Synthesis and antifungal activity of thiadiazole-functionalized chitosan derivatives

Article abstract of DOI:10.1016/j.carres.2013.03.001

A groups of novel water soluble chitosan derivatives containing 1,3,4-thiadiazole group were synthesized including 1,3,4-thiadiazole (TPCTS), 2-methyl-1,3,4-thiadiazole (MTPCTS), and 2-phenyl-1,3,4-thiadiazole (PTPCTS). Their antifungal activity against three kinds of phytopathogens was estimated by hypha measurement in vitro, and the fungicidal assessment shows that the synthesized chitosan derivatives have excellent activity against tested fungi. Of all the synthesized chitosan derivatives, MTPCTS inhibited the growth of the tested phytopathogens most effectively with inhibitory indices of 75.3%, 82.5%, and 65.8% against Colletotrichum lagenarium (Pass) Ell.et halst, Phomopsis asparagi (Sacc.) Bubak, and Monilinia fructicola (Wint.) Honey respectively at 1.0 mg/mL. These indices are higher than those of chitosan. These data also demonstrate that the hydrophobic moiety (alkyl and phenyl) and the length of alkyl substituent in thiadiazole tend to affect the antifungal activity of chitosan derivatives. It is hypothesized that thiadiazole groups enable the synthesized chitosan to possess obviously better antifungal activity and good solubility in water.

Full text of DOI:10.1016/j.carres.2013.03.001

Carbohydrate Research 373 (2013) 103–107
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and antifungal activity of thiadiazole-functionalized
chitosan derivatives
Qing Li a, Jianming Ren a,b, Fang Dong a, Yan Feng a,b, Guodong Gu c, Zhanyong Guo a,
⇑
a Key Laboratory of Coastal Biology and Bioresource Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai, Shangdong 264003, China
b Graduate School of Chinese Academy of Sciences, Beijing 100039, China
c C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, WV 26506, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A groups of novel water soluble chitosan derivatives containing 1,3,4-thiadiazole group were synthesized
including 1,3,4-thiadiazole (TPCTS), 2-methyl-1,3,4-thiadiazole (MTPCTS), and 2-phenyl-1,3,4-thiadia-
zole (PTPCTS). Their antifungal activity against three kinds of phytopathogens was estimated by hypha
measurement in vitro, and the fungicidal assessment shows that the synthesized chitosan derivatives
have excellent activity against tested fungi. Of all the synthesized chitosan derivatives, MTPCTS inhibited
the growth of the tested phytopathogens most effectively with inhibitory indices of 75.3%, 82.5%, and
65.8% against Colletotrichum lagenarium (Pass) Ell.et halst, Phomopsis asparagi (Sacc.) Bubak, and Monilinia
fructicola (Wint.) Honey respectively at 1.0 mg/mL. These indices are higher than those of chitosan. These
data also demonstrate that the hydrophobic moiety (alkyl and phenyl) and the length of alkyl substituent
in thiadiazole tend to affect the antifungal activity of chitosan derivatives. It is hypothesized that thiadi-
azole groups enable the synthesized chitosan to possess obviously better antifungal activity and good sol-
ubility in water.
Received 9 December 2012
Received in revised form 22 February 2013
Accepted 1 March 2013
Available online 21 March 2013
Keywords:
Chitosan derivatives
Antifungal activity
Thiadiazole
Water solubility
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
have been prepared such as N,N,N-trimethyl chitosan, N,O-
(acyl)chitosan, hydroxyethylacryl chitosan, acyl thiourea chitosan,
Plant pathogenic fungi negatively affect a large number of
important fruits and vegetables, and limit crop production world-
wide, especially in developing countries. For example, Phomopsis
asparagi can cause stem blight of asparagus and lead to yield loss;
the premature plant death caused by Colletotrichum lagenarium can
result in a yield loss of 6–48%.1,2 The loss caused by plant patho-
genic fungi has attracted wide attention and chemical pesticides
have been extensively used to control the growth of these fungi.
