Emulsification of oil-in-water emulsions with eggplant (Solanum melongena L.)

Journal Pre-proofs Emulsification of oil-in-water emulsions with eggplant (Solanum melongena L.) Yuxia Zhu, Xiaopu Ren, Yingjie Bao, Shun Li, Zengqi Peng, Yawei Zhang, Guanghong Zhou PII: DOI: Reference:

S0021-9797(19)31520-6 https://doi.org/10.1016/j.jcis.2019.12.055 YJCIS 25795

To appear in:

Journal of Colloid and Interface Science

Received Date: Revised Date: Accepted Date:

22 May 2019 11 December 2019 14 December 2019

Please cite this article as: Y. Zhu, X. Ren, Y. Bao, S. Li, Z. Peng, Y. Zhang, G. Zhou, Emulsification of oil-inwater emulsions with eggplant (Solanum melongena L.), Journal of Colloid and Interface Science (2019), doi: https://doi.org/10.1016/j.jcis.2019.12.055

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Emulsification of oil-in-water emulsions with eggplant (Solanum melongena L.) Yuxia Zhua, b, Xiaopu Rena, b, Yingjie Baoa, b, Shun Li a, b, Zengqi Penga, b*, Yawei Zhanga, b*, and Guanghong Zhoua, b a

College of Food Science and Technology, National Center of Meat Quality and Safety Control,

Nanjing Agricultural University, Nanjing 210095, China b

Synergetic Innovation Center of Food Safety and Nutrition, Nanjing, China

*Corresponding

authors: Zengqi Peng; Yanwei Zhang. Address: College of Food Science and

Technology, Nanjing Agricultural University, Nanjing 210095, China Telephone numbers: +86-25-8439-6558 E-mail addresses: [email protected]; [email protected]

Hypothesis: Eggplant is rich in polysaccharides. The mechanically homogenized eggplant flesh pulp (EFP) is expected to emulsify and stabilize o/w emulsions. The adsorption and network structure of the polysaccharides are hypothesized to contribute to the stability of emulsions. Experiments: Creaming index (CI) and droplet size distribution were observed to evaluate the stabilities of EFP emulsions at different EFP concentrations (0.50, 0.75, 1.00, 1.25 and 1.50% w/v). Optical and fluorescence microscopy, confocal laser scanning microscopy and cryogenic scanning electron microscopy were conducted to observe the emulsification properties of the EFP-stabilized emulsions. In addition, rheological measurements were performed to reveal the EFP emulsions’ rheological behaviours. Findings: The prepared oil-in-water emulsion emulsified by EFP remained stable at an EFP

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concentration of 1.50%. Rheological analysis illustrated that the emulsions had typical shear-thinning property and gel-like nature. The emulsification mechanisms were explained by the formation of an interfacial film adhered to the oil droplets and the coherent three-dimensional network formed by filament and sheet-like polysaccharide strands in the continuous phase. This finding may define a new kind of natural and dietary emulsifier for emulsion-based food, beverage, and pharmaceutical products. Key words: natural emulsifier; eggplant flesh pulp; microstructure; rheology

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1. Introduction Oil-in-water emulsions are an integral part of many commercial products used in food, personal care, cosmetic, detergent, and pharmaceutical industries [1]. In general, there are numerous kinds of synthetic and natural emulsifiers that can be utilized in the food industry. However, some commercial food-grade emulsifiers have been found to trigger an increased risk of some diseases. For instance, some commercial anionic and nonionic surfactants for food and biomedical applications were found to have chronic and sublethal toxicities [2]. Chassaing [3] reported that two commonly used emulsifiers in food products, carboxymethyl cellulose and polysorbate-80, can disturb the host microbiota relationship, resulting in a microbiota with enhanced mucolytic and pro-inflammatory activity, and ultimately induce inflammatory bowel disease, obesity/metabolic syndrome and promote robust colitis in mice. Moreover, mono- and diglycerides, the most widely used synthetic surfactants in food industry, can increase intestinal permeability, and thus cause allergic and autoimmune diseases [4]. So, there are increasing consumer demands for commercial food products, which are expected to be more natural and safer. Currently, there are several kinds of natural food-grade emulsifiers, including surface-active polysaccharides, proteins, phospholipids, and bio-surfactants [5]. Some proteins are surface active because they consist of strings of polar and non-polar amino acid units linked with polypeptide chains. Caseins and whey proteins are the most commonly used dairy protein emulsifiers in diverse food products due to their nutritional and functional properties [6, 7]. However, interesting research in emulsifiers is trending towards the partial or total replacement of animal-based proteins with natural plant sources, such as legume proteins [8], zein [9] and corn germ proteins [10], driven by pursuing food sustainability and security. Some natural phospholipids (usually called lecithin) and

