Accepted Manuscript

Studies on O-acetyl-glucomannans from Amorphophallus species: Comparison of

physicochemical properties and primary structures

Xiao-Dan Shi, Jun-Yi Yin, Liu-Jing Zhang, Xiao-Jun Huang, Shao-Ping Nie

PII: S0268-005X(18)30891-9
DOI: https://doi.org/10.1016/j.foodhyd.2018.11.013
Reference: FOOHYD 4754

To appear in: Food Hydrocolloids

Received Date: 16 May 2018

Revised Date: 16 October 2018
Accepted Date: 5 November 2018

Please cite this article as: Shi, X.-D., Yin, J.-Y., Zhang, L.-J., Huang, X.-J., Nie, S.-P., Studies on O-
acetyl-glucomannans from Amorphophallus species: Comparison of physicochemical properties and
primary structures, Food Hydrocolloids (2018), doi: https://doi.org/10.1016/j.foodhyd.2018.11.013.

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 ACCEPTED MANUSCRIPT

Graphical Abstract

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1 Studies on O-acetyl-glucomannans from Amorphophallus

2 species: comparison of physicochemical properties and
3 primary structures

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4 Xiao-Dan Shi a, Jun-Yi Yin a,b, Liu-Jing Zhang a, Xiao-Jun Huang a, Shao-Ping Nie a,*

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a
5 State Key Laboratory of Food Science and Technology, China-Canada Joint Lab of

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6 Food Science and Technology (Nanchang), Nanchang University, 235 Nanjing East

7 Road, Nanchang, Jiangxi Province, 330047, China

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8 Department of Applied Biology and Chemical Technology, The Hong Kong

9 Polytechnic University, Kowloon, Hong Kong, China

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10 *Corresponding author (E-mail: spnie@ncu.edu.cn)

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11 Abstract

12 Plants in Amorphophallus genus are rich in soluble dietary fibres and have long been

13 used as food and traditional medicine in Asian countries. In the present investigation,

14 natural O-acetyl-glucomannans were extracted from tubers of Amorphophallus rivirei

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15 (konjac), A. albus and two local types of A. bulbifer (wild type M and wild type B),

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16 which were designated as KGM, AGM, MGM, and BGM, successively. Comparative

17 studies were conducted on their physicochemical properties and primary structures.

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18 The four AcGMs had about 90% (w/w) neutral sugar, 11% (w/w) moisture, and

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19 1%–2% (w/w) ash. Relatively high contents of acetyl groups (17%–24%, w/w) were
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20 detected. Mannose and glucose as the main components existed in molar ratios of

21 1.41, 1.41, 1.21, and 1.58 for KGM, AGM, MGM, and BGM, respectively. Relative
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22 molecular weights of AcGMs increased in the order of: KGM<AGM<MGM<BGM.

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23 Apparent viscosities of AcGMs were concentration dependent. MGM and BGM with
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24 higher molecular weights and acetyl groups contents had higher viscosities. Further

25 structural analysis showed that the four AcGMs had similar primary structure. The
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26 branches probably linked at O-2,6 of mannosyl residues and/or O-3,6 of glucosyl

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27 residues. The acetyl groups mainly attached to O-2 and O-3 of mannosyl residues.
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29 Keywords: Amorphophallus; Glucomannan; Acetyl group; Physicochemical properties;

30 Rheological properties

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31 1. Introduction

32 Plants in the Amorphophallus genus are perennial herbs widely cultivated in the

33 tropical or subtropical Asian countries. They have rounded tubers and highly dissected

34 umbrella-shaped leaf blades. Based on the weight of dry powder, mature konjac

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35 tubers contain konjac glucomannan (KGM, 49%–60%), starch (10%–30%), fiber

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36 (2%–5%), crude protein (5%–14%), soluble sugars (3%–5%), ash (3.4%–5.3%), and

37 small amounts of alkaloid and saponin (Li, Xia, Wang, & Xie, 2005). Among these

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38 chemical components, KGM is a kind of water soluble dietary fiber and famous for its

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39 potentiality in the prevention and treatment of obesity (Kraemer, et al., 2007),
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40 diabetes (Vuksan, et al., 1999, 2000, 2001), and colonic disease (Chen, Cheng, Liu,

41 Liu, & Wu, 2006; Staiano, et al., 2000; Yong, et al., 2016). It has also been accepted
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42 as the generally regarded as safe (GRAS) food additive by FDA and European Union
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43 (Behera & Ray, 2016; Chua, Baldwin, Hocking, & Chan, 2010). It is commonly
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44 believed that KGM consists of randomly arranged β-(1→4)-mannose and

45 β-(1→4)-glucose in the main chain with ~8% of side chains linked at O-3 or O-6 of
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46 mannosyl and glucosyl residues. The acetyl groups substituted at O-6 of mannosyl
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47 residues in the backbone. The molecular weights of KGM range from 200 to 2000
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48 kDa (An, Thien, Dong, Le Dung, & Van, 2010; Behera & Ray, 2016, 2017; Katsuraya,

49 et al., 2003; Maeda, Shimahara, & Sugiyama, 1980).

50 As an important crop rich in functional dietary fiber (KGM), the tubers after

51 harvesting are usually washed, peeled, sliced, dried, and milled to produce konjac

52 flour. There are about 170 kinds of plants in the Amorphophallus genus around the

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53 world, of which only the flours extracted from A. rivirei and A. albus dominate the

54 market (Huang, et al., 2016). Investigations of glucomannans from new species are

55 helpful to the application and cultivation of plants in the Amorphophallus genus in

56 local areas. In recent years, studies exploring the O-acetyl-glucomannans (AcGMs)

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57 from other species such as A. guripingensis (An, et al., 2010; Huang, et al., 2016), A.

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58 paeoniifolius (Dey, et al., 2017; Dey, Wanjari, Kumar, Lomash, & Jadhav, 2016;

59 Dey, Ota, Srikanth, & Wanjari, 2012; Kumar, Ramakumar, Patel, Gupta, & Singh,

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60 2016), A. muelleri Blume (Yanuriati, Marseno, & Harmayani, 2017), and A.