The resulted pesticide residues, however, give rise to a potential
risk for environment and human health. Therefore, the search of
potential strategies to develop natural alternatives for chemical
pesticides has been one of the focuses of current research.3,4
Chitosan is one of the most abundant natural polysaccharides
and has been drawing broad attention due to its antifungal activity
against various groups of pathogenic fungi.5–10 The antifungal abil-
ity, coupled with its non-toxicity, biodegradability, and biocompat-
ibility, facilitates its emerging applications in food science,
agriculture, medicine, pharmaceuticals, and textile areas.11 An-
other advantage of chitosan, which makes it highly desirable, is
the capability of being chemically modified.12 By performing
chemical modification, a large number of chitosan derivatives
and N-heterocyclic chitosan to promote the antifungal activity of
chitosan.13–17 However, the application of chitosan is limited for
its poor solubility in water. Therefore, it is strongly desired, that,
chitosan derivatives with high antifungal ability and good water
solubility are developed.
1,3,4-Thiadiazole and its derivatives are known to possess var-
ious pharmacological activities such as antifungal, antibacterial,
anti-inflammatory, anticancer, and antitubercular.18–25 It is also re-
ported that antiviral and antibacterial activity of the thiourea
derivatives is due to the presence of –NH–C(S)–NH function in
the molecules and the change of the activity depends on the nature
of the substituent.19 These observations inspired us to modify
chitosan with thiadiazole as a substituent to improve antifungal
activity of chitosan derivatives. Moreover, as hydrophilic moiety
at the end of the molecular chains, thiadiazole can also enable
chitosan with better water solubility.
In this paper, we reported the synthesis and antifungal proper-
ties of a group of chitosan derivatives with 1,3,4-thiadiazole as
substituent including TPCTS, MTPCTS, and PTPCTS (Scheme 1).
Schiff bases (PCTS) was first synthesized by reaction between the
amino group of chitosan with 3-pyridinecarboxaldehyde. That
the Schiff bases functional groups were selected as bridges is also
by virtue of the antifungal activity of chitosan Schiff bases.26 Be-
sides, as a base with chemical properties similar to tertiary amines,
⇑
Corresponding author.
E-mail address: qdioqdio@yahoo.com.cn (Z. Guo).
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.03.001

104
Q. Li et al. / Carbohydrate Research 373 (2013) 103–107
CH2OH
O
CH2OH
CHO
O
OH
O
OH
O
N
n
n
N
NH2
N
C
H
PCTS
R
CH2OH
O
S
HN
O
H
S
Cl
S
NH2
N
R
N
R
Cl
N
PCTS
Cl
OH
O
N
Cl-
N
N
O
N
N
n
O
N
C
1
H
2
A: R = H
TPCTS: R = H
B: R = methyl
C: R = phenyl
MTPCTS: R = methyl
PTPCTS: R = phenyl
Scheme 1. Synthetic routes for PCTS, TPCTS, MTPCTS, and PTPCTS.
pyridine is easily attacked by alkylating agents to give N-alkylpy-
161.6 ppm (carbon of C@N). d 152.0, 150.2, 135.1, 131.7, and
124.4 ppm (pyridine ring carbons).30 d 60.1–102.0 ppm (pyranose
ridinium salts.27 Subsequently the thiadiazole groups were intro-
duced
into
chitosan
through
the
reaction
between
rings); FT-IR (thin film):
v
3424 (NH2 and OH),
v 1646 (C@N), v
chloroacetamide and pyridine of PCTS. The desired chitosan deriv-
atives designed in this way were expected to have advantageous
features such as high antifungal activity and good water solubility.
The chemical structures of the derivatives were characterized by
FT-IR and 13C NMR. Three common plant-threatening fungi, C.
lagenarium, P. asparagi, and Monilinia fructicola, were selected to
evaluate the antifungal properties of the derivatives by hypha
measurement in vitro.
1423, 1376 (C–H),
2).
