Gelatin-stabilized traditional emulsions: Emulsion forms, droplets,and storage stability

Mengzhen Ding, Ting Zhng, Hun Zhng, Ningping To, Xichng Wng,Jin Zhong,?

aNational R&D Branch Center for Freshwater Aquatic Products Processing Technology (Shanghai), Integrated Scientific Research Base on Comprehensive Utilization Technology for By-Products of Aquatic Product Processing, Ministry of Agriculture and Rural Affairs of the People’s Republic of China, Shanghai Engineering Research Center of Aquatic-Product Processing and Preservation, College of Food Science & Technology, Shanghai Ocean University, Shanghai 201306, China

bIowa State University, Ames Laboratory, Ames, IA 50011, USA

ABSTRACT

Fish oils are important substances in the field of food and drug delivery. Due to their unstable double bonds, fishy taste, and poor water solubility, it is pivotal to investigate novel dosage forms for fish oils,such as encapsulated droplets. In this work, we primarily prepared gelatin-stabilized fish oil-loaded traditional emulsions and investigated their emulsion forms, droplets, and storage stability under different preparation and storage conditions. Our results showed that higher gelatin solution pH, higher storage temperature in the range of 4–37 °C, and increased storage time induced the emulsion form switch from a liquid form to a redispersible gel form of the fish oil emulsion. The droplets had core-shell microstructures and a trimodal size distribution, which decreases linearly with increasing gelatin solution pH and homogenizing time, but decreases exponentially with increasing homogenizing speed. In addition, storage temperature showed a notably different effect on traditional emulsion storage. This work provides a fundamental knowledge for the formation, microstructure, and properties of gelatin-based traditional emulsions. It also provides a promising new application for fish oil-loaded emulsions in food beverages,soft candy, and other food products.

Keywords:

Emulsion form

Fish oil

Gelatin

Homogenizing conditions

pH

1. Introduction

Omega-3 unsaturated fatty acids are important nutrients for humans [1–5]. Fish oils are known to be rich in these fatty acids such as including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) [6]. Therefore, fish oils have been considered as one of promising materials in the food and pharmaceutical industries in recent years. However, fish oils can easily lose their nutritional properties due to the disruption of unstable double bonds after exposure to light, oxygen, or heat [7]. In addition, the taste of fish oils affects the flavor of food products, if added directly. Finally, fish oils cannot be directly added to aqueous foods because of their poor water solubility. Hence, novel dosage forms such as food emulsions need to be developed to shield the disadvantages of fish oils.

Collagens are the most prominent insoluble fibrous proteins in mamalian connective tissues and play pivotal physiological roles [8,9]. Gelatin is a product of native collagen by partial hydrolysis [10,11]. Collagens and gelatins have been widely studied and applied in food [9,12–14] and pharmaceutical industries[15,16], bioimaging [17,18], and tissue engineering [9,17,19–21].Gelatin-stabilized emulsions have been proved to inhibit fish oil oxidation [22]. Recently, gelatin has been used in emulsion preparation, as crosslinked gelatin particles [23], gelatin/chitosan complexes [24], or gelatin/glucomannan/tannic acid nanocomplexes [25] for encapsulating sun flower oil [23], corn oil [24], or medium-chain triacylglycerol oil [25]. Crosslinked gelatin nanoparticles [26,27] and hybrid gelatin/surfactants [28,29] have been explored to stabilize fish oil-loaded emulsions. Functional gelatins have also been developed to improve the stability of gelatin-stabilized emulsions [30]. Gelatin could also be applied to prepare oil-in-water multilayer emulsions stabilized by a gelatin-pectin bilayer [31,32] and a β-lactoglobulin-ι-carrageenan-gelatin trilayer [33,34]. Gelatin/gum-stabilized multilayer emulsions could be applied to prepare electrospun nanofibers [35]. However, these works did not systematically study the emulsions stabilized by pure gelatin. Therefore, the use of pure gelatin for traditional emulsion preparation is a better choice for the food industry, due to lower product costs and more suitable production processes.

