Preparation of Sesaminol from Sesaminol Triglucoside by β-Glucosidase and Cellulase Hydrolysis

Article abstract of DOI:10.1007/s11746-016-2819-4

Sesaminol triglucoside (i.e., 2,6-O-di(β-d-glucopyranosyl)-β-d-glucopyranosyl sesaminol, STG) is a physiologically active substance obtained abundantly from defatted sesame cake. Since, the industrial preparation of sesaminol from STG has not been reported previously, the aim of this research was to prepare sesaminol by hydrolysis of STG using β-glucosidase and cellulase. Under the optimal conditions of total enzyme dosage 100?μL (8,000.72?U), the ratio of β-glucosidase and cellulase 20:80 (v/v) (0.72:8,000, U/U), reaction time 24?h, substrate concentration 6?mg/mL, reaction temperature 50?°C, and reaction system pH 4.8, the yield of sesaminol was 48.9?%. Further, sesaminol and other hydrolysis products (sesaminol diglucoside and sesaminol monoglucoside) were successfully determined by high performance liquid chromatography and electrospray ionization/mass spectrometry.

Full text of DOI:10.1007/s11746-016-2819-4

J Am Oil Chem Soc
DOI 10.1007/s11746-016-2819-4
ORIGINAL PAPER
Preparation of Sesaminol from Sesaminol Triglucoside
by β‑Glucosidase and Cellulase Hydrolysis
1
1
1
1
1
Zhen Peng · Yayuan Xu · Qingran Meng · Husnain Raza · Xiaoqing Zhao ·
1
1
Bin Liu · Cao Dong
Received: 3 September 2015 / Revised: 6 March 2016 / Accepted: 7 March 2016
AOCS 2016
©
Abstract Sesaminol triglucoside (i.e., 2,6-O-di(β-d-
glucopyranosyl)-β-d-glucopyranosyl sesaminol, STG) is
a physiologically active substance obtained abundantly
from defatted sesame cake. Since, the industrial prepara-
tion of sesaminol from STG has not been reported previ-
ously, the aim of this research was to prepare sesaminol
by hydrolysis of STG using β-glucosidase and cellulase.
Under the optimal conditions of total enzyme dosage
their antioxidant, chemoprotective, anti-inflammatory,
and neuroprotective potential [2–4]. Sesaminol trigluco-
side (STG), a water-soluble lignan, is the richest lignan
glucoside in sesame seeds [5]. STG can be efficiently
obtained from de-fatted sesame cake (DSC) which is an
abundant by-product of the sesame oil industries [6, 7].
The content of STG in sesame seeds varies from 14.1
to 1,560 mg/100 g [2, 8], depending on sesame cultivars
and processing methods [9]. Many studies have revealed
that STG hardly shows oxidation resistance in vitro [10,
11]. However, it could be gradually hydrolyzed to pro-
duce sesaminol aglycones by microbiota in the intes-
tines which play a critical role in inhibition of oxidative
effects [12].
Undoubtedly, sesaminol aglycones produce higher
biological activities (e.g., antioxidant [1] and antitumor
activity [13]) than sesaminol glycosides. Sesaminol in
unroasted sesame oil is obtained mainly from the con-
version of sesamolin by acid catalysis during industrial
bleaching process rather than from STG hydrolysis [14].
Some β-glucosidases have been reported to hydrolyze
STG [2, 15–17]. Moreover, conversions of lignan glu-
cosides to their corresponding aglycones by microbial
fermentation have also been widely researched in recent
years [10, 13].
1
00 μL (8,000.72 U), the ratio of β-glucosidase and cel-
lulase 20:80 (v/v) (0.72:8,000, U/U), reaction time 24 h,
substrate concentration 6 mg/mL, reaction temperature
5
0 °C, and reaction system pH 4.8, the yield of sesami-
nol was 48.9 %. Further, sesaminol and other hydrolysis
products (sesaminol diglucoside and sesaminol monoglu-
coside) were successfully determined by high performance
liquid chromatography and electrospray ionization/mass
spectrometry.