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bio-surfactants (widely used saponin) are reported as promising emulsifiers in forming nano-scale emulsions due to their smaller molecules and excellent surface-active amphiphilic structure [11, 12]. Proteins, phospholipids and saponins are highly sensitive to the environmental stresses of pH, ionic strength, and/or temperature. However, polysaccharide-emulsified emulsions have been proved to be much less affected by changes in pH, ionic strength and temperature due to the primary stabilization caused by the steric repulsion [5]. Polysaccharides-based emulsifiers usually contain a mixture of non-polar and polar groups that adsorb to the oil droplet surfaces to form very thick interfacial films, meaning that steric repulsion alone is often sufficiently long-range to inhibit oil droplet aggregation. Gum arabic, the most widely used natural polysaccharide emulsifier [13], can be used to stabilize oil-in-water emulsions, particularly in flavoured beverages [14]. Many researchers have been working on the identification of new sources of polysaccharides that are suitable for use as emulsifiers, such as pectin extracted from beet and okra [15, 16], water-soluble yellow mustard mucilage [17], and corn fibre gum [18]. In addition, some researchers are focusing on environmentally-friendly approaches in the preparation of natural emulsifiers, such as the energy consumption, with or without chemical and/or enzymatic processing [19]. For instance, some starches become surface-active after being modified by chemical or enzymatic processing, but the resulting emulsifier would then not be considered natural, such as OSA-starch [20]. Additionally, the micro-fibrillated cellulose extracted from mangosteen rind [21] and cellulose nanocrystals prepared from defatted rice bran [22] can emulsify soybean oil oil-in-water (o/w) emulsions against coalescence. Further work is needed to develop natural emulsifiers, such as the identification of economical and sustainable sources, establishment of preparation approaches, and the characterization of

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emulsifier functionality. Moreover, the fate of emulsions within the human gastrointestinal tract has garnered vast interest [1]. To promote human health and wellness, it is very important to develop natural food-grade emulsifiers using dietary ingredients. Eggplant (Solanum melongena L.) is very popular vegetable worldwide and has an outstanding capability of absorbing large amounts of cooking fats and sauces in cuisine, which is attributed to the fibres in eggplant [23]. The American Diabetes Association recommends an eggplant-based diet as a choice for the management of type-2 diabetes because of the high-insoluble fibre and low soluble carbohydrate contents [24]. The eggplant fruit may exhibit excellent safety when used as a raw material to produce food emulsifiers, as all the components of eggplant fruit are edible, providing large amounts of dietary fibre for the human body. To the best of our knowledge, thus far, there has been no scientific report on the applications of eggplant and its derived materials in emulsion. The current study is aimed to exploit the feasibility of eggplant-based emulsifier and to investigate the effective emulsified constituents, microstructures and rheological properties of EFP emulsions. The research findings may provide an effective, safe, high-yield and environmentally friendly emulsifier for formulating emulsion-based food products entirely from natural ingredients.

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2. Materials and methods 2.1. Materials Purple eggplant fruits (Solanum melongena L.) with accordant maturity (height 20 cm and diameter 6 cm) and soybean oil were purchased from a local supermarket (Nanjing, China). Congo red (dye content, ≥ 35.0%), Nile red (for microscopy), Calcofluor white (for microbiology), Bromophenol blue (ACS reagent), isopropanol ( ≥ 99.0%) and Tween 80 (T80, Vetec™ reagent grade) were purchased from Sigma-Aldrich (Shanghai, China). Sudan IV (AR) was purchased from Aladdin (Shanghai, China). Sodium azide (NaN3, ≥99.5%) was purchased from Sigma Chemical Company (St Louis, MO, USA). Other common chemicals were analytical grade and purchased from Sinopharm Chemical Reagent Co, Ltd. (Shanghai, China). Distilled water was produced in the lab and used for the preparation of all solutions. 2.2. Chemical analysis Moisture, fat and ash contents were determined by using AOAC Official Methods [25]. Soluble protein content was determined by using a BCA Protein Assay Kit (A045-3, Jian Cheng, China). Soluble sugar content was determined by the anthrone-sulfuric acid method [26]. Cellulose, hemicellulose and lignin contents were determined according to the procedure described in Refs. [27-29]. Pectin content was determined following the method reported in Ref. [30]. All chemical analyses were measured in triplicate. Soluble sugar components were analysed using LC-MS equipped with an Agilent 1290 LC and a 6430 MS (Agilent Santa Clara, USA). Gradient chromatographic separation was performed on an Agilent BEH Amide column (1.7 µm, 2.1×150 mm). Mobile phase A was water (0.1% ammonium hydroxide), and mobile phase B was acetonitrile, and the elution gradient was performed with 80%

