Fractionation of polysaccharides by gradient non-solvent precipitation: A review

Accepted Manuscript Fractionation of polysaccharides by gradient non-solvent precipitation: A review Xiuting Hu, H. Douglas Goff

PII:

S0924-2244(18)30149-3

DOI:

10.1016/j.tifs.2018.09.011

Reference:

TIFS 2318

To appear in:

Trends in Food Science & Technology

Received Date: 4 March 2018 Revised Date:

30 July 2018

Accepted Date: 9 September 2018

Please cite this article as: Hu, X., Goff, H.D., Fractionation of polysaccharides by gradient non-solvent precipitation: A review, Trends in Food Science & Technology (2018), doi: 10.1016/j.tifs.2018.09.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Fractionation of polysaccharides by gradient non-solvent precipitation: A review

2 3

Xiuting Hua*, H. Douglas Goff b,**

RI PT

4 5

a

School of Food Science and Technology, Nanchang University, Nanchang 330047, China

6

b

Department of food science, University of Guelph, Guelph, Ontario N1G2W1, Canada

* Corresponding author

9

Xiuting Hu

M AN U

8

SC

7

School of Food Science and Technology, Nanchang University

11

Tel.: +86-791-88304753

12

E-mail address: [email protected]

13

H. Douglas Goff

14

Department of Food Science, University of Guelph

15

Tel.: +1- 519 824 4120

16

E-mail address: [email protected]

EP

AC C

17

TE D

10

1

ACCEPTED MANUSCRIPT 18

Abstract:

19

Background Almost all natural polysaccharides have wide molecular weight distribution and the complex

21

heterogeneous polysaccharides may also exhibit variations in proportions of sugar constituents,

22

linkage type, degree and arrangement of branching or degree of substitution. The heterogeneity of

23

molecular weight and chemical structure restricts basic research and application of

24

polysaccharides. Therefore, it is meaningful to fractionate polysaccharides into fractions with high

25

homogeneity in molecular weight and chemical structure.

26

Scope and Approach

M AN U

SC

RI PT

20

Compared with chromatography and ultrafiltration, gradient non-solvent precipitation is

28

inexpensive, has a wide application scope and can be easily scaled up or down as required.

29

Therefore, this work reviews fractionation of polysaccharides by gradient non-solvent

30

precipitation. Specifically, this work describes the commonly used non-solvents, fractionation

31

mechanism of gradient non-solvent precipitation, how to establish the fractionation procedure,

32

assessment of the fractionation effect and factors that affect fractionation.

33

Key Findings and Conclusions

EP

AC C

34

TE D

27

This paper provides comprehensive and detailed directions for how to fractionate a new

35

polysaccharide using this method. It is suggested to select a suitable non-solvent and establish the

36

fractionation procedure according to the curve of the polysaccharide recovery as a function of the

37

non-solvent concentration. Fractionation by gradient non-solvent precipitation is based on the

38

difference in solubility of polysaccharides. Therefore, fractionation may be affected by the

39

precipitation conditions and those factors that can affect the solubility of polysaccharides. During

2

ACCEPTED MANUSCRIPT 40

fractionation, those factors that negatively affect fractionation should be strictly controlled.

41

Keywords: polysaccharide; fractionation; gradient precipitation; organic solvents; ammonium

42

sulfate; polyethylene glycol

AC C

EP

TE D

M AN U

SC

RI PT

43

3

ACCEPTED MANUSCRIPT 44

1 Introduction Polysaccharides are widely distributed in plants, animals, fungi and microorganisms. They

46

serve organisms in crucial functions, such as energy storage, structure and defense orientation.

47

Natural polysaccharides and their derivatives are also widely used in food and pharmaceutical

48

industries as the gelling agent, stabilizer, thickener or disintegrator. Polysaccharides are divided

49

into homopolysaccharides and heteropolysaccharides. The former contains one kind of

50

monosaccharide unit and the latter contains two or more kinds of monosaccharide units. Unlike

51

small compounds made up of only one kind of molecule, polysaccharides consist of very different

52

species. In the simplest case of linear homopolymers, those polysaccharides exhibit a wide

53

distribution of molecular weight (chain length, or degree of polymerization). In addition to

54

molecular weight, complex heteropolysaccharides may exhibit variations in proportions of sugar

55

constituents, linkage type, or degree and arrangement of branching. In addition, the

56

monosaccharide units of some polysaccharides may be sulfated, acetylated, methylated or

57

substituted by other groups. Therefore, these polysaccharides may also have difference in the

58

degree of substitution.

EP

TE D

M AN U

SC

RI PT

45

Because relatively small differences in molecular weight and structure of polysaccharides

60

may result in substantial differences in their physicochemical properties, the availability of

61

samples with sufficiently high uniformity constitutes an indispensable requirement in the fields of

62

basic research and for analytical purposes (Eckelt, Haase, Loske, & Wolf, 2004). In the former

63

case, it is mandatory for the theoretical interpretation of measurements. In the latter, calibration of

64

analytical methods often requires high uniformity standards. For instance, size exclusion

65

chromatography (SEC) is frequently used for the measurement of molecular weight and molecular

66

weight distribution of polymers (Wang, Wood, Huang, & Cui, 2003). Unless the system is

AC C

59

4

ACCEPTED MANUSCRIPT equipped to measure molecular weight directly, such as with a multi-angle light scattering detector,

68

molecular weight standards have to be used to calibrate the SEC columns. Pullulan and dextran

69

are commercially available standards for polysaccharides. However, because the retention volume

70

in SEC is dependent on hydrodynamic volume rather than molecular weight, the use of pullulan or

71

dextran as standards can lead to inaccurate molecular weight determination of other

72

polysaccharides. On the other hand, some fractions sometimes account for a quite low proportion

73

in the polysaccharide sample, so it is difficult to identify these fractions by analyzing the whole

74

polysaccharide sample. Fractionation can enrich these fractions. Therefore, it is necessary to

75

fractionate them into more structurally homogeneous materials, particularly for unknown

76

polysaccharides, when examining their molecular structures or structure-property relationships.

77

Wide molecular weight distribution of polysaccharides also limits their industrial application. For

78

instance, the molar mass of the hydroxyethyl starch generally used in clinical applications ranges

79

40,000~450,000 g/mol and its molar degree of substitution ranges between 0.5 and 0.7 (Gosch,

80

Haase, Wolf, & Kulicke, 2002). Hydroxyethyl starch of higher molar mass may induce

81

anaphylactoid reactions. If the hydroxyethyl starch has an excessively low molar mass, it is

82

excreted too quickly through the kidneys. There are considerable similar examples. Therefore, it is

83

necessary to fractionate polysaccharides into fractions with high homogeneity.

SC

M AN U

TE D

EP

AC C

84

RI PT

67

Fractionation of polysaccharides is mainly performed by chromatography, ultrafiltration and

85

gradient non-solvent precipitation. Chromatography and ultrafiltration use columns and

86

membranes to fractionate polysaccharides, respectively. Chromatography columns and filtration

87

membranes have their specific molecular weight cut-off, and thus their ranges of application are

88

limited. Fouling of chromatography columns and filtration membranes often result in their short

5

ACCEPTED MANUSCRIPT lives. Therefore, the high cost of equipment investment and operation impedes the

90

industrialization of these two technologies. In addition, these methods are not suitable for

91

large-scale preparations. Fractionation by gradient non-solvent precipitation means that gradual

92

addition of non-solvent into the polysaccharide solution results in gradual precipitation of

93

polysaccharides from the solution. This method is independent of special equipment and only

94

requires simple equipment, such as stirred tanks and centrifuges, making it easily scalable up or

95

down according to the need. Therefore, gradient non-solvent precipitation has the most potential

96

for preparative fractionation of polysaccharides. In addition, this method has a large scope of

97

application for different polysaccharides with different molecular weights. Therefore, fractionation

98

of polysaccharides by gradient non-solvent precipitation is reviewed in this work.