Keywords Sesaminol triglucoside (STG) ·
β-Glucosidase · Cellulase · Sesaminol
Introduction
Sesame seed (Sesamum indicum L.) has always been an
oilseed of great importance worldwide, and it is rich in
various bioactive furofuran lignans, particularly sesa-
min, sesamol, sesamolinol and sesaminol glucosides [1],
which play many essential roles in human health due to
Sesaminol, a natural physiologically active ingredient
from STG hydrolysis, may be used as an important addi-
tive in functional foods. However, studies of the hydroly-
sis of STG by β-glucosidase and cellulase have not been
reported yet. So, the study was conducted to produce sesa-
minol through hydrolysis of STG using the enzymes men-
tioned above. Further, the identification of sesaminol and
other hydrolysis products [sesaminol diglucoside (SDG)
and sesaminol monoglucoside (SMG)] was also performed
using chromatographic techniques.
*
Cao Dong
caodong@jiangnan.edu.cn
1
School of Food Science and Technology, Jiangnan
University, Wuxi 214122, People’s Republic of China
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J Am Oil Chem Soc
Materials and Methods
De‑Fatted Sesame Cake
with several modifications [6]. Briefly, for the preparation
of STG, 50 g of DSC powder was extracted two times by
stirring with power mixer (Shanghai Specimen Model Fac-
tory, Shanghai, China) under the following conditions: eth-
anol concentration 75 % (v/v), solid–liquid ratio 0.05 g/mL,
temperature 25 °C, extraction time 10 h. After the superna-
tants were centrifuged at 3,583×g for 10 min, the super-
natants were combined, concentrated by rotary evaporation
(ShanghaiYarong Biochemistry Instrument Factory, Shang-
hai, China, RE-52A) under a certain reduced pressure, and
lyophilized to afford the crude extract of STG. Then 1.7 g
of the above crude extract was dissolved in 5 mL deionized
water, filtered through a 0.45 μm microporous membrane
(Teflon), and supplied to the polyamide chromatography
column (1.6 × 50 cm) after previous pretreatment. This
column was eluted using distilled water with flow veloc-
ity of 1.2 mL/min. Meanwhile, the elution fractions were
automatically collected in 10 mL per tube with the help of
automatic collector of BSZ-100 (Shanghai Jia Peng Tech-
nology Co., Ltd., Shanghai, China, No. 1205027). The elu-
tion fractions obtained were detected by reverse HPLC,
and characterized by liquid chromatography–electrospray
ionization–mass spectrometry (LC–ESI/MS) in a negative
mode (see below). The tubes with relatively high content
of STG were combined, concentrated by rotary evaporation
to remove solvent under a certain reduced pressure, and
lyophilized (Alpha 1-2 LD plus Lyophilizer: Marin Christ
Company, Osterode, Germany, No. 21298) at −40 °C and
50 Pa, providing relatively high purity of STG.
Sesame cake, a by-product acquired from sesame oil pro-
cessing, was supplied free of charge by Yicheng Silver Sea
Oil Co., Ltd. (Zhumadian, China). The sesame cake was
ground into powder with a pulverizer (Nine Yang Co., Ltd.,
Shanghai, China) after vacuum drying, passed through an
8
0-mesh sieve, and de-fatted thoroughly with n-hexane at
ambient temperature. The DSC obtained was air-dried in
the dark, and then stored at −20 °C. The oil contents of
sesame cake and DSC on dry basis were 1.3 and 0.14 %,
respectively, obtained by soaking the samples in petroleum
ether (30–60 °C) in a Soxhlet apparatus (Fusisainuo Ana-
lytical Instruments Co., Ltd., Suzhou, China) [18]. Analysis
of the resulting DSC indicated that the content of STG was
1
4.76 mg/100 g DSC.
