- Email: [email protected]
Cellulase-assisted extraction and antibacterial activity of polysaccharides from the dandelion Taraxacum officinale
Accepted Manuscript Title: Cellulase-assisted extraction and antibacterial activity of polysaccharides from the dandelion Taraxacum officinale Author: Hong-Bin Wang PII: DOI: Reference:
S0144-8617(13)01243-5 http://dx.doi.org/doi:10.1016/j.carbpol.2013.12.029 CARP 8405
To appear in: Received date: Revised date: Accepted date:
29-10-2013 3-12-2013 10-12-2013
Please cite this article as: Wang, H.-B.,Cellulase-assisted extraction and antibacterial activity of polysaccharides from the dandelion Taraxacum officinale, Carbohydrate Polymers (2013), http://dx.doi.org/10.1016/j.carbpol.2013.12.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Cellulase-assisted extraction and antibacterial activity of polysaccharides from
2
the dandelion Taraxacum officinale
ip t
3 4
Hong-Bin Wang*
cr
5
us
6
School of Marine Science and Technology, Huaihai Institute of Technology, 59
8
Changwu Road, Lianyungang 222005, China
an
7
9 10
*
11
Tel: +86-0518-85895427
12
Fax: +86-0518-85895428
13
E-mail: [email protected]
15 16 17
M d
te
Ac ce p
14
Corresponding author:
Running title: Polysaccharides from dandelion
1
Page 1 of 17
17 18
Abstract In the present study, we investigated the cellulase-assisted extraction and antibacterial activity of water-soluble polysaccharides from the dandelion Taraxacum
20
officinale. The extraction conditions, optimized for improving yield, were as follows:
21
time, 46.11 min; temperature, 54.87 °C; pH, 4.51 and cellulase enzyme, 4000 U/g.
22
Under these conditions, the yield of polysaccharides from dandelion (PD) reached
23
20.67% (w/w). The sugar content of PD was 95.6% (w/w), and it displayed high
24
antibacterial activity at a concentration of 100 mg/mL against Escherichia coli,
25
Bacillus subtilis and Staphylococcus aureus. These results indicate that PD may be a
26
viable option for use as a food preservative.
M
27
30 31 32
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29
Keywords Cellulase-assisted extraction; Polysaccharides; Antibacterial activities
Ac ce p
28
an
us
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ip t
19
2
Page 2 of 17
32 33
1. Introduction Dandelion (Taraxacum officinale), a member of the Asteraceae/Compositae plant family, is a perennial herb native to the Northern hemisphere (You et al., 2010).
35
Dandelion has been used as a phytomedicine for its choleretic, diuretic, anti-rheumatic,
36
anti-diabetic and anti-inflammatory properties (Bisset, 1994; Schütz, Carle, &
37
Sxhieber, 2006).
cr us
38
ip t
34
Extracts from different parts of the dandelion have been reported to exert various pharmacological effects. For instance, the aqueous extract from dandelion roots
40
reduce alcohol-induced oxidative stress (You et al., 2010); dandelion leaf extract
41
alleviated high-fat diet-induced non-alcoholic fatty liver (Davaatseren et al., 2013);
42
and dandelion flower extract suppressed both reactive oxygen species and nitric oxide
43
and prevented lipid oxidation in vitro (Hu, & Kitts, 2005). Moreover, peptides with
44
unusual cysteine motifs found in the flowers of T. officinale were reported to display
45
antimicrobial activity (Astafieva et al., 2012). However, insufficient data are available
47 48 49
M
d
te
Ac ce p
46
an
39
regarding the possible functions of the polysaccharides in dandelion (PD). In this study, a cellulase-assisted extraction procedure for PD was developed, the
extraction conditions were optimized using a response surface design and the antibacterial activity of PD was investigated.
50 51
2. Materials and methods
52
2.1. Materials
53
Dandelion shoots were purchased from a local pharmacy in Xinpu, China.
3
Page 3 of 17
Cellulase, with an enzymatic activity of 30,000 U/g, was purchased from Beijing
55
Shengshi Jiaming Technology Development Co. Ltd. (Beijing, China). Reagent-grade
56
chemicals were used.
ip t
54
57
2.2. PD extraction
cr
58
Dandelions were dried in a hot air oven (JK-OOI-240A, China) at 45 °C until a
60
constant weight was observed. The dried dandelions were then pulverized and sifted
61
through a 60 mesh sieve to obtain a fine powder with approximately 9% moisture
62
content (dry basis), which was stored in dark bags in a dry environment until use.
an
The dried dandelion powder was extracted with organic solvents (light petroleum,
M
63
us
59
acetone and methanol) in a Soxhlet apparatus, and then soaked in distilled water to
65
yield a 1% (w/v) suspension. The pH of the suspension was adjusted to 4.51, and
66
4000 U/g of cellulase was added. The reactor was maintained in a thermostatic water
67
bath at 54.87 °C for 46.11 min.
