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PREPARATION AND CHARACTERIZATION OF CARBAMOYLETHYLATED AND CARBOXYETHYLATED KONJAC MANNAN
PREPARATION AND CHARACTERIZATION OF CARBAMOYLETHYLATED AND CARBOXYETHYLATED KONJAC MANNAN Shoji Takigami ', Yoshifumi Suzuki ', Akira Igarashi' and Kiyoshi Mlyashita' 'Techni('(/I Research Centerfor lnstrunrentul Analvsis. Gnnm« Universitv, Kirvu. Gunma 376-8518. Japan
2Gllnl11a Prefecture Industrial Technology Research Laboratory. Maebashi. Glllll11a 371-0845. Japan
INTRODUCTION
Konjac mannan (KM) is the main component of konjac flour obtained from the tuber of the konjac plant ( Amorphohal/us Konjac K. Kock).
It is a heteropolysaccharide
consisting of I3-D-glucose (G) and I3-D-mannose (M), with a G/M ratio of I to 1.61.2 or 2 to 3 3 _ KM forms irreversible gels by alkali treatment. It also interacts synergistically with xanthan gum
4.5
and kappa carrageenan" and makes thermoreversible gels.
Chemical modification of konjac mannan (i.e., acetylation 7,8 methylation':", and nitration 10) has been carried out in order to study the chemical structure and molecular weight. KM,
However, there are few investigations on the gelation of chemically modified In this study, KM was reacted with acry\amide in the presence of NaOH and the
substitution reaction was investigated. The gelation behavior of the carbamoylethylated and carboxyethylated KM with various degree of substitution (D.S.) was also examined. EXPERIMENTAL Materials
Commercial konjac flour (Seiko) supplied by Ogino Shoten Co. Ltd. (Gunma, Japan) was used as a starting material.
The flour was made from konjac tubers of the Akagi
Ohodama species. The flour was washed with 30% methanol aqueous solution twice
62
Synthesis and derivatisation of biocompatible polymers
and then with 70% iricthanol aqueous solution hefoi-e air-drying.
The konjac inannan
sample was thus obtained. Acrylamide was a rcagcnt grade and other chemicals used were special grade (Wako Pure Chemical Industries, Ltd.. Japan). They were used without further purification.
Carbamoylethylation and carboxyethylation of konjac mannan Konjac mannan ( 2 2 ) w a s dissolved i n dihtilltd water (198g) at 30°C and 20% sodium hydroxide aqueous solution (7OOg) was added.
A solution of acrylamide (6.2 g in 10 g
of water) was added and the mixture was allowed to stand for a programmed time at
30 "C with stirring. After the fixed time, the reaction mixture was neutralized with 6
M hydrochloric acid.
The solution was dialyzed against deionized water until the
dialysate was free of chloride ions and then freeze-dried. The nitrogen content of the reacted product was determined using a CORDER MT-5 elemental analyzer (YANACO, Japan).
The amount of carboxyl groups in the reacted
KM was determined by conductometric titration using a CM-60s conductivity meter (TOA, Japan) with 0.01M sodium hydroxide aqueous solution.
The amount of
carbamoylethyl and carboxyethyl groups in the product was determined as the degree of substitution (D.S.) per pyranose unit.
Fourier transform infrared (FT-IR) microscopy
FT-IR reflection spectra of carbamoylethylated and carboxyethylated KM were analyzed by the attenuated total reflection (ATR) method.
The FT-IR measurements
were carried out using a Magna750 FT-IR spectrophotometer equipped with a Nic-Plan infrared microscope (Nicolet).
A Ge polarizer was used as a high refraclive index
material.
Gel to sol transition temperature The gel to sol transition temperature of a mixture of reacted KM and xanthan gum aqueous solution was determined by the falling-ball method.
0.5% reacted KM and
0.5% xanthan aqueous solutions with equivalent weight were mixed at 75°C.
The
mixture was put into a glass tube (IOmm in diameter) with a stainless steel ball (0.1Ig
63
Carbamoylethylated and carboxyethylated konjac mannan
and 7 mm in diamcter) and then it was sealed. The position of the ball was mcasured with elevating temperature at the heating rate of 0. I " C h i n using a cathetornetcr.
The
transition temperature was defined as the initial temperalure that the ball began to fall and was estimated by the extrapolation of change of the height.
RESULTS AND DISCUSSION Addition reaction of acrylamide onto konjac mannan FT-IR spectra of KM and carbamoylethylated and carboxyethylated KM are shown i n Figure 1.