Chemicals and Reagents
Naringenin (4′,5,7-trihydroxyflavanone >98 %), used as
internal standard [8], was purchased from Nanjing Goren
Bio-Technology Co., Ltd. (Nanjing, China). High perfor-
mance liquid chromatography (HPLC)-grade methanol was
purchased from Merck Serono Co. Ltd. (Darmstadt, Ger-
many). Ethanol, n-hexane, and methanol purchased from
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China)
were of analytical grade. Polyamide (100 mesh) for column
chromatography was purchased from Sinopharm Chemical
Reagent Co., Ltd. (Shanghai, China). An Automatic collec-
tor BSZ-100 was purchased from Huxi Analysis Instrument
Factory Co., Ltd. (Shanghai, China).
Determination and Identification of Sesaminol
Triglucoside and Its Hydrolysis Products
The determination STG and its hydrolysis products was car-
ried out with a Waters 1525 series HPLC system (Waters,
Milford, MA, USA), equipped with a UV detector at 290 nm
and a C18 (4.6 mm × 150 mm × 5 μm) column from Phe-
nomenex (Torrance, CA, USA) at 30 °C. The eluents of (A)
methanol and (B) distilled water were used in the following
conditions: 0–2 min (30 % A), 2–45 min (60 % A), and 45–58
(60 % A), and the flow rate was 1.0 mL/min. The quantifica-
tion of STG as well as its hydrolysis products was calculated
on the basis of the peak areas against the naringenin (internal
standard) [8]. The samples taken out from the reaction mix-
ture were centrifuged at 11,944×g for 5 min, and then filtered
through a 0.45 μm microporous membrane (Teflon) to get rid
of insoluble matters before HPLC analysis.
Identification of STG and its hydrolysis products pro-
duced in the process of enzyme reaction was achieved by
LC–ESI/MS analysis [7]. LC–ESI/MS analysis was per-
formed by using WATERS ACQUITY UPLC mass spec-
trometer (Waters, Milford, MA, USA) with a CSH C18
column (2.1 mm × 100 mm × 1.7 μm) and ESI in negative
Enzymes and Assays
β-Glycosidase (from Aspergillus niger, 30 mg/mL, 36 U/
mL) was obtained from Sigma-Aldrich Corporate (St. Louis,
USA). One unit of β-glycosidase activity corresponds to the
amount of enzyme which hydrolyzes 1 μmol p-nitrophenyl-
β-d-glucopyranoside per minute at pH 4.0 and 37 °C. Cellu-
lase (from Trichoderma reesei, 100,000 U/mL) was obtained
from Shandong Long Kurt Enzyme Co., Ltd. (Shandong,
China). One unit of cellulase activity is equivalent of 1 mg of
glucose produced by hydrolysis of carboxymethyl cellulose
per hour at pH 5.0 and 55 °C. All assays were carried out in
triplicate, with control groups to rectify for the background
in substrate and enzyme samples [19].
Preparation of Sesaminol Triglucoside
Extraction method of STG from de-fatted sesaminol cake
(
DSC) was developed according to the previous reports
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J Am Oil Chem Soc
mode. The eluents used were (A) acetonitrile and (B) 0.1 %
formic acid. The elution conditions were 0–6 min (5 %
A) and 6–8 min (60 % A), and the flow rate was 0.3 mL/
min. The main ESI ion source operating parameters were
Statistical Analysis
All experiments were done in triplicate and data in this
research are shown as the mean values with their stand-
ard deviations (means ± SD). Differences in the yield of
sesaminol between all assays were checked by a one-way
analysis of variance (ANOVA) test using SPSS 19.0 for
Windows (SPSS Inc., Chicago, USA). The statistical sig-
nificance was determined by post hoc comparison tests of
means at the level of P < 0.05.
as follow: drying gas, N ; flow rate of desolvation gas,
2
5
00 L/h; capillary pressure, 3.0 kV; cone pressure, 30 V;
source block temperature, 100 °C; desolvation tempera-
ture, 400 °C; collision energy (eV), 6 V; detector pressure,
1
,800 V and the scanning range was 100–1,500 m/z.