2-Amino-1,3,4-thiadiazole and its derivatives (1) were prepared
from substituted aryl acid and thiosemicarbazide in presence of
dehydrating agent phosphorus oxychloride (POCl3).31
v 1592, 806, 701 (pyridine ring) (Figures 1 and
2.3.1. General procedure for synthesis of 232
2-Chloroacetyl chloride (24 mmol) was slowly added drop wise
to a mixture of 1 (20 mmol) in anhydrous dioxane (20 mL) at 0 °C.
The reaction mixture was warmed to room temperature and stir-
red for an additional 12 h. The products were filtered and washed
with dichloromethane. The crude products were purified by crys-
tallization from a mixture solvent of ethanol/H2O.
2. Experimental
2.1. Materials
Chitosan was purchased from Qingdao Baicheng Biochemical
Corp. (China). Its degree of deacetylation is 97% and the viscos-
ity-average molecular weight is 7.0 ꢀ 104. 3-Pyridinecarboxalde-
hyde was purchased from Aladin Chemical Corp. The other
reagents are all of analytical grade and used without further
purification.
2.3.1.1. 2-Chloro-N-(1,3,4-thiadiazol-2-yl)acetamide (2A). Yield:
75.1%; white solid; MS (ESI, m/z): [Mꢁ1]+ calcd for C4H4ClN3OS:
176.98; found 175.96.
2.3.1.2. 2-Chloro-N-(5-methyl-1, 3, 4-thiadiazol-2-yl)acetamide
(2B).
C5H6ClN3OS: 190.99; found 192.30.
Yield: 67.3%; white solid; MS (ESI, m/z): [M+1]+ calcd for
2.2. Analytical methods
FT-IR spectra were measured on a Jasco-4100 Fourier Transform
Infrared Spectrometer (Japan, provided by JASCO Co., Ltd Shanghai,
China) with KBr disks. ESI mass spectra were collected on a Ther-
moFisher LCQ Fleet Mass Spectrometer (Thermo fisher, America).
13C Nuclear magnetic resonance (13C NMR) spectra were measured
with a Bruker AVIII-500 Spectrometer (Switzerland, provided by
Bruker Tech. and Serv. Co., Ltd Beijing, China.). The elemental anal-
yses (C, H, N, and S) were performed on a Vario Micro Elemental
Analyzer (Elementar, Germany). The Degree of Substitution (DS)
was calculated based on elemental analysis results, which is ac-
quired by following Fonseca et al.’s method.28
CTS
PCTS
TPCTS
MTPCTS
PTPCTS
2.3. The synthesis of the derivatives of chitosan
PCTS was synthesized as follows:29 Chitosan (3 g) was dissolved
in 200 mL 2% acetic acid-aqua and 60 mL ethanol at room temper-
ature, 2.7 mL 3-pyridinecarboxaldehyde was then added with stir-
ring. After 6 h, products were precipitated using excess ethanol and
filtered. The unreacted aldehydes were extracted in a Soxhlet
apparatus with ethanol and ether for 2 days. The products were
dried at 60 °C for 24 h, yield: 73.1%; DS: 67%; 13C NMR/DMSO: d
4000
3000
2000
1000
-1
Wavenumber (cm )
Figure 1. FT-IR spectra of chitosan, PCTS, TPCTS, MTPCTS, and PTPCTS.

Q. Li et al. / Carbohydrate Research 373 (2013) 103–107
105
Figure 2. 13C NMR spectra of PCTS, TPCTS, MTPCTS, and PTPCTS.
2.3.1.3. 2-Chloro-N-(5-phenyl-1,3,4-thiadiazol-2-yl)acetamide
(2C).
