*3.4. Rheological Property of Gelatin–Mulberry Leaf Polysaccharide (G-MLPs) Miscible System* 3.4.1. Effect of Concentration on Apparent Viscosity of G-MLPs

The change in and influence of apparent viscosity of four miscible systems with different concentrations and shear rates and the comparison with the apparent viscosity of the gelatin solution are shown in Figure 5. Under the condition of a low shear rate, the viscosity of the four miscible systems decreased with the increase in shear rate, and they showed the "shear thinning" behavior [20]. The main reason for the "shear thinning" behavior was that gelatin and polysaccharide were colloidal particles of macromolecules. Under the condition of a static or low shear rate, they entangled with each other and had

a higher viscosity, while with the increase in shear rate, the shear stress between the flow layers increased, which made the scattered gelatin–polysaccharide chain particles roll, rotate, and shrink into clusters, resulting in reducing the hooking effect between the chain molecules and the viscosity of gelatin–polysaccharide solutions. On the other hand, with the increase in flow rate, the molecular force between miscible systems decreased, the flow direction of gelatin–polysaccharide macromolecules changed from disorder to ordered, the flow direction appeared consistent, and the viscosity decreased [34]. This was a typical non-Newtonian fluid behavior, indicating that the miscible systems were a non-Newtonian fluid like pure polysaccharide solutions. As mentioned above, "Shear thinning" behavior is important in food processing industries for the development of various food products with desirable properties [20].

**Figure 4.** Particle size distribution of G-MLPs at different concentrations. (**a**) G-HBSS; (**b**) G-CHSS; (**c**) G-DASS; (**d**) G-CASS.

The apparent viscosity of the four miscible systems varied with the increase in mass concentration (Figure 5). Except for the apparent viscosity of the G-CHSS miscible system at the highest concentration, which was higher than that of the gelatin solution, the apparent viscosity of other miscible systems was lower than that of the gelatin solution, which indicated that the addition of polysaccharides reduced the apparent viscosity of the gelatin solution. Specifically, after the four mulberry leaf polysaccharide solutions were mixed with the gelatin solution, the apparent viscosity of G-HBSS, G-DASS, and G-CASS miscible systems decreased with the increase in mass concentration, while the apparent viscosity of the G-CHSS miscible system increased with the increase in mass concentration, which showed relatively appreciable thickening behavior of G-CHSS. One study reported that the apparent viscosity of polysaccharides increased with their concentration, while their apparent viscosity decreased after the addition of gelatin, which may be related to the fact that the particle size of the miscible system in the particle size experiment did not increase with the increase in solution concentration [35]. The previous studies [18,20] showed that the apparent viscosity increased with the increase in mass concentration, mainly because the fluid viscosity results from intermolecular internal friction. In this study, the particle size in the miscible system did not increase, resulting in the decrease in apparent viscosity. Within the studied concentration range, the fluid types of gelatin and mulberry leaf polysaccharide miscible systems did not change due to the change in concentration.

**Figure 5.** Effect of different concentrations on the viscosity of G-MLPs. (**a**) G-HBSS; (**b**) G-CHSS; (**c**) G-DASS; (**d**) G-CASS.

It can also be seen from Figure 5 that the rheological properties of the solutions of the miscible systems with different concentrations were the same as those of mulberry leaf polysaccharides. With the increase in concentration, the pseudoplastic flow was dominant. When the shear rate was 0.01–10 s<sup>−</sup>1, the viscosity of the four miscible systems of G-HBSS, G-CHSS, G-DASS, and G-CASS decreased rapidly with the increase in shear rate, and then became stable as the shear rate increased. With the increase in shear rate, the miscible system showed the properties of Newtonian fluid, which is characterized by the gradual stabilization of apparent viscosity. Compared with pure mulberry leaf polysaccharides, G-CHSS had the highest apparent viscosity, and G-DASS had the lowest apparent viscosity among the four miscible systems. The above results show that G-CHSS can be used as a food thickener, gelling agent, and binder in food, medicine, and cosmetics industries [18].

#### 3.4.2. Effect of pH on Apparent Viscosity of G-MLPs

The four miscible systems showed different apparent viscosity changes in low- and high-pH solutions (Figure 6). G-CHSS and G-DASS showed a decrease in apparent viscosity under acidic and alkaline conditions, which may be because the intermolecular force of the miscible system was weakened in the acidic or alkaline environment, resulting in the decomposition of the polymer of the polysaccharide and gelatin miscible system and a decrease in molecular weight and apparent viscosity [36]. G-HBSS and G-CASS had the highest apparent viscosity under acidic conditions, and the apparent viscosity decreased with the increase in pH. The reason for this may be that the two polysaccharide miscible systems belonged to an acidic polysaccharide miscible system, and their structure was much more stable under acidic conditions, while in the alkaline environment, the solvation effect of water increased with the increase in OH- in the solution, which may have broken the intramolecular and intermolecular hydrogen bonds of the molecules and reduced the interactions, resulting in making the structure of the miscible system loosen and decreasing the viscosity of G-MLPs [37]. Among the four miscible systems, the CHSS had the highest stability, and it can be used as an important ingredient in acidic or alkaline beverages and other products.

