Accepted Manuscript

Gelation mechanism of polysaccharides from Auricularia auricula-judae

Honghui Bao, Rui Zhou, SangGuan You, Shengfang Wu, Qi Wang, Steve W. Cui

PII: S0268-005X(17)31281-X

DOI: 10.1016/j.foodhyd.2017.07.023

Reference: FOOHYD 3992

To appear in: Food Hydrocolloids

Received Date: 20 September 2016

Revised Date: 11 July 2017

Accepted Date: 25 July 2017

Please cite this article as: Honghui Bao, Rui Zhou, SangGuan You, Shengfang Wu, Qi Wang,
Steve W. Cui, Gelation mechanism of polysaccharides from Auricularia auricula-judae, Food
Hydrocolloids (2017), doi: 10.1016/j.foodhyd.2017.07.023

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 HO HO HO O HO COOH
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OH O OH O OH C C
O C
CH2OH COOH
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O O formed OH O formed
H
H
HO COOH
O O
O
HOH2C HOOC C
C C
O O HO O HO O HOOC
HO O O O O
OH OH OH OH

Inter H-bond formed by uronic acid in AP chains intra H-bond formed extended stiff AP chain
network structure of AP formed by intra
single AP chain aggregated by inter H-bond and inter H-bond
o s e Addition of urea or
c
of glu
n glucuronic acid
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urea or glucuronic acid broke down inter H-bond

Addition of glucose made less water available resulting in the disconnection of the aggregates
to maintain the polymer chains in solution, into single chains, meanwhile, destroyed the intra
therefore, raised the density network of AP. H-bond of AP chain leading conformational
transition from rigid to flexible chains. Network of
AP collapsed.
 ACCEPTED MANUSCRIPT

1 Gelation mechanism of polysaccharides from Auricularia auricula-

2 judae

3 Honghui Bao a, Rui Zhou a,b, SangGuan You c, Shengfang Wu d, Qi Wang e and Steve W. Cui e,*

4 a College of Food Science, Heilongjiang Bayi Agricultural University, 2 Xinyang Road, Daqing,

5 Heilongjiang 163319, China

6 b Department of Food Processing and Distribution, Gangneung-Wonju National University, 120

7 Gangneung Daehangno, Gangneung, Gangwon, 210-702, Korea

8 c Department of Marine Food Science and Technology, Gangneung-Wonju National University,

9 120 Gangneung Daehangno, Gangneung, Gangwon, 210-702, Korea

10 d State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China

11 e Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Road W., Guelph,

12 Ontario N1G 5C9, Canada

13 * Corresponding author. Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93

14 Stone Road W., Guelph, Ontario N1G 5C9, Canada Tel.: +1 226 217 8076; fax: +1 226 217

15 8181.

16 E-mail address: cuis@agr.gc.ca (S. W. Cui), honghui_bao@163.com (H. H. Bao).

17

18 ABSTRACT

19

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1 In the current study, dynamic frequency sweep and stress relaxation tests were performed to

2 elucidate the possible gelation mechanisms of a unique thermal stable polysaccharides from

3 Auricularia auricula-judae (AP). Addition of NaCl, CaCl2 and Ca-chelating agent (EDTA) to

4 AP confirmed that electrostatic interaction was not the deciding force in forming the network of

5 AP dispersion and the intrinsic calcium of AP played a role of inhibition in AP network

6 formation. Addition of the hydrogen bond breaking agent, urea, highlighted the important role of

7 hydrogen bonding in the formation of gel networks. The results suggested that carboxyl group of

8 the uronic acid on AP chain are involved in the formation of inter/intra hydrogen bonding which

9 maintain AP gel network. Quantitative effect of concentration, pH, urea and glucuronic acid on

10 the network structure of AP dispersion were also be evaluated by entanglement network numbers

11 (ENN) based on stress relaxation.

12

13 Keywords:

14 Auricularia auricula-judae

15 Polysaccharides

16 Gelation mechanism

17 Hydrogen bond

18 Carboxyl group

19 Rheology

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1 1. Introduction

2 Polysaccharides have been used in food, pharmaceutical and cosmetic industry as thickeners,

3 stabilizers, gelling agents for several centuries. Finding new sources of polysaccharides with

4 potential industrial applications has become important for researchers because of the increasing

5 demand for hydrocolloids with specific functions. Auricularia auricula-judae, which is one of

6 the most popular mushrooms in Asian countries, has been used for food and medicine for

7 hundreds of years (Nguyen et al., 2012; Sone, Kakuta, & Misaki, 1978). The polysaccharides

8 from A. auricula-judae have been extensively studied due to its distinguished bioactivities. It

9 was reported that β-D-glucan from A. auricula-judae could inhibit the Acinar cell carcinoma

10 proliferation and induce apoptosis in S-180 tumor cell by up-regulating Bax and down-regulating

11 Bcl-2 (Ma, Wang, Zhang, Zhang & Ding, 2010). Zeng et al. claimed that a water extracted

12 polysaccharides from A. auricula-judae mycelium significantly decreased the levels of total

13 cholesterol and triglyceride of diet-induced hyperlipidemia mice (Zeng et al., 2013).