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to 5% B from 0 to 15 min; 5% B was maintained for 10 min; and re-equilibration with 80% B remained from 26 to 32 min. The flow rate was set to 0.5 mL/min with an injection volume of 5 µL and a column temperature of 40 °C. A Turbo Ion Spray source was operated in the positive electrospray ionization (ESI) mode using the following settings: ion spray voltage 4.5 KV, source gas 35 psi, drying gas temperature 350 °C and flow rate 9 L/min. The monosaccharide constituents of the polysaccharide were measured with an Agilent 1100 HPLC (Agilent Santa Clara, USA) equipped with a diode array detector (DAD) after hydrolysis and derivatization. Chromatographic separation was carried out on an Agilent Extend C18 column (5 µm, 4.6 × 250 mm), and samples were eluted with a mobile phase of 100 mM sodium phosphate buffer (PH 6.4) and acetonitrile (75:25). The flow rate was set to 1.0 mL/min with an injection volume of 20 µL and a column temperature of 30 °C. The detection wavelength was set to 250 nm. The monosaccharide constituents were identified by comparing the retention times with those of the reference substances. 2.3. Preparation of EFP Peeled eggplant flesh was cut into pieces with dimensions of 2×4×1 cm3. Eggplant flesh pieces together with distilled water (1:1, w/w) were blended in a Laboratory Blender (Waring, America) at a speed of 5000 rpm for 4 min to make EFP. 2.4. Preparation of o/w emulsions The EFP (4.0, 6.0, 8.0, 10.0 and 12.0 g) was diluted to 14.0 ml with distilled water and put them into 50-ml plastic centrifuge tubes. The samples were dispersed using an Ultra Turrax homogenizer (T25 digital model ULTRA-TURRAX, IKA, Germany) operating at 12,000 rpm for 1 min to obtain yellow-brown gel-like suspensions. Subsequently, 6.0 ml of soybean oil was added to the plastic

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centrifuge tubes and dispersed using an Ultra Turrax homogenizer at 12,000 rpm for 2 min in an ice-water bath to avoid overheating the samples. Prior to homogenization, NaN3 (0.05% w/v) was added to the emulsions as an antimicrobial agent. The EFP concentration in the emulsion was defined by the formula: EFP Concentration (%, w/v) = Dry Matter Content (%) × (EFP Weight×0.5/Total Volume)

(1)

where the dry matter content in peeled eggplant flesh was measured to be 4.99%. The emulsions achieved different EFP concentrations (0.50, 0.75, 1.00, 1.25 and 1.50% w/v) and were stored at ambient temperature for 24 h before analysis. In this study, the EFP was used directly after preparation in never-dried form, as an aqueous suspension. Emulsion emulsified with 0.50% T80 [31] was prepared according to the above method as a control. 2.5. Creaming index (CI) of the o/w emulsions The prepared emulsions were immediately transferred to 10 ml glass tubes. The sample glass tubes were kept at ambient temperature and the movement of the creaming boundaries was tracked for 7 days. During storage, most of the emulsions tended to separate into a strongly turbid or cream layer at the top and a serum layer at the bottom. The brown bottom phase was designated as serum and the top phase, which is a concentrated layer of droplets, was designated as emulsion cream. The total emulsion height (HT) and the serum layer height (HS) were measured. The extent of creaming was evaluated by creaming index (CI), defined by the formula: CI (%) = (HS / HT)×100%

(2)

2.6. Droplet size distribution The droplet size distribution of the emulsions in 1-day and 7-day storage were measured using a Mastersizer (Mastersizer 2000, Malvern Instruments Ltd., Malvern, England), where the stirring rate