Emulsion forms could be classified into liquid, redispersible emulsion gel, and unredispersible emulsion gel) [36]. Emulsion gel is a type of soft-solid material [37]. Redispersible emulsion gel is used to describe emulsion gel that can be redispersed into liquid emulsion under some conditions. Emulsion gels can be easily and stably transported and stored after their successful preparation. However, the form of the emulsion must be investigated, as it can affect its storage requirements and potential applications.The redispersibility of an emulsion gel should be considered prior to its practical application. Recent study demonstrated that the form and droplet size distribution of the emulsion were dependent on the emulsifier used and the storage time [28,36,38]. Therefore,this work mainly studies the effects of the preparation methods and storage conditions on the emulsion form, droplets, and storage stability of gelatin-stabilized fish oil-loaded traditional emulsions.

2. Materials and methods

2.1. Materials

Nile red and Nile blue dyes from Sangon Biotech Co., Ltd. (Shanghai, China) and deep-sea fish oils (food grade, DHA + EPA ≥70%)from Xi’an LvTeng Biological Technology Co., Ltd. (Xi’an, Shannxi Province, China) were stored at ?18°C. Bovine bone gelatin granules (type B, approximately 240 g bloom) from Aladdin Industrial Corp. (Shanghai, China) and other common chemicals from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China) were stored at room temperature.

2.2. Preparation of fish oil-loaded gelatin-stabilized traditional emulsions

Gelatin granules (4.0%, m/v) were dispersed in ultrapure water at room temperature. After 30 min, they were treated at 45°C for 30 min in an oscillating (180 r/min), constant-temperature water bath (Model SW22; Julabo, Seelbach, Germany). The diluted gelatin solutions (0.2%, 0.5%, 1.0%, and 2.0%) were obtained by diluting 4.0% gelatin solution with ultrapure water. The pH of the solutions was adjusted to 3, 5, 7, 9, and 11 using a 3 mol/L NaOH solution or an HCl solution. Fish oil and gelatin solution were mixed with a 1:1 vol ratio. The mixture was mechanically homogenized using an ULTRATURRAX?homogenizer (T 10 basic, IKA, Guangzhou, China) with a 10-mm head. Various homogenization times (15, 30, 60, 90, and 120 s) and speeds (8000; 9500; 11500; 14500; and 20500 r/min)were applied. The obtained emulsions were stored at 4°C for 3 days and were imaged by a digital camera at the designated time points. After letting them on stand, some liquid emulsions could form redispersible emulsion gels that could be redispersed to form liquid emulsions at 45°C for 5 min. After letting them on stand at some storage temperatures, some liquid emulsions could form unredispersible emulsion gels that could not be redispersed even when incubated at 45°C for 1 h. The creaming index (CI) of the emulsions at the designated time point was calculated as follows:

where Hsis the serum layer (the transparent and the turbid layers)height and Htis the total emulsion height.

2.3. Optical microscopy measurements

For liquid emulsions, 5 μL emulsion was placed on a glass microslide and covered with a cover glass. The samples were observed under a MS600 F inverted optical microscope (Shanghai Minz Precision Instruments Co. Ltd., China).

Redispersible emulsion gel was treated at 45°C for 5 min. Then,5 μL emulsion was used to prepare sample and then observed under a MS600 F inverted optical microscope.

For indispersible emulsion gel, approximately 0.005 g sample was placed on glass microslide and coved by a cover glass. The sample was observed under a ML8000 upright optical microscope(Shanghai Minz Precision Instruments Co. Ltd. China) or a MS600 F inverted optical microscope. The applied optical microscopes are indicated in the figure captions.

2.4. Confocal laser-scanning microscopy (CLSM) measurements

1 mL liquid emulsion was mixed with 40 μL fluorescent dye(0.1% Nile red and 1% Nile blue) 1,2-propanediol solution with 2% water. The mixture was vortexed for 1 min. Then, 5 μL sample was placed on a glass microslide.

The redispersible emulsion gel was treated at 45°C for 5 min.The resulting liquid emulsion was then dyed and placed on a glass microslide.

For indispersible emulsion gel, about 0.1 g sample was cut into small pieces (approximately 2 mm in diameter). Then, they were mixed with 40 μL of fluorescent dye (0.1% Nile red and 1% Nile blue)1,2-propanediol solution with 2% water for 5 min. The gel pieces were place on a glass microslide.