69 70 71 72
te
Ac ce p
68
d
64
The extract was filtered through a Whatman GF/A filter paper and concentrated
to approximately 20% (w/v). The proteins were removed using the Sevag method, and the extract was precipitated by adding 3 volumes of absolute ethanol, followed by filtration using a Whatman GF/A filter paper, and freeze drying. The % PD yield was calculated using Eq. (1) as follows: Yield = 100 W2/W1
(1),
73
where W1 and W2 represent the weights of the recovered PD and the original dried
74
dandelion powder, respectively.
4
Page 4 of 17
75 76 77
2.3. Analytical methods The pH of the solution was recorded using a digital pH meter (Model PHS-3C; CD Instruments, China). Ash, moisture, total sugar and protein contents of the samples
79
were determined according to standard methods (Hou, 2004).
cr us
80 81
ip t
78
2.4. Antibacterial activity assay
Antibacterial activity assay was performed according to the method described by
83
Qian (2014), with a slight modification. Common pathogens such as Bacillus subtilis,
84
Staphylococcus aureus and Escherichia coli were used for testing the antibacterial
85
activity of PD. The test strains were subcultured, incubated for 1 h at 37 °C, and
86
swabbed on to the surface of sterile, pre-solidified Mueller-Hinton agar plates. Sterile
87
paper discs (8 mm in diameter) were placed on the petri dishes, and 100 mg/mL PD
88
was added to each disc. Disc to which sterile water was added was used as an
90 91 92 93
M
d
te
Ac ce p
89
an
82
experimental control. The tests for assessing the antibacterial activity of PD were performed in duplicate, and the average values were reported. The presence of a zone of inhibition around the disc (8 mm diameter) indicates the sensitivity of the test bacteria to the PD sample. The total diameter of the zone of inhibition was measured for each test bacterium.
94 95 96
2.5. Experimental design A central composite design (CCD) was used to optimize the extraction conditions
5
Page 5 of 17
97
utilizing cellulase and for fitting a polynomial model as follows:
98
Y = β0 + β1X1 + β2X2 + β3X3 + β12X1 × X2 + β13X1 × X3 + β23X2 × X3 + β11X12 + β22X22
99
+ β33X32
(2),
where Y is the PD yield, β0 is the intercept term, β1, β2 and β3 are linear coefficients,
101
β12, β13 and β23 are interaction coefficients, β11, β22 and β33 are squared coefficients
102
and X1, X2 and X3 are coded independent variables. Design Expert software (version
103
7.1.6, State-Ease Inc., Minneapolis, USA) was used for the experimental design, data
104
analysis and model building.
an
us
cr
ip t
100
105
2.6. Statistical analysis
M
106
All data are presented as mean ± S.D. Statistical analysis was performed using
108
Statgraphics Centurion XV version 15.1.02. A multifactor ANOVA with posterior
109
multiple range test was used for determining statistical significance.
111 112 113 114
te
Ac ce p
110
d
107
3. Results and discussion
3.1. Effect of time, temperature and pH on PD extraction The optimum combination of the three variables, i.e., time, temperature and pH,
was investigated using a CCD. The design and results of the experiments conducted
115
using CCD are shown in Table 1. The results were analysed using ANOVA, and the
116
regression model was obtained as follows:
117
Y = −1067.35549 + 2.23414 × X1 + 24.87355 × X2 + 158.16926 × X3 − 0.021075 ×
118
X1X2 − 0.049750 × X1X3 − 0.46650 × X2X3 − 8.43687E-003 × X12 − 0.19886
6
Page 6 of 17
119
× X22 − 14.59653 × X32
(3),
where Y is the PD yield (%, w/w), X1 is the time (min), X2 is the temperature (°C) and
121
X3 is the pH. The statistical significance of Eq. (3) was verified through ANOVA for
122
the response surface quadratic model. The results (Table 2) indicate that the model
123
was highly significant, as demonstrated by the F and P values [(P > F) < 0.0001]. The
124
accuracy of fit was proven by the high value of multiple correlation coefficient (R2 =
125
97.38%), indicating that the response model could explain 97.38% of the total
126
variations. In general, a regression model with an R2 value >0.9 is considered to have
127
very high correlation (Haaland, 1989). The value of the adjusted multiple correlation
128
coefficient (R2Adj = 95.02%) was also sufficiently high to indicate the statistical
129
significance of the model.