99

2 The non-solvent

M AN U

SC

RI PT

89

Usually, the solvent of polysaccharides is water. In an aqueous solution, polysaccharide

101

molecules are linked with water molecules by hydrogen bonds, which keep polysaccharides

102

soluble in water. The non-solvent can induce the precipitation of polysaccharides from the solution,

103

thus the non-solvent is also named as the precipitant. The commonly used precipitants and the

104

corresponding polysaccharides are summarized in Table 1. The precipitants include organic

105

solvents (methanol, ethanol, isopropanol, acetone and 1-butanol), ammonium sulfate ((NH4)2SO4)

106

and polyethylene glycol (PEG).

EP

AC C

107

TE D

100

Different from other organic solvents, 1-butanol can selectively precipitate amylose due to

108

the formation of the amylose-1-butanol complex (Schoch, 1942). Therefore, 1-butanol can be

109

exclusively used to fractionate starch into amylose and amylopectin. However, 1-butanol cannot

110

fractionate other polysaccharides. Hence, fractionation by 1-butanol is not discussed in this paper.

6

ACCEPTED MANUSCRIPT The other organic solvents exhibit excellent miscibility with water and act as the dehydrating

112

agent. These organic solvents have much lower dielectric constant than water. Adding organic

113

solvents can lower the dielectric constant of the polysaccharide solution and promote

114

intra-molecular associations of water-soluble polysaccharides through competition for water, thus

115

inducing conformational changes, aggregation and precipitation of the polysaccharides (Jian, et al.,

116

2014). In other words, organic solvents decrease the polarity of water to precipitate

117

polysaccharides, because organic solvents have much lower polarity than water (Guo, Meng, Tang,

118

et al., 2016). According to the “like dissolves like” principle, the polysaccharides that are initially

119

soluble will be eventually precipitated if enough organic solvents are added to decrease the

120

polarity of water. The biggest difference of these organic solvents is the dielectric constant (or

121

polarity) and their dielectric constant (or polarity) is in the order of methanol > ethanol >

122

isopropanol > acetone. Therefore, the precipitation efficiency is in the order of methanol < ethanol

123

< isopropanol < acetone. Among these precipitants, ethanol is the most widely used due to its

124

safety and food purposes.

TE D

M AN U

SC

RI PT

111

Although the mechanism by which (NH4)2SO4 precipitates polysaccharides is not reported, it

126

can be inferred that it is similar to the mechanism by which (NH4)2SO4 precipitates proteins. In an

127

aqueous solution, the polysaccharide exposes its polar residues on the surface to maximize the

128

contact with water molecules (Huang, et al., 2013). Water molecules bind to the surface of the

129

biopolymer and form a hydration layer, which is essential to maintain the solubility and to prevent

130

the contact and aggregation of the polysaccharide molecules. The electrostatic repulsion of the

131

same charges in the charged polysaccharides also facilitates the interaction between the

132

polysaccharides and water. When NH4+ and SO42− ions are added into the aqueous polysaccharide

AC C

EP

125

7

ACCEPTED MANUSCRIPT solution, they can compete for the water molecules bound to the polymer surface and disrupt the

134

hydration layer, facilitating the interactions between the polysaccharide molecules to form

135

aggregates and precipitate. In addition, they are attracted to the opposite charges evident on the

136

polysaccharide. This attraction of opposite charges also leads to aggregation and precipitation of

137

polysaccharides.

RI PT

133

Two chemically different water-soluble polymers are often incompatible in aqueous solutions.

139

PEG is incompatible with dextran or dextrin. When the PEG concentration reaches the limiting

140

value, phase separation happens and dextran or dextrin is precipitated from the solution. In

141

summary, PEG precipitates dextran or dextrin based on their incompatibility. In addition to

142

dextran and dextrin, PEG has the potential to fractionally precipitate other polysaccharides, since

143

incompatibility widely exists between two chemically different water-soluble polymers. Actually,

144

PEG precipitation has been widely used to purify proteins (Atha & Ingham, 1981;

145

Hammerschmidt, Hobiger, & Jungbauer, 2016; Sim, et al., 2012).

146

3 The principle of fractionation by gradient non-solvent precipitation

TE D

M AN U

SC

138

Fractionation by gradient non-solvent precipitation is based on phase separation of

148

polysaccharide solutions. Polysaccharides of different molecular weights or chemical structures

149

have different solubility. Adding small amount of non-solvent initially leads to the precipitation of

150

the polysaccharides with low solubility and subsequent addition of non-solvent gradually

151

precipitates the polysaccharides with relatively high solubility, thereby achieving fractional

152

precipitation of polysaccharides based on differential solubility. Generally, the solubility of

153

polysaccharide is negatively correlated to its molecular weight (Hu et al., 2015; Hu, Liu, et al.,

154

2017; Hu, Wang, et al., 2017). Therefore, gradient non-solvent precipitation often induces

AC C

EP

147

8

ACCEPTED MANUSCRIPT precipitation of polysaccharides in the decreasing order of molecular weight (Fig. 1). However, in

156

addition to the molecular weight, most polysaccharides exhibit variations in proportions of sugar

157

constituents, linkage type, degree and arrangement of branching, and/or degree of substitution,

158

which also result in differential solubility (Izydorczyk & Biliaderis, 1996). Therefore, the mode of

159

fractionation is governed by not only the molecular weight but also the structural characteristics of

160

polysaccharides. Generally, higher precipitant concentration is required to precipitate the

161

polysaccharides with the structure characteristics that result in better solubility. For example, when

162

galactomannan was fractionated by gradient (NH4)2SO4 precipitation, fractions precipitated at the

163

lower saturation level of (NH4)2SO4 had less substituted mannan backbone compared with those

164

obtained at a higher salt concentration (Izydorczyk & Biliaderis, 1996). It was also found that the

165

less branched hemicelluloses were precipitated at lower ethanol percentages and more branched

166

hemicelluloses were obtained at higher ethanol concentrations (Bian et al, 2010). In general,

167

depending on the polysaccharide type, various factors, such as high molecular weight, low

168

branching levels and high substitution by hydrophobic groups, favor precipitation of

169

polysaccharides at low non-solvent concentrations.

170

4 Establishment of the fractionation procedure

SC

M AN U

TE D

EP

AC C

171

RI PT

155

Gradient non-solvent precipitation is often carried out as follows (Fig. 2). Non-solvent is

172

uniformly added to the polysaccharide solution. Afterward, the solution is held for a certain period

173

of time and then centrifuged to obtain the supernatant and precipitate. The residual non-solvent in

174

the polysaccharide precipitate should be removed. Organic solvents can be removed by drying.

175

After fractionation by (NH4)2SO4, the residual (NH4)2SO4 in the polysaccharide is often removed

176

by dialysis. On the other hand, traces of PEG in the polysaccharide precipitate are often removed

9

ACCEPTED MANUSCRIPT by chloroform. The final precipitate is designated as the 1st fraction. By continuing to add

178

non-solvent into the obtained supernatant, another polysaccharide fraction can be precipitated and

179

be designated as the 2nd fraction. The process of graded precipitation can proceed further to obtain

180

more fractions. The final supernatant is usually abandoned. In theory, the polysaccharides in the

181

final supernatant have high dispersity, since different polysaccharide species that are not

182

precipitated are left in the supernatant. In addition, it takes time and energy to remove the

183

cumulative non-solvents.