These samples were covered with square cover glass and observed by a confocal laser-scanning microscope (Leica TCS SP8,Wetzlar, Germany) with a scanning frequency of 200 Hz and a scanning density of 1024 × 1024 pixels. Nile red and Nile blue were excited by a 552-nm laser and a 633-nm laser, respectively [39].Three parallel specimens were investigated in this study. The objective was a 63× oil objective. The obtained images were analyzed by the Leica Application Suite X software.

2.5. Gaussian fitting of emulsion droplet sizes

For each type of sample, three images from three different batches were randomly selected and examined by optical microscopy. Approximately 500–5000 of droplet sizes were obtained for each emulsion using MeizsMcs 6.0 software (Shanghai Minz Precision Instruments Co., Ltd.). The frequency distributions were statistically analyzed with bin numbers of 16–22 prior to multiple Gaussian peak fitting [40,41]. Finally, peak values vs preparation parameters were fitted by linear or exponential equations.

2.6. Statistical analysis

Mean ± standard deviation (SD) was used to present the measured data in this work. One-way ANOVA was used for statistical comparison, with a P-value <0.05 considered statistically significant.

3. Results and discussion

3.1. Atomic force microscopy (AFM) observation of gelatin

AFM has been widely applied to observe the morphology of food proteins [42,43]. As shown in Fig. S1, gelatin can exist as several different nanostructures: nanoparticles, ring-like nanostructures, short nanofibers, and film-like structures (nanofilms).This gelatin polymorphism observed by us is consistent with previous findings [44,45]. AFM results also con firmed that film-like nanostructures could be formed by the gelatin molecules, which is consistent with previous work [36]. It may explain the interfacial gelatin layer formation of emulsions to encapsulate oils. In fish oil-loaded gelatin-stabilized emulsions, gelatin nanofilms might encapsulate oils as they decrease the interfacial tension and form a steric elastic film in [46].

Fig. 1. Observation of fish oil-loaded gelatin-stabilized (1.0%) traditional emulsions under different gelatin solution pH (3, 5, 7, 9, and 11). The traditional emulsions were prepared with a homogenizing time of 60 s and a homogenizing speed of 11500 r/min. (A-B): photographs of the traditional emulsions in glass vials at 0 h and after 3 days of storage at 4 °C, respectively. (C): inverted optical microscopy observation of the traditional emulsion droplets at 0 h and after 3 days of storage at 4 °C. Scale bars are 50 μm.Black arrows and black asterisks show redispersible emulsion gels. Other emulsions are liquid emulsions.

3.2. Effect of gelatin solution pH on fish oil-loaded gelatin-stabilized traditional emulsions

Traditional emulsions with different pH (3, 5, 7, 9, and 11) were homogenized for 60 s at 11500 r/min. After preparation, the pH showed no obvious change, suggesting that homogenization had no major effect on emulsion pH. These emulsions (Fig. 1) were milk white or light pink (pH 11) as a result of the distribution of gelatin in the strong basic solution. The freshly prepared emulsions consisted of droplets (Fig. 1C) that had a trimodal size distribution and decreased linearly in size with the increasing pH of the solution (Figs. 1C, S2). In the CLSM experiments, Nile red and Nile blue were used to stain fish oils and gelatins [39], respectively. CLSM results demonstrated that fish oils (red color) were encapsulated by gelatin (blue color) to form droplets in the water phase (Fig. 2A).Therefore, fish oil-loaded gelatin-stabilized traditional emulsions could be successfully prepared at pH 3?11. The effect of pH on droplet size may have resulted from the difference between the pH of the solution and the isoelectric point of gelatin. Previous work suggested solution had a major impact on the physical stability of lutein-enriched emulsions and caused droplet aggregation at pH 4 and 5 [47]. However, our work did not show the droplet aggregation. With an increase in the pH of the solution, the negative charge of gelatin increased, the hydrophilic region (in the shell layer)-to-hydrophobic region (in the oil core) ratio of the gelatin molecule increased, and the droplet curvature increased; therefore, the droplet size decreased, which is consistent with previous work [48]. However, it is contrary to pH effects of many oil-inwater emulsions [49]. After 3 days of storage at 4°C, the traditional emulsions showed creaming (Fig. 1B). The creaming index values were in the following order: pH 11 (CI: 34.6% ± 1.5%) > pH 3 (CI:19.4% ± 1.0%) > pH 5 (CI: 15.3% ± 1.0%) > pH 7 (CI: 6.7% ± 0.4%) > pH 9 (CI: 4.5% ± 0.5%). The traditional emulsions containing a gelatin solution at pH 7 and 9 changed to gel forms, which could be redispersed by incubation at 45°C for 5 min. Therefore, a higher gelatin solution pH induced a switch of the emulsions from a liquid form to a redispersible gel form. This would be beneficial for emulsion storage and transportation. For example, the emulsion could be shipped in a gel form from the raw-material factory to the food-processing factory and then be redispersed into a liquid form for food production. After 3 days of storage (Fig. 1C), the droplet sizes of the traditional emulsions at pH 9 showed no obvious changes, while the droplet sizes increased at pH 3, 5, 7, and 11. The droplets of traditional emulsions with gelatin pH values in the range of 7–11(Fig. 2) were relatively stable, whereas some holes appeared in the droplets of traditional emulsions with gelatin pH values of 3 and 5(Fig. 2B), suggesting that multiple emulsions had formed. Gelatin type B is an alkaline-processed gelatin with an isoelectric point of 4.8–5.1 [28]. The protein may aggregate when the emulsion pH approaches its isoelectric point. This could be the main reason for the relative instability of emulsions prepared with gelatin at pH 3 and pH 5. When the pH is far from the isoelectric point, gelatin type B tends not to aggregate. This may explain why the traditional emulsion transitioned between a liquid and a gel form. According to the optical microscopy images, the emulsion gels might be proteinstabilized emulsion gel – a type of aggregated particle gel [37].Considering creaming (Fig. 1) and droplet structure (Fig. 2), gelatinstabilized traditional emulsions at pH 9 were used for subsequent work.