14 Polysaccharides from A. auricula-judae also possess strong antioxidant and anticoagulant

15 activity (Yuan, He, Cui, & Takeuchi, 1998; Yoon et al., 2003; Wu et al., 2010). A water soluble

16 polysaccharide isolated from A. auricula-judae was identified as a β-(1→3)-D-glucan with two

17 β-(1→6)-D-glucosyl residues for every three main chain glucose residues showing a comb-

18 branched structure; this polysaccharide existed as a stiff chain conformation in water displaying

19 parallel self-orientation behaviour (Xu, Xu, & Zhang, 2012; Xu, Xu, & Zhang, 2013; Xu, Lin, Li,

20 Xu, & Zhang, 2013). It is reported that containing glucuronic acid and glucopyranosyl side

21 groups resulted in the increased stiffness of the chain conformation of the polysaccharides from

22 A. auricula-judae (Ma et al., 2010).

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1 Though the structural characteristics and bioactivities of polysaccharides from A. auricula-

2 judae have been extensively studied, however, there is a lack of understanding of the rheological

3 properties of these polysaccharides. In our previous studies (Bao et al., 2016), rheological

4 properties of water soluble polysaccharides from A. auricula-judae (AP) dispersion were

5 investigated by dynamic rheological approach to exhibit a strong gel structure and unique

6 thermal stability. The objective of current study was to elucidate the mechanism of gelation of

7 this polysaccharide using stress relaxation approach.

8 2. Materials and Methods

9 2.1. Materials

10 The fruiting bodies of A. auricula-judae, a commercial product cultivated in Heilongjiang

11 province (China), were washed with distilled water and air-dried at 60 °C. The dried sample was

12 milled using a blender, sieved (40-mesh) and stored at 4 °C before analyses. D-(+)-Glucose and

13 D-glucuronic acid were purchased from Sigma-Aldich Co. (St.Louis, MO, USA). All chemicals

14 and reagents used in this work were of analytical grade.

15 2.2. Extraction of the crude polysaccharide

16 The milled sample was refluxed with 80% ethanol (EtOH) at 60 °C for 2 h to remove the

17 lipophilic substances, then heated in boiling water for 2 h (×2) to extract the polysaccharides.

18 The supernatants were combined and deproteinated by the Sevag procedure (Sevag, Lackman, &

19 Smolens, 1938). Then, EtOH (99%) was added to the supernatants (get from Sevag procedure) to

20 obtain a final EtOH concentration of 70% (v/v). The crude polysaccharide (AP) was collected

21 by filtering the solution through a nylon membrane (0.45µm pore size, Whatman International,

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1 Maidstone, UK); the precipitate was washed with ethanol (99%) and acetone, and dried

2 overnight at room temperature.

3 2.3. Sample preparation

4 AP dispersions were first prepared at concentrations of 0.1%, 0.5%, 1% and 2% by dispersing

5 the required amount in deionized water at room temperature. Then, 2% AP dispersion was

6 loaded to base plate of rheometer and allowed to equilibrate at as set-point temperature for 1 h in

7 water bath. Experiments were carried out at 25, 45, and 75 oC respectively. 1.5% AP was

8 prepared in purified water, 0.01, 0.1, 0.5, 1.0, 2.0 M NaCl and 0.01, 0.1, 0.5, 1.0 M CaCl2

9 solution respectively to study the influence of ionic associations on the gelation of AP. To

10 evaluate the pH effect on networks of AP dispersion, 2% AP dispersion was prepared and

11 adjusted the pH values to around 2, 4, 6, 10, 12 by using 4 M NaOH and 4 M HCl before stress

12 relaxation measurement. A hydrogen bond breaking agent, urea, was added to 2% AP dispersion

13 at different concentrations (with and without heat) to elucidate whether the gelation of AP was

14 taken placed by hydrogen bond. Different concentrations of glucose and glucuronic acid were

15 added to 1.5% AP dispersion (with heat, 80 oC, 10 min, then cool) to investigate the possible site

16 for hydrogen bonding formation. All of the prepared dispersion were homogenously mixed for 8

17 h by constant stirring and stored over night at 4 oC. The pH of AP dispersion with glucuronic

18 acid was adjusted to 6 by 4 M NaOH before relaxation test.

19 2.4. Rheological measurements

20 The rheological measurements were performed on a strain controlled ARES Rheometer (TA

21 Instruments, New Castle, DE, USA), equipped with a parallel plate geometry (25 mm diameter

22 with a gap of 1 mm). During the experiments, the sample-hold region was covered with paraffin

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1 oil in order to prevent water loss. The data was collected after the sample was loaded on and the

2 temperature reached to set value.

3 The linear viscoelastic region (LVR) was established to set the strain of the rest of the

4 experiments (data not presented). The percent stain mode was used for LVB measurements in the

5 range of 0.01-1000% at 25 oC at low oscillation frequency (1 Hz).

