The Rheological Behavior of Polysaccharides from Mulberry Leaves ( Morus alba L.)

: In this study, mulberry leaves polysaccharides (MLPs) namely HBSS (extracted with hot bu ﬀ er soluble solids), CHSS (extracted with chelating agent soluble solids), DASS (extracted with diluted alkali soluble solids), and CASS (extracted with concentrated alkali soluble solids) were obtained using four di ﬀ erent solvents and examined for their rheological potential. Di ﬀ erent MLPs solutions harbored obvious disparity for viscosity and displayed a shear-thinning behavior at the tested range. Among all the fractions, DASS possessed the highest apparent viscosity at 0.5–2.5%. The apparent viscosity of MLPs solutions declined at acidic pH, alkaline pH, and higher temperature (90 ◦ C). The HBSS fraction showed the best heat stability of all the fractions. All the fractions displayed noticeable di ﬀ erences in apparent viscosity in response to Na + and Ca 2 + at 20 ◦ C. Both the modules such as G (cid:48) (storage modulus) and G” (loss modulus) showed augmentation with oscillation frequency. Initially, the value of G” was higher than G (cid:48) of MLPs at lower frequency and lower concentration, and the MLPS displayed stronger viscous nature; whereas, G (cid:48) was consistently higher at higher frequency and higher concentration, and the MLPS displayed stronger elastic characteristic. From our data, it was indicated that these MLPs can be used as promising natural materials (thickeners, gelling agents, binding agents, stabilizers) for their direct application to the food industry.


Effect of Different MLPs Concentrations on Apparent Viscosity
The different tested concentrations led to significant change in apparent viscosity measurements at 20 °C as shown in Figure 2. The concentrations of samples solutions were 0.5%, 0.8%, 1.0%, 1.5%, 2.0%, and 2.5%. Under the selected range, the apparent viscosity of MLPs fractions was decreased with the increasing shear rate. These results demonstrated the typical pseudoplastic fluid with shearthinning and non-Newtonian fluidic characteristics which was consistent with the previous report [33]. In general, polymer solutions had the same behavior which may be related to the molecular weight [34,35]. Moreover, the apparent viscosity increased with rising MLPs concentrations which might be due to the hydrodynamic and thermodynamic interactions of macromolecules and strengthening of aggregates [36]. At lower concentrations, previous studies have reported some entanglements in the polymer molecular chains; on the other hand, the easy formation of gel network at high concentrations was reported [37]. Hydrocolloids (pectin) had highly apparent viscosity at lower concentration [38]. Whereas, DASS showed the highest apparent viscosity and CASS showed the lowest viscosity value. These data indicated that the DASS fraction can be used as thickener, gelling agent, binding agent in the food, pharmaceutical, and cosmetic industries.

Effect of Different MLPs Concentrations on Apparent Viscosity
The different tested concentrations led to significant change in apparent viscosity measurements at 20 • C as shown in Figure 2. The concentrations of samples solutions were 0.5%, 0.8%, 1.0%, 1.5%, 2.0%, and 2.5%. Under the selected range, the apparent viscosity of MLPs fractions was decreased with the increasing shear rate. These results demonstrated the typical pseudoplastic fluid with shear-thinning and non-Newtonian fluidic characteristics which was consistent with the previous report [33]. In general, polymer solutions had the same behavior which may be related to the molecular weight [34,35]. Moreover, the apparent viscosity increased with rising MLPs concentrations which might be due to the hydrodynamic and thermodynamic interactions of macromolecules and strengthening of aggregates [36]. At lower concentrations, previous studies have reported some entanglements in the polymer molecular chains; on the other hand, the easy formation of gel network at high concentrations was reported [37]. Hydrocolloids (pectin) had highly apparent viscosity at lower concentration [38]. Whereas, DASS showed the highest apparent viscosity and CASS showed the lowest viscosity value. These data indicated that the DASS fraction can be used as thickener, gelling agent, binding agent in the food, pharmaceutical, and cosmetic industries.
The Ostwald-DeWaele equation η = m × γ × n −1 was fitted to the shear thinning region of the samples at various concentrations, where m was the consistency index and n was the flow behavior index. The value of n reflects the degree of deviation between solution fluid and Newtonian fluid (n = 1 is a significant characteristic of Newtonian fluid). The consistency index m of HBSS, CHSS, DASS, and CASS increased significantly (Table 1). However, the flow index n decreased significantly, which reflected the dependence of apparent viscosity on concentration (Table 1). In the present research, the n value of HBSS was less than the other three components, which may be due to the different extraction solvents and structures of the four polysaccharides.  (Table 1). However, the flow index n decreased significantly, which reflected the dependence of apparent viscosity on concentration (Table 1). In the present research, the n value of HBSS was less than the other three components, which may be due to the different extraction solvents and structures of the four polysaccharides.