SC

RI PT

177

The stepwise precipitant concentrations used to fractionally precipitate polysaccharides are

185

the key of the fractionation procedure. A suitable initial precipitant concentration helps to save

186

fractionation time. In order to save time and cost, it is expected to achieve the highest

187

polysaccharide yield by using the lowest dose of the precipitant. However, it seems that

188

polysaccharides cannot be completely precipitated from the solution, partially because there are

189

fractions which are soluble at high precipitant concentrations. Therefore, most of the reports try to

190

obtain the best fractionation effect rather than the biggest polysaccharide yield. As is shown in

191

Table 2, the final alcohol concentration reached about 80% (v/v) in most cases. Although the

192

alcohol concentration was high, the polysaccharide recovery was often less than 90%. One reason

193

for this result might be that the parent polysaccharides usually contained large amount of

194

impurities. However, the ethanol concentration of 33.3% or the isopropanol concentration of

195

28.6% resulted in about 90% galactomannan recovery (Jian, et al., 2014). This may be due to the

196

fact that galactomannan had very large molecular weight, which was confirmed by the result that

197

the galactomannan recovery was positively correlated with the molecular weight. When

198

fractionating sugar beet pectin, the highest isopropanol concentration was also very low, about

AC C

EP

TE D

M AN U

184

10

ACCEPTED MANUSCRIPT 40% (Karnik, et al., 2016; Karnik & Wicker, 2018). This might be due to the structure of sugar

200

beet pectin. Specifically, sugar beet pectin contained high amount of hydrophobic acetyl groups,

201

ferulic acids and proteins. In addition, the fractionation procedures in different studies were

202

diverse. A previous report that investigated the effect of structural diversity on ethanol

203

concentration of precipitating polysaccharide also suggested that the ethanol concentration must

204

be individually optimized for each type of polysaccharide during ethanol precipitation (Xu, et al.,

205

2014). Different reports even used different non-solvent concentrations to fractionate the same

206

polysaccharides. Therefore, it is difficult to select non-solvent concentrations for fractionating new

207

polysaccharides according to the previous reports. It was suggested that the non-solvent

208

concentrations could be selected based on the curves of the polysaccharide yield as a function of

209

alcohol concentration (Hu, et al., 2015). Hu et al. first precipitated dextrin solutions using different

210

alcohol concentrations to obtain the curves of the dextrin yield as a function of alcohol

211

concentration. According to these curves, the dextrin yield did not significantly increase once the

212

alcohol concentration was 86.7% (v/v). Therefore, fractionation was ended when the alcohol

213

concentration reached 86.7%. When the alcohol concentration was 33.3%, the dextrin yield

214

reached more than 30%. Therefore, the fractionation started from the alcohol concentration at

215

20.0%. On the other hand, Gonzaga et al. added ethanol into the polysaccharide solution until the

216

solution became turbid and stable to obtain the fractions (Gonzaga, et al., 2005). In addition to

217

fractionation, gradient alcohol precipitation can be used to purify polysaccharides. For instance,

218

heparin, dermatan sulfate and chondroitin sulfate in mixtures were successfully separated by

219

sequential precipitation with methanol, ethanol or propanol (Volpi, 1996). Similarly, it was found

220

that keratan sulfate could be precipitated prior to chondroitin sulfate (Galeotti, Maccari, & Volpi,

AC C

EP

TE D

M AN U

SC

RI PT

199

11

ACCEPTED MANUSCRIPT 221

2014). Therefore, keratan sulfate in chondroitin sulfate samples could be selectively removed by

222

sequential precipitation with ethanol. When (NH4)2SO4 is used to fractionate polysaccharides, the (NH4)2SO4 concentration is often

224

characterized by its saturation level. It seemed that (NH4)2SO4 had higher precipitation ability on

225

glucan than other kinds of polysaccharides. Li et al. reported that β-glucans could be precipitated

226

at a much lower saturation level of (NH4)2SO4 than arabinoxylans (Li, et al., 2006). Based on this

227

principle, wheat β-glucan was separated from wheat arabinoxylan by gradient (NH4)2SO4

228

precipitation.

M AN U

SC

RI PT

223

In summary, the precipitation efficiency of organic solvents is the lowest, partially because

230

they are liquid. Compared with addition of solid (NH4)2SO4 and PEG, addition of equal organic

231

solvents greatly increases the volume of the polysaccharide solution and decreases the

232

polysaccharide concentration. To fractionate a new polysaccharide, it is suggested to first obtain

233

the curve of the polysaccharide recovery as a function of the non-solvent concentration and then

234

establish the fractionation procedure according to the curve.

235

5 Assessment of the fractionation effect

EP

TE D

229

Sometimes, fractionation of polysaccharides just helps to enrich fractions and characterize

237

polysaccharides. In this case, the fractionation effect is not assessed. Sometimes, the aim of

238

fractionation is to obtain fractions with high homogeneity. Afterward, different fractions are used

239

for different specific purposes. In this case, the molecular weight homogeneity and structural

240

homogeneity of fractions obtained by fractionation should be determined to assess the

241

fractionation effect. The homogeneity of molecular weight could be indicated by the

242

molecular-weight dispersity (DM) (Stepto, 2010). The DM is calculated as the ratio of the

AC C

236

12

ACCEPTED MANUSCRIPT weight-average molecular weight (Mw) to the number-average molecular weight (Mn). The Mw of

244

the polysaccharides is larger than the Mn. Therefore, a lower DM indicates a narrower molecular

245

weight distribution of the polysaccharides. When most of fractions obtained by fractionation have

246

smaller DM than the parent polysaccharide, the fractionation is considered to be successful. One

247

sample is always fractionated into several fractions, and different fractionation conditions always

248

result in different mass distribution and molecular weight distribution of fractions. Therefore, the

249

average molecular-weight dispersity (DMa) was defined as follows to assess the fractionation

250

effect:

M AN U

SC

RI PT

243

251

DMa =

∑ (X

i

× DMi )

∑X

i

where DMa was the average dispersity of the fractions in one group, DMi was the dispersity of the

253

ith fraction, and the Xi was the mass fraction of the ith fraction (Hu, et al., 2016; Hu, Wang, et al.,

254

2017). The lowest DMa indicated the best fractionation effect. However, structural homogeneity,

255

including proportions of sugar constituents, linkage type, degree and arrangement of branching,

256

and the degree of substitution, is difficult to assess and so far no related research is reported.