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61 oncophyllus (Harmayani, Aprilia, & Marsono, 2014) are gradually emerging. These
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62 AcGMs showed great differences in their physicochemical properties including

63 chemical component, water holding capacity, viscosities, and so on. Structure plays a
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64 crucial role on the physicochemical properties of polysaccharides. Besides, the

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65 biological activities of biopolymers also depend on their structural features such as

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66 degree of acetylation, molecular weight, types of sugar, and glycosidic linkages (Ho,

67 et al., 2015; Jin, Zhang, Liang, & Zhang, 2015; Peng, Liu, Lei, & Wang, 2016; Sun,
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68 Chu, Sun, & Chen, 2016; Sun, Wang, & Zhou, 2012; Szopinski, Kulicke, & Luinstra,
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69 2015). Studies on the fine structures of AcGMs from various species may help explain
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70 the differences in their physicochemical properties. However, few of the previous

71 researches focused on these aspects when introducing or comparing their

72 physicochemical, processing, and biological properties.

73 Therefore, comparison of the physicochemical properties and elucidation of the

74 structures of AcGMs extracted from four Amorphophallus species are the main goals

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75 of our study. In this manuscript, chemical components, monosaccharide compositions,

76 molecular weights, and rheological properties were determined. Then,

77 Fourier-transform infrared spectroscopy (FT-IR), methylation analysis, and 1D/2D

78 NMR spectroscopy were applied to analyze the primary structural features of the four

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79 AcGMs. Enzymatic hydrolysis combined with NMR analysis was used to analyze the

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80 position of acetyl groups. Further analysis of the fine structures based on the

81 enzymatic hydrolysates and conformation of four AcGMs will be investigated in the

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82 future.

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83 2. Materials and methods
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84 2.1 Materials and reagents

85 Dried corms of Amorphophallus rivirei, fresh tubers of A. albus, and two local
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86 wild types of Amorphophallus bulbifer (type M in normal size and type B in big size)
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87 were bought from local market of Sichuan Province, China. Fresh tubers were washed
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88 carefully and then sliced, dried, and milled for further use. The monosaccharide

89 standards (L-fucose (Fuc), L-rhamnose (Rha), D-arabinose (Ara), D-galactose (Gal),

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90 D-glucose (Glc), D-mannose (Man), D-fructose (Fru), D-galacturonic acid (GalA) and
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91 D-glucuronic acid (GlcA)), methyl iodide, sodium borodeuteride (NaBD4),

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92 trifluoroacetic acid (TFA) were all purchased from Sigma-Aldrich Chemical Co. (St.

93 Louis, MO, USA). Dextran standards with different molecular weights (Mw of 2.0

94 ×106, 5.0×105, 7.0×104, 5.0× 104, 4.0× 104 and 1.0×104 Da) were bought from

95 Pharmacia Biotech (Uppsala, Sweden). Other reagents were of analytical grade

96 otherwise specified.

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97 2.2 Preparation of purified AcGMs

98 Pretreatment of raw tubers was conducted according to the previous report (Li &

99 Xie, 2006) with some modifications. To remove the fat-soluble components, raw flour

100 was immersed in anhydrous ether for 24 h. Then, 1 L ethanol solution (45%, v/v) was

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101 added to the de-fatted konjac flour (10 g) and kept stirring vigorously at room

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102 temperature for 1.5 h. The supernatants were removed by filtering through a 200 mesh

103 sieve. Concentrated water extract (~600 mL) was obtained by water extraction (1: 100

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104 g/mL, 2 h, 3 times) and ethanol precipitation at room temperature. Then thermostable

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105 α-amylase (0.15 g) and papain (0.15 g) were consecutively added to the concentrated
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106 extract at 80 oC and 60 oC for 2 h to remove starch and protein, respectively. After the

107 protein digestion, pH of the mixture was adjusted to pH 4.5, followed by

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108 centrifugation at 8000 rpm and 4 oC for 15 min. The supernatants were dialyzed
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109 against running tap water for 48 h and deionized water for 24 h. After dialysis,
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110 polysaccharide solution was concentrated and freeze-dried to obtain the purified

111 AcGMs (designated as KGM, AGM, MGM, and BGM for AcGMs from A. rivirei, A.
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112 albus, A. bulbifer in normal size, and A. bulbifer in big size, respectively).
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113 2.3 General analytical analysis

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114 Molecular weight distribution and relative weight-average molecular weights

115 (Mw) of AcGMs were detected by high performance gel permeation chromatography

116 (HPGPC) system coupled with UltrahydrogelTM Guard column (6 mm × 40 mm,

117 Waters, USA) and UltrahydrogelTM Linear column (7.8 mm × 300 mm, Waters, USA).

118 A refractive index detector (RID) and a variable wavelength detector (VWD) (Agilent

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119 Technologies, Amestelveen, Netherlands) were equipped in the HPGPC system.

120 Ultrapure water containing 0.02% (w/v) NaN3 was used as the mobile phase at a flow

121 rate of 0.6 mL/min. The AcGMs, glucose, and dextran standards (1.0 mg/mL) were

122 dissolved in the eluent and filtered through 0.22 µm filter prior to analysis.

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123 Neutral sugar content was determined using the phenol-H2SO4 method (Dubois,

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124 Gilles, Hamilton, Rebers, & Smith, 1956), with mannose as the standard. Uronic acid

125 was detected according to the m-hydroxydiphenyl assay (Blumenkrantz &

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126 Asboe-Hansen, 1973), using glucuronic acid as standard. Protein content was

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127 determined using bovine serum albumin (BSA) as standard (Bradford, 1976). The
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128 contents of ash and moisture were determined according to American Association of

129 Cereal Chemistry methods (AACC, 2003). Content of acetyl groups was determined
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130 by titration method and degree of acetylation (DA) was calculated as reported before
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131 (Han, Gao, Liu, Huang, & Zhang, 2013).