Preparation of Sesaminol from Sesaminol Triglucoside
by β‑Glucosidase and Cellulase Hydrolysis
Results and Discussion
A slight modification was performed according to the study
of Jan et al. [15]. In short, 0.8 mL STG aqueous solution (1,
Preparation and Identification of Sesaminol
Triglucoside
2, 4, 6, 10, 15 mg/mL) were incubated with different dos-
ages of two enzymes (25, 50, 100, 200 μL) at various ratios
of β-glucosidase and cellulose [0:100 (v/v), 20:80, 40:60,
The yield of STG crude extract was 19.4 % based on the
DSC used. Afterwards, STG with a purity of 86.5 %
was obtained by polyamide chromatography column
purification.
5
0:50, 60:40, 80:20, 100:0] in a final volume of 1 mL. All
enzymatic hydrolysis were, respectively, performed in
0 mM acetate buffer, pH (2.8, 3.8, 4.8, 5.8, 6.8), in an elec-
5
trical thermostatic oscillation sink (Shanghai Heng Technol-
ogy Co., Ltd., Shanghai, China) at a various temperatures
The compound F-1 was determined by HPLC
(t = 22.3 min, Fig. 1a). Then the chemical structure iden-
R
(30, 40, 50, 60, 70 °C) for 0–48 h. After incubation, the
tification of F-1 was carried out by ESI–MS (Fig. 1b).
−
reactions were stopped by heating at 100 °C for 3 min. The
samples were mixed with an equal volume of methanol to
dissolve hydrolysis products, and centrifuged at 11,944×g
for 5 min, then filtered through 0.45 µm microporous mem-
branes (Teflon). 20 µL samples were analyzed by HPLC
using the internal standard (naringenin) [8]. The yield of
sesaminol was calculated by the following formula:
Fragment at m/z 855.2 was the [M–H] ion (relative molec-
ular mass 856.2), fragments at m/z 891.1 and 892.2 were
−
−
− −
for [M+Cl ] ion, fragment m/z 902.2 was [M+HCOO ]
ion. The ions of m/z 693.1 and 369.1 indicated that F-1
missed one molecule of glucose residues (C H O ,
6
10
5
162 Da) and three molecules of glucose residues (482 Da).
Fragment at m/z 179.0 and 485.1 were from [C H O –H]
6
12
6
−
and the three glucose residues, respectively, suggesting
Yield of sesaminol (%) = (the amount of sesminol obtained)/
that the structure of F-1 at least has three molecules of glu-
cose residues. Thus, it can be deduced that the F-1 is STG.
(
the initial amount of STG) × 100.
A
B
00
855
1
0.16
F-1
O
O
Internal Standard
901
0
.12
O
O
CH2OH
OH
O
CH2
OH
O
O
O
OH
OH
OH
0.08
CH2OH
OH
O
O
O
OH
856
OH
902
0.04
903
0.00
837
973
61198 249
714 724
1
827
0
m/z
10
15
20
25
30
35
40
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300
Retention Time (min)
m/z
−
Fig. 1 a A typical reversed phase HPLC chromatogram of STG (t = 22.3 min); b ESI–MS spectrum of STG, m/z 855 [M–H]
R
1
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J Am Oil Chem Soc
0.015
A
4
693
100
Peak 2
B
O
0.010
O
O
2
CH
2
OH
O
O
O
OH
OH
O
0.005
CH
2
OH
O
O
3
694
OH OH
O
STG
OH
729
731
1387
0.000
117
249
0
m/z
20
25
30
35
40
45
50
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300
Retention Time (min)
m/z
369
100
C
577
O
D
Peak 3
Peak 4
O
369
O
577
O
O
O
O
O
CH
2
OH
O
OH
567
O
OH
5
77
OH
355
3
69
O
3
70
OH
5
78
O
O
370
O
1
63198 249
117
531
578
219
3
43 372 416
3
69 370
579 676
666
870
828 869
996
948 996
497
178
296
1197
1100 1156 1222
154
196263
326
488 501
1348 1361
137