Effect of pH on Apparent Viscosity
As known, pH could change the apparent viscosity of samples solutions which was revealed through the change of apparent viscosity of 1.0% samples solutions at different pH values as shown in Figure 3. Our results showed the decreased viscosity for all the four fractions with the rising shear rate at 0.01-1000 s −1 for all conditions. Particularly, the viscosity values of MLPs at pH 4 or pH10 were less than those at pH 7 which might be due to ionic interactions or composition modification (acid or

Effect of pH on Apparent Viscosity
As known, pH could change the apparent viscosity of samples solutions which was revealed through the change of apparent viscosity of 1.0% samples solutions at different pH values as shown in Figure 3. Our results showed the decreased viscosity for all the four fractions with the rising shear rate at 0.01-1000 s −1 for all conditions. Particularly, the viscosity values of MLPs at pH 4 or pH10 were less than those at pH 7 which might be due to ionic interactions or composition modification (acid or alkali) which can lead to conformational changes [39,40]. Therefore, the change of pH value could decrease apparent viscosity due to the influence on breaking the bond which can further lower the molecular weight [41].
alkali) which can lead to conformational changes [39,40]. Therefore, the change of pH value could decrease apparent viscosity due to the influence on breaking the bond which can further lower the molecular weight [41].

Effect of Temperature Range on Apparent Viscosity
For this, apparent viscosity of MLPs fractions was determined at the same range (1.0%) at a stable shear rate (100 s −1 ) as shown in Figure 4. The viscosity values of four fractions were lowered with the rising temperature (20 to 90 °C). Four samples solutions showed the change in the apparent viscosity, such as 53.42%, 68.73%, 59.25%, 83.87% for HBSS, CHSS, DASS, and CASS, respectively. The major alteration in the apparent viscosity was observed for CASS; whereas, HBSS displayed the least change of all samples. Moreover, HBSS possessed the stronger viscosity over the temperature range of 20-90 °C. From the above data, we can conclude that the larger change in the viscosity of samples led to the weaker heat stability. These results indicate that the HBSS fraction can preferably be applied in baking processing.

Effect of Temperature Range on Apparent Viscosity
For this, apparent viscosity of MLPs fractions was determined at the same range (1.0%) at a stable shear rate (100 s −1 ) as shown in Figure 4. The viscosity values of four fractions were lowered with the rising temperature (20 to 90 • C). Four samples solutions showed the change in the apparent viscosity, such as 53.42%, 68.73%, 59.25%, 83.87% for HBSS, CHSS, DASS, and CASS, respectively. The major alteration in the apparent viscosity was observed for CASS; whereas, HBSS displayed the least change of all samples. Moreover, HBSS possessed the stronger viscosity over the temperature range of 20-90 • C. From the above data, we can conclude that the larger change in the viscosity of samples led to the weaker heat stability. These results indicate that the HBSS fraction can preferably be applied in baking processing.
Agronomy 2020, 10, x FOR PEER REVIEW 5 of 14 alkali) which can lead to conformational changes [39,40]. Therefore, the change of pH value could decrease apparent viscosity due to the influence on breaking the bond which can further lower the molecular weight [41].

Effect of Temperature Range on Apparent Viscosity
For this, apparent viscosity of MLPs fractions was determined at the same range (1.0%) at a stable shear rate (100 s −1 ) as shown in Figure 4. The viscosity values of four fractions were lowered with the rising temperature (20 to 90 °C). Four samples solutions showed the change in the apparent viscosity, such as 53.42%, 68.73%, 59.25%, 83.87% for HBSS, CHSS, DASS, and CASS, respectively. The major alteration in the apparent viscosity was observed for CASS; whereas, HBSS displayed the least change of all samples. Moreover, HBSS possessed the stronger viscosity over the temperature range of 20-90 °C. From the above data, we can conclude that the larger change in the viscosity of samples led to the weaker heat stability. These results indicate that the HBSS fraction can preferably be applied in baking processing.