6 The dynamical oscillatory frequency sweep was carried out in controlled-strain mode. The

7 sample was subjected to frequency sweep over the frequency range of 0.1 to 30 Hz. The elastic

8 modulus (G') were measured as a function of frequency. A plot of frequency vs G' was obtained.

9 Stress relaxation test was conducted at strain of 20% at set temperature to evaluate the change

10 of the networks of the gelation sample. Data were collected as soon as the normal force was

11 close to zero and stopped when G(t) value was lower than 0.01 Pa.

12 3. Result and discussion

13 3.1. Effect of temperature and concentration on network formation

14 The effect of temperature and polysaccharide concentration on rheological properties of AP is

15 demonstrated in Fig. 1. Fig. 1a illustrated the stress relaxation spectra of AP at various

16 temperatures: the gel strength decreased with the temperature increased from 25 to 80 oC,

17 however, the pattern of curves are quite similar. These results are in agreement with our previous

18 study in dynamic experiments (Bao et al., 2016) that AP exhibited excellent thermal stability.

19 The decay of the log relaxation modulus G(t) at different AP concentrations is shown in Fig. 1b.

20 The result indicated that the relaxation modulus G(t) of AP dispersion increased with increment

21 of AP concentration, suggesting increase of gel strength. This is because with the increment of

22 AP concentration, individual molecules start to overlap, increasing the formation of

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1 intermolecular junctions, hence improved the strength of the networks of AP dispersion (Bao et

2 al., 2016; Freitas et al., 2009), consequently, the gel strength raised up as evidenced by

3 prolonged the relaxation time.

4 3.2. Effect of pH on stress relaxation of AP

5 Solution pH is one of the important factors for affecting gelation due to changes in molecular

6 conformation, the strength of hydrogen bonds and electrostatic interactions (Li, Al-Assaf, Fang,

7 & Phillips, 2013). The relaxations curves of 2% AP dispersion at different pH conditions at 25

8 oC are presented in Fig. 2. The results showed that at both acidic conditions (pH 2-6) and

9 alkaline conditions (pH 6-10) the network formation of AP dispersion decreased, which is in

10 agreement with previous results based on dynamic measurements (Bao et al., 2016). Compared

11 with alkali condition, acid condition obviously disrupted the network of AP dispersion; this is

12 not only because of slight Mw degradation in acid condition (Zhang, Chen, Ma, & Zhang, 2013)

13 but also due to breakdown of hydrogen bond and rupture the interaction of macromolecular of

14 AP since H+ is a hydrogen bond breaking agent (Wu, Ai, Chen, Kang, & Cui, 2013b). However,

15 low-methoxy pectin formed a crosslinked network at pH 3.0 in the absence of Ca2+ (Gilsenan,

16 Richardson, & Morris, 2000). Hyaluronan (HA) formed the putty structure at low pH (2.5) due to

17 the formation of a large quantity of hydrogen bonds at the isoelectric point of carboxyl groups

18 (Wu et al., 2013b). The effect of pH on gelation of polysaccharides is significantly different

19 depending on the molecular characteristics and gelation mechanism of gelling agent.

20 3.3 Effect of ionic strength on network formation of AP dispersion

21 The effect of electrostatic interaction on the network formation of AP dispersion was

22 confirmed by addition of NaCl, CaCl2 and Ca-chelating agent, EDTA (ethylene-diaminetetra-

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1 acetic acid). At first, we use NaCl, CaCl2 to examine the contribution of Na+ and Ca2+ to the

2 network formation of AP. The results showed that the effect of salt concentration on the network

3 formation of AP dispersion was not crucial. As showed in Fig.3, the relaxation modulus (G(t))

4 was slightly increased when the NaCl or CaCl2 concentration was added to 0.01M, however,

5 decreased with further addition of salt from 0.1 to 1 M. Secondly, we used Ca-chelating agent,

6 Na2EDTA to investigate the contribution of intrinsic Ca2+ to the network formation of AP. The

7 result (Fig. 3b) showed that the network density was increased after addition of Na2EDTA. This

8 is quite different from that of alginate. In comparison, we added 0.1 M CaCl2 into an alginate

9 solution which formed a hard gel; this strong gel was broken down with the addition of 8%

10 Na2EDTA. It was reported that the chains of alginate associated into fibrils in the presence of

11 Ca2+ and its network density increased with increasing Ca2+ ion concentration (Liu, Li, Tang, Bi,

12 & Lin, 2016; Morris, Rees, & Thom, 1973). However, our results suggested that, the calcium in

13 AP did not support but inhibited the network formation of AP dispersion. The above

14 observations in stress relaxation are in accordance with the data from dynamic analysis (Fig. 4).

15 Fig. 4 shows the frequency dependence of the storage modulus (G') of AP with different

16 concentrations of Calcium and AP with 8% EDTA. The gel strength was slightly increased when

17 the CaCl2 concentration was added to 0.01 M and decreased with further addition of salt from 0.1

18 to 1 M. The gel strength was also significantly enhanced after EDTA chelation. The value of G'

19 for 1% AP at 1 Hz was 13.68, compared with 6.84 and 22.78 for that of AP with 1 M CaCl2 and

20 with 8% EDTA, respectively. These observations suggested that electrostatic interaction was not

21 the deciding force in forming the network of AP dispersion.