**Figure 6.** Effect of pH on the viscosity of G-MLPs. (**a**) G-HBSS; (**b**) G-CHSS; (**c**) G-DASS; (**d**) G-CASS.

3.4.3. Effects of Na+ Concentration on Apparent Viscosity of G-MLPs

Figure 7 shows the effect of Na<sup>+</sup> concentration on the apparent viscosity of 10 mg/mL gelatin–polysaccharide miscible systems. It can be seen from the figure that the apparent viscosity of G-CASS (Figure 7b) increased with the increase in Na<sup>+</sup> concentration. The reason for this phenomenon may be that the addition of Na<sup>+</sup> affected the molecular conformation of the miscible system and improved the structural entanglement, resulting in the increase in apparent viscosity [38,39]. The apparent viscosity of G-CHSS decreased with the increase in Na+ concentration (Figure 7b). This may be due to the salting out of the polysaccharide solution of the miscible system due to the addition of Na+, which would reduce the concentration of the polysaccharide solution and the apparent viscosity. The higher the NaCl concentration, the more serious the salting out phenomenon and the greater the viscosity drop [18,20]. The apparent viscosity of G-HBSS and G-DASS did not change regularly with the concentration of Na+, which may have been caused by the particularity of the structure of the HBSS and DASS. The action law of Na+ on their miscible systems was different from that of G-CHSS and G-CASS. In other words, the low concentration of Na+ increased the structural entanglement between molecules, resulting in the increase in apparent viscosity, while the high concentration of Na<sup>+</sup> salted out the system, resulting in the decrease in apparent viscosity [18]. The inconsistency of the rheological behavior of samples may also have been due to the different intensity of the interaction between gelatin

and the HBSS, CHSS, DASS, and CASS in different concentrations of sodium chloride solution. Therefore, during the food processing, the content of salt ions needs to be strictly controlled to ensure the desired products.

**Figure 7.** Effect of Na+ on the viscosity of G-MLPs (10 mg/mL). (**a**) G-HBSS; (**b**) G-CHSS; (**c**) G-DASS; (**d**) G-CASS.

#### 3.4.4. Effect of System Temperature on Apparent Viscosity of G-MLPs

The different effects of cold treatment and heat treatment on the apparent viscosity of the four miscible systems are shown in Figure 8. Gelatin is thermo-reversible, meaning that the viscosity of a gelatin solution would increase with decreasing temperature to form a gel, and vice versa. Thus, heating and freezing followed by reversing the system temperature to room temperature will have no significant effect on gelatin viscosity [39]. The rheological properties of the four miscible systems after cold and heat treatment were different. After cold treatment, the apparent viscosity of G-HBSS decreased, while the apparent viscosity of G-CHSS, G-DASS, and G-CASS was increased. After heat treatment, the apparent viscosity of G-HBSS was decreased. The increase in the apparent viscosity of G-DASS was higher than that of cold treatment, while the increase in the apparent viscosity of G-CASS was lower than that of cold treatment, and the apparent viscosity of G-CASS after freezing treatment was the highest among the four miscible systems, which indicated that G-CASS was more suitable as a stabilizer in the freezing process. After heat treatment, the apparent viscosity of the G-CHSS sample generally showed an upward trend with the increase in shear rate and changed from "shear thinning" to "shear thickening" expansive fluid [40]. The reason for this phenomenon may be that the interaction between gelatin and the CHSS was strengthened during the heat treatment, resulting in the very stable miscible system. When the temperature returned to room temperature, it was still in an orderly distribution. Therefore, with the increase in shear rate, the order in the system was disturbed and transformed into disorder, and the apparent viscosity increased with the increase in shear force. The above results show that the four miscible systems, except G-HBSS, had good freeze–thaw stability and could be applied to the products that need to

be stabilized after freezing, while the "shear thickening" characteristic of G-CHSS marks its suitability as a thickener in heat processing.

**Figure 8.** Effect of freezing and heating on the viscosity of G-MLPs. (**a**) G-HBSS; (**b**) G-CHSS; (**c**) G-DASS; (**d**) G-CASS.