250 341
541
837
1346
9
1
m/z
0
m/z
00 200 300 400 500 600 700 800 900 1000 1100 1200 1300
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300
m/z
m/z
−
Fig. 2 a Reversed-phase HPLC analysis of STG and its hydrol-
ysis products. Peak 2, SDG (tR = 31.2 min); peak 3, SMG
of STG hydrolysis products: b peak 2, SDG, m/z 693 [M–H] ; c peak
−
−
3, SMG, m/z 531 [M–H] ; d peak 4, sesaminol, m/z 369 [M–H]
(
t = 38.2 min); peak 4, sesaminol (t = 47.1 min); ESI–MS spectra
R
R
Determination and Identification of Sesaminol
Triglucoside Hydrolysis Products
Table 1 Sesaminol triglucoside and its hydrolysis products obtained
by HPLC and LC–ESI/MS
Peak Retention time
min)
ESI/MS m/z
[M–H]
Assignment
−
(
The process of the hydrolysis of STG catalyzed by
β-glucosidase and cellulase at pH 4.8 and 50 °C was moni-
tored by analytical reversed-phase HPLC (Fig. 2a). Sesa-
minol and other hydrolysis products (SDG and SMG) were
identified by ESI–MS (Fig. 2). MS data (Table 1) was
achieved from LC–ESI/MS analysis.
STG 22.3
855
693
531
369
Sesaminol triglu-
coside
1
2
3
31.2
38.2
47.1
Sesaminol diglu-
coside
Sesaminol mono-
glucoside
Typical retention times of peaks 2, 3, 4 resulting
from the hydrolysis of STG (Fig. 1) under the standard
HPLC conditions were as follows: peak 2, 31.2 min;
peak 3, 38.2 min; peak 4, 47.1 min (Fig. 2). The LC–
ESI/MS analysis of these hydrolysis products provided
Sesaminol
Retention time refers to HPLC analysis in Fig.
1 (STG,
tR = 22.3 min) and Fig. 2a (peak 2, SDG, t = 31.2 min; peak 3,
R
SMG, tR = 38.2 min; peak 4, sesaminol, tR = 47.1 min)
−
fragments [M–H] at m/z 369 indicating that the com-
pound 4 was sesaminol (Fig. 2d, relative molecular mass
3
70). Thus, we can concluded that sesaminol is the main
peaks 2 and 3, which clearly suggested that these two
products were SDG (Fig. 2b, relative molecular mass
694) and SMG (Fig. 2c, relative molecular mass 532),
respectively.
hydrolysis products of STG due to the gradual hydroly-
sis with β-glucosidase and cellulase. The LC–ESI/MS
analysis gave [M–H] ions at m/z 693 and m/z 531 for
−
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J Am Oil Chem Soc
5
00000
00000
Meanwhile, the changes of these compounds produced
during the enzymatic hydrolysis are shown in Fig. 3. We
found a gradual increase in sesaminol and glucose pro-
duced during the enzymatic hydrolysis of STG, with a tran-
sient yield of SDG at the early stage. Quite low amounts of
SMG were detected during the enzymatic hydrolysis. ESI–
MS analysis indicated that there were no dimers as well as
trimers of glucoses in the enzymatic reaction. Sesaminol
stereoisomers including 2-episesaminol, 6-episesaminol,
as well as diasesaminol were not characterized during the
enzymatic hydrolysis. After 6 h, sesaminol could be sub-
stantially produced from STG hydrolysis, and the maxi-
mum yield of sesaminol could be achieved at 12 h during
hydrolysis. In contrast, a gradual increase of the SMG level
was observed throughout the entire time course of reaction.
These results suggested that sesaminol could be success-
fully prepared by the enzymatic hydrolysis of STG with
β-glucosidase and cellulase, which was consistent with the
previous studies [1, 20–22].