257

6 Factors that affect fractionation

EP

AC C

258

TE D

252

In theory, fractionation by gradient non-solvent precipitation is affected by the precipitation

259

conditions and those factors which affect the solubility of polysaccharides. The molecular weight

260

of polysaccharide affects its solubility, so it also affects fractionation. It was found that the higher

261

the level of high-molecular-weight dextrin, the broader the molecular weight distribution of the

262

different dextrin fractions (Gelders, et al., 2003). Definitely, the effect of the molecular weight and

263

structure of polysaccharides on fractionation cannot be eliminated. Therefore, more effort should

13

ACCEPTED MANUSCRIPT 264

be made to eliminate the negative effect of the external factors. The external factors are as follows:

265

6.1 The rate of adding the precipitant It is easy to understand that the rate of adding the precipitant affects the precipitation process,

267

particularly gradient precipitation during which limited precipitants are used to precipitate the

268

polysaccharides. It was reported that the dextrin yield was higher when alcohol was quickly added

269

than when it was slowly added (Hu, et al., 2015). It was postulated that adding alcohol fast

270

resulted in extremely high alcohol concentration in certain parts of the dextrin solution, thus

271

resulting in co-precipitation of the dextrin. As a result, the rate of adding alcohol should be

272

carefully controlled during fractionation of dextrin by gradient alcohol precipitation. Similar

273

phenomena occur to precipitation by (NH4)2SO4. Therefore, (NH4)2SO4 is often ground into

274

powders. Alternatively, a saturated (NH4)2SO4 solution is used to avoid extremely high (NH4)2SO4

275

concentration in certain parts. Similarly, (NH4)2SO4 powder or solution should be slowly added

276

into

277

PEG-polysaccharide-water system over the phase separation temperature. On the other hand, PEG

278

is not quickly dissolved in water like these organic solvents and (NH4)2SO4. Therefore, combined

279

with heating and stirring, quick addition of PEG is feasible during fractionation by PEG (Hu, et al.,

280

2016). Alternatively, a PEG solution may be used in future.

281

6.2 The precipitant

SC

M AN U

TE D

polysaccharide

solution.

The

phase

separation

does

not

occur

to

the

EP

the

AC C

282

RI PT

266

Due to non-specificity, the three types of precipitants can be widely used to fractionate

283

different kinds of polysaccharides. Basedow & Ebert used ethanol and PEG to fractionate dextran

284

and found that the fractionation results of the two precipitants had no significant difference

285

(Basedow & Ebert, 1979). However, Hu et al. found gradient alcohol precipitation had better

14

ACCEPTED MANUSCRIPT fractionation effect but lower dextrin recovery than gradient PEG precipitation (Hu, et al., 2016).

287

In addition, the fractionation effect of alcohols was in the order of methanol > ethanol >

288

isopropanol, while the precipitation efficiency was in the reverse order (Hu, et al., 2015). With

289

regard to the choice of non-solvent, it should be noted that the non-solvent cannot have very

290

strong precipitation ability (Zhang, et al., 2011). Otherwise, the fractionation will be difficult to

291

control. For example, if one polysaccharide can be completely precipitated by adding ethanol at

292

the concentration of 5% (v/v), it is almost impossible to control such a fractionation process. A

293

suitable non-solvent is the one that can provide a board range of concentration to use. In this case,

294

it is easy to control the fractionation process and adjust the non-solvent concentration for best

295

fractionation. The suitable non-solvent may also be found from the curves of the polysaccharide

296

recovery as a function of the non-solvent concentration.

297

6.3 The precipitation temperature

TE D

M AN U

SC

RI PT

286

The precipitation temperature mainly affects the precipitation efficiency. Low fractionation

299

temperature decreases the polarity of solvents and solubility of polysaccharides, thus improving

300

the precipitation efficiency. Because the precipitation efficiency of organic solvents is relatively

301

low, it is suggested to preform fractionation by gradient organic solvent precipitation at low

302

temperature (Gelders, et al., 2003). Therefore, fractionation by organic solvents is often performed

303

at 4 oC. However, the solubility of (NH4)2SO4 and PEG is low at low temperature and their

304

precipitation efficiency is relatively high at room temperature. Therefore, fractionation by

305

(NH4)2SO4 and PEG is often carried out at room temperature.

306

6.4 The precipitation time

307

AC C

EP

298

The precipitation time may affect the polysaccharide recovery. It was found that the solubility

15

ACCEPTED MANUSCRIPT of polysaccharides in ethanol was so poor that the precipitation of polysaccharides was always

309

quickly finished within minutes (Xu, et al., 2014). Similarly, it was inferred that precipitation of

310

polysaccharides by (NH4)2SO4 was quickly finished. However, after the previous studies usually

311

kept the polysaccharide solution added with organic solvents or (NH4)2SO4 at the precipitation

312

temperature for 1 h or overnight to fully precipitate the polysaccharides. The phase separation of

313

the polymer solution induced by another polymer is very slow. Therefore, the precipitation time of

314

gradient PEG precipitation was 24 h (Hu, et al., 2016; Hu, Wang, et al., 2017).

315

6.5 The initial polysaccharide concentration

M AN U

SC

RI PT

308

As stated above, fractionation by gradient non-solvent precipitation is based on differential

317

solubility of the polysaccharides. The solubility of polysaccharides is dependent on their

318

molecular weight and chemical structure. Undoubtedly, the polysaccharide solubility also depends

319

on its concentration. Therefore, the initial polysaccharide concentration may affect fractionation of

320

polysaccharide. The key of graded precipitation is to avoid co-precipitation of different

321

components. Therefore, the polysaccharide concentration cannot be too high, as co-precipitation

322

easily happens at high concentration, especially in the first fraction. The fractionation result is

323

usually better when the concentration of the polysaccharide solution is lower. But if the

324

polysaccharide concentration is too low, the recovery of polysaccharide will decrease and the

325

consumption of the non-solvent will greatly increase. In general, the initial polysaccharide

326

concentration often ranges from 0.1% to 4% (w/v), which is usually negatively correlated with the

327

molecular weight. However, the most suitable initial concentration may depend on many factors.

328

Hu et al. found that when dextrin was fractionally precipitated by ethanol, the optimal initial

329

concentration was in the range of 1.8%–2.7% (Hu, et al., 2015). However, when fractionating the

AC C

EP

TE D

316

16

ACCEPTED MANUSCRIPT 330

same dextrin by gradient PEG precipitation, the initial dextrin concentration, which ranged from

331

0.9% to 3.6%, did not affect the DMa of the dextrin fractions (Hu, Wang, et al., 2017).

332

6.6 The pH Hydroxyl groups in polysaccharide chain are ionized at high pH (Hedayati, Shahidi,

334

Koocheki, Farahnaky, & Majzoobi, 2016). Alkali can also break the intermolecular hydrogen

335

bonds of polysaccharide. Thus, pH can affect the polysaccharide solubility. In addition, the

336

solubility of charged polysaccharides is more sensitive to pH, because pH can affect the ionization

337

of dissociable groups in charged polysaccharides, such as carboxyl groups and amino groups. It

338

was also reported that pH had a significant effect on the alcoholic precipitation of sugar beet

339

pectin (Guo, Meng, Tang, et al., 2016). Therefore, pH is likely to affect fractionation of

340

polysaccharides. Hu et al. found that neutral environment was the most suitable for fractionation

341

of dextrin by gradient alcohol precipitation, while a weakly alkaline environment was optimal for

342

fractionation of dextrin by gradient PEG precipitation (Hu, Liu, et al., 2017; Hu, Wang, et al.,

343

2017). The pH could change the solubility of dextrin in alcohol or PEG solution. Theoretically, the

344

most suitable pH for dextrin fractionation was the one that could provide the greatest solubility

345

difference between high-molecular-weight dextrin and low-molecular-weight dextrin. Therefore, it

346

was likely that neutral and slightly alkaline environment provided the greatest solubility difference

347

between high-molecular-weight dextrin and low-molecular-weight dextrin in the alcohol and PEG

348

solution, respectively. However, the effect of pH on fractionation of other polysaccharides has not

349

been reported yet. Previous reports often fractionated polysaccharides in a neutral environment.