#### 3.4.5. Effect of G-MLPs on Viscoelasticity

At 25 ◦C, the storage modulus G' and loss modulus G" of gelatin and mulberry leaf polysaccharide miscible systems changed with frequency, as shown in Figure 9. By dynamically measuring the G' and G" of the samples, the advantages of solid elasticity or liquid viscosity of the sample could be quantified [41]. Within the range of shear oscillation frequency measured in this study, the storage modulus G' and loss modulus G" of the four miscible systems had a certain dependence on the shear oscillation frequency, which showed that they continued to increase with the increase in shear oscillation frequency and indicated that gelatin and mulberry leaf polysaccharides solutions were a viscoelastic material [18,20]. The storage modulus G' and loss modulus G" of G-HBSS were different from those of the other three miscible systems. At 5 mg/mL and 10 mg/mL, G' was greater than G" under the low shear vibration frequency of G-HBSS, and solution mainly showed the elastic properties of a solid. With the increase in frequency, G' was less than G", and the main property of the solution changed to the viscosity of the liquid. The frequency of shear oscillation further increased until the G' of the solution was greater than G". The elastic properties of the solid were dominant [42]. The changes between the storage modulus G' and loss modulus G" of G-HBSS at 15 mg/mL and 20 mg/mL were the same as those of the other three miscible systems. At low frequency, the storage modulus G' was less than the loss modulus G", and the solution showed the viscosity of the liquid. With the increase in frequency, a crossover value is reported [43]. At this time, the loss modulus G' curve and the storage modulus G" curve was close to or intersected, which also meant that the crossover value was negatively correlated with the elastic contribution at the beginning of the elastic behavior [44]. With the further increase in oscillation frequency, G' began to exceed G", which indicated that the elastic properties were dominant. Additionally, the miscible system mainly showed the elastic properties of solids. The crossover values of four gelatin and mulberry leaf polysaccharide miscible systems varied with different concentrations. At 5 mg/mL and 15 mg/mL, the crossover value of G-CHSS was lower than that of the other three, while at 10 mg/mL and 20 mg/mL, the crossover value of G-DASS was lowest.

**Figure 9.** Frequency dependence of storage (G') and loss (G") modulus of G-HBSS (**a**–**d**), G-CHSS (**e**–**h**), G-DASS (**i**–**l**), and G-CASS (**m**–**p**) at different concentrations.

#### **4. Conclusions**

The zeta potential, turbidity, rheological properties, and particle size of gelatin– mulberry leaf polysaccharide miscible systems were evaluated. Under acidic conditions, the four miscible systems were in a clear state. Under alkaline conditions, G-HBSS and G-CHSS were in a clear state, while G-DASS and G-CASS changed from a clear to cloudy state. With the increase in pH, the potential of the four miscible systems changed from positive to negative and decreased gradually. At pH 7.0, the zeta potential of the four miscible systems increased with the increase in MLP concentration, but it was still negative. The particle size distribution of the four miscible systems showed polydispersity at different concentrations, but the particle size did not change much. The four miscible systems showed "shear thinning" behavior, and the addition of gelatin reduced the apparent viscosity of four polysaccharides solutions. The apparent viscosity of G-HBSS, G-DASS, and G-CASS decreased with the increase in mass concentration, while the apparent viscosity of the G-CHSS miscible system increased with the increase in mass concentration, and it had relatively appreciable thickening. The effect of pH value on the stability of the

miscible system was in the order of G-CHSS > G-HBSS > G-CASS > G-DASS. Na+ had different effects on the apparent viscosity of the solutions of the four miscible systems. With the increase in Na+ concentration, the apparent viscosity of G-CASS increased, while G-HBSS, G-CHSS, and G-DASS decreased. After cold and heat treatment, G-CHSS changed from a "shear thinning" to "shear thickening" expansive fluid. Dynamic oscillatory shear data revealed that the miscible systems exhibited viscous properties with the increase in oscillation frequency.

**Author Contributions:** J.-G.Z. and Z.-J.W. proposed and designed the experiment; X.-X.Z., B.-Y.L. and Z.-J.G. experimented; X.-X.Z., B.-Y.L. and K.T. wrote the paper; M.R.K. and R.B. processed the data; R.B., J.-G.Z. and Z.-J.W. corrected the language of the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the key research and development projects in Ningxia Province (2021BEF02013), the National Natural Science Foundation of Ningxia Province (2021AAC02019), the youth talent cultivation project of North Minzu University (2021KYQD27, FWNX14), the Researchers Supporting Project No. (RSP-2021/138) King Saud University (Riyadh, Saudi Arabia), and the Major Projects of Science and Technology in Anhui Province (201903a06020021, 202004a06020042, 202004a06020052).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** All related data and methods are presented in this paper. Additional inquiries should be addressed to the corresponding author.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

