the
enzyme.

Journal of Food Biochemistry •• (2011) ••–•• © 2011 Wiley Periodicals, Inc. 1

GARLIC FRUCTAN EXOHYDROLASE
MATERIALS AND METHODS
Materials and Reagents
Materials. Garlic heads (passed hibernation) that were har-
vested from Jinxiang County of Shandong Province (China)
were purchased from Shipai Vegetable Market of Guangzhou
City (Guangdong, China). Garlic fructans were prepared
according to previously published protocols (Huang et al.
2004; Jiang et al. 2005), with the following quality control cri-
teria: monosaccharide <5% (w/w), total sugar >92% (w/w),
disaccharides <4% (w/w), polymer degree 2–60, total ash
>2% (w/w) and total water content <3% (w/w).
Chemicals and Reagents. Ammonium sulfate, acetic
acid, sodium acetate, citric acid, barium chloride and 3,5-
dinitrosalicylic acid (DNS) were all purchased from Sigma
Aldrich Inc. (St. Louis, MO) and were of analytical purity.
Main Equipments. High-speed centrifuge with tempera-
ture control (Anting GG322-GL-20M1, Shanghai Anting
Scientific Instrument Factory,Shanghai,China),Spectrumlab
53 Spectrophotometer (Shanghai Lengguang Technology Co.
Ltd., Shanghai, China) and ICS 22500 ion chromatography
machine (Dionex Inc., Sunnyvale, CA) were used in this study.
Methods
Isolation of FEH Crude Extract. Garlic cloves (300 g)
were added to an appropriate amount of 50 mM sodium
acetate solution (pH 5) and homogenized. The homogenate
was then filtered through a cheese cloth before the filtrate
was centrifuged at 4,000 rpm for 10 min to obtain ~ 260 mL
supernatant containing FEH crude extract.
Ammonium Sulfate Fractional Precipitation. Crystal
ammonium sulfate was added to the enzyme crude extract to
20% saturation. The mixture was dissolved well before it was
centrifuged at 12,000 rpm and 4C for 15 min. The precipi-
tated pellet was stored at 4C and more ammonium sulfate was
added to the supernatant. The above procedures were then
repeated with the ammonium sulfate concentration reaching
40, 60 and 80% saturation, respectively. Finally, pellets from
different fractions were dissolved in an appropriate amount
of 50 mM (pH 5) sodium acetate buffer.
Desalting by Dialysis. The resuspended pellets were
transferred to dialysis bags with a molecular weight cutoff of
3.5 kDa and dialyzed against the same sodium acetate buffer
2 Journal of Food Biochemistry •• (2011) ••–•• © 2011 Wiley Periodicals, Inc.
K.L. CHEONG, F. YAN and X. HUANG
at 4C. The dialysis buffer was changed every 4 h until there
was no precipitation detected in the dialysis buffer using the
barium chloride method. The solutions in the dialysis bags
were then transferred to new tubes for determination of enzy-
matic activity. The total protein content was 0.225 mg/mL,
which was determined by Coomassie Brilliant Blue G-250
(Sigma Aldrich Inc., St. Louis, MO).
Determination of the FEH Enzyme Activity. To deter-
mine the enzyme activity of FEH, garlic fructan solution was
prepared by dissolving appropriate amount of laboratory-
made garlic fructan (Huang et al. 2004) in a 100 mL volumet-
ric flask. The enzyme solution was then added. The mixture
was incubated at 35C for 1 h before the hydrolyzed product,
reducing sugar, was measured via the DNS colorimetric
method. One unit FEH was defined as the amount of enzyme
that hydrolyzed garlic fructan to produce 1 mg reducing
sugar within 1 h at 35C.
Determination of Hydrolysis Site. An appropriate
amount of garlic fructan was mixed with the isolated enzyme
solutions containing FEH (different fractions isolated above)
and allowed to react. The products of reaction were then
spotted on a silica gel GF254 Thin Layer Chromotography plate.
The developing solvent was chloroform : acetic acid : water
= 6:7:1.a-Naphthol was used as the color reagent.Based on the
movement and size of the enzymolysis products, the reducing
sugars and hydrolysis site were preliminarily determined.
To further characterize the hydrolysis site of FEH and the
enzymolysis products, garlic fructan solutions were separated
in an AminoPac PA 10 (2 ¥ 250 mm) ion chromatography
separation column before and after enzymolysis. The mobile
phase was 10.0 mM NaOH. Au was used as the working elec-
trode, while Ag/AgCl was used as the reference electrode of
the pulse ampere detector. Composition of the enzymolysis
product was calculated based on the peak area integrations of
the ion chromatogram.
Effect of Temperature on Garlic FEH Activity. After
