**1. Introduction**

Mulberry, belonging to the mulberry genus of the mulberry family, is widely distributed throughout the world [1]. In particular, the planting area of mulberry trees in China ranks first in the world, and the yield of mulberry leaves is rich [2]. Mulberry leaves are mainly used for silkworm breeding, and a few are used for the preparation of tea and fruit juice [3,4]. In China, mulberry leaf is also a medicinal resource which is widely used in traditional Chinese medicine [5]. Mulberry leaves contain a variety of functional components, such as alkaloids, polyphenols and flavonoids, proteins, amino acids, and carbohydrates [6–9]. A variety of functional components endow mulberry leaves with different biological activities, such as hypoglycemic, anti-atherosclerotic, antioxidant, and antibacterial activities [10–13]. Though there are many studies on the active components of alkaloids, flavonoids, and polyphenols in mulberry leaves, in recent years, more studies have emerged on the activity of polysaccharides.

**Citation:** Zhang, X.-X.; Liao, B.-Y.; Guan, Z.-J.; Thakur, K.; Khan, M.R.; Busquets, R.; Zhang, J.-G.; Wei, Z.-J. Interaction between Gelatin and Mulberry Leaf Polysaccharides in Miscible System: Physicochemical Characteristics and Rheological Behavior. *Foods* **2022**, *11*, 1571. https://doi.org/10.3390/ foods11111571

Academic Editors: Jianhua Xie, Yanjun Zhang and Hansong Yu

Received: 22 April 2022 Accepted: 24 May 2022 Published: 26 May 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Plant polysaccharides have attracted great attention in recent years because of their various biological activities, such as antitumor, antiviral, hypoglycemic, and antioxidant activities [14–17]. Because of their rheological properties, polysaccharides can also be used as adhesives and gelling agents in the food and cosmetics industries [18]. Previous studies have shown that plant polysaccharides can be used as a source of natural antioxidants in the food, pharmaceutical, and other industries [19]. At the same time, because of their rheological properties, plant polysaccharides can be used as thickeners and adhesives in natural materials and food industries [20].

The food system is a complex system composed of compounds with different properties, such as polysaccharides, proteins, and minerals. The current research focus is on regulating and controlling the interaction of biological macromolecules in the process of food processing to change their characteristics or structure [21]. Polysaccharides and proteins are the main nutrients in food formulas and can be miscible under certain conditions and change the system by forming the electrostatic complex [22]. This change affects the food structure and rheological properties, which is of great significance in food processing. As a kind of protein macromolecule, gelatin has been widely used in food processing [23].

In our previous study, four mulberry leaf polysaccharides (MLPs) were extracted continuously with a thermal buffer, a chelating agent, dilute alkalis, and concentrated alkali extractant to obtain thermal buffer soluble solids (HBSSs), chelating agent soluble solids (CHSSs), dilute alkali soluble solids (DASSs), and concentrated alkali soluble solids (CASSs), respectively [19,20]. Four MLPs had a shear-thinning characteristic and the properties of non-Newtonian fluid [20], and exhibited significant differences in the physicochemical and antioxidant activities. The CASS displayed higher thermal properties and suitability as a supplement in hot processed foods [19]; The DASS showed the highest apparent viscosity at different tested concentrations [20]. However, there are no reports on the interaction between mulberry leaf polysaccharides and gelatin in the miscible system. In this study, the effects of gelatin on zeta potential, turbidity, particle size, distribution, and rheological properties of gelatin–polysaccharide miscible systems were studied.

#### **2. Materials and Methods**

#### *2.1. Materials and Chemicals*

The mulberry leaves (*Morus alba* L.) used in the experiment were provided by Anhui Academy of Agricultural Sciences (Anhui Province, Hefei, China). Four types of mulberry leaf polysaccharides (HBSS, CHSS, DASS, and CASS) were obtained by continuous extraction with four solvents [19,20]. All chemicals were analytical-grade.

#### *2.2. Preparation of Gelatin–Polysaccharide Miscible Solution*

A total of 0.5 g gelatin was dissolved in 100 mL water at 40 ◦C, 1 h, then stored at 4 ◦C for 24 h, and soaked in water at 25 ◦C for 18 h to prepare a stable gelatin solution with a mass concentration of 5 mg/mL [24]. Then, 0.1 g of mulberry leaf polysaccharides was added to 100 mL of 5 mg/mL gelatin solution and stirred until it was completely dissolved to obtain gelatin–polysaccharide miscible solutions (G-MLPs) with a concentration of 1 mg/mL for the phase diagram experiment, in which 2.3. 5 mg/mL gelatin solution was used as the solvent for the preparation of gelatin–polysaccharide solutions with different concentrations.

#### *2.3. Phase Diagram*

The gelatin–polysaccharide miscible solutions prepared in Section 2.2. were used as a mother liquor to prepare gelatin–polysaccharide miscible systems with different pHs (3–10, interval 0.5). At first, the miscible system was placed at room temperature for 1 h, and then the solution state was observed. The state was divided into the following three types: clear solution, cloudy solution, precipitation, and cloudy solution [25]. The phase diagram was drawn according to the change in pH.