Yield: 54.3%; white solid; MS (ESI, m/z): [M+1]+ calcd
for C10H8ClN3OS: 253.01; found 254.33.
film):
(C@N),
dine ring),
v
3424 (NH2 and OH),
1425, 1365 (C–H),
620 (C–S).
v
1689 (C@O of acylamide),30,34
v 1596, 806, 705 (benzene ring and pyri-
v
1646
v
v
2.3.2. TPCTS, MTPCTS, and PTPCTS were synthesized as follows30
A solution of PCTS (10 mmol) and 2 (20 mmol) in 50 mL di-
methyl sulfoxide was stirred for 48 h at 60 °C. The solution was
collected by precipitation with excess acetone. The unreacted alde-
hyde was extracted in a Soxhlet apparatus with ethanol for two
days. The products were lyophilized.
2.4. Antifungal assays
Antifungal assays were performed by following the plate
growth rate method described by Guo et al.29 Briefly, the com-
pounds were dispersed in distilled water at a concentration of
5.0 mg/mL. Then, each sample (chitosan, TPCTS, MTPCTS, and
PTPCTS) solution was added to sterilized potato dextrose agar to
give a final concentration of 0.1, 0.5, and 1.0 mg/mL. After the mix-
ture was cooled in the plate (6.0 cm diameter), the mycelium of
fungi of 5.0 mm diameter was transferred to the test plate and
incubated at 27 °C for 2–3 days. When the mycelium of fungi
reached the edges of the control plate (without the presence of
samples), the antifungal index was calculated as follows:
2.3.2.1. TPCTS.
DS: 41.6%; 13C NMR/DMSO: d 169.3 ppm (C@O of
acylamide). d 164.1 ppm (carbon of C@N). d 121.1–158.8 ppm (pyri-
dine ring and thiadiazole ring carbons),30,33 d 53.2–98.6 ppm (pyranose
rings). The peak of methylene carbon in –NHCOCH2– was covered by
peaks of DMSO at 38–40 ppm; FT-IR (thin film):
OH), 1635 (C@N),
1700 (C@O of acylamide),30,34
1380 (C–H), 802 (pyridine ring), 628 (C–S).
v
3409 (NH2 and
1554 (N–H),
v
v
v
v
v
v
Antifungal index ð%Þ ¼ ð1 ꢁ Da=DbÞ ꢀ 100
DS: 35.7%; 13C NMR/D2O: d 174.7 ppm (C@O of
where Da is the diameter of the growth zone in the test plates and
Db is the diameter of the growth zone in the control plate. Each
experiment was performed three times, and the data were shown
with mean S.D. The Scheffe method was used to evaluate the dif-
ferences in antifungal index in the antifungal tests. Results with P
<0.05 were considered statistically significant.35
2.3.2.2. MTPCTS.
acylamide). d 164.8 ppm (carbon of C@N). d 127.4–145.8 ppm (pyri-
dine ring and thiadiazole ring carbons).30,33 d 55.9–97.7 ppm (pyranose
rings). d 38.8 ppm (methylene carbon in –NHCOCH2–). d 22.2 ppm
(methylene carbon in thiadiazole ring); FT-IR (thin film):
(NH2 and OH), 1639 (C@N),
1727 (C@O of acylamide),30,34
(N–H), 1419, 1376 (C–H), 802 (pyridine ring), 620 (C–S).
v
v
3397
1531
v
v
v
v
v
3. Results and discussion
2.3.2.3. PTPCTS.
DS: 15.4%; 13C NMR/DMSO: d 169.8 ppm (C@O
of acylamide). d 162.8 ppm (carbon of C@N). d 127.4, 129.9, 131.4 and
3.1. Antifungal activity and solubility
136.9 ppm (benzene ring carbons). 125.0, 126.87, 144.6, 147.9, 151.8,
154.7 and 158.4 (pyridine ring and thiadiazole ring carbons).31,35
56.2–98.6 ppm (pyranose rings). The peak of methylene carbon in –
NHCOCH2– was covered by peaks of DMSO at 38–40 ppm; FT-IR (thin
d
Chitosan has poor solubility in water, so we used water-soluble
low molecular chitosan in antifungal activity test. PCTS also has
poor solubility in water, and its antifungal activity was soft against

106
Q. Li et al. / Carbohydrate Research 373 (2013) 103–107
C. lagenarium as chitosan concerned. PCTS also has poor solubility
in water and low antifungal activity against C. lagenarium com-
pared to chitin. TPCTS, MTPCTS, and PTPCTS have favorable water
solubility probably due to thiadiazole as the hydrophilic moiety at
the end of the molecular chains.