Fig. 2. CLSM images of fish oil-loaded gelatin-stabilized (1.0%) traditional emulsions at different gelatin solution pH (3, 5, 7, 9, and 11). The traditional emulsions were prepared with a homogenizing time of 60 s and a homogenizing speed of 11500 r/min. (A): Traditional emulsion droplets at 0 h. (B): Traditional emulsion droplets after 3 days of storage at 4 °C. Scale bars are 25 μm. Black asterisks show redispersible emulsion gels and other emulsions are liquid emulsions.

3.3. Redispersion process of fish oil-loaded gelatin emulsion gels

After letting them on stand for 3 days of storage at 4°C, the traditional emulsions at pH 7–9 could form emulsion gels (indicated by black arrows and black asterisks in Figs. 1 and 2), which could be redispersed to form liquid emulsions at 45°C for 5 min. This redispersion process was further analyzed. After 1 day of storage at 4°C, liquid emulsions (Fig. 3A, 3B) changed into gel emulsions(Fig. 3C), consisting of droplets (Fig. 3F). The gel formation process did not obviously change the shape or size of the droplets (data not shown). The gels changed into a liquid form (Fig. 3D) after 5 min of incubation at 45°C. The redispersed emulsion (Fig. 3G) had similar droplets to those of the freshly prepared emulsion (Fig. 3E) and the emulsion gel (Fig. 3F). These results demonstrated that neither the gel formation process nor the redispersion process affected droplet structure (shape and size). It should be noted that our redispersed emulsion gels were solid, which was different to redispersed emulsion powders using spray-drying techniques in other work [50]. The preparation of redispersed emulsion gels only needs to let them on stand for some times without the use of complex instruments.Moreover, the shapes of the emulsion gels could be controlled by designing the liquid emulsion containers. Therefore, the redispersed emulsion gels might have good potential in the emulsion industry.

Fig. 3. Redispersion process of fish oil-loaded gelatin (1.0%) emulsion gels. The preparation parameters for the traditional emulsions were as follows: homogenizing time, 60 s; homogenizing speed, 11500 r/min; and gelatin solution pH 9. (A):photograph of the traditional emulsion in a glass vial at 0 h. (B): photograph of the traditional emulsion in an upside-down glass vial at 0 h. (C): photograph of the traditional emulsion gel in an upside-down vial after 1 day of storage at 4°C. (D):photograph of the traditional emulsion in an upside-down glass vial after redispersion at 45°C for 5 min. (E): Inverted optical microscopy images of the emulsion droplets at 0 h. (F): Upright optical microscopy images of the emulsion droplets in the traditional emulsion gel corresponding to image (C). (G): Inverted optical microscopy image of the emulsion droplets in the redispersed emulsion (D). Scale bars are 50 μm. Black arrows indicate the emulsion gel.