Effect of Various Temperature Treatments on Apparent Viscosity
As shown in Figure 5, various temperature treatments caused significant change in the apparent viscosity measurements. The apparent viscosity was noticeably changed after heating or freezing treatment and the change in the viscosity values of CHSS and CASS was higher than the remaining fractions. The apparent viscosity of samples solutions was increased after the freezing process (−20 • C) as compared to heating (100 • C). The apparent viscosity value of DASS was the highest among all samples after the freezing treatment, which indicated that DASS can be more suitable as a stabilizer in freeze processing. As previously reported, high temperature could increase the thermal motion of molecules and intermolecular distance, which could further weaken the interactions and recede the apparent viscosity [42]. The change in the apparent viscosity might be due to transitions in the consequent phase of water and the change in composition as described in the previous studies [43].

Effect of Various Temperature Treatments on Apparent Viscosity
As shown in Figure 5, various temperature treatments caused significant change in the apparent viscosity measurements. The apparent viscosity was noticeably changed after heating or freezing treatment and the change in the viscosity values of CHSS and CASS was higher than the remaining fractions. The apparent viscosity of samples solutions was increased after the freezing process (−20 °C) as compared to heating (100 °C). The apparent viscosity value of DASS was the highest among all samples after the freezing treatment, which indicated that DASS can be more suitable as a stabilizer in freeze processing. As previously reported, high temperature could increase the thermal motion of molecules and intermolecular distance, which could further weaken the interactions and recede the apparent viscosity [42]. The change in the apparent viscosity might be due to transitions in the consequent phase of water and the change in composition as described in the previous studies [43].

Effect of Various Salts on Apparent Viscosity
The effect of Na + on apparent viscosity was displayed in Figure 6. The apparent viscosity of MLPs was reduced with the growing shear rate at 0.01-1000 s −1 with the addition of different concentrations of Na + . The results indicated that four kinds of samples solutions showed completely different behaviors from each other. Initially, the viscosity value for HBSS was declined followed by an increase at higher concentration of Na + . On the contrary, CHSS showed the opposite trend with the increased viscosity which was followed by a decrease with the increase in concentration of Na + . Furthermore, the apparent viscosity of DASS was declined after the addition of Na + and the values were gradually decreased with the increasing Na + concentration. The anionic or cationic polysaccharides had higher apparent viscosity due to the negative contacts in the branches [44]. The observed decrease in the apparent viscosity might be due to the charge shielding effect which could increase the concentrations of counter-ions and lead to molecules contractions [45].

Effect of Various Salts on Apparent Viscosity
The effect of Na + on apparent viscosity was displayed in Figure 6. The apparent viscosity of MLPs was reduced with the growing shear rate at 0.01-1000 s −1 with the addition of different concentrations of Na + . The results indicated that four kinds of samples solutions showed completely different behaviors from each other. Initially, the viscosity value for HBSS was declined followed by an increase at higher concentration of Na + . On the contrary, CHSS showed the opposite trend with the increased viscosity which was followed by a decrease with the increase in concentration of Na + . Furthermore, the apparent viscosity of DASS was declined after the addition of Na + and the values were gradually decreased with the increasing Na + concentration. The anionic or cationic polysaccharides had higher apparent viscosity due to the negative contacts in the branches [44]. The observed decrease in the apparent viscosity might be due to the charge shielding effect which could increase the concentrations of counter-ions and lead to molecules contractions [45]. The effect of Ca 2+ on apparent viscosity of four fractions' solutions was shown in Figure 7. The prominent differences in apparent viscosity were observed due to the presence of Ca 2+ for all the samples. In the case of HBSS and CASS, the viscosity was improved with the addition of Ca 2+ and it was constantly increased with the rising concentration of Ca 2+ . On the other hand, CHSS and DASS showed decreased viscosity values in the presence of high concentration of Ca 2+ which was followed by a slight increase in the viscosity. The apparent viscosity might increase due to the increased intermolecular association in the case of HBSS and CASS fractions [40]. The effect of Ca 2+ on apparent viscosity of four fractions' solutions was shown in Figure 7. The prominent differences in apparent viscosity were observed due to the presence of Ca 2+ for all the samples. In the case of HBSS and CASS, the viscosity was improved with the addition of Ca 2+ and it was constantly increased with the rising concentration of Ca 2+ . On the other hand, CHSS and DASS showed decreased viscosity values in the presence of high concentration of Ca 2+ which was followed by a slight increase in the viscosity. The apparent viscosity might increase due to the increased intermolecular association in the case of HBSS and CASS fractions [40].

The Linear Viscoelastic Region Measurements of MLPs
MLPs with the concentration of 0.5%, 1.0%, and 2.0% were measured with the stress ranging from 0.1% to 1000% in order to make sure that the structure of samples was protected [46]. The obtained results were depicted in Figure 8 which indicated that the 1% shape change could be used as dynamic oscillatory measurements.