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132 Monosaccharide composition was analyzed according to a published method

133 with slight modifications (Mopper, et al., 1992). AcGMs (5 mg) were dissolved in 0.5
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134 mL ultrapure water and then 0.5 mL of 12 M H2SO4 solution was added and stirred
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135 for 0.5 h in the ice bath. The mixture was hydrolyzed at 100 oC for 2 h after being
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136 diluted with 2.0 mL ultrapure water. The hydrolysate was then diluted 50 times before

137 injection. High performance anion-exchange chromatograph with pulsed

138 amperometric detector (HPAEC-PAD) system (Dionex ICS-5000 System, Dionex

139 Corporation, CA) equipped with a CarboPacTM PA20 guard column (3 mm×30 mm)

140 and a CarboPacTM PA20 analytical column (3 mm×150 mm) were used to analyze the

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141 monosaccharide composition (Li, Cui, Nie, & Xie, 2014).

142 All the above chemical analysis was conducted in three times and data were

143 expressed as mean ± standard deviation.

144 2.4 Rheological measurements

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145 For rheological measurements, AcGMs were prepared by constant stirring at 60

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C for 6 h in ultrapure water. All dissolved samples were kept in 25 oC for 12 h before

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147 test. Measurements were carried out on an ARES-G2 strain controlled rheometer (TA

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148 instruments, New Castle, DE, USA) equipped with a parallel plate geometry (40 mm

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149 diameter, 1.0 mm gap).
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150 Steady shear tests were applied for determination of flow behaviors of the

151 AcGMs at concentrations of 0.1%, 0.25%, 0.5%, 1.0%, 1.5%, and 2.0% (w/v), with
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152 shear rate ranging from 0.1 to 1000 s-1 at 25.0 oC. All measurements were repeated
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153 three times. In dynamic shear mode, the concentration of AcGMs was fixed at 2.0%
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154 (w/v) and the testing temperature was kept at 25.0 oC. Strain sweep measurements

155 were performed at 1.0 Hz to determine the linear viscoelastic regime of the AcGMs
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156 solution with strain varying from 0.01% to 100%. Storage modulus and loss modulus
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157 were detected at a strain of 10% and frequency from 0.1 to 50 Hz at 25.0 oC.
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158 2.5 Scanning electron microscopy (SEM)

159 To observe the morphology of the four AcGMs, a JSM-6701F scanning electron

160 microscopy (SEM) (JEOL Ltd., Japan) was used. Samples (0.5 mg/mL) dissolved in

161 ultrapure water were freeze-dried, dispersed onto an aluminum stub and gold coated

162 with an ionic sputter coater. The acceleration voltage was 5 kV and the magnifications

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163 were set at 1000-, 2000-, 5000-fold under high vacuum condition.

164 2.6 Primary structure analysis

165 2.6.1 Fourier-transform infrared spectroscopy (FT-IR)

166 FT-IR spectra of AcGMs were obtained by Thermo Nicolet 5700 infrared

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167 spectrophotometer (Thermo-Electron, Madison, WI, US). Spectra were recorded at

absorbance mode, with wavenumbers from 400 cm-1 to 4000 cm-1 and a resolution of

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169 4 cm-1. Spectrum of each sample was collected from 32 co-added scans.

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170 2.6.2 Methylation and GC-MS analysis

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171 Methylation of the AcGMs was carried out according to the method by Ciucanu
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172 and Kerek (1984) with slight modifications. Briefly, dried sample (3 mg) was

173 dissolved in 2 mL anhydrous dimethyl sulfoxide (DMSO) with constant stirring at

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174 room temperature for 12 h to ensure a complete dissolved solution. Then, dried
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175 powder of NaOH (20 mg) was added, and the mixture was stirred vigorously for 3 h
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176 at room temperature. Methyl iodide (0.8 mL) was added into the solution drop by

177 drop in the dark. The methylation reaction was stopped by adding one drop of water
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178 after 2.5 h of constantly stirring. The methylated polysaccharides were extracted with
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179 2 mL dichloromethane (CH2Cl2). The organic extracts were washed by equal volume
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180 of water for three times and passed through a Na2SO4 column (0.5×15 cm) to remove

181 water, and then evaporated by a stream of nitrogen gas. Finally, methylated sample

182 was hydrolyzed, reduced using sodium borodeuteride and acetylated with acetic

183 anhydride to obtain the partial methylated alditol acetates (PMAAs). The PMAAs

184 were then analyzed using GC-MS system (Agilent Technology 7890/7000 QQQ, USA)

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185 equipped with an SP-2330 (Supelco, Bellefonte, Pa) capillary column (30 m × 0.25

186 mm, 0.2 mm film thickness) programmed as described before (Wang, et al., 2017).

187 2.6.3 Enzymatic hydrolysis and NMR spectroscopy

188 Enzymatic hydrolysis of AcGMs by endo-β-1,4-mannanase (Aspergillus niger,

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189 Megazyme, Ireland, 600 U/mL) was conducted according to method modified from

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190 the previous report (Xing, et al., 2015). Four AcGMs (2 g) were completely dissolved

191 in 100 mL de-ionized water by stirring at 80 °C for 2 h. After cooling to 37 °C, 25 µL

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192 of the endo-β-1,4-mannanase was added to each polysaccharide solution except BGM.

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193 The volume of mannanase added to the BGM solution was 50 µL, due to its high
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194 viscosities. The mixture was kept stirring at 37 °C for 40 min and then subjected to a

195 boiling water bath for 20 min to de-active the enzyme. The enzymatically hydrolyzed
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196 products (KGM-EH, AGM-EH, MGM-EH, and BGM-EH) were obtained after
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197 ethanol precipitation and freeze-drying as described by Xing, et al. (2015). The four
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198 hydrolyzed fractions were applied to 1H NMR spectroscopy.

199 NMR spectra of the native and hydrolyzed AcGMs were recorded on the Bruker
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200 Avance 600 M and 400 M spectrometer, respectively (Bruker, Rheinstetten, Germany).
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201 All AcGMs were deuterium-exchanged for three times by freeze-drying method. The
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202 recovered sample was dissolved in the D2O (99.9% D atom, Sigma-Aldrich, USA)

203 and transferred into a regular NMR tube and subjected to analysis. 1D 1H and 13
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204 NMR experiments and a series of 2D NMR experiments including 1H-1H correlation

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205 spectroscopy (COSY), H-1H total correlation spectroscopy (TOCSY), 1
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206 heteronuclear single-quantum coherence spectroscopy (HSQC) were performed

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207 according to the standard Bruker pulse sequences.