90
80
70
60
50
40
30
20
10
0
0.1 mg/mL
0.5 mg/mL
1.0 mg/mL
We tested the antifungal activity of the starting chitosan,
TPCTS, MTPCTS, and PTPCTS against three common plant-threat-
ening fungi C. lagenarium, P. asparagi, and M. fructicola. Control of
these plant-threatening fungi could benefit the production of re-
lated vegetables and fruits. The results are shown in Figures 3–5.
As seen in Figure 3, chitosan inhibited the growth of C. lagena-
rium and the inhibitory index is 31.6% at 1.0 mg/mL. The inhibitory
indices of all the samples enhanced with increasing concentration.
Compared with the antifungal activity of chitosan, the chitosan
derivatives had better activity, and the inhibitory indices of TPCTS,
MTPCTS, and PTPCTS are 58.6%, 75.3%, and 75.1% at 1.0 mg/mL,
respectively. It is obvious that TPCTS, MTPCTS, and PTPCTS show
much better antifungal activity due to the introduction of func-
tional groups—thiadiazole groups. As illustrated by the data, the
antifungal activity of MTPCTS is better than that of TPCTS. The
observation is in agreement with the results obtained by Sajom-
sang, who found that the antifungal activity of chitosan derivatives
against the plant pathogenic fungi increased with an increase in
the chain length of alkyl substituent.36 Meanwhile, it is apparent
that the inhibitory indices of PTPCTS with DS of 15.4% were almost
the same as that of MTPCTS with DS of 35.7%, suggesting that a
higher inhibitory index of PTPCTS should be possible if MTPCTS
and PTPCTS have the same DS. Results above indicate that the
hydrophobic moiety (methyl and phenyl) in thiadiazole is an
important factor in affecting the antifungal activity of chitosan
derivatives. It would be reasonable to presume that phenyl in thia-
diazole contributes more to the antifungal activity than methyl
since phenyl has better hydrophobicity.
Figures 4 and 5 show the antifungal activity of chitosan and all
the derivatives against P. asparagi and M. fructicola. All the samples
showed antifungal activity against P. asparagi and M. fructicola, and
the inhibitory indices of them mounted up with increasing concen-
tration. The inhibitory indices of chitosan, TPCTS, MTPCTS, and
PTPCTS against P. asparagi are 31.3%, 68.8%, 82.5%, and 77.6% at
1.0 mg/mL, respectively. And the inhibitory indices of chitosan,
TPCTS, MTPCTS, and PTPCTS against M. fructicola are 9.8%, 51.3%,
65.8%, and 60.6% at 1.0 mg/mL, respectively. Similar to the anti-
fungal activity against C. lagenarium, three chitosan derivatives
give much stronger antifungal activity than unmodified chitosan,
and the inhibitory indices of MTPCTS and PTPCTS are higher than
chitosan
TPCTS
MTPCTS
PTPCTS
Figure 4. The antifungal activity of chitosan, TPCTS, MTPCTS, and PTPCTS against P.
asparagi.
0.1 mg/mL
70
0.5 mg/mL
1.0 mg/mL
60
50
40
30
20
10
0
chitosan
TPCTS
MTPCTS
PTPCTS
Figure 5. The antifungal activity of chitosan, TPCTS, MTPCTS, and PTPCTS against
M. fructicola.
those of TPCTS at the same concentration. The results further con-
firm that (1) thiadiazole groups grafted into the synthesized chito-
san derivatives contribute a lot to the antifungal action and
consequently increase the antifungal activity of them; (2) the anti-
fungal activity of the synthesized chitosan derivatives increase
with an increase in the chain length of alkyl substituent; (3) the
antifungal activity should be affected by the hydrophobic moiety
at the periphery of the synthesized chitosan derivatives.