3.4. Effect of homogenizing parameters on fish oil-loaded gelatin-stabilized traditional emulsions

It is generally accepted that droplet size decreases and the traditional emulsion stability increases with increasing homogenizing time and speed. However, the quantitative relationships between droplet size and homogenizing parameters and their effects on emulsion stability remain unclear.

Traditional emulsions were successfully prepared at five homogenizing times (Fig. 4A, 4C) and speeds (Fig. 5A, 5C). The emulsions mainly consisted of droplets with a trimodal size distribution (Figs. S3, S4). Moreover, the droplet size decreased linearly with increasing homogenizing time (Fig. S3) and exponentially with increasing homogenizing speed (Fig. S4). After 3 days of storage at 4°C, all the traditional emulsions had changed to gel forms (Figs. 4B and 5 B). They showed different creaming index values in the following order: 15 s (CI: 16.4% ± 1.5%) > 30 s(CI: 8.6% ± 0.8%) > 60 s (CI: 4.4% ± 0.5%) > 90 s (CI: 3.1% ± 0.3%) ≈120 s (CI: 2.7% ± 0.6%); 8000 r/min (CI: 21.3% ± 1.0%) > 9500 r/min(CI: 17.4% ± 1.0%) > 11500 r/min (CI: 4.4% ± 0.5%) > 14500 r/min(CI: 2.1% ± 0.3%) > 20500 r/min (CI: 1.2% ± 0.4%). After redispersion, no obvious droplet changes (3 days and 0 h, Figs. 4C and 5 C) were observed. Therefore, homogenizing time and speed clearly affected droplet size and creaming stability of the traditional emulsions, whereas they did not obviously change the stability of the droplet size after 3 days of storage of the traditional emulsions. It is consistent with previous work on gelatin nanoparticle-stabilized emulsions [41]. Therefore, the effects of homogenizing time and speed might not depend on the gelatin forms (molecules or nanoparticles).

Fig. 4. Observation of fish oil-loaded gelatin-stabilized (1.0%) emulsions with different homogenizing times. The traditional emulsions were prepared with a homogenizing speed of 11500 r/min and a gelatin solution pH 9. (A-B): photographs of the traditional emulsions in glass vials at 0 h and after 3 days of storage at 4°C, respectively. (C):Inverted optical microscopy images of the emulsion droplets at 0 h and after 3 days of storage at 4 °C. Scale bars are 50 μm. Black arrows and black asterisks indicate redispersible emulsion gels. Other emulsions are liquid emulsions.

Fig. 5. Observation of fish oil-loaded gelatin-stabilized (1.0%) emulsions with different homogenizing speeds. The traditional emulsions were prepared with a homogenizing time of 60 s and a gelatin solution pH 9. (A-B): photographs of the traditional emulsions in glass vials at 0 h and after 3 days of storage at 4°C, respectively. (C): Inverted optical microscopy images of the emulsion droplets at 0 h and after 3 days of storage at 4 °C. Scale bars are 50 μm. Black arrows and black asterisks indicate redispersible emulsion gels. Other emulsions are liquid emulsions.