addition of 0.25 mL enzyme solution and reaction for 1 day
in 3 mg/mL garlic fructan solution at various temperature
(30, 35, 40, 45 and 50 ⫾ 0.5C) controlled by water bath, the
enzymatic activity of FEH was measured. Each sample was
triplicated.
Effect of pH on Garlic FEH Activity. The enzymatic
activity of FEH was measured in citric acid and disodium
phosphate-buffered solution of different pH (3.6, 4.2, 4.8, 5.4,
6.0, 6.6 and 7.2). All other reaction conditions were the same
as above. Every sample were triplicated and the results were
expressed as the mean ⫾ standard error.
K.L. CHEONG, F. YAN and X. HUANG
Effect of Ionic Strength on Garlic FEH Activity. After
reaction for 1 day in 3 mg/mL garlic fructan solution contain-
ing various concentrations of NaCl (0,0.375,0.75,1.5,3.0 mol/
L),the enzymatic activity of FEH in pH 5.5,45C was measured.
Each sample was triplicated and results were expressed as
mean ⫾ standard error.
Effect of Substrate Concentration on Garlic FEH
Activity. After reaction for 1 day in garlic fructan solution of
different concentrations (0, 1, 2, 3, 4, 6 mg/mL) in pH 5.5,
45C, the enzymatic activity of FEH was measured. All other
reaction conditions were the same as above. Each sample was
triplicated and results were expressed as mean ⫾ standard
error.
Statistics. Values in the graphs represent means of three
replicates on one enzyme preparation. Data processing was
performed using SPSS 11.5 software (SPSS Inc., Chicago, IL).
The corresponding standard error was indicated. Experi-
ments were repeated on different enzyme preparations with
consistent results.
RESULTS AND ANALYSIS
Distribution of Garlic FEH in Different
Ammonium Sulfate Fractions
As seen in Fig. 1, the 20% ammonium sulfate precipitation
fraction possessed high FEH activity, while no FEH activity
was observed in the 20–80% ammonium sulfate fractions.
FIG. 1. FRACTIONAL PRECIPITATION OF GARLIC FRUCTAN
EXOHYDROLASE BY AMMONIUM SULFATE
Data are means of three replicates.
Journal of Food Biochemistry •• (2011) ••–•• © 2011 Wiley Periodicals, Inc.
GARLIC FRUCTAN EXOHYDROLASE
However, it was not possible to determine whether the hydro-
lase was an endohydrolase or an exohydrolase. The cellular
and tissue-level localizations of garlic FEH also need to be
investigated.
Determination of Hydrolysis Site
Figure 2a showed the thin-layer chromatography (TLC)
separation of the garlic fructan hydrolysis solution. As shown,
the unhydrolyzed garlic fructan (I) did not move from the
spotting site on the TLC plate. However, when garlic fructan
was reacted with FEH (III), a spot with the same Rf value as
the fructose (II) standard appeared on the TLC plate. More-
over, there was no sign of oligofructose formation in sample
(III). Because endohydrolase reaction produces oligofructose
and such a spot was not present in the FEH and garlic fructan
mixture, the isolated fructan hydrolase was not an endohy-
drolase. Rather, we preliminarily determined that it was an
exohydrolase.
Ion chromatography results of garlic fructan before and
after FEH enzymolysis are shown in Fig. 2c,d, respectively. As
can be seen, the main enzymolysis product was fructose.
Based on the integrated peak areas in Fig. 2c,d, fructose
content increased from 3 to 55 mg/L after the hydrolysis of
garlic fructan.
In summary, both TLC and ion chromatography results
suggested that the garlic FEH isolated in this study was an
exohydrolase.
In addition, compared with the chromatogram of Fig. 2c
with that of Fig. 2d, the latter possessed an extra peak at reten-
tion time 10 min, and its peak area was bigger than that of the
fructose peak. However, the chemical composition of this
peak still needs to be determined.
Effect of Temperature on FEH Activity
As shown in Fig. 3a, temperature had a relatively huge impact
on enzyme reaction rate. At a low temperature, the reaction
rate was low. As the temperature increased, the rate increased
and more reducing sugars were produced, indicating
increased enzyme activity. The optimum temperature was
found to be 45C. Temperatures above 45C would cause
reduced enzyme activity.
Effect of pH on FEH Activity
As seen in Fig. 3b, garlic FEH activity increased in the pH
range of 4–5.5. Thereafter, further increase of pH reduced the
enzyme activity. Therefore, the optimum pH for garlic FEH is
pH 5.5.
3
GARLIC FRUCTAN EXOHYDROLASE K.L. CHEONG, F. YAN and X. HUANG