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was 1200 rpm and shading degree was set to 10-15%. Regarding the optical properties of the samples, the refractive indices of oil and water were 1.33 and 1.46, respectively. The droplet sizes of the sample were evaluated through the surface-weighted mean droplet diameter D [3, 2]. 2.7. Optical and fluorescence microscopy The observations of EFP and emulsions were carried out as previously described, but with a slight modification [32]. The optical and fluorescence photographs of o/w emulsions were captured utilizing an Eclipse microscope equipped with a digital camera (Olympus BX51, Japan). Calcofluor white was used as a fluorescent dye for polysaccharide at the concentration of 0.1% w/v. Congo red, Bromophenol blue and Sudan IV (both 0.1% w/v in ethanol) were used as dyes for polysaccharide, protein and soybean oil, respectively. A drop of staining solution was added to a slide following the addition of a drop of emulsion, and the samples were incubated for 3 min before observation. The emulsions were prepared with double staining according to the procedure described by Zhang, et al [33]. 2.8. Confocal laser scanning microscopy (CLSM) The distribution of EFP in emulsion was observed by confocal laser scanning microscopy (Zeiss, LSM710, Gottingen, Germany). Nile red (0.1% w/v in isopropanol), Calcofluor white (0.1% w/v) and the prepared emulsions were mixed in 1:1:1 v/v, and a 20 μL mixture was placed on the microscope slide immediately. Following 1 min of incubation after coverslip addition, a confocal laser scanning micrograph was the taken using Zeiss, LSM710. Samples were excited with two laser beams at 514 nm (for Nile red) and 405 nm (for Calcofluor white). 2.9. Cryogenic-scanning electron microscopy (Cryo-SEM) The microstructure and network structure of the emulsion were visualized using cryo-scanning

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electron microscopy (SU8010, Hitachi, Japan). First, an emulsion sample was selected and placed in a slot on a stub and was rapidly frozen in liquid nitrogen at the temperature of -196ºC for 2 min. Second, these specimens were transferred to preparation chamber cold stage (PP3010T, Quorum Technologies, UK) and fractured. Finally, the samples were sublimated at -70ºC for 5 min under controlled vacuum conditions and sputtered with platinum using a current of 10 mA for 60 s. The samples were scanned by Cryo-SEM at the stage temperature of -140ºC and an accelerated voltage of 3 kV. 2.10. Rheological measurements The rheological behaviours of the prepared emulsions were performed on a rotational physical MCR301 rheometer (Anton Paar, Graz, Austria). All the rheological measurements were carried out at 25ºC on a 50-mm-diameter parallel plate with a gap fixed at a 0.5-mm gap. Shear viscosity was tested with shear rates ranging from 0.01 to 1000 s-1. Strain sweeps were conducted to ascertain the linear viscoelastic region (LVR) with the strain from 0.01 to 1000% at a fixed frequency of 1.0 Hz. After that, the dynamic frequency sweeps of the storage modulus (G') and loss modulus (G") were conducted by applying a constant strain of 0.1%, which was within the linear viscoelastic region, over frequencies ranging from 0.1 to 100.0 Hz. All rheological tests were performed in duplicate without applying high shear forces before the rheological measurements. 2.11. Statistical analysis All measurements were performed in triplicate for each sample with the exception of rheological measurements, which were performed in duplicate. The data were analysed using SAS v9.2 Windows program by an analysis of variance (one-way ANOVA) and Duncan’s multiple-range test. The results are expressed as the mean values ± standard deviations, and the differences were

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considered to be significant when p < 0.05.

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3. Results and discussion 3.1. Chemical compositions of EFP Table 1 Chemical compositions of peeled eggplant flesh Composition (% w/w, dry basis) Moisture a

95.01 ± 0.36

Fat

4.61 ± 0.76

Soluble protein

11.65 ± 0.21

Ash

6.09 ± 0.11

Cellulose

18.64 ± 1.95

Lignin

5.86 ± 1.02

Hemicellulose

9.29 ± 1.89

Pectin

5.16 ± 0.17

Soluble sugar

35.25±1.19

a%

w/w, wet basis. Results are expressed as means ± standard deviations (n=3).