3.5. Effect of storage temperature on fish oil-loaded gelatin-stabilized traditional emulsions

Temperatures of ?18, 4, room temperature (16–18°C), 37,and 60°C were chosen to simulate frozen-storage temperature, refrigerated-storage temperature, ambient-storage temperature, human-body temperature, and Pasteurization temperature,respectively. After 3 days of storage, the traditional emulsions at different temperatures showed different behaviors (Fig. 6). Their creaming index values were as follows: 60°C (CI: 25.4% ± 2.0%) >37°C (CI: 16.6% ± 1.2%) > room temperature (CI: 7.7% ± 1.0%) > 4°C (CI: 4.4% ± 0.5%) > ?18°C (CI: 0%, Fig. 6A). The traditional emulsion stored at ?18°C did not show obvious creaming, which was expected because they were frozen at this temperature. The samples stored at 4, 37, and 60°C changed to gel forms (Figs. 6C, 6 E,and 6 F). The samples stored at 4°C could be redispersed at 45°C for 5 min, but they still consisted of droplets (indicated by a black asterisk in Fig. 6G). The samples stored at 37 and 60°C could not be redispersed, even when incubated at 45°C for 1 h, suggesting that higher storage temperatures induced a loss of redispersion ability.The irreversible gelation may have resulted from the adhesion of the gelatin nanofilm. These nonredispersible samples (indicated by white squares in Fig. 6G) demonstrated that samples stored at 37°C still consist of droplets (Fig. 6G), whereas those stored at 60°C have no obvious droplets. The sample stored at ?18°C changed to flocculent precipitates (Fig. 6B) with irregular droplets (Fig. 6G).For the sample stored at room temperature, the inner component was still in liquid form, but the outer component that was close to the vial wall changed to a gel form (Fig. 6D). This demonstrated that its rate of gel formation was slower than those of the emulsions stored at 4 and 37°C. The inner liquid part still consisted of droplets under these conditions (Fig. 6G). The CLSM images (Fig. 7) showed that the traditional emulsions stored at ?18, 4, room temperature,and 37°C still consisted of droplets, whereas the droplets disappeared after storage at 60°C. Therefore, storage temperature clearly affects the traditional emulsions. The optimal temperature for the storage of fish oil-loaded gelatin-stabilized traditional emulsions was found to be 4°C. It is consistent with previous work on gelatin nanoparticle-stabilized emulsions [41]. However, it is inconsistent with the effect of storage temperature on the lutein-enriched emulsions [47]. Therefore, the effect of storage temperature might not depend on the gelatin forms (molecules or nanoparticles).

Fig. 6. Effect of storage temperature on fish oil-loaded gelatin-stabilized (1.0%) traditional emulsions. The traditional emulsions were prepared with a homogenizing time of 60 s, a homogenizing speed of 11500 r/min, and a gelatin solution pH 9. Then, the emulsions were stored at different temperature for 3 days. (A): Photograph of the traditional emulsions in glass vials. (B-F): Photographs of the traditional emulsion in lie-down glass vials. (G): Inverted optical microscopy images of the traditional emulsion droplets.Scale bars are 50 μm. Black arrows, black asterisk, black squares indicate emulsion gel, redispersible emulsion gel, and unredispersible emulsion gels, respectively.

Fig. 7. CLSM images of fish oil-loaded gelatin-stabilized (1%) emulsions. The preparation parameters of the traditional emulsions were as follows: homogenizing time, 60 s;homogenizing speed, 11500 r/min; and gelatin solution pH, 9. Then, the emulsions were stored at different temperature for 3 days. Scale bars are 25 μm. Black arrows indicate emulsion gels. Black arrows, black asterisk, black squares indicate emulsion gel, redispersible emulsion gel, and unredispersible emulsion gels, respectively.

4. Conclusions

In this study, the effects of preparation methods and storage conditions on the emulsion form, droplets, and storage stability of gelatin-stabilized fish oil-loaded traditional emulsions were studied. The redispersion process was also analyzed. The results showed that the traditional emulsions could be successfully prepared and modified. The increase in gelatin solution pH, storage temperature, and storage time induced a switch from a liquid form to a redispersible gel form for these emulsions. The droplets mainly had a trimodal size distribution. Their sizes linearly decreased with increasing gelatin solution pH and homogenizing time and exponentially decreased with increasing homogenizing speed. The multiple forms (liquid emulsion, redispersible emulsion gel, and unredispersible emulsion gel) of these emulsions allow for multiple potential applications in food beverages, soft candy, and other food products. Knowledge of these forms would be beneficial for determining the optimal emulsion storage and transportation conditions. For example, fish oil-loaded emulsions could be shipped in a gel form from the raw material factory to the food-processing factory and then redispersed into a liquid form for food production. The multiple forms of the emulsions may be associated with the properties of gelatin, such as its film formation behaviors and its easy gelation ability. Any companies interested in using these emulsions could further analyze the liquid-to-gel transition times of different emulsions prior to their final application.

Declaration of Competing Interest

All the authors declared no conflict of interest.

Acknowledgements

This research has been supported by research grants from the National Key R & D Program of China (No. 2019YFD0902003-2)and Shanghai Municipal Education Commission - Gaoyuan Discipline of Food Science & Technology Grant Support (Shanghai Ocean University).

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fshw.2020.04.007.