cr
us
an
M
d
The interaction between time and temperature and that between time and pH were
te
130
ip t
120
significant, unlike that between temperature and pH (Table 2). On the basis of the
132
model, the optimum conditions of time, temperature and pH for obtaining maximum
133 134 135 136
Ac ce p
131
PD yield were calculated as 46.11 min, 54.87 °C and 4.51, respectively. By substituting values of the factors into the regression equation, the maximum predictable response for the PD yield was calculated and experimentally verified. The maximum PD yield obtained experimentally using the optimized conditions was
137
20.67% (w/w), which was consistent with the predicted value of 20.83% (w/w) that
138
was obtained using a response surface methodology regression analysis.
139 140
3.2. Effect of cellulase amount on PD extraction
7
Page 7 of 17
141
The amount of cellulase enzyme may also play an important role in governing the efficiency of PD extraction, which was investigated next. Optimum reaction
143
conditions were obtained using 4000 U/g of cellulase (Fig. 1), in contrast to a
144
previous study, in which optimum conditions for cellulase-assisted extraction of
145
polysaccharides included a pH of 5.0, temperature of 56 °C, 3500 U/g cellulase and
146
an extraction time of 40 min. These differences in the reported optimum pH,
147
temperature, cellulase amount and extraction time could be due to differences in
148
substrates and enzyme sources.
an
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ip t
142
149
3.3. Product characterization
M
150
The ash, moisture and total sugar contents of the product samples were 1.1%,
152
1.6% and 95.6% (w/w), respectively. The product samples were water-soluble and did
153
not contain any protein, which was consistent with the results of a previous study
154
(Gao, Sun, Teng, and Hou, 2010).
156 157 158
te
Ac ce p
155
d
151
3.4. Antibacterial activity of PD The results of the antibacterial activity assay using PD are shown in Fig. 2. The
diameter of the zone of inhibition formed by PD was found to be 11.02 mm, 15.26
159
mm and 12.47 mm for B. subtilis, S. aureus and E. coli, respectively, indicating a high
160
antibacterial activity of PD. These results agree with those of a previous study (Song,
161
Liu, Zhang, and Liu, 2012). Those authors observed that PD could inhibit the growth
162
of E. coli, S. aureus, Staphylococcus epidermidis, Salmonella typhimurium and
8
Page 8 of 17
163
bacteria belonging to the genus Streptococcus.
164
166
4. Conclusions In the present study, cellulase-assisted extraction of water-soluble PD was
ip t
165
performed. The PD yield was affected by extraction time, temperature, pH and the
168
amount of cellulase enzyme. Partial characterization of PD was performed, and its
169
antibacterial activity against B. subtilis, S. aureus and E. coli was determined. Our
170
results show that PD has high antibacterial activity and may be a viable option for use
171
as a food preservative.
an
us
cr
167
175 176 177 178 179 180 181 182
d
This research was supported by A Project Funded by the Priority Academic
te
174
Acknowledgments
Program Development of Jiangsu Higher Education Institutions.
Ac ce p
173
M
172
References
Astafieva, A. A., Rogozhin, E. A., Odintsova, T. I., Khadeeva, N. V., Grishin, E. V., & Egorov, Ts. A. (2012). Discovery of novel antimicrobial peptides with unusual cysteine motifs in dandelion Taraxacum officinale Wigg. flowers. Peptides, 36, 266–271 Bisset, N.G., (1994). Taraxaci radix cum herba. In: Herbal Drugs and
183
Phytopharmaceuticals: A Handbook For Practice in Scientific Basis (pp.
184
486–489). Boca Raton, FL: CRC Press.
9
Page 9 of 17
185
Davaatseren, M., Hur, H. J., Yang, H. J., Hwang, J.T., Park, J. H., Kim, H. J., Kim, M. J., Kwon, D. Y., & Sung, M. J. (2013).Taraxacum official (dandelion) leaf
187
extract alleviates high-fat diet-induced nonalcoholic fatty liver. Food and
188
Chemical Toxicology, 58, 30–36.
Gao, J. B., Sun L. N., Teng, Y., & Hou, W. (2010). Isolation, purification and analysis
cr
189
ip t
186
of glycoprotein from Taraxacum polysaccharides. Chinese Journal of Modern
191
Applied Pharmacy, 27, 905-908. (in Chinese).