80
0.1 mg/mL
0.5 mg/mL
1.0 mg/mL
70
60
50
40
30
20
10
0
4. Conclusion
The environmental issues caused by pesticides, especially
chemical antifungal agents, cannot be ignored. New environ-
ment-friendly effective substitutes of chemical fungicides need to
be explored. In this paper, three novel chitosan derivatives with
1,3,4-thiadiazole as substituent were designed and synthesized,
and their antifungal activity against three kinds of phytopathogens
was estimated by hypha measurement in vitro. All the chitosan
derivatives have good solubility in water, and exhibit higher inhi-
bition indices than chitosan. These data demonstrate that the
chemical modification of chitosan with thiadiazole would lead to
an enhancement of the biological activity against some plant path-
ogenic fungi and better water solubility. In addition, the antifungal
activity of these chitosan derivatives is enhanced by the hydropho-
bic moiety (alkyl and phenyl) at the periphery of molecular chains,
chitosan
TPCTS
MTPCTS
PTPCTS
Figure 3. The antifungal activity of chitosan, TPCTS, MTPCTS, and PTPCTS against C.
lagenarium.

Q. Li et al. / Carbohydrate Research 373 (2013) 103–107
107
15. Ma, G. P.; Yang, D. Z.; Zhou, Y. S.; Xiao, M.; Kennedy, J. F.; Nie, J. Carbohydr.
Polym. 2008, 74, 121–126.
16. Zhong, Z. M.; Xing, R. E.; Liu, S.; Wang, L.; Cai, S. B.; Li, P. C. Carbohydr. Res. 2008,
343, 566–570.
17. Badawy, M. E. I. Polym. Int. 2008, 57, 254–261.
18. Pandey, A.; Rajavel, R.; Chandraker, S.; Dash, D. Eur. J. Chem. 2012, 9, 2524–
2531.
19. Shiv, K.; Gupta, P. K.; Sharma, M. B.; Kumar, B. Eur. J. Chem. 2011, 8, 594–597.
20. Shabban, M. R.; Saleh, T. S.; Farag, A. M. Heterocycles 2009, 78, 151–159.
21. Demirayak, S.; AbuMohsen, U.; Karaburun, A. C. Eur. J. Med. Chem. 2002, 37,
255–260.
which suggests that the hydrophobic properties of the grafted moi-
ety help improve the antifungal activity of chitosan derivatives.
Further study will be carried out to ascertain this hypothesis. These
findings mentioned above bring further evidence that chitosan
derivatives are active and have the potential of becoming alterna-
tives for disease control instead of some harmful pesticides.
Acknowledgments
22. Thomasco, M. L.; Godwood, C. R.; Weaver, A. E.; Ochoada, M. J.; Fard, W. C.;
Zurenko, G. E.; Hamel, C. J.; Stapert, D.; Moerman, K. J.; Schadt, D. R.; Yogi, H. B.
Bioorg. Med. Chem. Lett. 2003, 13, 4193–4196.
23. Gazzar, A. E.; Hegab, M. I. Bioorg. Med. Chem. Lett. 2008, 18, 4538–4543.
24. Alagarsamy, V.; Pathak, U. S. Indian J. Heterocycl. Chem. 2003, 12, 335–338.
25. Verma, K.; Jan, K.; Milan, P.; Jiri, S. Eur. J. Med. Chem. 2002, 37, 409–418.
26. Guo, Z. Y.; Ren, J. M.; Dong, F.; Wang, G.; Li, P. C. J. Appl. Polym. Sci. 2013, 127,
2553–2556.
We thank the National Natural Science Foundation of China
(41206152) and the project of National Science & Technology Pillar
Program (2011 BAC02B00) for financial support of this work.