208 3. Results and discussion

209 3.1 Homogeneity, chemical components and monosaccharide composition

210 In the present study, 45% (v/v) of ethanol solution was used to remove the

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211 impurities attached on the surface of glucomannan granules before extraction.

Thermo-stable α-amylase and papain were added to remove starch and protein in the

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212

213 suspension of re-dissolved ethanol precipitate, respectively. Curves of molecular

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214 weight distributions of four AcGMs by HPGPC are shown in Fig. 1. The elution

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215 curves are almost symmetrical, indicating that the fractions with varying molecular
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216 weights distributed evenly. The calibration curve of dextran was fitted using linear

217 regression by the equation of Kav = -0.2885 Log Mw + 1.6369 (R2=0.9937). After
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218 calculation, relative weight-average molecular weights were found decreasing in the
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219 order of: BGM (2400 kDa) >MGM (2120 kDa) >AGM (1110 kDa) >KGM (919 kDa).
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220 The molecular weights of KGM and AGM in the present study were close to those of

221 glucomannans isolated from A. paeoniifolius, A. panomensis, and A. tonkinensis

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222 (1043–1115 kDa) (An, et al., 2010), A. guripingensis (1236 kDa), and A. rivirei (1669
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223 kDa) (Huang, et al., 2016).

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224 Space for Fig. 1

225 Yields and contents of chemical components of four AcGMs are shown in Table

226 1. Obviously, yields of AGM and BGM were close to that of MGM, but lower than

227 that of KGM. Contents of neutral sugar in the four AcGMs were all above 87% (w/w),

228 which were higher than those of AGF (61.347%) from A. guripingensis and ARF

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229 (72.80%) from A. rivirei (Huang, et al., 2016). Differently, the moisture accounted for

230 11%–12% (w/w) in the literature (Huang, et al. 2016), which was close to our results,

231 and the ash contents were slightly lower than their data (4%–5%, w/w). No protein

232 and uronic acid were detected in all AcGMs. The quality of purified konjac flour or

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233 KGM is highly dependent on the content of KGM. To improve the quality of konjac

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234 flour, mechanical (dry techniques), chemical (wet techniques), and bioprocessing

235 methods have been developed (Behera & Ray, 2017). The highly purified AcGMs

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236 suggested our procedure is very efficient for KGM preparation, which may be applied

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237 into the commercial production of KGM. The acetyl groups accounted for ~17% for
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238 KGM and AGM, and ~24% for MGM and BGM, which were much higher than

239 results of KGMs from AGF and ARF (Huang, et al., 2016). Accordingly, values of
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240 degree of acetylation (DA) were 0.80, 0.78, 1.12, and 1.25 for KGM, AGM, MGM,
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241 and BGM, respectively.

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242 Results of monosaccharide composition (Table 2) revealed that glucose and

243 mannose were the major sugars in the AcGMs. Molar ratios of mannose to glucose
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244 (M/G) of the four AcGMs varied from 1.21 to 1.58 in the present investigation and
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245 they were all lower than 1.60 as most studies reported (Nishinari, Williams & Phillips,
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246 1992; Sugiyama, Shimahara, Andoh, Takemeto, & Kamata, 1972). Trace amounts of

247 galactose were also found in all AcGMs. In summary, the AcGMs from different

248 species prepared using the same procedure in our experiments had similar chemical

249 components and monosaccharide composition but different molecular weights. The

250 differences existed between our results and the data reported before may be attributed

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251 to the varying cultivars, origins and processing methods (Chua, et al., 2010; Nishinari,

252 Williams & Phillips, 1992; Smith & Srivastava, 1959).

253 Space for Table 1

254 Space for Table 2

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255 3.2 Rheological properties of four AcGMs

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256 3.2.1 Steady shear behavior

257 Double logarithmic flow curves of four AcGMs from the Amorphophallus

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258 species at different concentrations were obtained and provided as supporting

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259 information (Fig. S1). Obviously, the apparent viscosities of AcGMs depended on the
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260 concentrations. The polymers performed as near-Newtonian fluids at lower

261 concentrations (0.1–0.5 wt% for KGM & AGM, 0.1–0.25 wt% for MGM & BGM)
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262 and pseudoplastic fluids at higher concentrations. The apparent viscosity of

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263 pseudoplastic fluids remained constant at lower shear rates, and decreased with the
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264 increase of shear rate at higher shear rates region. The remarkable shear-thinning

265 behavior was more obvious for MGM and BGM. Similar flow behavior of KGM was
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266 observed before (Huang, et al., 2016; Wang, et al., 2012), which can be explained as
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267 follows. At low shear rate region, the viscosity kept constant because the rate of
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268 disruption of existing entanglement fell behind the rate of re-entanglement. At high

269 shear rate region, the intra- and intermolecular associative bonding system were

270 highly broken in the polymer network as a result of decrease in viscosity (Bhandari,

271 Singhal, & Kale, 2002; Hua, Wang, Yang, Kang, & Zhang, 2015). With the

272 concentration of AcGMs increased from 0.1% (w/v) to 2.0% (w/v), the apparent

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273 viscosity increased as well, resulting from the enhancement of intermolecular

274 interactions such as the stronger hydrogen bonds caused by massive hydroxyl groups.

275 With concentration increasing, the molecular chains within the unit space increased,

276 resulting in obvious stronger intermolecular hydrogen bonds and then more entangled

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277 points between molecular chains appeared. Finally, the more entangled

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278 three-dimensional network formed, which led to higher viscosity. The above result

279 was a common phenomenon for polysaccharide solutions (Hua, et al., 2015; Torres,

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280 Hallmark, & Wilson, 2014; Wu, Ding, Jia, & He, 2015; Yu, Xu, Zhou, Lv, & Chen,

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281 2017).
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282 A comparative figure of apparent viscosity of four AcGMs at a concentration of

283 1.5% (w/v) was presented in Fig. 2. Four AcGMs had decreasing apparent viscosities
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284 in the order of: BGM>MGM>AGM>KGM. Since BGM and MGM with higher
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285 viscosities had higher molecular weights and DA, the difference in the apparent
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286 viscosity among the four AcGMs may be partly attributed to the two structural