The Linear Viscoelastic Region Measurements of MLPs
MLPs with the concentration of 0.5%, 1.0%, and 2.0% were measured with the stress ranging from 0.1% to 1000% in order to make sure that the structure of samples was protected [46]. The obtained results were depicted in Figure 8 which indicated that the 1% shape change could be used as dynamic oscillatory measurements.

The Linear Viscoelastic Region Measurements of MLPs
MLPs with the concentration of 0.5%, 1.0%, and 2.0% were measured with the stress ranging from 0.1% to 1000% in order to make sure that the structure of samples was protected [46]. The obtained results were depicted in Figure 8 which indicated that the 1% shape change could be used as dynamic oscillatory measurements.

Oscillatory M
Viscoelastic properties were determined to evaluate the viscous and elastic characteristics of samples [47]. The change in storage modulus (G ) and loss modulus (G") of MLPs (5, 10, and 20 mg/mL) was determined based on the above mentioned linear viscoelastic region measurements at 1% oscillation intensity in Figure 9. The curves of G and G" ascended with the growing shear oscillation frequency (0.01-100 Hz). Initially, the value of G" was higher than G at lower frequency and lower concentration; whereas, G was consistently higher at higher frequency and higher concentration, which showed the characteristic of samples solutions. The phenomenon of G increased on the account of increasing the number and average size of junction points [48]. The crossover frequency displayed the viscoelastic behavior of samples. As reported previously, the lower crossover value often leads to the larger elastic nature [49]. The fractions displayed stronger viscous nature when G" was higher than G ; whereas, a stronger elastic characteristic was observed at higher G as compared to G". Our data affirmed the gel-like characteristics of the tested samples in the case of G > G". Moreover, a liquid-like behavior was noticed in the case of G < G". The weak gel properties were observed when G was just above or on the G". All these findings were in agreement with the previously reported data [50]. Our results suggest that MLPs with large viscosity and elasticity could contribute to the food processing.
Agronomy 2020, 10, x FOR PEER REVIEW 10 of 14 Figure 9. Frequency sweeps at 1% strain to determine the G' (storage modulus) and G'' (loss modulus) of MLPs at different concentrations.

Materials and Chemicals
Initially, dried mulberry leaves (Morus alba L.) were procured as mentioned in our previous study [32] and grounded into a powder (60 mesh) before the extraction of polysaccharides.

Materials and Chemicals
Initially, dried mulberry leaves (Morus alba L.) were procured as mentioned in our previous study [32] and grounded into a powder (60 mesh) before the extraction of polysaccharides.

Sequential Extraction and Purification of MLPs
Four MLPs (HBSS, CHSS, DASS, and CASS) were sequentially extracted by our previous method [24]. The purified samples were obtained by using a DEAE-Cellulose column (DEAE Cellulose-52 (Sigma-Aldrich, St. Louis, MO, USA) using which eluent was 0.1 mol/L of NaCl solutions at a flow rate of 1 mL/min. The obtained fractions were dialyzed (4 • C), freeze-dried, and studied for their rheological behavior.

Effect of Acidic and Alkaline pH on Apparent Viscosity of MLPs
Briefly, 1.0% of MLPs solutions were added to NaOH or HCl to adjust the pH values from 4.0 to 10.0, respectively to measure the apparent viscosity under the selected range of 0.01-1000 s −1 at 20 • C [33,52].

Effect of Temperature on Apparent Viscosity of MLPs
One percent of MLPs solutions was selected to evaluate the effects of different temperature ranges (20-90 • C) followed by the measurement of apparent viscosity at a shear rate of 100 s −1 [33].

Effect of Various Temperature Treatments on Apparent Viscosity of MLPs
The MLPs samples were exposed to different temperature conditions such as room temperature (25 • C for 2 h), very high temperature (100 • C for 2 h), and freezing temperature (−20 • C for 2 h), respectively. Subsequently, the MLPs solutions were placed at room temperature before the experiment. The apparent viscosity of 1.0% samples solutions after different temperature treatments was measured under the selected range of 0.01-1000 s −1 at 20 • C [33,52].

Effect of Various Salts on Apparent Viscosity of MLPs
MLPs were dissolved in NaCl, CaCl2 at the concentrations of 0, 0.1, 0.2, and 0.4 mol/L. The apparent viscosity of 1.0% samples solutions was determined under the selected range of 0.01-1000 s −1 at 20 • C [33,52].

Oscillatory Shear Measurements of MLPs
To measure the storage modulus (G ) and loss modulus (G") of MLPs fractions at different concentrations of 5, 10, and 20 mg/mL, oscillatory shear determinations were used as per the previous