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1 3.4. The effect of urea on network formation of AP

2 Hydrogen bond (H-bond) played an important role in the gelation of polysaccharides from

3 Konjac glucomannan, curdlan and iota-carrageenan (Nishinari & Zhang, 2004; Zhang &

4 Nishinari, 2009; Patel, Campanella, & Janaswamy, 2013). As for AP, we demonstrated that

5 electrostatic strength is not the deciding force for its gelation. It is logic to ask if it is the

6 hydrogen bond that develop the network of AP dispersion. To answer this question, the gelation

7 properties of AP dispersion were evaluated with the hydrogen bond breaking agent, urea, using

8 stress relaxation technique. Urea is known as hydrogen bond disrupting agent which can break

9 the intermolecular hydrogen bonding between polysaccharides chains (Liu et al., 2013;

10 McQueen-Mason & Cosgrove, 1994; Wu, Ai, Chen, Kang, & Cui, 2013a; Wu et al., 2013b). The

11 network formation of polysaccharides could be effectively demonstrated by the stress relaxation

12 experiment in which the relaxation modulus G(t) was measured against time (Ferry, 1980). Fig. 5

13 shows the stress relaxation spectrum of AP at different concentrations of urea with or without

14 heat treated. It was interesting to observe that addition of 20% urea did not change the relaxation

15 behaviour when it was not heated. However significant decline of relaxation modulus was

16 observed after the mixture was heated at 80 oC for few minute. For the heating treated mixture,

17 the relaxation modulus decreased gradually with the increase of urea concentration, indicating

18 weakening of the networks of AP. The decrease of G(t) became more significant when urea

19 concentration reached at 10% which suggested the complete breakdown of the gel structure. The

20 relaxation modulus fell to zero immediately when urea concentration reached to 20%. This result

21 confirmed that urea could collapse the network of AP dispersion which leads to the conclusion

22 that hydrogen bonds contributed to the development of networks of AP dispersion. High

23 temperatures promote the breakup of hydrogen bond (Ventura & Bianco-Peled, 2015; Patel et al.,

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1 2013). Our result also suggested that heat energy is important in promoting the interactions of

2 urea molecules with the AP chains indicating the hydrogen bonds interaction between AP chain

3 is not easy to be broken down which could be one of the reason for the unique thermal stability

4 of AP dispersion. The further evidence for this conclusion is showed in Fig. 6. The frequency

5 sweep result suggested that the storage modulus (G') decreased significantly with the increasing

6 urea concentration. When the urea concentration reached to 10%, the gel structure was

7 completely destroyed. Hydrogen bonds are the ruling forces in the establishment of the AP gel

8 structure.

9 3.5. Identification of the possible hydrogen bond donor

10 AP is rich of –OH and –COOH group. Therefore, it was reasonable to infer that inter/intra

11 hydrogen bonding may occur among the numerous –COOH and –OH groups on the back bone of

12 AP. To identify which group was involved in the hydrogen bonding of AP chains, the small

13 molecules possessing active hydrogen-bond donors and/or acceptors was added to the AP

14 dispersion. Glucose was first selected as a competitive small molecular in which hydroxyl group

15 possessed similar structure and movability with that of AP chain. If the hydroxyl group of AP

16 chain was the contributor of the hydrogen bond which forming the network of AP dispersion, the

17 strength of network will be decreased with the addition of glucose in AP dispersion. The

18 hydroxyl groups of glucose would compete with the hydroxyl group of AP chain to form the

19 hydrogen bond, hence, weaken the inter/intra interaction of AP chain, therefore, weaken the gel

20 strength and density of network of AP dispersion. However, the result (Fig. 6) showed that after

21 mixed glucose with AP, the storage modulus G' was not decreased but increased with the

22 increasing concentration of glucose, indicating the gel strength was increased by addition of

23 glucose. Similar results were also demonstrated by the stress relaxation data (Fig. 7). The density

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1 of network of AP dispersion was significant increased with the increasing concentration of

2 glucose. The reason for glucose promoting gelation of AP may because glucose replaced a large

3 amount of water, making less water available to maintain the polymer chains in solution,

4 therefore, raised the density of network of AP (Tsoga, Richardson, & Morris, 2004). Our result

5 indicated that, the hydroxyl group in AP chain was not actively involved in inter/intra hydrogen

6 bond formation in AP dispersion.