287 features. To explore the dependence of steady shear viscosity on the shear rate, the
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288 power law model was applied. The resultant rheological parameters were summarized
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289 in Table S1 as supplementary data. The power law model fitted well as the correlation
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290 coefficients for all AcGMs were almost 1.0. Generally, the Newtonian fluid has a flow

291 behavior index (n) of 1. The lower n value is, the higher degree of pseudoplastic

292 properties the fluid has (Pongsawatmanit, Temsiripong, Ikeda, & Nishinari, 2006). In

293 the present study, MGM and BGM tended to show more typical pseudoplastic flow

294 behavior than KGM and AGM, due to the values of n were 0.6–0.7 for KGM and

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295 AGM, 0.3–0.4 for MGM and BGM. In addition, the consistency coefficient (K) is an

296 indicator of the viscous nature of the tested system. The parameters of MGM and

297 BGM were significantly higher than those of KGM and AGM, confirming the high

298 viscosity of the former detected above.

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299 Cross model (Cross, 1965) and Carreau model (Carreau, 1972) were further

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300 used to describe the shear thinning behavior of AcGMs solutions in the present study

301 (Razavi, Cui, & Ding, 2016). Relevant rheological parameters in the two models were

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302 shown in Table 3. The apparent viscosity versus shear rate curves of four AcGMs

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303 fitted both models well. The magnitude of zero-shear rate viscosity (η0) can reflect the
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304 microstructural nature of biopolymer during storage, that is, higher value of η0

305 represents greater number of linkages between the macromolecules (Razavi, Cui, &
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306 Ding, 2016). BGM and MGM showed much higher values than KGM and AGM, and
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307 the estimated value of BGM was approximately twice of that of MGM. These results
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308 suggested that interactions among polymer chains in BGM were stronger than those

309 of other three fractions.

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310 Space for Fig. 2

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311 Space for Table 3

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312 3.2.2 Frequency sweep

313 The frequency dependence of storage modulus (G′) and loss modulus (G″) of

314 four AcGMs at a concentration of 2.0% (w/v) and 25.0 oC were obtained and shown in

315 Fig. 3. As the frequency increased from 0.1 Hz to 50 Hz, line of storage modulus (G′)

316 was always lower than those of loss modulus (G″) for KGM and AGM, indicating a

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317 liquid-like behavior. However, MGM and BGM performed in different way. On the

318 one hand, G″ was higher than G′ at lower frequencies because the entanglement of

319 molecular chains disentangled during a long period oscillation. On the other hand, G′

320 was higher than G″ at higher frequencies, showing a solid-like behavior, which may

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321 be explained by that the molecular chains had no enough time to disentangle during a

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322 short period of oscillation at higher frequencies (Huang, et al., 2016; Wang, et al.,

323 2012). The frequency at crossover point of BGM (2.0 Hz) was smaller than MGM

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324 (3.2 Hz), illustrating that the entangled polymer network of BGM was obviously

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325 stronger than that of MGM. This phenomenon may be explained by the higher
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326 molecular weights of BGM (Huang, et al., 2016; Wang, et al., 2015; Zhong, et al.,

327 2015).
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328 Comparing to the previous study, where 1.0 wt.% of KGM with lower molecular
D

329 weight (747 kDa) showed crossover point at about 0.5 Hz (Du, Li, Chen, & Li, 2012),
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330 the four samples we obtained had much weaker gelation properties. The discrepancy

331 may be attributed to the high contents of acetyl groups, as acetyl groups confer water
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332 solubility on KGM and removal of acetyl groups makes gelation of KGM become
C

333 easier (Du, Li, Chen, & Li, 2012; Gao, & Nishinari, 2004).
AC

334 Space for Fig. 3

335 3.3 Surface morphology characterization

336 The SEM images of the four AcGMs at magnifications of 1000-, 2000-, and

337 5000-fold are shown in Fig. 4. Randomly distributed sheet-like and flaky appearance

338 could be found in the four AcGMs under the 1000 fold exaggeration condition (Fig.

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339 4a). AGM exhibited much more irregular micro-spherical shapes along the filaments

340 than KGM and MGM at 2000 and 5000 fold augmentations (Fig. 4b). BGM mainly

341 performed as flake-like and filamentary structures. No obvious spherical structures

342 were observed in BGM. The surface morphology together with the rheological

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343 properties results may be related to the different structural features in molecular

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344 weight and functional groups aspects.

345 Space for Fig. 4

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346 3.4 Primary structures of four AcGMs

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347 3.4.1 FT-IR spectra
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348 FT-IR spectra of four AcGMs are shown in Fig. 5. The broad absorption at

349 3500–3000 cm-1 was caused by O-H stretching vibrations of the inter- and
M

350 intra-molecular hydrogen bonds of hydroxyl groups (Silverstein, Bassler, & Morril,
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351 1991). Bands at around 2950 cm-1 (3000–2800 cm-1) were related to the C-H
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352 stretching and bending vibrations of the methyl group (Kaèuráková, Capek, Sasinkova,

353 Wellner, & Ebringerova, 2000; Kang, et al., 2011). Peaks with strong intensity at
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354 around 1730, 1374, and 1242 cm-1 were assigned to the valence vibration of C=O, the
C

355 symmetric C–H bending vibration of the methyl group, and the C–O vibration of
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356 O-acetyl groups, successively (Femenia, Garcıá -Pascual, Simal, & Rosselló, 2003;

357 Widjanarko, Nugroho, & Estiasih, 2011; Xing, et al., 2014). The peak at 807 cm-1 in

358 the FT-IR spectra, could be assigned to the in-phase ring stretching of the mannose

359 pyranosyl ring and signal at 890 cm-1 indicated the β-configuraion of glucose

360 (Mohaček-Grošev, Božac, & Puppels, 2001). Based upon the aforementioned

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361 literature reports, the FT-IR data in the current study confirmed the existence of

362 O-acetyl-glucomannans and no difference is observed for FT-IR spectra of all

363 samples. The result indicated that the species in Amorphophallus genus induced no

364 significant changes in the primary composition and linkage patterns of sugar residues

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365 of AcGMs. Whether there are any differences in the saccharide chain sequence need

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366 further analysis.