350

6.7 Other factors

351

AC C

EP

TE D

M AN U

SC

RI PT

333

When alcohol was used as the precipitant, salt also affected fractionation (Hu, Liu, et al.,

17

ACCEPTED MANUSCRIPT 2017). When dextrin was fractionated in KCl solutions, salt was co-precipitated with dextrin. KCl

353

resulted in earlier precipitation of dextrin compared to salt-free solution and co-precipitation of

354

dextrin, which was in accordance with the conclusion that salts decreased the solubility of starch

355

(Zhou, et al., 2014). In this case, salt might decrease the solubility difference of dextrin with

356

different sizes, thus negatively affecting fractionation of dextrin. A similar phenomenon may occur

357

to other polysaccharides. In addition, the cation-pectin electrostatic interaction significantly

358

weakened the hydration degree of sugar beet pectin, especially when divalent cations were used,

359

and the combined effect of added counter cations and ethanol led to a significant increase in the

360

precipitation of negatively-charged sugar beet pectin (Guo, Zhang, Meng, & Yu, 2017). Therefore,

361

divalent cations may affect fractionation of pectin and other negatively charged polysaccharides

362

through electrostatic interaction.

M AN U

SC

RI PT

352

When PEG was used as the precipitant, fractionation was also affected by the molecular

364

weight and molecular weight distribution of PEG (Hu, et al., 2016). PEG with high molecular

365

weight (over 20000 Da) could induce very high viscosity of the solution, which impeded

366

fractionation. Furthermore, fractionation was best accomplished by the most narrowly-distributed

367

PEG.

368

7 Conclusions

EP

AC C

369

TE D

363

Gradient non-solvent precipitation provides a simple and facile way for preparative

370

fractionation of polysaccharides. The non-specificity of the non-solvents makes this method

371

suitable for fractionating almost all kinds of polysaccharides. The fractionation result is affected

372

by various factors and difficult to predict. In theory, enlarging the solubility difference among

373

polysaccharides facilitates fractionation by gradient precipitation. However, the non-specificity of

18

ACCEPTED MANUSCRIPT these precipitants makes it difficult to obtain products with enough uniformity. Therefore, it is still

375

a long-standing challenge to get access to structurally-homogeneous polysaccharides, especially

376

polysaccharides with high molecular weight. Repeated fractionation by one non-solvent or

377

combination of different non-solvents may help to obtain polysaccharides with high uniformity. To

378

extend industrial application of this method, further research is needed to shorten the fractionation

379

time and design special equipment for improving its continuity of operation.

380

Acknowledgements

SC

This work was supported by the National Natural Science Foundation of China (31601425)

M AN U

381

RI PT

374

382

and Project of Jiangxi Province Education Department (GJJ160187).

383

References

384

Atha, D. H., & Ingham, K. C. (1981). Mechanism of precipitation of proteins by polyethylene glycols. Journal of Biological Chemistry, 256, 12108-12117.

TE D

385

Basedow, A. M., & Ebert, K. H. (1979). Production, characterization and solution properties of

387

dextran fractions of narrow molecular weight distributions. Journal of Polymer Science:

388

Polymer Symposium, 66, 101-115.

390

Bertoft, E. (1989). Fractional precipitation of amylopectin alpha-dextrins using methanol.

AC C

389

EP

386

Carbohydrate research, 189, 169-180.

391

Bian, J., Peng, F., Peng, P., Xu, F., & Sun, R. C. (2010). Isolation and fractionation of

392

hemicelluloses by graded ethanol precipitation from Caragana korshinskii. Carbohydrate

393

research, 345, 802-829.

394

Cardoso, S. M., Silva, A. M. S., & Coimbra, M. A. (2002). Structural characterisation of the olive

395

pomace pectic polysaccharide arabinan side chains. Carbohydrate research, 337,

19

ACCEPTED MANUSCRIPT 396

917-924. Chen, C., Zhang, B., Huang, Q., Fu, X., & Liu, R. H. (2017). Microwave-assisted extraction of

398

polysaccharides from Moringa oleifera Lam. leaves: Characterization and hypoglycemic

399

activity. Industrial crops and products, 100, 1-11.

RI PT

397

Cornell, H. J., McGrane, S. J., & Melbourne, C. J. R. (1999). A novel and rapid method for the

401

partial fractionation of starch using 1-butanol in the presence of thiocyanate.

402

Starch/Stärke, 51, 335-341.

404

Defloor, I., Vandenreyken, V., Grobet, P. J., & Delcour, J. A. (1998). Fractionation of

M AN U

403

SC

400

maltodextrins by ethanol. Journal of Chromatography A, 803, 103-109. Dervilly, G., Saulnier, L., Roger, P., & Thibault, J. F. (2000). Isolation of homogeneous fractions

406

from wheat water-soluble arabinoxylans. influence of the structure on their

407

macromolecular characteristics. Journal of agricultural and food chemistry, 48, 270-278.

410 411

Macromolecular materials and engineering, 289, 393-399. Eckelt, J., Sugaya, R., & Wolf, B. A. (2006). Large scale fractionation of pullulan and dextran.

EP

409

Eckelt, J., Haase, T., Loske, S., & Wolf, B. A. (2004). Large scale fractionation of macromolecules.

Carbohydrate polymers, 63, 205-209.

AC C

408

TE D

405

412

Feng, L., Yin, J., Nie, S., Wan, Y., & Xie, M. (2016). Fractionation, physicochemical property and

413

immunological activity of polysaccharides from Cassia obtusifolia. International journal

414 415 416 417

of biological macromolecules, 91, 946-953.

Frigard, T., Andersson, R., & Aman, P. (2002). Grandual enzmymatic modification of barley and potato amylopectin. Carbohydrate polymers, 47, 169-179. Galeotti, F., Maccari, F., & Volpi, N. (2014). Selective removal of keratan sulfate in chondroitin

20

ACCEPTED MANUSCRIPT 418

sulfate samples by sequential precipitation with ethanol. Analytical biochemistry, 448,

419

113-115. Gelders, G., Bijnens, L., Loosveld, A., Vidts, A., & Delcour, J. (2003). Fractionation of starch

421

hydrolysates into dextrins with narrow molecular mass distribution and their detection by

422

high-performance anion-exchange chromatography with pulsed amperometric detection.

423

Journal of Chromatography A, 992, 75-83.

RI PT

420

Gong, G., Dang, T., Deng, Y., Han, J., Zou, Z., Jing, S., Zhang, Y., Liu, Q., Huang, L., & Wang, Z.

425

(2017). Physicochemical properties and biological activities of polysaccharides from

426

Lycium barbarum prepared by fractional precipitation. International journal of biological

427

macromolecules, 109, 611-618.

M AN U

SC

424

Gonzaga, M. L. C., Ricardo, N. M. P. S., Heatley, F., & Soares, S. d. A. (2005). Isolation and

429

characterization of polysaccharides from Agaricus blazei Murill. Carbohydrate polymers,

430

60, 43-49.

TE D

428

Gosch, C. I., Haase, T., Wolf, B. A., & Kulicke, W. M. (2002). Molar mass distribution and size of

432

hydroxyethyl starch fractions obtained by continuous polymer fractionation. Starch/Stärke,

433

54, 375-384.

AC C

EP

431

434

Guan, Y., Zhang, B., Qi, X. M., Peng, F., Yao, C. L., & Sun, R. C. (2015). Fractionation of

435

bamboo hemicelluloses by graded saturated ammonium sulphate. Carbohydrate polymers,

436

129, 201-207.