7 Similarly, glucuronic acid was used to examine the role of carboxyl groups in the formation of

8 hydrogen bonds. The viscoelastic behaviour and stress relaxation of AP affected by glucuronic

9 acid are showed in Fig. 6 and Fig. 8. The gel strength was significantly decreased by addition of

10 glucuronic acid even the glucuronic acid was just 2.5%. The value of G' for 1% AP at 1 Hz was

11 15.62, compared with 11.03, 10.83 and 8.47 for AP with 2.5%, 5% and 10% glucuronic acid,

12 respectively. This decline of gel strength was more effectively detected by the stress relaxation

13 experiment (Fig. 8). Addition of glucuronic acid resulted in obviously decreased in density of

14 network of AP dispersion. When the concentration of glucuronic acid reached at 10%, the

15 network of AP collapsed. The observed results suggested that during the gelation procedure of

16 AP dispersion, the added carboxyl group of free glucuronic acid competed with that of the uronic

17 acid residue on the AP chain to form the hydrogen bond. Compared with the free glucuronic acid

18 molecules, the movability of the uronic acid residues on AP chain was quite limited by its own

19 conformation; on the other hand, the coefficient of self-diffusion of the added free glucuronic

20 acid molecules was quite higher than that of uronic acid on the AP chain (Wu et al., 2013a).

21 Thus, it was easier for the carboxyl group of the free glucuronic acid to form hydrogen bond with

22 the residue of uronic acid on AP chain which inhibited the inter/intra hydrogen bond formation

23 between/in AP chain resulted in the disconnection of AP chain and led its conformational

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1 transition from rigid to flexible chain which weaken the network of AP dispersion. Excessive

2 added free glucuronic acid disrupted the network of AP. Previous result suggested that the

3 carbonyl oxygen of the carboxylic group and the hydrogen of that were contributed to form inter

4 and intra H-bond of AP dispersion. It was assumed that polysaccharide from A. auricula-judae

5 existed as stiff chain and could self-assembly into nanofibers might because of inter hydrogen

6 bond (Xu et al., 2013). Our work supported Xu’s explanation, and further illustrated the

7 mechanism of hydrogen bond forming is through the uronic acid on molecular chain of AP. Fig.

8 9 illustrated the possible mechanisms of the network formation of AP stiff chain and the effect of

9 adding urea, glucuronic acid and glucose on the formation/disruption of the network structure of

10 AP.

11 3.6. Quantitative determination of the viscoelastical characteristics of AP dispersion

12 Both of the quality and the quantity of the entanglement networks are important for the

13 viscoelastical properties of a system (Ferry, 1979). Wu and co-workers developed a new method

14 to quantitatively calculate the entanglement networks of a hydrogel system which does not

15 contain cross-linked networks using stress relaxation approach (Wu et al., 2013b). This method

16 is also suitable for quantitative determination of the viscoelastical characteristics of AP

17 dispersion which existed as a stiff chain conformation and displayed parallel self-orientation

18 behavior in aqueous solution (Xu et al, 2013). In the method, entanglement networks number

19 (ENN) was introduced to easily calculate the relative quantity of entanglement networks. The

20 effect of AP concentrations, temperature, pH and urea concentration on ENN of AP will be

21 evaluated and the quantitative relationships were also established (Table 1). A quantitative

22 relationship between AP concentration and the ENN could be illustrated by following equation:

23 𝑁 = 0.1965𝑒3.1081𝑐, where N is the ENN and c is AP concentrations. The R2 of this equation is

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1 0.9774, indicated the equation fitted well with the experiment data. From this equation, we

2 observed that the concentration of AP is a major effect on the ENN. The quantitative relationship

3 between temperature and ENN (table 1) showed that the ENN decreased with the increase of

4 temperature in a quantitative manner. However, the temperature was not the major factor

5 influencing the ENN compared with concentration. Changes of the ENN with pH of AP

6 dispersion are also shown in Table 1. When pH values were increased from 6 to 12, there was

7 not too much changing of the ENN; on the contrary, the ENN sharply decreased when pH

8 decreased from 6 to 2 indicating network disruption. Table 1 also gives the quantitative

9 relationship between urea concentration and ENN. The ENN decreased sharply with the increase

10 of urea concentration indicating the reduction of AP network. The ENN for 2% AP is 86,

11 compared with 18, 3 and 1.85 for that of AP with 2.5%, 5% and 10% urea, respectively. Further

12 increase of urea concentration continued to break down the remaining hydrogen bonds. At 20%

13 of urea in AP, the ENN was only 0.07 suggested the complete collapse of the network structure.

14 All the quantitative determination of the viscoelastical characteristics was agreement with the

15 previous quality measurement of AP dispersion.

16 3. Conclusions

17 This study interpreted the gelation mechanism of polysaccharides from Auricularia auricula-

18 judae for the first time. A systematic approach was used to investigate the possible mechanisms

19 of network formation of AP. The addition of salt, urea, glucose and glucuronic acid into the AP

20 dispersion elucidated the individual roles of these components. According to current study,

21 following conclusion could be made: ionic force was not the driving force to maintain the

22 network of AP dispersion and the presence of intrinsic calcium in AP inhibited its network

23 formation. Hydrogen bond was confirmed to be the main force supporting the AP gel network.