367 Space for Fig. 5

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368 3.4.2 Methylation and GC-MS analysis

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369 Results of methylation and GC-MS of the original AcGMs are summarized in
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370 Table 4. The AcGMs showed no difference in the linkage patterns but in relative

371 molar ratio of individual linkages. Assignments of individual sugar residues were
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372 based on the relative retention time, mass spectra of the corresponding partial
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373 methylated alditol acetates (PMAAs) and data in literatures (Behera & Ray, 2016;
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374 Manna & McAnalley, 1993; Maeda, et al., 1980; Guo, et al., 2012; Ishrud, Zahid,

375 Ahmad, & Pan, 2001; Smith & Srivastava, 1959; Wang, et al, 2014). Molar ratio of
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376 each sugar residues was calculated according to the relative percentage of peak area.
C

377 Eight kinds of sugar residues were assigned from the total ion chromatograms
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378 according to the corresponding peaks. The major →4)-Manp-(1→ and

379 →4)-Glcp-(1→ residues accounted for 69.82%, 68.84%, 74.32%, and 68.53% in

380 KGM, AGM, MGM, and BGM, respectively, indicating the presence of 4-linked

381 glucomannan. Small amounts of →3)-Manp-(1→ and →3)-Glcp-(1→ residues were

382 also identified, which were not common in the previous reports. Both mannose and

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383 glucose were found as terminal units. The values of ratio of terminal mannosyl to

384 glucosyl units for KGM, AGM, MGM, and BGM were 0.9, 0.6, 0.7, and 0.8,

385 respectively, which were higher than that (0.5) determined by Smith & Srivastava

386 (1959), but lower than the value (2.0) reported by Katsuraya, et al., (2003). As for

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387 branches, earlier studies showed that konjac glucomannan had branches linked

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388 through O-3 or O-6 of glucosyl and mannosyl residues (Maeda, et al., 1980; Kato &

389 Matsuda, 1973; Smith & Srivastava, 1959). Afterwards, Katsuraya, et al, (2003)

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390 identified the O-6 carbon of glucosyl units as the branching points by methylation and

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13
391 C NMR methods. In our study, the mass spectrum of the last peak showed both the
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392 characteristics peaks produced from the PMAA of 1,2,4,6- and 1,3,4,6-linked glycosyl

393 residues. According to the relative retention time reported before (Shea & Carpita,
M

394 1988), signals of →4)-2,6-Manp-(1→ and →4)-3,6-Glcp-(1→ may overlap.

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395 Therefore, the side chains in the four AcGMs may attached to the O-2,6 positions of
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396 →4)-Manp-(1→ and/or O-3,6 positions of →4)-Glcp-(1→ in the backbone.

397 Space for Table 4

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398 3.4.3 NMR spectroscopy

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399 3.4.3.1 NMR spectroscopy of the original AcGMs

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400 The concentration, viscosity, and transparency of a polysaccharide solution

13
401 have great influence on the quality of NMR spectra, especially the C correlated

402 ones. It was reported that a high-quality 13C NMR spectrum could be obtained when

403 polysaccharides are able to dissolve in the solvents at concentrations of 3–4% (w/v)

404 with low viscosity (Xing, et al., 2014). In our experiment, the original AcGMs

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405 performed as weak gels when the polysaccharide concentrations come to 2.0% (w/v)

406 or above. Therefore, the NMR spectra of the original AcGMs were in low resolution,

407 which could only provide structural information of the major sugar residues. The fine

408 structures of the acetyl groups will be given based on the analysis of the

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409 enzymatically hydrolyzed samples.

The 1H and 13C spectra of the native AcGMs were shown in Fig. S2. The HSQC,

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410

411 COSY, and TOCSY NMR spectra of the two lower molecular weights fractions

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412 (KGM and AGM) were given in Fig. S3. The 2D NMR spectra of BGM and MGM

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413 were not given since no useful structural information is available. Two main peaks at
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13
414 around 101.3 ppm and 103.7 ppm were determined in the anomeric regions of C

415 NMR spectra of the four samples (Fig. S2), which were obviously derived from the
M

416 major 4-linked-β-mannosyl and 4-linked-β-glucosyl residues, respectively (Deng, et

D

417 al., 2018; Shi, et al., 2017, 2018). Even though part of the anomeric proton signals
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418 were overlapped with that of HOD in the 1H spectra, the chemical shifts of

419 intra-residue correlated protons and/or carbons of the two major residues can be
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420 clearly distinguished in the 2D NMR spectra (Fig. S3). Chemical shifts of the
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421 non-anomeric carbons (C-2~C-6) of the two sugar residues were marked and
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422 summarized in Table S2 according to the previous data (Hannuksela, & du Penhoat,

423 2004; Teleman, Nordström, Tenkanen, Jacobs, & Dahlman, 2003; Xing, et al., 2015).

424 As the important non-sugar functional groups, existence of acetyl groups was

425 confirmed by the 1H signals of methyl groups at 2.13–2.23 ppm in the 1H spectra

426 (Fig. S2) and correlated signal (21.2/2.18 ppm) in the HSQC spectra (Fig. S3, a & d),

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427 referring to data reported previously (Deng, et al., 2018; Hannuksela & du Penhoat,

428 2004). According to the literature (Hannuksela & du Penhoat, 2004), substitution of

429 acetyl groups to a carbon in the mannose could induce great increase of the chemical

430 shift of proton directly linked to the carbon. Thus, the peaks in the region of

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431 5.40–5.50 ppm in the downfield of 1H spectrum (Fig. S2) and 4.86/4.00, 4.86/3.96,

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432 4.86/3.75 ppm in the TOCSY spectrum (Fig. S3c) were attributed to the H-2,

433 H-1/H-4, H-1/H-6a, and H-1/H-6b of →4)-2-OAc-Manp-(1→ (designated as 2M4)

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434 residues, successively (Xing, et al., 2015).