KM shows absorption due to the stretching vibration of the C=O bond the in
acetyl group at 1730 cm" and of OH groups of bound water and the pyranose ring at 1650 cm.' and near 1100 cm.' (spectrum a), respectively.
The acetyl group was
released under alkali conditions and a peak of the stretching vibration of the C=O amide bond appeared at 1670 cm-' (spectra b and c).
After 1 h, a new absorption due to
stretching vibration of the C=O bond in the carboxyl group appeared at 1720 cm" (spectrum d) and the intensity of the peak increased with increasing reaction time (spectra e to g).
The absorption due to the amide group was not observed after 6 h
(spectrum h) and the IR spectra of the KM reacted more than 12 h showed the same
2000
1500
1000
Wave number (cm-') Figure 1. FT-IR spectra of carbamoylethylated and carboxyethylated konjac mannan reacted for various time. a: original KM, reacted for: b: 15min. c: 30min, d: I h. e: 1.5 h, f: 2 h, g: 3 h, h: 6h, i: 12h, j: 24 h
64
Synthesis and derivatisation of biocompatible polymers
0
5
10
15
20
25
Reaction time (h) Relationships between degree of substitutions and reaction time for carbamoylethylated and carboxyethylated kon.jac mannan. 3:carbamoylethyl group, 0: carboxyethyl group. A:total
Figure 2 .
pattern (spectra i and j).
The amide group and carboxyl group belong to the
carbamoylethyl group and the carboxyethyl group, respectively. Figure 2 shows the relationships between degree of substitution (D.S.) and the reaction time
for carbamoylethylated and carboxyethylated KM.
The
D.S. of the
carbamoylethyl group increased with reaction time and showed a maximum at 2 h and then decreased rapidly. On the other hand, the carboxyethyl group was detected after 30 min.
The D.S. of carboxyethyl group increased remarkably and became almost
constant values after 6 h. This is due to hydrolysis of carbamoylethyl group to a carboxyethyl group. values after 3 h. complete in 3 h.
'
The total D.S. increased with reaction time and reached constant This means the addition reaction of acrylamide onto KM was
Carbamoylethylatlon and carboxyethylation of KM are carried out as
follows' .
KM-OH
+
CH?=CHCONH2
NaOH
______)
(carbamoylethylated KM)
(konjac mannan) KM-O-CH,CH,CONH,
KM-O-CHlCH2CONH2
NaOH
t-
KM-O-CHlCH2COOI-1 (carboxyethylated KM)
Carbamoylerhylatedand carboxyethylated konjac mannan
65
Gel formation of reacted konjac mannan with xanthan gum The mixture of 0S% KM and 0.5% xanthan gum aqueous solutions formed a thei-inoreversible elastic gel and showed
; I
gel to sol Iransition tempei-ature (Tsol) a1
61 "C. The gel strength and T S Ofor ~ the mixture of reacted KM and xanthan gum with the same composition decreased with reaction time and the KM reacted for more than 3 h could not form a gel with xanthan gum.
Figure 3 shows the relationship between
TSOIand total D.S. of reacted KM. The Tsol decreased a little with increasing total D.S and then decreased rapidly when the total D.S. became greater than 0.1. presumed to be the influence of carboxyethyl group.
The relationship between Tsoi
and the D.S. of the carboxyethyl group is shown in Figure 4. remarkably
This can be
The TSOIdecreased
in the presence of a small amount of carboxyethyl
group and
carboxyethylated KM could not form a synergistic gel with xanthan gum. The gel formation is restricted by the electrostatic repulsion between the carboxyethyl groups on the reacted KM and the carboxyl groups on xanthan gum. However, since Tsol of the mixture of carbamoylethylated KM which is a nonelectrolyte and xanthan gum decreased a little, it was presumed that the gel formation is 70 60 0
v
f
'k a
50
U
40
2
30
*=
20
C
.-0 2 2
t-r . 10
0
I
0
I
0.05
I
0.10
I
0.15
0.20
Total D.S. per pyranose unit Figure 3. Relationship between gel to sol transition temperature and total degree of substitution for carbamoylethylated and carboxyethylated konjac mannan.
0
0.02 0.04 0.06 0.08 0.10 D.S. of carboxyethyl group per pyranose unit
Figure 4. Relationship between gel to sol transition temperature and degree of substitution of carboxyethyl group for carbamoylethylated and carboxyethylated konjac mannan.
66
Synthesis and denvatisation of biocornpalible polymers
a l w affected by the steric hindrance of the suhstituent groups introduced on KM by thc
addition reaction.