4
300000
200000
100000
0
0
10
20
30
40
50
Time (h)
Fig. 3 Changes in STG and its hydrolysis products during hydroly-
sis with β-glucosidase and cellulase. 1.5 mL STG (8 mg/mL) was
reacted with 200 μL β-glucosidase and cellulase (1:4, v/v) at pH
4
.8 and 50 °C for 48 h. STG (squares), SDG (circles), SMG (tri-
angles up), sesaminol (triangles down). Each point represents the
mean ± SD (n = 3)
Effect of Total Enzyme Dosage on the Yield
of Sesaminol
3
2
1
0
0
0
0
200
00
1
5
0
The yields of sesaminol determined by HPLC are shown
in Fig. 4, in which the enzyme ratio of β-glucosidase and
cellulase used was 1:4 (v/v). The reaction rate signifi-
cantly increased (P < 0.05), reaching a maximum yield of
sesaminol up to 28.2 % for an enzyme dosage of 200 μL,
then decreasing significantly at a reaction time above 6 h
25
(
P < 0.05), indicating that sesaminol might readily undergo
demethylenation, reduction or further oxidation into other
products [23]. It was noticeable that the total enzyme dos-
age of 50 μL led to the highest yield of 16.8 % at the end of
this enzymatic hydrolysis (48 h) which might be due to the
lower transformation of sesaminol to its byproducts. This
conversion was related to factors such as reaction time, the
binding capacity of enzymes with substrate, sesaminol con-
centration, especially the dynamic synergistic interaction
between β-glucosidase and cellulase [24]. The yields of
0
10
20
30
40
50
Time (h)
Fig. 4 Effect of total enzyme dosage on yield of sesaminol. The
enzyme ratio of β-glucosidase and cellulase used was 1:4 (v/v). All
reactions were carried out under the conditions of substrate concen-
tration (0.8 mL, 6 mg/mL), pH 4.8, 50 °C after an appropriate time
interval. Each point represents the mean ± SD (n = 3)
2
8.2 and 26.6 % of sesaminol were achieved at the enzyme
dosages of 200 and 100 μL, respectively, which were sig-
nificantly higher than those obtained from enzyme dos-
ages of 50 and 25 μL (P < 0.05). However, no significant
difference for sesaminol yields was observed for the total
enzyme dosage of 200 and 100 μL (P > 0.05). Thus, the
total enzyme dosage of 100 μL was selected considering
the cost of enzyme and the yield efficiency.
in the absence of β-glucosidase or cellulase, little sesami-
nol was obtained. For example, cellulase alone at 0:100
(v/v) or β-glucosidase alone at 100:0 released only 18.7
and 31.7 % of the total sesaminol from STG, respectively,
but when β-glucosidase and cellulase were simultaneously
added at a ratio of 60:40 and 20:80, the yields of sesami-
nol significantly increased to 43.5 and 41.3 % (P < 0.05),
respectively.
These results might suggest a dynamic synergistic inter-
action between β-glucosidase and cellulase on hydroly-
sis of STG, which was in accordance with the findings of
Kuriyama and Murui [25]. These authors showed that when
Effect of Ratio of Two Enzymes and Reaction Time
on the Yield of Sesaminol
As shown in Fig. 5, relatively high yields of sesaminol were
achieved with both β-glucosidase and cellulose. However,
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J Am Oil Chem Soc
5
0
0:100
0:80
0:60
0:50
0:40
0:20
00:0
45
2
4
5
40
4
3
3
2
0
5
0
5
6
8
1
30
20
10
0
0
10
20
30
40
50
20
0
5
10
15
20
Time (h)
substrate concentration (mg/mL)
Fig. 5 Effect of the ratio of two enzymes and the reaction time on
yield of sesaminol. During the reaction, the ratio of β-glucosidase
and cellulase were 0:100 (v/v) (squares), 20:80 (circles), 40:60 (tri-
angles up), 50:50 (triangles down); 60:40 (triangles left); 80:20 (tri-
angles right); 100:0 (diamond). All reactions were carried out under
the conditions of substrate concentration (0.8 mL, 6 mg/mL), pH 4.8,
Fig. 6 Effect of substrate concentration (1, 2, 4, 6, 10, 15 mg/mL) on
yield of sesaminol. The reaction was carried out under the conditions
of β-glucosidase and cellulose (20:80, v/v) at pH 4.8, 50 °C after 24 h
incubation. Data was expressed as the mean ± SD (n = 3)
5
0 °C after an appropriate time interval. Each point represents the
mean ± SD (n = 3)
and cellulose (20:80, v/v) at 50 °C after 24 h incubation is
presented in Fig. 6. The relation between initial rates and
substrate concentration followed a hyperbolic curve. Thus,
lower substrate concentrations led to first order kinetics.