As shown in Table 1, the chemical composition of EFP based on dry material was 33.79% total fibre (including 18.64% cellulose, 9.29% hemicelluloses and 5.16% pectin), 35.25% soluble sugar, 11.65% soluble protein, 5.86% lignin, 4.61% fat and 6.09% ash, which are comparable to the contents in eggplant, 38.96% dietary fibre, 45.8% sugars and 12.78% protein, reported by Gürbüz, et. al [34]. 3.2. Overall appearance of EFP emulsified o/w emulsions The photographs of the emulsions during storage at ambient temperature are shown in Fig. 1a. After 1 day of storage, obvious creaming of the freshly prepared emulsions could be observed, and a sharp boundary between the top cream layer and the bottom serum layer appeared when the concentrations of EFP were less than 1.25%. The serum was initially cloudy due to the presence of a small residual amount of suspended individual or aggregated oil droplets [21] and became clear after 7 days of storage, which may be caused by gravity-induced upward movement of the residual oil droplets. The freshly prepared emulsions were flowable, but this fluidity was lost after storage for 7 days, as demonstrated by turning the test tubes upside-down. This loss in fluidity was caused by the increased 12

viscosity, and the droplets were trapped within the network with no mobilization [35]. Thus, the droplet-droplet coalescence, gravity-induced creaming sedimentation and macroscopic phase separation were retarded or stopped during long- term storage.

Fig. 1. (a) Photographs of o/w emulsions emulsified by EFP stored for 1 day and 7 days. From left to right, the concentrations of EFP are 0.50, 0.75, 1.00, 1.25 and 1.50%, respectively. (b) Creaming index during storage at room temperature for 7 days. Results are expressed as means ± standard deviations (n=3).

The creaming index of the emulsions was decreased by increasing the EFP concentration (Fig. 1b). After storage for 7 days, the CI values of emulsions emulsified by 0.50%, 0.75%, 1.00%, 1.25% and 1.50% EFP reached their plateaus of 51.7%, 32.3%, 24.0%, 10.7% and 0%, respectively. The emulsion creamed easily when the EFP concentration was below 1.25%, while it no longer creamed again throughout the entire storage period when the concentration was increased to 1.50%. According to Stoke's law and gravity effect, the creaming stability of emulsion can be improved by reducing the droplet size, increasing the viscosity of the continuous phase, or minimizing density differences between the droplets and the continuous phase [1]. The viscosity will be strengthened when the EFP concentration increases. In addition, the adsorption of EFP components on the droplet surface increases the effective density of oil droplets, thus minimizing the density difference between the droplets and the continuous phase. Both factors may contribute to improving the stability against

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creaming and flocculation during storage [32]. 3.3. Droplet sizes of the o/w emulsions

Fig. 2. Droplet size distributions of emulsions emulsified by EFP at concentrations of (a) 0.50%, (b) 0.75%, (c) 1.00%, (d) 1.25% and (e) 1.50% w/v. [The insets represent typical optical micrographs for 1 day and 7 days, the scale bar is 50μm.]. (f) Average droplet size (D [3, 2]) after storage for 1 day and 7 days. Different capital letters (A-E) and lowercase letters (a-b) show significantly different (p < 0.05) droplet sizes with EFP concentrations and storage times, respectively. Results are expressed as means ± standard deviations (n=3).

Pronounced trimodal distributions that are characterized by three peaks (Fig. 2) were encountered in 14

the prepared EFP emulsions, especially when the EFP concentration was above 0.75% (Figs. 2b-e). The lower peak representing droplet size remained stable with the EFP concentration variation and storage time, while the medium peak representing primary droplet size distribution moved towards lower values with the increase of EFP concentration, indicating that the average droplet size in emulsions decreased significantly (p < 0.05) (Fig. 2f). Larger droplet size and fluctuation contributed to the upper peak in the emulsion at the 0.50% EFP concentration (Fig. 2a). This is likely to be attributed to the insufficient amount of EFP needed to provide complete coverage to the oil droplets during homogenization. Such phenomena can also be observed from the typical optical micrographs seen as the insets in Figs. 2a-e. The average droplet size at 7 days of storage was 15 μm larger than that at 1 day of storage at the concentration of 0.50% (p < 0.05), whereas differences in the average droplet sizes between 7 days and 1 day of storage were not significant for the emulsions at concentrations ranging from 0.75 to 1.50%. Thus, excellent stabilization can be achieved in long term storage with EFP concentrations above 0.75%. Additionally, the distance between the peaks decreased with the increase of EFP concentration, indicating that a more homogeneous system was produced. Such results coincide with the results reported in Ref. [36] and can be interpreted by Stock’s law. Assuming that the emulsion has a volume of V and an internal phase volume fraction of F, the emulsion interface S can be calculated by: S  6 V  F d