REFERENCES I. K. Kato and K. Matsuda. 'Chemical structure o f konjac mannan. I. Isolation and
characterization of oligosaccharides from the partial acid hydrolyzatc of manniln'. A y . . Biof. CImi.. 1969. 35, 1446-53.
2. H. Shiniahara, H. Suzuki. N. Sugiyama and K. Nishizawa. 'Mannan and related compounds. IV. Isolation and characterization of oligosaccharides from an enzymic hydrolysate of konjac glucomannan', Agc Biol. Cheru., 1975, 39. 293-9.
3. F. Smith and H. Srivastava. 'Constitutional studies on the glucomannan of konjac flour, J. AJIZ.Chern. Soc., 1959, 81. 1715-18.
4. G. Brownsey. P. Cairns, M.J. Miles, V.J. Morris, 'Evidence for intermolecular binding between xanthan and the glucomannan konjac mannan', Curhohvdrute Research, 1988, 176, 329-34. 5. P. A. Williams, D. H. Day, M. J. Langdon, G. 0. Phillips and K. Nishinari,
'Synergistic interaction of xanthan gum with glucomannans and galactomannans'. Food Hydrocolloids, 199I , 4,489-93. 6. K. Kohyama, H. Iida and K. Nishinari, 'A mixed system composed of different
molecular weights konjac glucomannan and kappa carrageenan: large deformation and dynamic viscoelastic study' Food Hydrocolloids. 1993, 7, 2 13-26.
7. N. Sugiyama, H. Shimahara, T. Andoh, M. Takemoto and T. Kamata, 'Molecular weights of konjac mannans of various sources', Agr. B i d . Chern., 1972, 36, 1381-87.
8. K. Kato and K. Matsuda, 'Isolation of oligosaccharides corresponding to the branching-point of konjac mannan' Agr. Biol. Chern., 1973,37, 2045-5 I .
9. N. Kishida, S. Okimasu and T. Kamata, 'Molecular weight and intrinsic viscosity of konjac gluco-mannan', Agr. Biol. Chem., 1978, 42, 1645-50. 10. H. Torigata, H. Inagaki and N. Kitano, 'Study of konjac mannan IV.
Molecular
weight and molecular form of nitrated konjac mannan', Nippotz Kngnku Znsslzi, 195 1,
73,30-32. I I . M. Shimada, H. Kuribara, S. Takigami and Y. Nakamura, 'Fine structure of
carbamoylethylated and carboxyethylated cotton cellulosic fibers', Sen-i Gnkkuishi,
1977.33, T- 109- 14.
2Gllnl11a Prefecture Industrial Technology Research Laboratory. Maebashi. Glllll11a 371-0845. Japan
INTRODUCTION
Konjac mannan (KM) is the main component of konjac flour obtained from the tuber of the konjac plant ( Amorphohal/us Konjac K. Kock).
It is a heteropolysaccharide
consisting of I3-D-glucose (G) and I3-D-mannose (M), with a G/M ratio of I to 1.61.2 or 2 to 3 3 _ KM forms irreversible gels by alkali treatment. It also interacts synergistically with xanthan gum
4.5
and kappa carrageenan" and makes thermoreversible gels.
Chemical modification of konjac mannan (i.e., acetylation 7,8 methylation':", and nitration 10) has been carried out in order to study the chemical structure and molecular weight. KM,
However, there are few investigations on the gelation of chemically modified In this study, KM was reacted with acry\amide in the presence of NaOH and the
substitution reaction was investigated. The gelation behavior of the carbamoylethylated and carboxyethylated KM with various degree of substitution (D.S.) was also examined. EXPERIMENTAL Materials
Commercial konjac flour (Seiko) supplied by Ogino Shoten Co. Ltd. (Gunma, Japan) was used as a starting material.
The flour was made from konjac tubers of the Akagi
Ohodama species. The flour was washed with 30% methanol aqueous solution twice
62
Synthesis and derivatisation of biocompatible polymers
and then with 70% iricthanol aqueous solution hefoi-e air-drying.
The konjac inannan
sample was thus obtained. Acrylamide was a rcagcnt grade and other chemicals used were special grade (Wako Pure Chemical Industries, Ltd.. Japan). They were used without further purification.
Carbamoylethylation and carboxyethylation of konjac mannan Konjac mannan ( 2 2 ) w a s dissolved i n dihtilltd water (198g) at 30°C and 20% sodium hydroxide aqueous solution (7OOg) was added.