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435 3.4.3.2 1H NMR analysis of hydrolyzed AcGMs
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436 To compare the characteristics of O-acetyl groups in the AcGMs from

437 Amorphophallus species, the four samples were subjected to the

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438 endo-β-1,4-mannannase and then analyzed by NMR spectroscopy. The 1H NMR

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439 spectra at different temperatures of the four hydrolyzed AcGMs are shown in Fig. 6.
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440 In the 1H NMR spectra of the mannanase-treated products, strong peaks at 5.17 and

441 4.90 ppm of H-1 of reducing end →4)-α-Manp (αM4) and →4)-β-Manp (βM4)
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442 residues were distinguished and no obvious evidence of the corresponding glucose in
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443 the reducing end was found. This phenomenon was consistent with the conclusion
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444 that mannannase tends to hydrolyze the M4-M4 and M4-G4 dimer rather than G4-M4

445 and G4-G4 dimers (Xing, et al., 2015). Splitting of signals in the anomeric region of

1
446 H spectra (Fig. 6) indicated that there were various possible combinations of sugar

447 residues, which may resulted from the random distribution of O-acetyl groups (van

448 Hazendonk, Reinerik, de Waard, & van Dam, 1996). The protons attached to the

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449 O-acetylated carbon atoms at 5.38–5.50 and 5.06–5.11 ppm were assigned to H-2 of

450 O-2-acetylated 4-linked-Manp residue (designated 2M4) and H-3 of O-3-acetylated

451 4-linked-Manp (designated 3M4), respectively. No acetyl groups were found linking

452 to the glucosyl residues. In particular, the peaks at around 5.50 and 5.40 ppm were

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453 attributed to the 2M4 residues building up the 2M4-S (the non-acetylated sugar

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454 residues) and 2M4-AcM4 (acetylated M4 residues) units, respectively. (Teleman, et al.,

455 2003; van Hazendonk, et al., 1996; Xing, et al., 2015). As shown in Fig. 6, the

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456 2M4-AcM4 units were obviously observed in the spectra of KGM-EH and AGM-EH,

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457 but not in the MGM-EH and BGM-EH. By integrating the peaks of methyl group of
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458 acetyl groups (2.1–2.2 ppm), H-2 of 2M4 (5.35–5.50 ppm) and H-2 of 3M4 (4.0 ppm)

459 residues in the fingerprint region (Fig. 6), almost equal amounts of O-acetyl groups
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460 were found at O-2 and O-3 positions of mannosyl residues in the four hydrolyzed
D

461 samples. The substituted positions of acetyl groups in the glucomannan from
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462 Amorphophullas species detected in the present investigation were slightly different

463 from the previous report, which reported that acetyl groups linked to O-2, O-3, and
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464 O-6 position of mannose units (Lin, et al., 2010). Interestingly, the same distribution
C

465 of acetyl groups at O-2 and O-3 had been identified in the (galacto)glucomannans
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466 from spruce (Lundqvist, et al., 2002) and aspen wood (Teleman, et al., 2003).

467 Space for Fig. 6

468 4. Conclusion

469 In conclusion, a simple, convenient, and environmentally friendly processing

470 method of AcGM from Amorphophallus genus was provided. The four AcGMs (KGM,

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471 AGM, MGM, and BGM) showed high purity and acceptable yields. Chemical

472 components, physicochemical properties, and primary structure were determined and

473 compared in the current study. Contents of the major chemicals (neutral sugar,

474 moisture, and ash) and mannose to glucose (M/G) molar ratios of the four AcGMs

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475 were close to each other, but molecular weights showed noticeable discrepancy.

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476 MGM and BGM had higher apparent viscosities and potentially stronger gelation

477 abilities than KGM and AGM. The results may improve the production of

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478 high-quality KGM and optimize the growing conditions according to the agricultural

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479 demands. As for structures, FT-IR spectra confirmed that the AcGMs obtained from
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480 different species had similar main structural features. Methylation/GC-MS and NMR

481 spectra suggested that four AcGMs had similar main chain consisting of randomly
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482 arranged β-(1→4)-mannose and β-(1→4)-glucose. Acetyl groups in the four AcGMs
D

483 mainly linked to O-2 and O-3 positions at a ratio of 1:1 in the mannanase-hydrolyzed
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484 products. Side chains in the four AcGMs may attached to the O-2,6 positions of

485 →4)-Manp-(1→ and/or O-3,6 positions of →4)-Glcp-(1→ in the main chain. The
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486 complex structure and conformation of O-acetyl-glucomannans from Amorphaphullas

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487 species need further investigations.

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488

489 Acknowledgements

490 The financial supports for this study by the National Key R&D Program of

491 China (2017YFD0400203), Project of Academic Leaders of the Major Disciplines in

492 Jiangxi Province (20162BCB22008) and Outstanding Science and Technology

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493 Innovation Team Project in Jiangxi Province (20165BCB19001) and Innovation Fund

494 Project of Nanchang University (cx2016008) were gratefully acknowledged.

495 Notes

496 The authors declare no competing financial interest.

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643 Sun, L., Wang, L., & Zhou, Y. (2012). Immunomodulation and antitumor activities of

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646 Szopinski, D., Kulicke, W. M., & Luinstra, G. A. (2015). Structure-property relationships of

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665 (2001). Konjac-Mannan and American ginsing: emerging alternative therapies for type 2 diabetes
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667 Wang, C., Xu, M., Lv, W. P., Qiu, P., Gong, Y. Y., & Li, D. S. (2012). Study on rheological
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669 Wang, J., Nie, S., Cui, S. W., Wang, Z., Phillips, A. O., Phillips, G. O., et al. (2017). Structural
670 characterization and immunostimulatory activity of a glucan from natural Cordyceps sinensis.
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672 Wang, X., Wang, X., He, S., Sun, H., Wang, P., Zhang, P., & Zhang, M. (2015). The rheological
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675 Wang, Y., Liu, D., Chen, S., Wang, Y., Jiang, H. & Yin, H. (2014). A new glucomannan from
676 Bletilla striata: Structural and anti-fibrosis effects. Fitoterapia, 92, 72-78.

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684 structural characterization. Bioactive Carbohydrates and Dietary Fibre, 4(1), 74-83.