FIG. 2. ANALYSIS OF THE FRUCTAN EXOHYDROLASE (FEH) ENZYMATIC HYDROLYSATE. (a) THIN-LAYER CHROMOTOGRAPHY OF THE HYDROLYSATE
OF GARLIC FEH: (1) GARLIC FRUCTAN (Rf = 0); (2) FRUCTOSE (Rf = 0.56); (3) ENZYMATIC HYDROLYSATE OF GARLIC FRUCTAN (Rf = 0.56). (b) ION
CHROMATOGRAPHY OF AN 8 PPM FRUCTOSE SOLUTION STANDARD. (c) ION CHROMATOGRAPHY OF THE SUBSTRATE. (d) ION CHROMATOGRAPHY
OF THE ENZYMATIC HYDROLYSATE AFTER 10-FOLD DILUTION

Effect of Ionic Strength on FEH Activity Effect of Substrate Concentration on

As seen in Fig. 3c, the enzyme activity increased as the ionic
strength of the reaction mixture increased and a plateau was
reached at 1.5 M, suggesting that the optimum ionic strength
was 1.5 M.
FEH Activity
As seen in Fig. 3d, the enzyme activity increased
as the substrate concentration increased from 1 to
4 mg/mL. However, when the substrate concentra-

4 Journal of Food Biochemistry •• (2011) ••–•• © 2011 Wiley Periodicals, Inc.

K.L. CHEONG, F. YAN and X. HUANG
FIG. 3. EFFECTS OF ENVIRONMENTAL CONDITIONS ON FRUCTAN EXOHYDROLASE ACTIVITY. (a). TEMPERATURE; (b). pH; (c). ION STRENGTH;
(d). SUBSTRATE CONCENTRATION
Data are means of three replicates.
tion was higher than 4 mg/mL, the enzyme activity
remained relatively unchanged. Therefore, 4 mg/mL
is the optimum substrate concentration for the garlic
FEH.
DISCUSSION
Garlic fructan accounts for more than 75% (w/w) of dry
substance (Wang et al. 2004), which is key to garlic quality.
During garlic storage, shrinkage, softening, drying, decreased
Journal of Food Biochemistry •• (2011) ••–•• © 2011 Wiley Periodicals, Inc.
GARLIC FRUCTAN EXOHYDROLASE
crispiness and other quality-degrading signs of the cloves
often occur, with germination being the most significant
and representative characteristic. If garlic germination can be
inhibited, the quality deterioration will be controlled. The
rudimental cause of germination are the enzymes in garlic,
particularly the garlic FEH, as FEH could hydrolyze fructan to
produce monosaccharide for energy metabolism, therefore
providing an ideal condition for garlic germination. Thus,
to solve the problem of garlic quality deterioration, the key is
to control the activity of garlic FEH.
5
GARLIC FRUCTAN EXOHYDROLASE
Fructans are claimed to enhance the cold and drought tol-
erance of plants as reserve carbohydrates (Maria et al. 2008;
Yoshihiro et al. 2010). In addition, interests in fructans have
continued to increase because of their prebiotic function
(Tita and Sjef 2003). However, there is little known about
garlic fructans, and a limited understanding of FEH may be
one of the reasons for this. Preliminary experiments sug-
gested that FEH not only helps to stabilize garlic fructans, but
it also plays a significant role in improving the prebiotic func-
tion of the garlic fructan by decreasing their polymerization
(Zeng et al. 2009).
In this study, we discovered that garlic FEH was present
in the 0–20% ammonium sulfate precipitation fraction.
Through analysis of the enzymolysis product, we further
demonstrated that FEH belonged to the exohydrolase
family. Enzymatic characterization of FEH showed that the
optimum condition for its activity was 45C, pH 5.5, ionic
strength of 1.5 M and substrate concentration of 4 mg/mL.
Enzymology characterization of garlic FEH is the basic scien-
tific step toward the understanding of garlic metabolism,
inhibition of its germination during storage and improve-
ment of the garlic fructan product quality. Results obtained
from this study will provide insights for manufacturing
homogeneous-quality garlic fructan products.
ACKNOWLEDGMENTS
This research was supported by National Advanced Tech-
nology Research and Development Program (Grant no.
2007AA10Z340), National Natural Science Foundation of
China (No. 30671472) and Natural Science Foundation of
China (No. 31171722).
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