A solution of acrylamide (6.2 g in 10 g
of water) was added and the mixture was allowed to stand for a programmed time at
30 "C with stirring. After the fixed time, the reaction mixture was neutralized with 6
M hydrochloric acid.
The solution was dialyzed against deionized water until the
dialysate was free of chloride ions and then freeze-dried. The nitrogen content of the reacted product was determined using a CORDER MT-5 elemental analyzer (YANACO, Japan).
The amount of carboxyl groups in the reacted
KM was determined by conductometric titration using a CM-60s conductivity meter (TOA, Japan) with 0.01M sodium hydroxide aqueous solution.
The amount of
carbamoylethyl and carboxyethyl groups in the product was determined as the degree of substitution (D.S.) per pyranose unit.
Fourier transform infrared (FT-IR) microscopy
FT-IR reflection spectra of carbamoylethylated and carboxyethylated KM were analyzed by the attenuated total reflection (ATR) method.
The FT-IR measurements
were carried out using a Magna750 FT-IR spectrophotometer equipped with a Nic-Plan infrared microscope (Nicolet).
A Ge polarizer was used as a high refraclive index
material.
Gel to sol transition temperature The gel to sol transition temperature of a mixture of reacted KM and xanthan gum aqueous solution was determined by the falling-ball method.
0.5% reacted KM and
0.5% xanthan aqueous solutions with equivalent weight were mixed at 75°C.
The
mixture was put into a glass tube (IOmm in diameter) with a stainless steel ball (0.1Ig
63
Carbamoylethylated and carboxyethylated konjac mannan
and 7 mm in diamcter) and then it was sealed. The position of the ball was mcasured with elevating temperature at the heating rate of 0. I " C h i n using a cathetornetcr.
The
transition temperature was defined as the initial temperalure that the ball began to fall and was estimated by the extrapolation of change of the height.
RESULTS AND DISCUSSION Addition reaction of acrylamide onto konjac mannan FT-IR spectra of KM and carbamoylethylated and carboxyethylated KM are shown i n Figure 1.
KM shows absorption due to the stretching vibration of the C=O bond the in
acetyl group at 1730 cm" and of OH groups of bound water and the pyranose ring at 1650 cm.' and near 1100 cm.' (spectrum a), respectively.
The acetyl group was
released under alkali conditions and a peak of the stretching vibration of the C=O amide bond appeared at 1670 cm-' (spectra b and c).
After 1 h, a new absorption due to
stretching vibration of the C=O bond in the carboxyl group appeared at 1720 cm" (spectrum d) and the intensity of the peak increased with increasing reaction time (spectra e to g).
The absorption due to the amide group was not observed after 6 h
(spectrum h) and the IR spectra of the KM reacted more than 12 h showed the same
2000
1500
1000
Wave number (cm-') Figure 1. FT-IR spectra of carbamoylethylated and carboxyethylated konjac mannan reacted for various time. a: original KM, reacted for: b: 15min. c: 30min, d: I h. e: 1.5 h, f: 2 h, g: 3 h, h: 6h, i: 12h, j: 24 h
64
Synthesis and derivatisation of biocompatible polymers
0
5
10
15
20
25
Reaction time (h) Relationships between degree of substitutions and reaction time for carbamoylethylated and carboxyethylated kon.jac mannan. 3:carbamoylethyl group, 0: carboxyethyl group. A:total
Figure 2 .
pattern (spectra i and j).
The amide group and carboxyl group belong to the
carbamoylethyl group and the carboxyethyl group, respectively. Figure 2 shows the relationships between degree of substitution (D.S.) and the reaction time
for carbamoylethylated and carboxyethylated KM.
The
D.S. of the
carbamoylethyl group increased with reaction time and showed a maximum at 2 h and then decreased rapidly. On the other hand, the carboxyethyl group was detected after 30 min.
The D.S. of carboxyethyl group increased remarkably and became almost
constant values after 6 h. This is due to hydrolysis of carbamoylethyl group to a carboxyethyl group. values after 3 h. complete in 3 h.
'
The total D.S. increased with reaction time and reached constant This means the addition reaction of acrylamide onto KM was
Carbamoylethylatlon and carboxyethylation of KM are carried out as
follows' .