194 195
an
193
Haaland, P. D. (1989). Separating Signals from the Noise. In P. D. Haaland (Ed.), Experimental Design in Biotechnology (pp.61-83). New York: Marcel Dekker. Hou, M. L. (2004). Food Analysis. Chemical Industry Press, Beijing, China. (in
M
192
us
190
chinese).
Hu, C., & Kitts, D. D. (2005). Dandelion (Taraxacum officinale) flower extract
197
suppresses both reactive oxygen species and nitric oxide and prevents lipid
198
oxidation in vitro. Phytomedicine, 12, 588–597.
200 201 202 203 204
te
Ac ce p
199
d
196
Qian, Z. G. (2014). Cellulase-assistied extraction of polysaccharides from Cucurbita moschata and their antibacterial activity. Carbohydrate Polymers, 101, 432-434.
Schütz, K., Carle, R., & Sxhieber, A. (2006). Taraxacum – a review on its phytochemical and pharmacological profile. Journal of Ethnopharmacology, 107, 313–323. Song X. Y., Liu, Q, Zhang Y. Z., & Liu, Y.X. (2010). Study on extraction technology
205
and antibacterial activity of polysaccharides from Herba Taraxaci. China
206
Pharmacy Publishing House, 21, 4453-4455. (in Chinese).
10
Page 10 of 17
You, Y., Yoo, S., Yoon, H. G., Park, J., Lee, Y. H., Kim, S., Oh, K. T., Lee, J., Cho,
208
H. Y., & Jun, W. (2010). In vitro and in vivo hepatoprotective effects of the
209
aqueous extract from Taraxacum officinale (dandelion) root against
210
alcohol-induced oxidative stress. Food and Chemical Toxicology, 48,
211
1632–1637.
cr
ip t
207
us
212 213
an
214
Ac ce p
te
d
M
215
11
Page 11 of 17
215
Table 1 The central-composite design for optimizing extraction conditions
216
218
ip t cr
7.69 6.34 13.39 5.82 12.36 9.34 14.59 22.36 9.87 11.36 20.58 19.57 20.32 19.91 11.25 19.81 12.41 20.56 15.34 6.23
us
5.34 4.50 4.00 4.00 3.66 5.00 5.00 4.50 5.00 4.00 4.50 4.50 4.50 4.50 5.00 4.50 4.00 4.50 4.50 4.50
Yield (%)
an
55.00 63.41 60.00 50.00 50.00 50.00 50.00 55.00 60.00 60.00 55.00 55.00 55.00 55.00 60.00 55.00 50.00 55.00 55.00 46.59
M
40.00 40.00 50.00 30.00 40.00 30.00 50.00 40.00 30.00 30.00 56.82 40.00 40.00 40.00 50.00 40.00 50.00 40.00 23.18 40.00
X3
d
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
X2
te
X1
X1= Time (min), X2= Temperature (°C), X3= pH.
Ac ce p
217
Run
12
Page 12 of 17
218
Table 2 Analysis of variance for the experimental results of the central-composite design
219
1 1 1 1 1 1 1 1 1 9 5 5
27.77 0.73 1.60 6.72 233.30 125.70 5.82 0.32 7.13 41.29 1.97
*Statistically significant at 95% of probability level.
221
**Statistically significant at 99% of probability level.
222
X1= Time (min), X2= Temperature (°C), X3= pH.
225 226
0.0004 0.4136 0.2340 0.0269 <0.0001 <0.0001 0.0365 0.5816 0.0235 <0.0001 0.2380
*
* ** ** *
* **
d
te Ac ce p
224
Significance
M
220
223
P>F
ip t
42.40 1.11 2.45 10.26 356.18 191.90 8.88 0.50 10.88 567.38 10.12 5.15
F value
cr
X1 X2 X3 X1 2 X2 2 X3 2 X1×X2 X1×X3 X2×X3 Model Lack of Fit Pure Error
Degree of freedom
us
Sum of square
an
Factora
13
Page 13 of 17
Figure captions
227
Fig. 1. Effect of cellulase amount on extraction of dandelion polysaccharides. Bars represent the
228
standard deviation. Data are shown as mean ± SD (n = 3).