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1 The competition of glucose and glucuronic acid with AP chains demonstrated that the structure

2 of network of AP was formed by hydrogen bonds between carboxyl group of the uronic acid on

3 AP chains.

4 Acknowledgements

5 This research was supported by the National Natural Science Foundation of China (Grant No.

6 31201333), the project of Overseas Talent of Education Department of Heilongjiang Province

7 (Grant No. 1253HQ010), Scientific Research Starting Foundation for the Introduced Talents in

8 Heilongjiang Bayi Agricultural University (Grant No. 2012YB-11) and Postdoctoral Science

9 Foundation of Heilongjiang Bayi Agricultural University. Honghui Bao is a visiting scientist of

10 Agricultural and Agri-Food Canada (AAFC) funded by China Scholarship Council. The authors

11 also would like to thank Cathy Wang of AAFC for her technical assistance.

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1 References

2 Agoub, A. A., Giannouli, P., & Morris, E. R. (2009). Gelation of high methoxyl pectin by

3 acidification with D-glucono-δ-lactone (GDL) at room temperature. Carbohydrate Polymers, 75,

4 269-281.

5 Bao, H., You, S., Cao, K., Zhou, R., Wang, Q., & Cui, S. W. (2016). Chemical and rheological

6 properties of polysaccharides from fruit body of Auricularia auricula-judae. Food Hydrocolloids,

7 57, 30-37.

8 Ferry, F. D. (1980). Viscoelastic properties of polymer. Singapore: John Wiley & Sons.

9 Freitas, F., Alves, V. D. Carvalheira, M., Costa, N., Oliveira, R. & Reis, M. A. M. (2009).

10 Emulsifying behaviour and rheological properties of the extracellular polysaccharide produced

11 by Pseudomonas oleovorans grown on glycerol byproduct. Carbohydrate Polymers, 78, 549-556.

12

13 Gilsenan, P. M., Richardson, R. K., & Morris, E. R. (2000). Thermally reversible acid-induced

14 gelation of low-methoxy pectin. Carbohydrate Polymers, 41, 339-349.

15 Katopo, L., Kasapis, S., & Hemar, Y. (2012). Segregative phase separation in agarose/whey

16 protein systems induced by sequence-dependent trapping and change in pH. Carbohydrate

17 Polymers, 87, 2100-2108.

18 Li, X., Al-Assaf, S. A., Fang, Y., & Phillips, G. O. (2013). Characterisation of commercial LM-

19 pectin in aqueous solution. Carbohydrate Polymers, 92, 1133-1142.

15
 ACCEPTED MANUSCRIPT

1 Liu, H., Li, Q., Zhu, D., Li, J., Liu, J., Geng, P., & He, Y. Effects of sucrose and urea on soy hull

2 pectic polysaccharide gel induced by D-glucono-1,5-lactone. Carbohydrate Polymers, 98, 542-

3 545.

4 Liu, S., Li, H., Tang, B., Bi, S., & Lin, L. (2016). Scaling law and microstructure of alginate

5 hydrogel. Carbohydrate Polymers, 135, 101-109.

6 Ma, Z., Wang, J., Zhang, L., Zhang, Y., & Ding, K. (2010). Evaluation of water soluble β-D-

7 glucan from Auricularia auricular-judae as potential anti-tumor agent. Carbohydrate Polymers,

8 80(3), 977-983.

9 McQueen-Mason, S., & Cosgrove, D. J. (1994). Disruption of hydrogen bonding between plant

10 cell wall polymers by proteins that induce wall extension. Proceedings of the National Academy

11 of Sciences, 91, 6574-6578.

12 Morris, E. R., Rees, D. A., & Thom, D. (1973). Characterization of polysaccharide structure and

13 interactions by circular dichroism: Order-disorder transition in the calcium alginate system.

14 Journal of the Chemical Society, Chemical Communications, 7, 245-246.

15 Nguyen, T. L., Chen, J., Hu, Y., Wang, D., Fan, Y., Wang, J., Abula, S., Zhang, J., Qin, T., Chen,

16 X. Y., Chen, X. L., Khakame, S. K., & Dang, B. K. (2012). In vitro antiviral activity of sulfated

17 Auricularia auricular polysaccharides. Carbohydrate Polymers, 99, 1254-1258.

18 Nishinari, K., & Zhang, H. (2004). Recent advances in the understanding of heat set gelling

19 polysaccharides. Trends in Food Science & Technology, 15, 305-312.

20 Norziah, M. H., Foo, S. L., & Karim, A. A. (2006). Rheological studies on mixtures of agar

21 (Gracilaria changii) and κ-carrageenan. Food Hydrocolloids, 20, 204-217.

16
 ACCEPTED MANUSCRIPT

1 Patel, B. K., Campanella, O. H., & Janaswamy, S. (2013). Impact of urea on the three-

2 dimensional structure, viscoelastic and thermal behavior of iota-carrageenan. Carbohydrate

3 Polymers, 92, 1873-1879.

4 Roth, L. E., Vega, D. A., Valles, E. M., & Villar, M. A. (2004). Viscoelastic properties of

5 networks with low concentration of pendant chains. Polymer, 45(17), 5923-5931.