KM-OH
+
CH?=CHCONH2
NaOH
______)
(carbamoylethylated KM)
(konjac mannan) KM-O-CH,CH,CONH,
KM-O-CHlCH2CONH2
NaOH
t-
KM-O-CHlCH2COOI-1 (carboxyethylated KM)
Carbamoylerhylatedand carboxyethylated konjac mannan
65
Gel formation of reacted konjac mannan with xanthan gum The mixture of 0S% KM and 0.5% xanthan gum aqueous solutions formed a thei-inoreversible elastic gel and showed
; I
gel to sol Iransition tempei-ature (Tsol) a1
61 "C. The gel strength and T S Ofor ~ the mixture of reacted KM and xanthan gum with the same composition decreased with reaction time and the KM reacted for more than 3 h could not form a gel with xanthan gum.
Figure 3 shows the relationship between
TSOIand total D.S. of reacted KM. The Tsol decreased a little with increasing total D.S and then decreased rapidly when the total D.S. became greater than 0.1. presumed to be the influence of carboxyethyl group.
The relationship between Tsoi
and the D.S. of the carboxyethyl group is shown in Figure 4. remarkably
This can be
The TSOIdecreased
in the presence of a small amount of carboxyethyl
group and
carboxyethylated KM could not form a synergistic gel with xanthan gum. The gel formation is restricted by the electrostatic repulsion between the carboxyethyl groups on the reacted KM and the carboxyl groups on xanthan gum. However, since Tsol of the mixture of carbamoylethylated KM which is a nonelectrolyte and xanthan gum decreased a little, it was presumed that the gel formation is 70 60 0
v
f
'k a
50
U
40
2
30
*=
20
C
.-0 2 2
t-r . 10
0
I
0
I
0.05
I
0.10
I
0.15
0.20
Total D.S. per pyranose unit Figure 3. Relationship between gel to sol transition temperature and total degree of substitution for carbamoylethylated and carboxyethylated konjac mannan.
0
0.02 0.04 0.06 0.08 0.10 D.S. of carboxyethyl group per pyranose unit
Figure 4. Relationship between gel to sol transition temperature and degree of substitution of carboxyethyl group for carbamoylethylated and carboxyethylated konjac mannan.
66
Synthesis and denvatisation of biocornpalible polymers
a l w affected by the steric hindrance of the suhstituent groups introduced on KM by thc
addition reaction.
REFERENCES I. K. Kato and K. Matsuda. 'Chemical structure o f konjac mannan. I. Isolation and
characterization of oligosaccharides from the partial acid hydrolyzatc of manniln'. A y . . Biof. CImi.. 1969. 35, 1446-53.
2. H. Shiniahara, H. Suzuki. N. Sugiyama and K. Nishizawa. 'Mannan and related compounds. IV. Isolation and characterization of oligosaccharides from an enzymic hydrolysate of konjac glucomannan', Agc Biol. Cheru., 1975, 39. 293-9.
3. F. Smith and H. Srivastava. 'Constitutional studies on the glucomannan of konjac flour, J. AJIZ.Chern. Soc., 1959, 81. 1715-18.
4. G. Brownsey. P. Cairns, M.J. Miles, V.J. Morris, 'Evidence for intermolecular binding between xanthan and the glucomannan konjac mannan', Curhohvdrute Research, 1988, 176, 329-34. 5. P. A. Williams, D. H. Day, M. J. Langdon, G. 0. Phillips and K. Nishinari,
'Synergistic interaction of xanthan gum with glucomannans and galactomannans'. Food Hydrocolloids, 199I , 4,489-93. 6. K. Kohyama, H. Iida and K. Nishinari, 'A mixed system composed of different
molecular weights konjac glucomannan and kappa carrageenan: large deformation and dynamic viscoelastic study' Food Hydrocolloids. 1993, 7, 2 13-26.
7. N. Sugiyama, H. Shimahara, T. Andoh, M. Takemoto and T. Kamata, 'Molecular weights of konjac mannans of various sources', Agr. B i d . Chern., 1972, 36, 1381-87.
8. K. Kato and K. Matsuda, 'Isolation of oligosaccharides corresponding to the branching-point of konjac mannan' Agr. Biol. Chern., 1973,37, 2045-5 I .
9. N. Kishida, S. Okimasu and T. Kamata, 'Molecular weight and intrinsic viscosity of konjac gluco-mannan', Agr. Biol. Chem., 1978, 42, 1645-50. 10. H. Torigata, H. Inagaki and N. Kitano, 'Study of konjac mannan IV.
Molecular
weight and molecular form of nitrated konjac mannan', Nippotz Kngnku Znsslzi, 195 1,
73,30-32. I I . M. Shimada, H. Kuribara, S. Takigami and Y. Nakamura, 'Fine structure of
carbamoylethylated and carboxyethylated cotton cellulosic fibers', Sen-i Gnkkuishi,
1977.33, T- 109- 14.