437

Guo, X., Meng, H., Tang, Q., Pan, R., Zhu, S., & Yu, S. (2016). Effects of the precipitation pH on

438

the ethanolic precipitation of sugar beet pectins. Food hydrocolloids, 52, 431-437.

439

Guo, X., Meng, H., Zhu, S., Tang, Q., Pan, R., & Yu, S. (2016). Stepwise ethanolic precipitation of

21

ACCEPTED MANUSCRIPT sugar beet pectins from the acidic extract. Carbohydrate polymers, 136, 316-321.

441

Guo, X., Zhang, T., Meng, H., & Yu, S. (2017). Ethanol precipitation of sugar beet pectins as

442

affected by electrostatic interactions between counter ions and pectin chains. Food

443

hydrocolloids, 65, 187-197.

RI PT

440

Hammerschmidt, N., Hobiger, S., & Jungbauer, A. (2016). Continuous polyethylene glycol

445

precipitation of recombinant antibodies: Sequential precipitation and resolubilization.

446

Process Biochemistry, 51, 325-332.

SC

444

He, P. F., He, L., Zhang, A. Q., Wang, X. L., Qu, L., & Sun, P. L. (2017). Structure and chain

448

conformation of a neutral polysaccharide from sclerotia of Polyporus umbellatus.

449

Carbohydrate polymers, 155, 61-67.

M AN U

447

Hedayati, S., Shahidi, F., Koocheki, A., Farahnaky, A., & Majzoobi, M. (2016). Physical

451

properties of pregelatinized and granular cold water swelling maize starches at different

452

pH values. International journal of biological macromolecules, 91, 730-735.

TE D

450

Hu, X. T., Liu, C. M., Jin, Z. Y., & Tian, Y. Q. (2015). Fractionation of starch hydrolysate into

454

dextrin fractions with low dispersity by gradient alcohol precipitation. Separation and

455

Purification Technology, 151, 201-210.

457

AC C

456

EP

453

Hu, X. T., Liu, C. M., Jin, Z. Y., & Tian, Y. Q. (2016). Fractionation of dextrin by gradient polyethylene glycol precipitation. Journal of chromatography. A, 1434, 81-90.

458

Hu, X. T., Liu, C. M., Jin, Z. Y., & Tian, Y. Q. (2017). Preparative fractionation of dextrin by

459

gradient alcohol precipitation. Separation science and technology, 52, 2704-2714.

460

Hu, X. T., Wang, Y., Liu, C. M., Jin, Z. Y., & Tian, Y. Q. (2017). Preparative fractionation of

461

dextrin by polyethylene glycol: Effects of initial dextrin concentration and pH. Journal of

22

ACCEPTED MANUSCRIPT 462

chromatography. A, 1530, 226-231. Huang, Q. L., Siu, K. C., Wang, W. Q., Cheung, Y. C., & Wu, J. Y. (2013). Fractionation,

464

characterization and antioxidant activity of exopolysaccharides from fermentation broth

465

of a Cordyceps sinensis fungus. Process Biochemistry, 48, 380-386.

468 469

properties of wheat arabinoxylan. Carbohydrate polymers, 17, 237-247.

Izydorczyk, M. S., & Biliaderis, C. G. (1996). Gradient ammonium sulphate fractionation of galactomannans. Food hydrocolloids, 10, 295-300.

SC

467

Izydorczyk, M. S., & Biliaderis, C. G. (1992). Influence of structure on the physicochemical

M AN U

466

RI PT

463

470

Izydorczyk, M. S., Biliaderis, C. G., Macri, L. J., & MacGregor, A. W. (1998). Fractionation of oat

471

(1→3), (1→4)-β-D-glucans and characterisation of the fractions. Journal of cereal

472

science, 27, 321-325.

Jian, H. L., Lin, X. J., Zhang, W. A., Zhang, W. M., Sun, D. F., & Jiang, J. X. (2014).

474

Characterization of fractional precipitation behavior of galactomannan gums with ethanol

475

and isopropanol. Food hydrocolloids, 40, 115-121.

TE D

473

Kang, J., Cui, S. W., Chen, J., Phillips, G. O., Wu, Y., & Wang, Q. (2011). New studies on gum

477

ghatti (Anogeissus latifolia) part I. Fractionation, chemical and physical characterization

AC C

478

EP

476

of the gum. Food hydrocolloids, 25, 1984-1990.

479

Karnik, D., Jung, J., Hawking, S., & Wicker, L. (2016). Sugar beet pectin fractionated using

480

isopropanol differs in galacturonic acid, protein, ferulic acid and surface hydrophobicity.

481 482 483

Food hydrocolloids, 60, 179-185. Karnik, D., & Wicker, L. (2018). Emulsion stability of sugar beet pectin fractions obtained by isopropanol fractionation. Food hydrocolloids, 74, 249-254.

23

ACCEPTED MANUSCRIPT 484

Klucinec, J. D., & Thompson, D. B. (1998). Fractionation of high-amylose maize starches by

485

differential alcohol precipitation and chromatography of the fractions. Cereal Chemistry,

486

75, 887-896.

488

Li, D., & Zhu, F. (2018). Characterization of polymer chain fractions of kiwifruit starch. Food

RI PT

487

chemistry, 240, 579-587.

Li, H., Dai, Q., Ren, J., Jian, L., Peng, F., Sun, R., & Liu, G. (2016). Effect of structural

490

characteristics of corncob hemicelluloses fractionated by graded ethanol precipitation on

491

furfural production. Carbohydrate polymers, 136, 203-209.

M AN U

SC

489

492

Li, J. E., Wang, W. J., Zheng, G. D., & Li, L. Y. (2017). Physicochemical properties and

493

antioxidant activities of polysaccharides from Gynura procumbens leaves by fractional

494

precipitation. International journal of biological macromolecules, 95, 719-724.

496

Li, W., Cui, S., & Kakuda, Y. (2006). Extraction, fractionation, structural and physical

TE D

495

characterization of wheat β-D-glucans. Carbohydrate polymers, 63, 408-416. Nagel, A., Winkler, C., Carle, R., Endress, H. U., Rentschler, C., & Neidhart, S. (2017). Processes

498

involving selective precipitation for the recovery of purified pectins from mango peel.

499

Carbohydrate polymers, 174, 1144-1155.

501 502 503

AC C

500

EP

497

Neuchl, C., & Mersmann, A. (1995). Fractionation of polydisperse dextran using ethanol. Chemical engineering science, 50, 951-958.

Neuchl, C., & Mersmann, A. (1996). Comparison of the non-solvents ethanol and isopropanol for the fractionation of dextran. Fluid phase equilibria, 116, 133-139.

504

Paseephol, T., Small, D., & Sherkat, F. (2007). Process optimisation for fractionating Jerusalem

505

artichoke fructans with ethanol using response surface methodology. Food chemistry, 104,

24

ACCEPTED MANUSCRIPT 506

73-80. Peng, F., Bian, J., Peng, P., Xiao, H., Ren, J. L., Xu, F., & Sun, R. C. (2012). Separation and

508

characterization of acetyl and non-acetyl hemicelluloses of Arundo donax by ammonium

509

sulfate precipitation. Journal of agricultural and food chemistry, 60, 4039-4047.

510

Peng, F., Ren, J. L., Xu, F., Bian, J., Peng, P., & Sun, R. C. (2009). Comparative study of

511

hemicelluloses obtained by graded ethanol precipitation from sugarcane bagasse. Journal

512

of agricultural and food chemistry, 57, 6305-6317.