6 Round, A., Rigby, N. M., MacDougall, A. J., & Morris, V. J. (2010). A new view of pectin

7 structure revealed by acid hydrolysis and atomic force microscopy. Carbohydrate Research, 345,

8 487-497.

9 Sevag, M. G, Lackman, D. B., & Smolens, J. (1938). The isolation of the components of

10 streptococcal nucleoproteins in serologically active form. The Journal of biological Chemistry,

11 124, 425-436.

12 Shao, P., Qin, M., Han, L., & Sun, P. (2014). Rheology and characteristics of sulfated

13 polysaccharides from chlorophytan seaweeds Ulva fasciata. Carbohydrate Polymers, 113, 365-

14 372.

15 Tsoga, A., Richardson, R. K., & Morris, E. R. (2004). Role of cosolutes in gelation of high-

16 methoxy pectin. Part 1. Comparison of sugars and polyols. Food Hydrocolloids, 18, 907-919.

17 Ventura, I., & Bianco-Peled, H. (2015). Small-angle X-ray scattering study on pectin-chitosan

18 mixed solutions and thermoreversible gels. Carbohydrate Polymers, 123, 122-129.

19 Wu, Q., Tan, Z., Liu, H., Gao, L., Wu, S., Luo, J., Zhang, W., Zhao, T., Yu, J., & Xu, X. (2010).

20 Chemical characterization of Auricularia auricular polysaccharides and its pharmacological

17
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1 effect on heart antioxidant enzyme activities and left ventricular function in aged mice.

2 International Journal of Biological Macromolecules, 46(3), 284-288.

3 Wu, S., Ai, L., Chen, J., Kang, J., & Cui, S. W. (2013a). Study of the mechanism of formation of

4 hyaluronan putty at pH 2.5: Part I, Experimental measurements. Carbohydrate Polymers, 98,

5 1677-1682.

6 Wu, S., Ai, L., Chen, J., Kang, J., & Cui, S. W. (2013b). Study of the mechanism of formation of

7 hyaluronan putty at pH 2.5: Pat II-Theoretical analysis. Carbohydrate Polymers, 98, 1683-1688.

8 Xu, S., Lin, Y., Huang, J., Li, Z., Xu, X., & Zhang, L. (2013). Construction of high strength

9 hollow fibers by self-assembly of a stiff polysaccharide with short branches in water. The

10 Journal of Physical Chemistry A, 1, 4198-4206.

11 Xu, S., Xu, X., & Zhang, L. (2012). Branching structure and chain conformation of water-

12 soluble glucan extracted from Auricularia auricula-judae. Agricultural and Food Chemistry, 60,

13 3498-3506.

14 Xu, S., Xu, X., & Zhang, L. (2013). Effect of heating on chain conformation of branched β-

15 Glucan in water. The Journal of Physical Chemistry B, 117, 8370-8377.

16 Yamamoto, F., & Cunha, R. L. (2007). Acid gelation of gellan: Effect of final pH and heat

17 treatment conditions. Carbohydrate Polymers, 68, 517-527.

18 Yoon, S., Yu, M., Pyun, Y., Hwang, J., Chu, D., Juneja, L. R., & Mourao, P. A. S. (2003). The

19 nontoxic mushroom Auricularia auricular contains a polysaccharide with anticoagulant activity

20 mediated by antithrombin. Thrombosis Research, 112(3), 151-158.

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1 Yuan, Z., He, P., Cui, J., & Takeuchi, H. (1998). Hypoglycemic effect of water-soluble

2 polysaccharide from Auricularia auricular-judae Quel. on genetically diabetic KK-Ay mice.

3 Bioscience, Biotechnology and Biochemistry, 62(10), 1898-1903.

4 Zeng, F., Zhao, C., Pang, J., Lin, Z., Huang, Y., & Liu, B. (2013). Chemical properties of a

5 polysaccharide purified from solid-state fermentation of Auricularia auricular and its biological

6 activity as a hypolipidemic agent. Food Science, 78(9), 1470-1475.

7 Zhang, H., & Nishinari, K. (2009). Characterization of the conformation and comparison of

8 shear and extensional properties of curdlan in DMSO. Food Hydrocolloids, 23, 1570-1578.

9 Zhang, N., Chen, H., Ma, L., & Zhang, Y. (2013). Physical modifications of polysaccharide from

10 Inonotus obliquus and the antioxidant properties. International Journal of Biological

11 Macromolecules, 54, 209-215.

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1 Legend of Table

2 Table 1. The relationships between entanglement networks number (N) and AP concentration,

3 urea concentration, temperature and pH of AP dispersion.