It was shown that the reaction rate significantly increased
(P < 0.05), reaching a maximum yield of sesaminol up
to 40.8 % and then decreasing slightly following a sub-
strate inhibition kinetics, at concentrations above 6 mg/
mL of STG (P > 0.05). To the best of our knowledge, few
researches are available to explain the affinity of the two
enzymes for the substrate of STG. However, it seems that
the two enzymes are capable of hydrolyzing the glycosidic
bonds at a high hydrolysis rate in the beginning, and then a
substrate inhibition effect might mainly influence the reac-
tion rate of the two enzymes.
cellulase was used with β-glucosidase, more abundant sesa-
minol was obtained by hydrolysis of STG. Based on these
results, it could be deduced that two enzymes should be
used together in order to efficiently prepare sesaminol from
STG. The enzymatic hydrolysis was supposed to alter the
physical and (or) chemical properties of STG or its inter-
mediate products and made it easier for further enzymatic
attack, since combination of enzymes and substrates might
lead to the changes of glucose configuration in STG. How-
ever, the synergistic mode might vary according to the
hydrolysis rate of STG by β-glucosidase and cellulase, the
binding capacity of enzymes employed and substrate as
well as the specificity of the two enzymes. There was no
significant difference between the yields of sesaminol at
the ratio of 60:40 and 20:80 (P > 0.05). Thus, the ratio of
β-glucosidase and cellulase of 20:80 was chosen consider-
ing the relatively expensive cost of β-glucosidase compared
with cellulase.
Therefore, the substrate concentration of 6 mg/mL was
chosen for the optimization.
Effect of Reaction Temperature and pH on the Yield
of Sesaminol
When the ratio of β-glucosidase and cellulase was 20:80
(
4
v/v), the yield of sesaminol significantly increased up to
1.3 % (P < 0.05) at the initial stage. However, no sig-
The maximum yield of sesaminol obtained was 40.8 %
by both β-glycosidase and cellulase (20:80, v/v) at 50 °C
(Fig. 7a) and a pH value of 4.8 (Fig. 7b), which could be
justified with the fact that β-glycosidase and cellulase
display the highest activity at pH 4.0 (37 °C) and pH 5.0
nificant increase was observed as the reaction time went
beyond 24 h (P > 0.05). Therefore, the reaction time of
2
4 h was selected.
(55 °C), respectively. This also suggested that enzymes
Effect of Substrate Concentration on the Yield
of Sesaminol
activity was sensitive to temperature and pH values. Too
high or too low temperatures and pH values made the
enzymes inactive, thereby led to the decline in the hydro-
lytic reaction rate. Therefore, the optimum temperature and
pH was 50 °C, pH 4.8.