SC

RI PT

507

Peng, F., Ren, J. L., Xu, F., Bian, J., Peng, P., & Sun, R. C. (2010). Fractional study of

514

alkali-Soluble hemicelluloses obtained by graded ethanol precipitation from sugar cane

515

bagasse. Journal of agricultural and food chemistry, 58, 1768–1776.

M AN U

513

Peng, H., Zhou, M., Yu, Z., Zhang, J., Ruan, R., Wan, Y., & Liu, Y. (2013). Fractionation and

517

characterization of hemicelluloses from young bamboo (Phyllostachys pubescens Mazel)

518

leaves. Carbohydrate polymers, 95, 262-271.

TE D

516

Peng, P., Peng, F., Bian, J., Xu, F., & Sun, R. (2011). Studies on the starch and hemicelluloses

520

fractionated by graded ethanol precipitation from bamboo Phyllostachys bambusoides f.

521

shouzhu Yi. Journal of agricultural and food chemistry, 59, 2680-2688.

AC C

EP

519

522

Pourfarzad, A., Habibi Najafi, M. B., Haddad Khodaparast, M. H., Hassanzadeh Khayyat, M., &

523

Malekpour, A. (2014). Fractionation of Eremurus spectabilis fructans by ethanol:

524 525

Box-Behnken design and principal component analysis. Carbohydrate polymers, 106, 374-383.

526

Ragaee, S. M., Wood, P. J., Wang, Q., Tosh, S. M., Brummer, Y., & Huang, X. (2008). Isolation,

527

fractionation, and structural characteristics of alkali-extractable β-glucan from rye whole

25

ACCEPTED MANUSCRIPT 528 529 530

meal. Cereal Chemistry Journal, 85, 289-294. Schoch, T. J. (1942). Fractionation of starch by selective precipitation with butanol. Journal of the American Chemical Society, 64, 2957-2961. Shi, J. B., Yang, Q. L., Lin, L., & Peng, L. C. (2013). Fractionation and characterization of

532

physicochemical and structural features of corn stalk hemicelluloses from yellow liquor

533

of active oxygen cooking. Industrial crops and products, 44, 542-548.

RI PT

531

Shi, X. D., Nie, S. P., Yin, J. Y., Que, Z. Q., Zhang, L. J., & Huang, X. J. (2017). Polysaccharide

535

from leaf skin of Aloe barbadensis Miller: Part I. Extraction, fractionation,

536

physicochemical properties and structural characterization. Food hydrocolloids, 73,

537

176-183.

M AN U

SC

534

Sim, S. L., He, T., Tscheliessnig, A., Mueller, M., Tan, R. B., & Jungbauer, A. (2012). Protein

539

precipitation by polyethylene glycol: a generalized model based on hydrodynamic radius.

540

Journal of biotechnology, 157, 315-319.

543 544 545

international, 59, 23-24.

EP

542

Stepto, R. F. T. (2010). Dispersity in polymer science (IUPAC Recommendation 2009). Polymer

Volpi, N. (1996). Purification of heparin, dermatan sulfate and chondroitin sulfate from mixtures

AC C

541

TE D

538

by sequential precipitation with various organic solvents. Journal of Chromatography B,

685 27-34.

546

Wang, J., Nie, S., Cui, S. W., Wang, Z., Phillips, A. O., Phillips, G. O., Li, Y., & Xie, M. (2017).

547

Structural characterization and immunostimulatory activity of a glucan from natural

548

Cordyceps sinensis. Food hydrocolloids, 67, 139-147.

549

Wang, Q., Wood, P. J., Huang, X., & Cui, W. (2003). Preparation and characterization of

26

ACCEPTED MANUSCRIPT 550

molecular

weight

standards

of

low

polydispersity

551

(1→3)(1→4)-β-D-glucan. Food hydrocolloids, 17, 845-853.

from

oat

and

barley

Xu, J., Yue, R. Q., Liu, J., Ho, H. M., Yi, T., Chen, H. B., & Han, Q. B. (2014). Structural diversity

553

requires individual optimization of ethanol concentration in polysaccharide precipitation.

554

International journal of biological macromolecules, 67, 205-209.

RI PT

552

Xue, B. L., Wen, J. L., Xu, F., & Sun, R. C. (2012). Structural characterization of hemicelluloses

556

fractionated by graded ethanol precipitation from Pinus yunnanensis. Carbohydrate

557

research, 352, 159-165.

M AN U

SC

555

Zha, X. Q., Xiao, J. J., Zhang, H. N., Wang, J. H., Pan, L. H., Yang, X. F., & Luo, J. P. (2012).

559

Polysaccharides in Laminaria japonica (LP): Extraction, physicochemical properties and

560

their hypolipidemic activities in diet-induced mouse model of atherosclerosis. Food

561

chemistry, 134, 244-252.

562

TE D

558

Zhang, M., Zhu, L., Cui, S. W., Wang, Q., Zhou, T., & Shen, H. (2011). Fractionation, partial characterization

and

bioactivity

of

water-soluble

polysaccharides

and

564

polysaccharide-protein complexes from Pleurotus geesteranus. International journal of

565

biological macromolecules, 48, 5-12.

AC C

EP

563

566

Zhou, H., Wang, C., Shi, L., Chang, T., Yang, H., & Cui, M. (2014). Effects of salts on

567

physicochemical, microstructural and thermal properties of potato starch. Food chemistry,

568 569 570

156, 137-143.

Zief, M., Brunner, G., & Metzendorf, J. (1955). Fractionation of partially hydrolyzed dextran. Industrial and engineering chemistry, 48, 119-121.

571

27

ACCEPTED MANUSCRIPT Figures captions

573

Fig. 1 Schematic representation of fractionating polysaccharides by gradient non-solvent

574

precipitation in the descending order of molecular weight.

575

Fig. 2 The fractionation procedure by gradient non-solvent precipitation.

AC C

EP

TE D

M AN U

SC

RI PT

572

28

ACCEPTED MANUSCRIPT

RI PT

Table 1 The commonly used precipitants and the corresponding polysaccharides. Polysaccharide

Reference

Methanol

Dextran

John Eckelt, Sugaya, & Wolf, 2006; Zief, Brunner, & Metzendorf, 1955

Dextrin

Bertoft, 1989; Gelders, Bijnens, Loosveld, Vidts, & Delcour, 2003; Hu, Liu, Jin, & Tian,

M AN U

SC

Precipitant

2015

Basedow & Ebert, 1979; Neuchl & Mersmann, 1995, 1996

Dextrin

Defloor , Vandenreyken, Grobet, & Delcour, 1998; Frigard, Andersson, & Aman, 2002;

TE D

Dextran

Gelders, et al., 2003; Hu, Liu, Jin, & Tian, 2017; Hu, et al., 2015

Arabinan

Dervilly, Saulnier, Roger, & Thibault, 2000

EP

Wheat arabinoxylan

AC C

Ethanol

Cardoso, Silva, & Coimbra, 2002

Polysaccharides from Agaricus blazei Murill fructan

Gonzaga, Ricardo, Heatley, & Soares, 2005

Fructan

Paseephol, Small, & Sherkat, 2007; Pourfarzad, Habibi Najafi, Haddad Khodaparast, Hassanzadeh Khayyat, & Malekpour, 2014

ACCEPTED MANUSCRIPT

Bian, Peng, Peng, Xu, & Sun, 2010; Li, et al., 2016; Peng, et al., 2009; Peng, et al., 2010;

RI PT

Hemicellulose

Peng, et al., 2013; Peng, Peng, Bian, Xu, & Sun, 2011; Shi, Yang, Lin, & Peng, 2013;