(3)

where d is the average diameter of the emulsion droplets. By increasing the EFP concentration, a larger emulsion interface S can be obtained in principle [37, 38]. Thus, a smaller average droplet size appeared in the emulsion with a higher EFP concentration. Moreover, a higher packing density can

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be achieved at the EFP concentration of 1.50%, which effectively prevents the droplets from flocculation and coalescence and results in long-term stabilization of the dispersed droplets. T80 emulsion had a smaller average droplet size with the maximum usage of 0.50% in food [31] compared with those of EFP emulsions at both 1 day and 7 days of storage (p < 0.05). Such a difference in the droplet size is likely caused by the different molecules and structures of the two types of emulsifiers, as well as the rate at which the emulsifier attaches to oil droplet surfaces [1, 39]. 3.4. Microstructures of the EFP and o/w emulsions To observe the microstructures of EFP emulsions using optical and fluorescence microscopy, polysaccharide and protein staining protocols were implemented. In the microscope field, red spherical films were observed when the emulsion sample was stained with Congo red (Fig. 3a), and the spherical films showed bright fluorescent signal when stained with Calcofluor white (Fig. 3b). In addition, the continuous phase around the oil-droplet was lightly stained in red (Fig. 3a). However, there was no visible blue observed in the field with Bromophenol blue staining (Fig. 3c). In addition, no obvious blue spherical films were observed surrounding the red-stained oil droplets when counterstained with Bromophenol blue + Sudan IV (Fig. 3f). Thus it can be inferred that the protein is likely covered by polysaccharides on the oil surface, which is similar to the emulsification of pectin together with protein reported by Leroux [40]. After drying the emulsions stained with Congo red and Calcofluor white (Figs. 3d and e), the oil droplets deformed from a spherical shape to a polyhedral one as the water evaporated, and the polysaccharides in continuous phase were trapped on the polyhedral droplet surface. Based on Fig. 3, it was likely that the surface films were attributed to the adsorption of massive polysaccharides associated with small amount of protein, thus leading to a steric effect to emulsify the o/w emulsions. The formed surface films on the oil droplets remained

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very stable when squeezed, indicating that the films were both physically and chemically robust.

Fig. 3. Optical and fluorescence micrographs (200×) of 1.00% EFP emulsified emulsions stained with (a) Congo red, (b) Calcofluor white and (c) Bromophenol blue. Optical and fluorescence micrographs (200×) of 1.00% EFP emulsified emulsions dried in air for 4 h before being stained with (d) Congo red and (e) Calcofluor white. (f) Optical micrographs (200×) of 1.00% EFP emulsified emulsions counterstained with Bromophenol blue + Sudan IV. The scale bars are 50 μm.

To further investigate the effective emulsification components of the EFP emulsions, confocal laser scanning micrographs (CLSMs) were taken, which showed that there was a blue casing around the red droplet (Fig. 4). Adsorption of the EFP polysaccharides formed a round-shape bright fluorescence on droplet surfaces. This round-shaped bright fluorescence formed thin films to prevent droplet coalescence. The CLSMs also indicate that the average droplet size of the emulsion decreases with the increase of EFP concentration. The emulsion with an EFP concentration of 0.50% had the largest average droplet size (Fig. 4a), while that with a 1.50% EFP concentration had the smallest average droplet size (Fig. 4c).

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Fig. 4. Confocal laser scanning micrographs of EFP emulsified emulsions with EFP concentrations of (a) 0.50%, (b) 1.00% and (c) 1.50% w/v. The scale bars are 20 μm.

Fig. 5. Cryogenic-scanning electron micrographs of EFP with scale bars of (a) 50 μm and (b) 5 μm; 1.00% EFP emulsified emulsions with scale bars of (c) 100 μm and (d) 20 μm.