229
Fig. 2. Antibacterial activity of dandelion polysaccharides. Bars represent the standard
230
deviation. Different letters on the bars indicate the significant differences (P<0.05). Data are
231
shown as mean ± SD (n = 3).
us
cr
ip t
226
232
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233
14
Page 14 of 17
233
Fig. 1
234
ip t
25
cr
15
10
us
Yield (%)
20
0 1000
2000
3000
4000
Cellulase amount (U/g)
5000
6000
M
235
an
5
Ac ce p
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236
15
Page 15 of 17
236
Fig. 2
237 18
a
cr
12 10 8
us
Inhibition zone diameter (mm)
b
14
ip t
c
16
6 4
an
2 0 Bacillus subtilis
Staphylococcus aureus
M
238
Escherichia coli
239
Ac ce p
te
d
240
16
Page 16 of 17
240
Highlights
241
> The PD can be effectively extracted by cellulase-assisted extraction. > The PD
243
product was partially characterized by component. > The PD product has high
244
antibacterial ability.
cr
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242
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17
Page 17 of 17
S0144-8617(13)01243-5 http://dx.doi.org/doi:10.1016/j.carbpol.2013.12.029 CARP 8405
To appear in: Received date: Revised date: Accepted date:
29-10-2013 3-12-2013 10-12-2013
Please cite this article as: Wang, H.-B.,Cellulase-assisted extraction and antibacterial activity of polysaccharides from the dandelion Taraxacum officinale, Carbohydrate Polymers (2013), http://dx.doi.org/10.1016/j.carbpol.2013.12.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Cellulase-assisted extraction and antibacterial activity of polysaccharides from
2
the dandelion Taraxacum officinale
ip t
3 4
Hong-Bin Wang*
cr
5
us
6
School of Marine Science and Technology, Huaihai Institute of Technology, 59
8
Changwu Road, Lianyungang 222005, China
an
7
9 10
*
11
Tel: +86-0518-85895427
12
Fax: +86-0518-85895428
13
E-mail: [email protected]
15 16 17
M d
te
Ac ce p
14
Corresponding author:
Running title: Polysaccharides from dandelion
1
Page 1 of 17
17 18
Abstract In the present study, we investigated the cellulase-assisted extraction and antibacterial activity of water-soluble polysaccharides from the dandelion Taraxacum
20
officinale. The extraction conditions, optimized for improving yield, were as follows:
21
time, 46.11 min; temperature, 54.87 °C; pH, 4.51 and cellulase enzyme, 4000 U/g.
22
Under these conditions, the yield of polysaccharides from dandelion (PD) reached
23
20.67% (w/w). The sugar content of PD was 95.6% (w/w), and it displayed high
24
antibacterial activity at a concentration of 100 mg/mL against Escherichia coli,
25
Bacillus subtilis and Staphylococcus aureus. These results indicate that PD may be a
26
viable option for use as a food preservative.
M
27
30 31 32
d
te
29
Keywords Cellulase-assisted extraction; Polysaccharides; Antibacterial activities
Ac ce p
28
an
us
cr
ip t
19
2
Page 2 of 17
32 33
1. Introduction Dandelion (Taraxacum officinale), a member of the Asteraceae/Compositae plant family, is a perennial herb native to the Northern hemisphere (You et al., 2010).
35
Dandelion has been used as a phytomedicine for its choleretic, diuretic, anti-rheumatic,
36
anti-diabetic and anti-inflammatory properties (Bisset, 1994; Schütz, Carle, &
37
Sxhieber, 2006).
cr us
38
ip t
34
Extracts from different parts of the dandelion have been reported to exert various pharmacological effects. For instance, the aqueous extract from dandelion roots
40
reduce alcohol-induced oxidative stress (You et al., 2010); dandelion leaf extract
41
alleviated high-fat diet-induced non-alcoholic fatty liver (Davaatseren et al., 2013);
42
and dandelion flower extract suppressed both reactive oxygen species and nitric oxide
43
and prevented lipid oxidation in vitro (Hu, & Kitts, 2005). Moreover, peptides with
44
unusual cysteine motifs found in the flowers of T. officinale were reported to display
45
antimicrobial activity (Astafieva et al., 2012). However, insufficient data are available
47 48 49
M
d
te
Ac ce p
46
an
39
regarding the possible functions of the polysaccharides in dandelion (PD). In this study, a cellulase-assisted extraction procedure for PD was developed, the
extraction conditions were optimized using a response surface design and the antibacterial activity of PD was investigated.
50 51
2. Materials and methods
52
2.1. Materials
53
Dandelion shoots were purchased from a local pharmacy in Xinpu, China.