#### *2.4. Measurement of Zeta Potential, Particle Size, and Distribution*

The zeta potential, particle size, and distribution of gelatin–polysaccharide miscible solution at different pHs (3.5, 7, 10) with a concentration of 20 mg/mL and different concentrations (5, 10, 15, and 20 mg/mL) at pH 7 were determined by Malvin particle size analyzer (Zetasizer Nano-ZSE, Malvern Instruments, Worcestershire, UK) [26]. The calculations of zeta potential and particle size were as follows:

$$
\zeta \left( \times 10^3 \right) = \frac{3 \text{ } \eta \text{ U}\_{\text{E}}}{2\_{\text{cf}} \text{ } (\text{k}\alpha)} \tag{1}
$$

ε (F/M) is numerically equal to εrε0; εr is the relative permittivity of the mixture (25 ◦C water is about 78.2); <sup>ε</sup>0 is the vacuum relative permittivity (about 8.854 × <sup>10</sup>−<sup>12</sup> F/m); <sup>η</sup> (Pa·s) is the viscosity of the dispersion system (25 ◦C water is about 8.937 × <sup>10</sup>−<sup>4</sup> Pa·s); f (κα) is the ratio of the particle radius α to Debye's length in Henry's equation and approximately 1.5 in the Smoluchowski model.

$$\mathrm{d}\left(\times 10^{9}\right) = \frac{\mathrm{K\_{B}T}}{\mathrm{3\pi\,\eta\,\,D}}\tag{2}$$

<sup>η</sup> (Pa·s) is the viscosity of the dispersion system (25 ◦C water is about 8.937 × <sup>10</sup>−<sup>4</sup> Pa·s); KB is Boltzmann's constant, and T is absolute temperature (k); the refractive index was set as follows: water 1.33, G 1.45, G-MLPs 1.59; the absorbance of the material was 0.01.

#### *2.5. Determination of Rheological Properties*

2.5.1. Effect of Concentration, pH, Na+ Concentration, and Temperature on Apparent Viscosity of G-MLPs

The rheological properties of G-MLPs were determined using the previously described method [20]. The rheological properties of the gelatin–polysaccharide miscible system were mainly studied through the effects of concentration, pH, Na<sup>+</sup> concentration, and temperature on the apparent viscosity of G-MLPs. In short, gelatin–polysaccharide aqueous solutions with concentration gradients of 5, 10, 15, and 20 mg/mL were prepared to study the effect of concentration on apparent viscosity, and 5 mg/mL gelatin solution was used as control. The pH value of 10 mg/mL gelatin–polysaccharide aqueous solution was adjusted to 3.5, 7.0, and 10.0 with HCl and NaOH solution to study the effect of pH on apparent viscosity. NaCl solutions with concentrations of 0, 0.1, 0.2, 0.4, and 0.8 mol/L were prepared, respectively, and then gelatin was dissolved and prepared in 5 mg/mL gelatin–NaCl solution, and finally polysaccharides were added to the solution to make the concentration of polysaccharides 10 mg/mL. When studying the effect of temperature on the apparent viscosity of the miscible system, we selected −20, 25, and 100 ◦C for cold and heat treatment of 10 mg/mL gelatin–polysaccharide miscible systems. Rheometer (DHR-3, TA instruments, New Castle, DE, USA) conditions were as follows: steady-state shear mode; clamp: diameter 40 mm; cone plate: cone angle 2◦; shear speed range: 0.01–1000 s<sup>−</sup>1; temperature: 20 ◦C.

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

The changes in apparent viscosity of gelatin–polysaccharide aqueous solution with mass concentrations of 5, 10, 15, and 20 mg/mL were measured under different shear oscillation frequencies. Test conditions were as follows: DHR-3: clamp: diameter 40 mm; cone plate: cone angle 2◦; shear speed range: 0.01–100 Hz; temperature: 20 ◦C [20].

#### *2.6. Statistical Analysis*

All the tests reported were repeated three times. One-way ANOVA through SPSS 21.0 was used for the data analysis at *p* < 0.05, and origin 8.0 software was used for graphs.

#### **3. Results and Discussion**

#### *3.1. Phase Diagram of Gelatin–Mulberry Leaf Polysaccharides (G-MLPs) Miscible System*

Table 1 shows the effect of pH on the phase behavior of the gelatin–mulberry leaf polysaccharide miscible system. The electrical properties of proteins were reported to vary according to the pH of their environment. When it was in an electronegative state, proteins co-dissolved with anionic polysaccharides due to electrostatic repulsion, and under electropositive conditions, proteins aggregated due to the electrostatic binding reaction with anionic polysaccharides, resulting in a cloudy and precipitated solution [27]. At pH < 7, the dispersions of the four miscible systems were clear. When pH was 7–8.5, G-HBSS and G-CHSS were still clear, indicating that the reaction between G-HBSS and G-CHSS affected the intermolecular aggregation of polysaccharides [22]. G-DASS and G-CASS were the alkaline extracts; therefore, their resulting systems changed from clear to cloudy. When the pH was close to alkaline conditions, the repulsion force between G-DASS and G-CASS molecules decreased, resulting in aggregation, and the miscible system changed from clear to cloudy [25]. With the continuous increase in pH value, both the G-HBSS and G-CHSS miscible systems changed from clear to cloudy, while G-DASS and G-CASS changed to cloudy and precipitated systems. At pH 9, the four samples were unstable, indicating that the system environment with high pH was conducive to the formation of an insoluble gelatin–polysaccharide complex, which ultimately increased the instability of the system [25].