SC

Xue, Wen, Xu, & Sun, 2012 Kang, et al., 2011

Exopolysaccharides from Cordyceps sinensis fungus

Huang, Siu, Wang, Cheung, & Wu, 2013

Polysaccharides

Zha, et al., 2012

from

Laminaria

japonica

Jian, et al., 2014

Pectin

Guo, Meng, Zhu, et al., 2016

Lycium

barbarum

AC C

from

Feng, Yin, Nie, Wan, & Xie, 2016

EP

Polysaccharides from Cassia obtusifolia Polysaccharides

TE D

Galactomannan gum

M AN U

Gum ghatti

Gong, et al., 2017

Polysaccharides from Moringaoleifera Lam. Leaves

Chen, Zhang, Huang, Fu, & Liu, 2017

Glucan

Wang, et al., 2017

Polysaccharide

form

Polyporus

umbellatus He, et al., 2017

ACCEPTED MANUSCRIPT

Li, Wang, Zheng, & Li, 2017

Dextran

Neuchl & Mersmann, 1996

Dextrin

Gelders, et al., 2003; Hu, et al., 2015

Galactomannan gum

Jian, et al., 2014

Pectin

Karnik, Jung, Hawking, & Wicker, 2016; Karnik & Wicker, 2018; Nagel, et al., 2017

Acetone

Pullulan

John Eckelt, et al., 2006

1-Butanol

Starch

Cornell, McGrane, & Melbourne, 1999; Klucinec & Thompson, 1998; Li & Zhu, 2018;

SC

M AN U

TE D

Isopropanol

RI PT

Polysaccharides from Gynura procumbens

Schoch, 1942 Izydorczyk & Biliaderis, 1992

EP

Arabinoxylan Galactomannan Glucan

AC C

(NH4)2SO4

Mushroom polysaccharides

Izydorczyk, &Biliaderis, 1996 Izydorczyk, Biliaderis, Macri, & MacGregor, 1998; Li, Cui, & Kakuda, 2006; Ragaee, et al., 2008; Wang, et al., 2003 Zhang, et al., 2011

ACCEPTED MANUSCRIPT

Guan, et al., 2015; Peng, et al., 2012

Polysaccharide from leaf skin of Aloe barbadensis

Shi, et al., 2017

RI PT

Hemicellulose

Basedow & Ebert, 1979

Dextrin

Hu, Liu, Jin, & Tian, 2016; Hu, Wang, Liu, Jin, & Tian, 2017

EP

TE D

M AN U

Dextran

AC C

PEG

SC

Miller

ACCEPTED MANUSCRIPT

Non-solvent concentrations

Polysaccharide

Methanol (v/v)

20%, 33%, 50%, 66.7%, 75%, 80% and 83%

Dextrin

Ethanol (v/v)

20%, 33%, 50%, 66.7%, 75%, 80% and 83%

Dextrin

20%, 30%, 40%, 50%, 60% and 70% 50%, 65% and 80%

Reference Hu, et al., 2015 Hu, et al., 2015; Hu, Liu, et al., 2017

Arabinoxylan

Dervilly, et al., 2000

Arabinan

Cardoso, et al., 2002

Hemicellulose

Peng, et al., 2009; Peng, et al., 2010

TE D

15%, 30% and 60%

SC

Non-solvent

M AN U

RI PT

Table 2 Examples of the non-solvent concentrations used during gradient precipitation.

Hemicellulose

Bian, et al., 2010

Hemicellulose

Peng, et al., 2011

Hemicellulose

Xue, et al., 2012

Hemicellulose

Peng, et al., 2013

50%, 67%, 75%, 83% and 88%

Hemicellulose

Shi, et al., 2013

15%, 30%, 45% and 60%

Hemicellulose

Li, et al., 2016

10%, 20%, 30%, 45%, 60% and 80%

EP

15%, 30%, 45%, 60% and 75%

20%, 40%, 60% and 80%

AC C

15%, 60% and 90%

ACCEPTED MANUSCRIPT

17%, 29%, 50%, 67% and 84%

Polysaccharide from Cordyceps sinensis

Huang, et al., 2013

40%, 60% and 80%

Polysaccharide from Laminaria japonica

Zha, et al., 2012

23.1%, 28.6% and 33.3%

Galactomannan gum

Jian, et al., 2014

50%, 67%, 71%, 75%, 78% and 80%

Pectin

Guo, Meng, Zhu, et al., 2016

Polysaccharide from Lycium barbarum

Gong, et al., 2017

Polysaccharide from Moringaoleifera Lam

Chen, et al., 2017

SC

RI PT

Gum ghatti

M AN U

Kang, et al., 2011

50%, 65% and 80%

30%, 50% and 70%

TE D

40%, 60% and 80%

Glucan

Wang, et al., 2017

Polysaccharide from Gynura procumbens

Li, et al., 2017

Dextrin

Hu, et al., 2015

Galactomannan

Jian, et al., 2014

10%, 18%, 25%, 31%, 36% and 40%

Sugar beet pectin

Karnik, et al., 2016

10%, 18%, 25% and 31%

Sugar beet pectin

Karnik & Wicker, 2018

30%, 50% and 70%

EP

20%, 40%, 60% and 80% 20%, 33%, 50%, 66.7%, 75%, 80% and 83%

(v/v)

16.7%, 23.1% and 28.6%

AC C

Isopropanol

ACCEPTED MANUSCRIPT

Arabinoxylan

saturation level

20%, 30%, 45%, 80% and 100%

Galactomannan

70%, 80% and 100%

Galactomannan

30%, 35%, 40% and 50%

Glucan

15%, 15.5%, 16%, 16.3%, 16.6%, 17.2% and 21% 15.5%, 16.7%, 17.8%, 18.3%, 20%, 21.1% and 24%

and 33.5%

PEG

AC C

5%, 15%, 25%, 40%, 55% and 70%

EP

16.4%, 16.8%,17.5%, 18.5%, 19.5%, 23.2%, 28.5%

the initial addition 5 g, stepwise 5 g, the final addition 80 g (For 100 mL solution)

SC

Izydorczyk, &Biliaderis, 1996 Izydorczyk, &Biliaderis, 1996 Izydorczyk, et al., 1998

Glucan

Wang, et al., 2003

Glucan

Wang, et al., 2003

Glucan

Li, et al., 2006

TE D

16.2%, 17.01%, 18.01%, 19.44%, 24% and 40%

RI PT

60%, 70%, 80% and 95%

M AN U

Izydorczyk & Biliaderis, 1992

(NH4)2SO4

Glucan

Ragaee, et al., 2008

Polysaccharide from Aloe barbadensis Miller

Guan, et al., 2015

Dextrin

Hu, et al., 2016; Hu, Wang, et al., 2017

ACCEPTED MANUSCRIPT Fig. 1 Schematic representation of fractionating polysaccharides by gradient non-solvent

AC C

EP

TE D

M AN U

SC

RI PT

precipitation in the descending order of molecular weight.

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

Fig. 2 The fractionation procedure by gradient non-solvent precipitation.

ACCEPTED MANUSCRIPT Gradient precipitation is based on solubility differences of polysaccharides. Polysaccharides are generally precipitated in descending order of molecular weight. Non-solvents of polysaccharides mainly include organic solvents, (NH4)2SO4 and PEG.

AC C

EP

TE D

M AN U

SC

RI PT

Fractionation of polysaccharides by this method is affected by various factors.

1