3
Page 3 of 17
Cellulase, with an enzymatic activity of 30,000 U/g, was purchased from Beijing
55
Shengshi Jiaming Technology Development Co. Ltd. (Beijing, China). Reagent-grade
56
chemicals were used.
ip t
54
57
2.2. PD extraction
cr
58
Dandelions were dried in a hot air oven (JK-OOI-240A, China) at 45 °C until a
60
constant weight was observed. The dried dandelions were then pulverized and sifted
61
through a 60 mesh sieve to obtain a fine powder with approximately 9% moisture
62
content (dry basis), which was stored in dark bags in a dry environment until use.
an
The dried dandelion powder was extracted with organic solvents (light petroleum,
M
63
us
59
acetone and methanol) in a Soxhlet apparatus, and then soaked in distilled water to
65
yield a 1% (w/v) suspension. The pH of the suspension was adjusted to 4.51, and
66
4000 U/g of cellulase was added. The reactor was maintained in a thermostatic water
67
bath at 54.87 °C for 46.11 min.
69 70 71 72
te
Ac ce p
68
d
64
The extract was filtered through a Whatman GF/A filter paper and concentrated
to approximately 20% (w/v). The proteins were removed using the Sevag method, and the extract was precipitated by adding 3 volumes of absolute ethanol, followed by filtration using a Whatman GF/A filter paper, and freeze drying. The % PD yield was calculated using Eq. (1) as follows: Yield = 100 W2/W1
(1),
73
where W1 and W2 represent the weights of the recovered PD and the original dried
74
dandelion powder, respectively.
4
Page 4 of 17
75 76 77
2.3. Analytical methods The pH of the solution was recorded using a digital pH meter (Model PHS-3C; CD Instruments, China). Ash, moisture, total sugar and protein contents of the samples
79
were determined according to standard methods (Hou, 2004).
cr us
80 81
ip t
78
2.4. Antibacterial activity assay
Antibacterial activity assay was performed according to the method described by
83
Qian (2014), with a slight modification. Common pathogens such as Bacillus subtilis,
84
Staphylococcus aureus and Escherichia coli were used for testing the antibacterial
85
activity of PD. The test strains were subcultured, incubated for 1 h at 37 °C, and
86
swabbed on to the surface of sterile, pre-solidified Mueller-Hinton agar plates. Sterile
87
paper discs (8 mm in diameter) were placed on the petri dishes, and 100 mg/mL PD
88
was added to each disc. Disc to which sterile water was added was used as an
90 91 92 93
M
d
te
Ac ce p
89
an
82
experimental control. The tests for assessing the antibacterial activity of PD were performed in duplicate, and the average values were reported. The presence of a zone of inhibition around the disc (8 mm diameter) indicates the sensitivity of the test bacteria to the PD sample. The total diameter of the zone of inhibition was measured for each test bacterium.
94 95 96
2.5. Experimental design A central composite design (CCD) was used to optimize the extraction conditions
5
Page 5 of 17
97
utilizing cellulase and for fitting a polynomial model as follows:
98
Y = β0 + β1X1 + β2X2 + β3X3 + β12X1 × X2 + β13X1 × X3 + β23X2 × X3 + β11X12 + β22X22
99
+ β33X32
(2),
where Y is the PD yield, β0 is the intercept term, β1, β2 and β3 are linear coefficients,
101
β12, β13 and β23 are interaction coefficients, β11, β22 and β33 are squared coefficients
102
and X1, X2 and X3 are coded independent variables. Design Expert software (version
103
7.1.6, State-Ease Inc., Minneapolis, USA) was used for the experimental design, data
104
analysis and model building.
an
us
cr
ip t
100
105
2.6. Statistical analysis
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106
All data are presented as mean ± S.D. Statistical analysis was performed using
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Statgraphics Centurion XV version 15.1.02. A multifactor ANOVA with posterior
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multiple range test was used for determining statistical significance.
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3. Results and discussion
3.1. Effect of time, temperature and pH on PD extraction The optimum combination of the three variables, i.e., time, temperature and pH,
was investigated using a CCD. The design and results of the experiments conducted
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using CCD are shown in Table 1. The results were analysed using ANOVA, and the
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regression model was obtained as follows:
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Y = −1067.35549 + 2.23414 × X1 + 24.87355 × X2 + 158.16926 × X3 − 0.021075 ×
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X1X2 − 0.049750 × X1X3 − 0.46650 × X2X3 − 8.43687E-003 × X12 − 0.19886
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× X22 − 14.59653 × X32
(3),
where Y is the PD yield (%, w/w), X1 is the time (min), X2 is the temperature (°C) and
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X3 is the pH. The statistical significance of Eq. (3) was verified through ANOVA for
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the response surface quadratic model. The results (Table 2) indicate that the model
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was highly significant, as demonstrated by the F and P values [(P > F) < 0.0001]. The
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accuracy of fit was proven by the high value of multiple correlation coefficient (R2 =
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97.38%), indicating that the response model could explain 97.38% of the total
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variations. In general, a regression model with an R2 value >0.9 is considered to have
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very high correlation (Haaland, 1989). The value of the adjusted multiple correlation
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coefficient (R2Adj = 95.02%) was also sufficiently high to indicate the statistical
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significance of the model.