Influence of substrate concentration (0.8 mL, 1–15 mg/
mL) on the yield of sesaminol from STG by β-glucosidase
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J Am Oil Chem Soc
A
45
B
4
3
2
1
5
6
7
8
4
3
3
2
0
5
0
5
9
0
2
0
30
40
50
60
70
80
2
4
6
8
Temperature (°C)
pH
Fig. 7 a Effect of reaction temperature (30, 40, 50, 60, 70 °C) on
yield of sesaminol. The reaction was carried out under the conditions
of substrate concentration (0.8 mL, 6 mg/mL), β-glucosidase and cel-
lulose (20:80, v/v) at pH 4.8 after 24 h incubation. b Effect of reac-
tion system pH (2.8, 3.8, 4.8, 5.8, 6.8) on yield of sesaminol. The
reaction was carried out under the conditions of substrate concen-
tration (0.8 mL, 6 mg/mL), β-glucosidase and cellulose (20:80, v/v)
at 50 °C after 24 h incubation. Data are expressed as means ± SD
(n = 3)
in functional foods & nutraceuticals. Recent Pat Food Nutr Agric
Conclusion
3:17–29
5
6
7
.
.
.
Katsuzaki H, Kawakishi S, Osawa T (1933) Structure of novel
antioxidative lignan triglucoside isolated from sesame seed. Het-
erocycles 36:933–936
Sarkis JR, Michel I, Tessaro IC, Marczak LDF (2014) Optimi-
zation of phenolics extraction from sesame seed cake. Sep Purif
Technol 122:506–514
Zhu XL, Zhang X, Sun YK, Su D, Sun Y, Hu B, Zeng XX (2013)
Purification and fermentation in vitro of sesaminol triglucoside
from sesame cake by human intestinal microbiota. J Agric Food
Chem 61:1868–1877
Moazzami AA, Andersson RE, Kamal-Eldin A (2006) HPLC
analysis of sesaminol glucosides in sesame seeds. J Agric Food
Chem 54:633–638
In conclusion, a 19.4 % yield of STG crude extract based on
the amounts of DSC used was successfully achieved. More-
over, relatively high-purity of STG (86.5 %) was obtained
with polyamide column chromatography purification pro-
cedures. In addition, this study also demonstrated that sesa-
minol can be prepared from STG by β-glucosidase and cel-
lulase hydrolysis. The optimal conditions for preparation of
sesaminol (final volume 1 mL) were total enzyme dosage
8
9
.
.
100 μL (8,000.72 U), the ratio of β-glucosidase and cellulase
20:80 (v/v), reaction time 24 h, substrate concentration 6 mg/
Hemalatha S, Ghafoorunissa (2004) Lignans and tocopherols in
Indian sesame cultivars. J Am Oil Chem Soc 81:467–470
mL, reaction temperature 50 °C, and reaction system pH 4.8.
Under these conditions, the experimental yield of sesaminol
1
0. Miyake Y, Fukumoto S, Okada M, Sakaida K, Nakamura Y,
Osawa T (2005) Antioxidative catechol lignans converted from
sesamin and sesaminol triglucoside by culturing with Aspergil-
lus. J Agric Food Chem 53:22–27
−
was 48.9 %. STG (m/z 855 [M–H] ) and its hydrolysis prod-
−
−
ucts (SDG m/z 693 [M–H] , SMG m/z 531 [M–H] and ses-
−
aminol m/z 369 [M–H] ) were also successfully identified by
1
1. Suja KP, Jayalekshmy A, Arumughan C (2004) Free radical
scavenging behavior of antioxidant compounds of sesame (Ses-
amum indicum L.) in DPPH center dot system. J Agric Food
Chem 52:912–915
LC–ESI/MS. This study provided a possibility for the large-
scale enzymatic preparation of sesaminol from STG and the
production of functional foods rich in sesaminol.
1
1
2. Katsuzaki H, Kawakishi S, Osawa T (1994) Sesaminol gluco-
sides in sesame seeds. Phytochemistry 35:773–776
3. Miyahara Y, Hibasami H, Katsuzaki H, Imai K, Osawa T, Ina K,
Komiya T (2001) Sesaminol from sesame seed induces apopto-
sis in human lymphoid leukemia Molt 4B cells. Int J Mol Med
7:485–488
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