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697 Zhong, K., Zhang, Q., Tong, L., Liu, L., Zhou, X., & Zhou, S. (2015). Molecular weight
698 degradation and rheological properties of schizophyllan under ultrasonic treatment. Ultrasonics
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700 Figure captions

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701
702 Fig. 1 Molecular weight distribution of four AcGMs by HPGPC
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703

704 Fig. 2 Comparison of the apparent viscosity of four AcGMs from Amorphophallus
species at concentration of 1.5% (w/v) at 25.0 oC
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706

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707 Fig. 3 Frequency dependence (0.1-50 Hz) of storage modulus (G′) and loss modulus
708 (G″) of four AcGMs from Amorphophallus species at concentration of 2.0% (w/v)
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709 with the strain fixed at 10% and temperature at 25.0 oC
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710

711 Fig. 4 Scanning electron micrographs of four AcGMs with magnification of 1000× (a)
712 2000× (b), and 5000× (c)
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713
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714 Fig. 5 FT-IR spectra of four AcGMs
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715
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716 Fig. 6 1H NMR spectra of the enzymatically hydrolyzed AcGMs from
Amorphophallus species at around 295 K. The labels in the spectra refer to the
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718 corresponding protons or carbons of each sugar residue, with the following encoding:
719 M4, non-acetylated →4)-β-Manp-(1→ residue; 2M4, 2-O-acetylated
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720 →4)-β-Manp-(1→ residue; 3M4, 3-O-acetylated →4)-β-Manp-(1→ residue; Mt,

721 non-reducing end β-Manp-(1→ residue; αM4: reducing end →4)-α-Manp residue;
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722 βM4: reducing end →4)-β-Manp residue; G4, →4)-β-Glcp-(1→ residue; Gt,
723 non-reducing end β-Glcp-(1→ residue.
724
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725 Tables

726 Table 1
727 Yields, chemical components of AcGMs from Amorphophallus genusa

Acetyl group
Samples Yield (%) Neutral sugar (%) Moisture (%) Ash (%)
content (%)

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KGM 33.2 87.1 ± 0.1 10.8 ± 0.3 1.3 ± 0.1 17.6 ± 1.1
AGM 18.7 87.9 ± 0.7 12.5 ± 0.1 1.4 ± 0.1 17.3 ± 1.8
MGM 15.7 92.4 ± 0.3 10.8 ± 0.1 1.5 ± 0.2 24.1 ± 2.9

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BGM 17.6 89.0 ± 0.1 10.4 ± 1.3 1.5 ± 0.5 24.3 ± 1.5
a
: Data other than yield were calculated based on the weight of freeze-dried AcGMs and

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presented as the mean ± standard deviation (n=3).

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728 Table 2
729 Monosaccharide compositions of AcGMs from Amorphophallus speciesa

Monosaccharide composition (%) Total sugar

Samples M/G
Gal Glc Man (%)
KGM 0.21±0.04 31.72 ± 0.51 44.77 ± 1.81 76.70 ± 2.83 1.41 ± 0.04
AGM 0.25±0.02 30.02 ± 0.75 42.40 ± 0.91 72.70 ± 2.02 1.41 ± 0.01

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b
MGM tr. 33.29 ± 0.31 40.25 ± 0.36 73.54 ± 0.66 1.21 ± 0.02
b
BGM tr. 30.98 ± 0.66 49.01 ± 1.31 79.99 ± 2.41 1.58 ± 0.01
a
: Data are calculated based on the weight of freeze-dried AcGMs and presented as the mean ±

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standard deviation (n=3).
b
: Lower than the limit of detection.

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730 Table 3
731 Rheological parameters from the shear thinning models determined at 1.5% (w/v) and
732 25.0 oCa

Cross model b Carreau model b

Samples
η0 (Pa.s) k (s) R2c η0 (Pa.s) k (s) R2
KGM 0.56 ± 0.04 0.004 ± 0.001 0.9997 0.54 ± 0.03 0.08 ± 0.01 0.998

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AGM 0.89 ± 0.06 0.006 ± 0.001 0.9999 0.86 ± 0.06 0.09 ± 0.02 0.999
MGM 9.68 ± 0.76 0.09 ± 0.01 0.9999 8.53 ± 0.61 0.31 ± 0.04 0.998
BGM 17.24 ± 0.66 0.13 ± 0.01 1.0000 15.09 ± 0.42 0.33 ± 0.01 0.998

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a
: Results are expressed as means ± SD (n=3).
b
: Cross model: η = η∞ + (η0-η∞/(1+(kγ̇)m); Carreau model: η = η∞ + (η0-η∞/((1+(kγ̇)2)m) where η0,
zero-shear rate viscosity; η∞, infinite-shear rate viscosity; γ̇, shear rate; k, consistency; m, the

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power law index.
c
: R2, correlation coefficient.

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733 Table 4
734 Linkage pattern analysis of four AcGMs from Amorphophallus species

Relative percentage of peak area (%)

Linkage type Mass fragments (m/z)
KGM AGM MGM BGM
Manp-(1→ 43,71,88,102,118,129,145,161,162,205 7.65 5.43 5.51 7.50
Glcp-(1→ 43,71,88,102,118,129,145,161,162,205 8.82 9.38 7.42 9.95

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→4)-Manp-(1→ 43,87,102,118,129,162,233 38.40 35.88 37.49 36.49
→3)-Manp-(1→ 43,59,88,102,118,131,162,191 4.52 5.64 3.67 4.03
→3)-Glcp-(1→

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43,59,87,99,118,131,162,191 3.46 4.40 3.42 3.72
→4)-Glcp-(1→ 43,71,87,99,118,131,173,233 31.42 32.96 36.83 32.04
→4)-2,6-Manp-(1→

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43,103,115,128,145,157,187,217,259,289 5.74 6.32 5.67 6.26
→4)-3,6-Glcp-(1→
Total amount of →4)-Manp-(1→ and →4)-Glcp-(1→ residues 69.82% 68.84% 74.32% 68.53%
Ratios of terminal mannosyl to glucosyl units 0.9 0.6 0.7 0.8

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Highlights

Four purified O-acetyl-glucomannans (AcGMs) were extracted and

characterized.

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The AcGMs with high contents of acetyl groups had similar primary

structure.

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AcGMs from Amorphophallus bulbifer had higher molecular weights

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and viscosities.

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Acetyl groups substituted at O-2 & O-3 positions of 4-linked
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mannose.
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