Note: The solubility or insolubility was evaluated by visual observation. (-: clear solution; : cloudy solution; •: precipitation and cloudy solution).

### *3.2. Zeta Potential Analysis of Gelatin–Mulberry Leaf Polysaccharides (G-MLPs) Miscible System*

The change in the zeta potential of gelatin and mulberry leaf polysaccharide miscible systems with respect to pH is shown in Figure 1a. With the increase in pH value, the zeta potential of the four miscible systems G-HBSS, G-CHSS, G-DASS, and G-CASS changed from positive to negative. Moreover, the zeta potential of the miscible system also decreased with the increase in pH. The zeta potential of the miscible system was negatively charged with the increase in pH, which may have been due to the electrostatic binding reaction between gelatin and mulberry polysaccharides, and the deprotonation reaction of amino and carboxyl functional groups on gelatin molecules and carboxyl groups in the mulberry polysaccharide structure with the increase in pH [28]. At pH 7.0, with the increase in mulberry leaf polysaccharide concentration, the zeta potential of the miscible system gradually increased, but it was still negative, as shown in Figure 1b. This may be due to the fact that the combination of mulberry leaf polysaccharides and gelatin resulted in the movement of the net charge of the miscible system to the positive direction [29,30].

#### *3.3. Particle Size Analysis of Gelatin–Mulberry Leaf Polysaccharides (G-MLPs) Miscible System*

The particle size and distribution of the gelatin solution and four mulberry leaf polysaccharide solutions at the same concentration (10 mg/mL) are shown in Figure 2. The particle size of the HBSS, CHSS, DASS, and CASS had two, one, three, and two peaks, respectively. Unlike the DASS, the other three mulberry leaf polysaccharides were monodisperse, indicating that the composition of the HBSS, CHSS, and CASS was relatively uniform and may have formed electrostatic complexes with the aqueous solution. This result is consistent

with the particle size distribution of water-soluble polysaccharides extracted from kidney beans [31]. The particle size of gelatin had two peaks, and the particle size was larger, which may have been due to the high pH value of the solution, which led to its positive or negative charge and increased the interaction between gelatin and water molecules [32].

**Figure 1.** Zeta potential of G-MLPs at 20 mg/mL as a function of pH (**a**); zeta potential of G-MLPs as a function of concentration at pH 7 (**b**).

**Figure 2.** Particle size distribution of MLPs and gelatin (G).

Figure 3 shows the particle size and distribution of four miscible systems at pH 3.5, 7.0, and 10.0. At pH 3.5, G-HBSS, G-CHSS, G-DASS, and G-CASS formed two, one, two, and three main peaks, respectively. When the pH was 7, there were two, one, three, and two peaks, respectively. When the pH was 10, there were three, two, three, and two peaks, respectively. Compared with the particle size of gelatin and four polysaccharides, the particle size distribution of the four miscible systems of gelatin and mulberry leaf polysaccharides changed significantly. Under different pH conditions, the G-CHSS solution still maintained high monodispersity, which may have been caused by the electrostatic complex formed after the miscibility of the CHSS and gelatin [30]. The three miscible systems of G-HBSS, G-DASS, and G-CASS showed polydispersity, which may have been due to the charge repulsion between gelatin and these three mulberry leaf polysaccharides [33]. With the increase in pH, the change trend of particle size of the four miscible systems was also different. The particle size of G-HBSS and G-CHSS miscible systems decreased with the increase

in pH, while the particle size of G-DASS and G-CASS increased with the increase in pH. These phenomena may have been caused by the different extractants of the HBSS, CHSS, DASS, and CASS. Specifically, the reason for the change trend of G-HBSS and G-CHSS may be that with the increase in pH, gelatin and two polysaccharides produced repulsion, which reduced the particle size of the complex [30], while the reason for the change of G-DASS and G-CASS may be that both were alkaline extracts. Under alkaline conditions, the electrostatic complexation reaction intensity between gelatin and polysaccharides increased, and the formed complex was easy to aggregate, resulting in the increase in particle size, which was the same as the change of the solution state reflected in the above phase diagram [33].

**Figure 3.** Particle size distribution of G-MLPs at different pHs. (**a**) G-HBSS; (**b**) G-CHSS; (**c**) G-DASS; (**d**) G-CASS.

The particle size distribution and size of the four miscible systems at different concentrations are shown in Figure 4. It can be seen from the figure that the miscible system showed polydispersity, but the particle size did not change much with the change in concentration, indicating that the change in concentration only affected the distribution of particle size, and had little effect on its size. This may be because with the increase in the polysaccharide concentration, it had little effect on the intermolecular force of the miscible system of gelatin and polysaccharides [32].