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The interaction between time and temperature and that between time and pH were
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significant, unlike that between temperature and pH (Table 2). On the basis of the
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model, the optimum conditions of time, temperature and pH for obtaining maximum
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PD yield were calculated as 46.11 min, 54.87 °C and 4.51, respectively. By substituting values of the factors into the regression equation, the maximum predictable response for the PD yield was calculated and experimentally verified. The maximum PD yield obtained experimentally using the optimized conditions was
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20.67% (w/w), which was consistent with the predicted value of 20.83% (w/w) that
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was obtained using a response surface methodology regression analysis.
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3.2. Effect of cellulase amount on PD extraction
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The amount of cellulase enzyme may also play an important role in governing the efficiency of PD extraction, which was investigated next. Optimum reaction
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conditions were obtained using 4000 U/g of cellulase (Fig. 1), in contrast to a
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previous study, in which optimum conditions for cellulase-assisted extraction of
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polysaccharides included a pH of 5.0, temperature of 56 °C, 3500 U/g cellulase and
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an extraction time of 40 min. These differences in the reported optimum pH,
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temperature, cellulase amount and extraction time could be due to differences in
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substrates and enzyme sources.
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3.3. Product characterization
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The ash, moisture and total sugar contents of the product samples were 1.1%,
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1.6% and 95.6% (w/w), respectively. The product samples were water-soluble and did
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not contain any protein, which was consistent with the results of a previous study
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(Gao, Sun, Teng, and Hou, 2010).
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3.4. Antibacterial activity of PD The results of the antibacterial activity assay using PD are shown in Fig. 2. The
diameter of the zone of inhibition formed by PD was found to be 11.02 mm, 15.26
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mm and 12.47 mm for B. subtilis, S. aureus and E. coli, respectively, indicating a high
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antibacterial activity of PD. These results agree with those of a previous study (Song,
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Liu, Zhang, and Liu, 2012). Those authors observed that PD could inhibit the growth
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of E. coli, S. aureus, Staphylococcus epidermidis, Salmonella typhimurium and
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bacteria belonging to the genus Streptococcus.
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4. Conclusions In the present study, cellulase-assisted extraction of water-soluble PD was
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performed. The PD yield was affected by extraction time, temperature, pH and the
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amount of cellulase enzyme. Partial characterization of PD was performed, and its
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antibacterial activity against B. subtilis, S. aureus and E. coli was determined. Our
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results show that PD has high antibacterial activity and may be a viable option for use
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as a food preservative.
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This research was supported by A Project Funded by the Priority Academic
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Acknowledgments
Program Development of Jiangsu Higher Education Institutions.
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References
Astafieva, A. A., Rogozhin, E. A., Odintsova, T. I., Khadeeva, N. V., Grishin, E. V., & Egorov, Ts. A. (2012). Discovery of novel antimicrobial peptides with unusual cysteine motifs in dandelion Taraxacum officinale Wigg. flowers. Peptides, 36, 266–271 Bisset, N.G., (1994). Taraxaci radix cum herba. In: Herbal Drugs and
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Phytopharmaceuticals: A Handbook For Practice in Scientific Basis (pp.
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486–489). Boca Raton, FL: CRC Press.
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Davaatseren, M., Hur, H. J., Yang, H. J., Hwang, J.T., Park, J. H., Kim, H. J., Kim, M. J., Kwon, D. Y., & Sung, M. J. (2013).Taraxacum official (dandelion) leaf
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extract alleviates high-fat diet-induced nonalcoholic fatty liver. Food and
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Chemical Toxicology, 58, 30–36.
Gao, J. B., Sun L. N., Teng, Y., & Hou, W. (2010). Isolation, purification and analysis
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of glycoprotein from Taraxacum polysaccharides. Chinese Journal of Modern
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Applied Pharmacy, 27, 905-908. (in Chinese).