To study the microstructure-function relationship, Cryo-SEM was employed to acquire the distribution characteristics of EFP polysaccharides on the droplet surface and in the continuous phase. The EFP microstructure was first observed by Cryo-SEM, which showed a coherent “three-dimensional network” in very large scale (Fig. 5a). Upon increasing the magnification, the

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local structural network of the polysaccharides presented in filament and sheet-like morphology (Fig. 5b). The spatial distribution of oil droplets and the microstructure of droplets surface associated with insoluble polysaccharides were seen in Fig. 5c. The droplets were randomly trapped within the network. The local adsorption of the dense network could be clearly observed in a higher power field (Fig. 5d). The filament and sheet-like EFP polysaccharides adsorbed tightly on the surface film, and simultaneously entangled with those adsorbed on the surface of adjacent droplets, forming a strong entanglement and junction zone for the emulsified droplets. The adsorption can form a stable surface film to coat the oil droplets, functioning as a steric barrier to protect emulsion droplets against flocculation and coalescence, while the network can increase the viscosity of the continuous phase and prevent the flow of oil droplets. Thus, the emulsification mechanism can likely be interpreted by the combination effects of the steric barrier caused by surface films and droplet restraint by the three-dimensional network. The surface films are likely caused by adsorption of soluble sugars and associated proteins, and insoluble polysaccharides are connected with the surface films to form the three-dimensional network. The soluble sugars were determined to be monosaccharides and disaccharides by LC-MS, while the polysaccharides with a viscosity-average molecular weight (MV) of 19,897 were found to consist of galacturonic acid (33.3%), galactose (25.6%), glucose (11.9%), xylose (9.9%), rhamnose (3.3%) and mannose (2.7%) through HPLC. 3.5. Rheological properties of emulsions The apparent viscosity of the emulsion increased with the increase of EFP concentration during storage (Fig. 6). For 1- and 7-day storage, the apparent viscosity of all emulsions decreased by increasing the shear rate, indicating that the emulsions had shear thinning properties regardless of the emulsifier concentration [41]. The zero-shear viscosity increased rapidly with the increase of EFP

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concentration due to its enhanced thickening ability. Therefore, the test tubes could be turned upside-down after 7 days of storage (Fig. 1a), and the unstable liquid-like emulsions were transformed into stable gel-like ones. When comparing the apparent viscosity values of 7-day storage (Fig. 6b) with those of freshly prepared emulsions (Fig. 6a), a significant increase of these values was observed, in agreement with that reported in Ref. [32]. It should be pointed that the flocculating of droplets and rearrangement of EFP occurred in the continuous phase during storage, leading to increased apparent viscosity.

Fig. 6. The apparent viscosity of EFP emulsified emulsions at different concentrations for (a) 1 day and (b) 7 days of storage.

Rheology is broadly applied in the assessment of emulsion flocculation and coalescence [41], and the viscoelastic properties were measured to relate the LVR to the strain amplitude (Fig. 7). The G' and G" gradually increased by increasing the EFP concentration, and remained virtually constant up to a relative strain of 1% at all concentrations. All the samples at a strain of 0.1% were kept constant to conduct the frequency sweep within the LVR (Fig. 8). All emulsions displayed higher G' values than G". Both G' and G" were less dependent on the frequency with the increased concentration of EFP, presenting a typical gel-like behaviour [41]. By increasing the concentrations of EFP, both G' and G" values become larger, indicating a strong network [21]. Moreover, both G' 20

and G" become larger after 7 days of storage (Fig. 8b), since the EFP underwent entanglement or cross-linking at the droplet interface and surface film with a high interfacial viscosity and/or elastic modulus may be formed [1, 42]. Furthermore, when the EFP is insufficient to cover the droplet surface at a 0.5% EFP concentration, coalescence will occur following the flocculation, which make the average droplet size become larger (Fig. 2f) (p < 0.05). Based on these rheological measurements and microscopic observations, it was concluded that the polysaccharide components in EFP exhibit their functionalities by adsorbing on the oil-water surface and developing a quasi-gel microstructure composed of flocculated oil droplets and a viscoelastic network in the aqueous phase.

Fig. 7. Strain sweep test of o/w emulsions emulsified with EFP at different concentrations for 1 day of storage.

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Fig. 8. Storage modulus (filled symbols) and loss modulus (empty symbols) as a function of frequency for o/w emulsions emulsified with EFP at different concentrations for (a) 1 day and (b) 7 days of storage.