References
27. Sajomsang, W. Carbohydr. Polym. 2010, 80, 631–647.
28. dos Santos, Z. M.; Caroni, A. L. P. F.; Pereira, M. R.; da Silva, D. R.; Fonseca, J. L. C.
Carbohydr. Res. 2009, 344, 2591–2595.
29. Guo, Z. Y.; Xing, R.; Liu, S.; Zhong, Z. M.; Ji, X.; Wang, L.; Li, P. C. Carbohydr. Res.
2007, 342, 1329–1332.
30. Li, R. C.; Guo, Z. Y.; Jiang, P. G. Carbohydr. Res. 2010, 345, 1896–1900.
31. Alegaon, S. G.; Alagawadi, K. R. Med. Chem. Res. 2012, 21, 816–824.
32. Ma, L.; Xie, C. F.; Ma, Y. H.; Liu, J.; Xiang, M. L.; Ye, X.; Zheng, H.; Chen, Z. Z.; Xu,
Q. Y.; Chen, T.; Chen, J. Y.; Yang, J. C.; Qiu, N.; Wang, G. C.; Liang, X. L.; Peng, A.
H.; Yang, S. Y.; Wei, Y. Q.; Chen, L. J. J. Med. Chem. 2011, 54, 2060–2068.
33. Jha, A.; Murthy, Y. L. N.; Sanyal, U.; Durga, G. Med. Chem. Res. 2012, 21, 2548–
2556.
34. Huang, R. H.; Du, Y. M.; Zheng, L. S.; Liu, H.; Fan, L. H. React. Funct. Polym. 2004,
59, 41–51.
35. de Rodriguez, D. J.; Hemandez-Castillo, D.; Rodriguez-Garcia, R.; Angulo-
Sanchez, J. L. Ind. Crop. Prod. 2005, 21, 81–87.
36. Sajomsang, W.; Gonil, P.; Saesoo, S.; Ovatlarnporn, C. Int. J. Biol. Macromol.
2012, 50, 263–269.
1. Chen, Y. J.; Dai, G. H. J. Sci. Food Agric. 2012, 92, 1937–1943.
2. Elena, K. Plant. Pathol. 2006, 55, 300.
3. Gan-Mor, S.; Matthews, G. A. Biosyst. Eng. 2003, 84, 119–125.
4. Kuchler, F.; Ralston, K.; Unnevehr, L. J. Food Policy 1997, 22, 119–132.
5. Teli, M. D.; Sheikh, J. Int. J. Biol. Macromol. 2012, 50, 1195–1200.
6. Ai, H.; Wang, F. R.; Xia, Y. Q.; Chen, X. M.; Lei, C. L. Food Chem. 2012, 132, 493–
498.
7. Chang, H. L.; Chen, Y. C.; Tan, F. J. Food Chem. 2011, 124, 589–595.
8. Chung, Y. C.; Kuo, C. L.; Chen, C. C. Bioresour. Technol. 2005, 13, 1473–1482.
9. Lin, S. B.; Lin, Y. C.; Chen, H. H. Food Chem. 2009, 116, 47–53.
10. Qin, C. Q.; Zhou, B.; Zeng, L. T.; Zhang, Z. H.; Liu, Y.; Du, Y. M.; Xiao, L. Food
Chem. 2004, 84, 107–115.
11. Gao, Y.; Cranston, R. Text. Res. J. 2008, 78, 60–72.
12. De Britto, D.; De Moura, M. R.; Aouada, F. A.; Mattoso, L. H. C.; Assis, O. B. G.
Food Hydrocolloids 2012, 27, 487–493.
13. Jia, Z.; Shen, D.; Xu, W. Carbohydr. Res. 2001, 333, 1–6.
14. Sashiwa, H.; Kawasaki, N.; Nakayama, A.; Muraki, E.; Yamamoto, N.; Zhu, H.;
Nagano, H.; Omura, Y.; Saimoto, H.; Shigemasa, Y.; Aiba, S. I. Biomacromolecules
2002, 3, 1120–1125.