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an
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Haaland, P. D. (1989). Separating Signals from the Noise. In P. D. Haaland (Ed.), Experimental Design in Biotechnology (pp.61-83). New York: Marcel Dekker. Hou, M. L. (2004). Food Analysis. Chemical Industry Press, Beijing, China. (in
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us
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chinese).
Hu, C., & Kitts, D. D. (2005). Dandelion (Taraxacum officinale) flower extract
197
suppresses both reactive oxygen species and nitric oxide and prevents lipid
198
oxidation in vitro. Phytomedicine, 12, 588–597.
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Qian, Z. G. (2014). Cellulase-assistied extraction of polysaccharides from Cucurbita moschata and their antibacterial activity. Carbohydrate Polymers, 101, 432-434.
Schütz, K., Carle, R., & Sxhieber, A. (2006). Taraxacum – a review on its phytochemical and pharmacological profile. Journal of Ethnopharmacology, 107, 313–323. Song X. Y., Liu, Q, Zhang Y. Z., & Liu, Y.X. (2010). Study on extraction technology
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and antibacterial activity of polysaccharides from Herba Taraxaci. China
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Pharmacy Publishing House, 21, 4453-4455. (in Chinese).
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You, Y., Yoo, S., Yoon, H. G., Park, J., Lee, Y. H., Kim, S., Oh, K. T., Lee, J., Cho,
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H. Y., & Jun, W. (2010). In vitro and in vivo hepatoprotective effects of the
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aqueous extract from Taraxacum officinale (dandelion) root against
210
alcohol-induced oxidative stress. Food and Chemical Toxicology, 48,
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1632–1637.
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Table 1 The central-composite design for optimizing extraction conditions
216
218
ip t cr
7.69 6.34 13.39 5.82 12.36 9.34 14.59 22.36 9.87 11.36 20.58 19.57 20.32 19.91 11.25 19.81 12.41 20.56 15.34 6.23
us
5.34 4.50 4.00 4.00 3.66 5.00 5.00 4.50 5.00 4.00 4.50 4.50 4.50 4.50 5.00 4.50 4.00 4.50 4.50 4.50
Yield (%)
an
55.00 63.41 60.00 50.00 50.00 50.00 50.00 55.00 60.00 60.00 55.00 55.00 55.00 55.00 60.00 55.00 50.00 55.00 55.00 46.59
M
40.00 40.00 50.00 30.00 40.00 30.00 50.00 40.00 30.00 30.00 56.82 40.00 40.00 40.00 50.00 40.00 50.00 40.00 23.18 40.00
X3
d
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
X2
te
X1
X1= Time (min), X2= Temperature (°C), X3= pH.
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Run
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Table 2 Analysis of variance for the experimental results of the central-composite design
219
1 1 1 1 1 1 1 1 1 9 5 5
27.77 0.73 1.60 6.72 233.30 125.70 5.82 0.32 7.13 41.29 1.97
*Statistically significant at 95% of probability level.
221
**Statistically significant at 99% of probability level.
222
X1= Time (min), X2= Temperature (°C), X3= pH.
225 226
0.0004 0.4136 0.2340 0.0269 <0.0001 <0.0001 0.0365 0.5816 0.0235 <0.0001 0.2380
*
* ** ** *
* **
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Significance
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P>F
ip t
42.40 1.11 2.45 10.26 356.18 191.90 8.88 0.50 10.88 567.38 10.12 5.15
F value
cr
X1 X2 X3 X1 2 X2 2 X3 2 X1×X2 X1×X3 X2×X3 Model Lack of Fit Pure Error
Degree of freedom
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Sum of square
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Factora
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Figure captions
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Fig. 1. Effect of cellulase amount on extraction of dandelion polysaccharides. Bars represent the
228
standard deviation. Data are shown as mean ± SD (n = 3).
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Fig. 2. Antibacterial activity of dandelion polysaccharides. Bars represent the standard
230
deviation. Different letters on the bars indicate the significant differences (P<0.05). Data are
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shown as mean ± SD (n = 3).
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Fig. 1
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Yield (%)
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0 1000
2000
3000
4000
Cellulase amount (U/g)
5000
6000
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Fig. 2
237 18
a
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Inhibition zone diameter (mm)
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6 4
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2 0 Bacillus subtilis
Staphylococcus aureus
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Escherichia coli
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Highlights
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> The PD can be effectively extracted by cellulase-assisted extraction. > The PD
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product was partially characterized by component. > The PD product has high
244
antibacterial ability.
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