4. Conclusions In this paper, a new kind of natural emulsifier, EFP, was developed to satisfy the demands from consumers for “all-natural” foods and replacements for animal-based ingredients with more label-friendly plant-based ones. The EFP was prepared with mature purple eggplant fruits (Solanum melongena L.), a sustainable natural resource, using homogenization without any chemical or enzymatic process. Such a physically prepared emulsifier with no additional processing and solvents added to separate and purify the desired fractions falls into green manufacture and is very environmentally friendly. Inspired by its outstanding oil-holding capability, it was supposed that the EFP could work as a good natural emulsifier. It was then demonstrated that the prepared EFP could effectively emulsify o/w emulsions at concentrations of 1.50%, a relatively low level compared with that of other polysaccharide emulsified emulsions [13-16]. The emulsification principle can be jointly interpreted by the combination of the barrier effect of the surface film adhered to the oil droplets and the coherent three-dimensional network formed by filament and sheet-like polysaccharide strands in the continuous phase. Compared with other amphiphilic polymers or synthetic surfactants [2-3, 11-12], EFP has the advantages of being a natural source that can be 22

economically acquired, eligibility for green manufacture using physical preparation, and possessing health benefits in food application. In a case study of meatball, it tasted better, and the colour was not affected when the eggplant powder was less than 4.5% (see the supplementary file). Therefore, EFP may function as a natural food-grade emulsifier in food, cosmetic and biomedical areas. Further studies are important to investigate the stability to environmental stresses, such as pH, ionic strength, and temperature changes, and the gastrointestinal fate of EFP emulsions in food applications. Acknowledgements This work was supported by Herbivore Livestock fattening and Technique of High Quality Meat Produce Research of South China (201303144). Declarations of interest: none References [1] D.J. McClements, Food Emulsions: Principles, Practice and Techniques, CRC Press, Boca Raton, 2016. [2] M.A. Lewis, Chronic and sublethal toxicities of surfactants to aquatic animals: a review and risk assessment, Water Res. 25 (1991) 101-113. [3] B. Chassaing, O. Koren, J.K. Goodrich, A.C. Poole, S. Srinivasan, R.E. Ley, A.T. Gewirtz, Dietary emulsifiers impact the mouse gut microbiota promoting colitis and metabolic syndrome, Nature. 519 (2015) 92-96. [4] K.F. Csaki, Synthetic surfactant food additives can cause intestinal barrier dysfunction, Med Hypotheses. 76 (2011) 676-681. [5] D.J. McClements, C.E. Gumus, Natural emulsifiers-Biosurfactants, phospholipids, biopolymers, and colloidal particles: Molecular and physicochemical basis of functional performance, Adv Colloid Interface Sci. 234 (2016) 3-26. [6] M. Li, M.A.E. Auty, J.A. O’ Mahony, A.L. Kelly, A. Brodkorb, Covalent labelling of β-casein and its effect on the microstructure and physico-chemical properties of emulsions stabilized by β-casein and whey protein, Food Hydrocolloids. 61 (2016) 504-513. [7] R.S.H. Lam, M.T. Nickerson, The effect of pH and temperature pre-treatments on the physicochemical and emulsifying properties of whey protein isolate, Lwt-Food Sci Technol. 60 (2015) 427-434. [8] O. Benjamin, P. Silcock, J. Beauchamp, A. Buettner, D.W. Everett, Emulsifying Properties of Legume Proteins Compared to beta-Lactoglobulin and Tween 20 and the Volatile Release from Oil-in-Water Emulsions, J Food Sci. 79 (2014) 2014-2022. [9] M. Rutkevičius, S. Allred, O.D. Velev, K.P. Velikov, Stabilization of oil continuous emulsions with colloidal particles from water-insoluble plant proteins, Food Hydrocolloids. 82 (2018) 89-95. [10] M.P. Hojilla-Evangelista, Improved Solubility and Emulsification of Wet-Milled Corn Germ Protein Recovered by Ultrafiltration-Diafiltration, J Am Oil Chem Soc. 91 (2014) 1623-1631. [11] J. Li, J.N. Pedersen, S. Anankanbil, Z. Guo, Enhanced fish oil-in-water emulsions enabled by rapeseed lecithins 23

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Competing Interests: The authors declare no competing interests.

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Author Contributions: Yu-Xia Zhu designed the work and prepared the manuscript; Xiao-Pu Ren, Ying-Jie Bao and Shun Li carried out the experiments and data processing; Dr. Ya-Wei Zhang provided some data analysis in this work; Profs. Zeng-Qi Peng and Guang-Hong Zhou provided some helpful discussions and revised the manuscript.

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