20
Table 1

AP concentration (c) urea concentration (c)

0.5% 1% 1.5% 2% 0% 2.5% 5% 10% 20%

ENN (N)* 0.7±0 6.7±0 21±1 85±1 86±1 18±2 7±0 1.85±0 0.07±0

Quantitative relationship 𝑁 = 0.1965𝑒3.1081𝑐 (R2=0.9774) 𝑁 = 53.631𝑒 ‒ 0.338𝑐 (R2=0.9856)

Temperature (t) pH

25 35 45 75 2 4 6 10 12

ENN (N) 148±1 139±4 133±1 111±3 2.97±1 27.0±1 40±0 41±1 49±1

Quantitative relationship 𝑁 = 170.74𝑒 ‒ 0.006𝑡 (R2=0.998)

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1 Legend of Figures

2 Fig.1. Effect of temperature (a) on stress relaxation of 2% AP at pH 6.4 and effect of

3 concentration (b) on stress relaxation of AP at 25 oC.

4 Fig.2. Effect of pH on stress relaxation of 2% AP dispersion at 25 oC.

5 Fig.3. Stress relaxation of 1.5% AP dispersion with different concentrations of salts and with 8%

6 EDTA at pH 6.4.

7 Fig.4. Double logarithmic plot of the storage modulus (G') against frequency (at 2% strain) for

8 1.5% AP with different concentration of Ca2+ and with 8% EDTA.

9 Fig.5. Shear relaxation spectra of 2% AP dispersion with different concentrations of urea at pH

10 6.4 with and without heat treatment.

11 Fig.6. Influence of urea, glucose, glucuronic acid on the storage modulus G' of 1.5% AP

12 dispersion at pH 6.4, at 25 oC at 1 Hz.

13 Fig.7. Stress relaxation spectra of 1.5% AP dispersion with different concentration of glucose at

14 pH 6.4 at 25 oC.

15 Fig. 8. Stress relaxation spectra of 1.5% AP dispersion with different concentration of glucuronic

16 acid at pH 6.4 at 25 oC.

17 Fig. 9. Schematic diagram of the formation and disruption of AP network.

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102
a
25 oC
45 oC
80 oC
G(t) (Pa)

101

100
0 10 20 30 40 50 60 70

Time (min)

103
b 0.1%
0.5%
102 1.0%
1.5%
2.0%
101
G(t) (Pa)

100

10-1

10-2

10-3
0 10 20 30 40 50 60 70

Time (min)

Fig.1

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103
pH=6.4
pH=4.4
102 pH=2.0
pH=10.1
pH=12.4
101
G(t)(Pa)

100

10-1

10-2

10-3
0 10 20 30 40 50 60 70

Time (min)

Fig.2

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102
a 0 M Na
0.01M Na
0.1M Na
0.5M Na
101 1M Na
G(t) (Pa)

100

10-1

10-2
0 10 20 30 40 50 60 70

Time (min)

103
b 0 M Ca
0.01M Ca
102 0.1M Ca
0.5M Ca
1M Ca
8% EDTA
101
G(t) (Pa)

100

10-1

10-2
0 10 20 30 40 50 60 70

Time (min)

Fig.3

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0 M Ca
102
0.01 M Ca
0.1 M Ca
0.5 M Ca
1 M Ca
8% EDTA
101
G' (Pa)

30
Ca
EDTA
20
100
10

0
0 0.01 0.1 0.5 1 EDTA
10-1
10-3 10-2 10-1 100 101 102

Frequency (Hz)

Fig. 4

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103

0% urea
2.5% urea
102 5.0% urea
10% urea
20% urea (after heated)
20% Urea (before heated)
101
G(t)(Pa)

100

10-1

10-2
0 10 20 30 40 50 60

Time (min)

Fig. 5

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50
urea with 2% AP
glucose with 1.5% AP
40 glucuronic acid with 1.5% AP

30
G' (Pa)

20

10

0
0 2.5% 5% 10%

Concentration of small molecular weight (%)

Fig. 6

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103

0% glucose
2.5% glucose
5% glucose
102 10% glucose
G(t)(Pa)

101

100

10-1
0 10 20 30 40 50 60 70

Time (min)

Fig. 7

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103

0% glucuronic acid
2.5% glucuronic acid
102 5.0% glucuronic acid
10% glucuronic acid

101
G(t) (Pa)

100

10-1

10-2
0 10 20 30 40 50 60 70

Time (min)

Fig. 8

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HO HO HO
O O O
O OH OH
O OH O O
C
COOH CH2OH
O O formed
H
H

O O
HOH2C HOOC
C
O O HO O HO O
HO O O O
OH OH OH

Inter H-bond formed by uronic acid in AP chains Network structure of AP formed by

intra and inter H-bond
e
lu cos Addition of urea or
nofg
itio glucuronic acid
Add

Addition of glucose made less water available

to maintain the polymer chains in solution,
therefore, raised the density network of AP.
Fig. 9
Urea or glucuronic acid broke down inter H-bond
resulting in the disconnection of the aggregates
into single chains, meanwhile, destroyed the intra
H-bond of AP chain leading conformational
transition from rigid to flexible chains. Network of
AP collapsed.

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Highlights

 Gelation mechanism of thermal stable polysaccharides was elucidated

 Not the ionic interaction, but hydrogen bond was the main force forming the gel network;

 Carboxyl group formed inter/intra hydrogen bond forming AP gel network.

 Quantity of the entanglement networks of AP dispersion was evaluated.