Influences of Ulva fasciata polysaccharide on the rheology and stabilization of cinnamaldehyde emulsions

Carbohydrate Polymers 135 (2016) 27–34

Contents lists available at ScienceDirect

Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Influences of Ulva fasciata polysaccharide on the rheology and stabilization of cinnamaldehyde emulsions Ping Shao a , Jiamei Shao a , Yike Jiang b , Peilong Sun a,∗ a b

Department of Food Science and Technology, Zhejiang University of Technology, Zhejiang, Hangzhou 310014, PR China Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901, USA

a r t i c l e

i n f o

Article history: Received 19 May 2015 Received in revised form 27 June 2015 Accepted 21 August 2015 Available online 28 August 2015 Keywords: Ulva fasciata polysaccharide Cinnamaldehyde Emulsification property Rheology

a b s t r a c t Emulsifying properties of water soluble polysaccharides from Ulva fasciata (UFP) were evaluated in cinnamaldehyde/water emulsions in terms of droplet size distribution, rheological properties, visual phase separation, and zeta-potential. The cinnamaldehyde/water (10%, wt/wt) emulsions were formulated and stabilized by different concentrations of UFP (0.1–4%, wt/wt). The obtained emulsions showed monomodal droplet size distributions with average droplet size (D[3,2]) below 1.0 ␮m, when 3% (wt/wt) UFP was added as the emulsifying agent under a homogenization pressure of 75 MPa. The rheological properties and zeta-potential of the emulsions appeared to be dependent on the UFP concentration. Furthermore, the UFP exhibited better emulsifying and stabilizing properties in the investigated system when compared to other commercial polysaccharides of gum Arabic and gum Ghatti. The results also suggested that the emulsifying and stabilizing mechanism of the UFP may not only be ascribed to its surface-active protein moiety, but also to the hydrophobicity of the polysaccharide itself. These findings provided a theoretical basis for potential utilization of UFP as a novel hydrocolloid emulsifying agent. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Food hydrocolloids are high-molecular-weight hydrophilic biopolymers and are widely used as functional ingredients in the food industry for the control of microstructure, texture, flavor and shelf-life (Dickinson, 2003; Funami, 2011). Food hydrocolloids include a large group of polysaccharides such as extractions from plants, seaweeds and microorganisms, biopolymers from chemically or enzymatically modified starches or cellulose, as well as gums derived from plant exudates (Chivero, Gohtani, Yoshii, & Nakamura, 2015; Choy, May, & Small, 2012). Most hydrocolloids can be used as stabilizers (stabilizing agents) in oil-in-water emulsions, but only a few can act as emulsifiers (emulsifying agents) due to their strong hydrophilic character. The latter functionality requires substantial surface activity at the oil/water interface, and hence the ability to facilitate the formation and stabilization of fine droplets during and after the emulsification process (Dickinson, 2003, 2009). The emulsifying capacity of the widely used polysaccharide emulsifier of gum Arabic is attributed to its molecular origin in the presence of a small amount of protein, which

∗ Corresponding author. E-mail address: [email protected] (P. Sun). http://dx.doi.org/10.1016/j.carbpol.2015.08.075 0144-8617/© 2015 Elsevier Ltd. All rights reserved.

is covalently linked with highly branched polysaccharide structures (Jafari, Beheshti, & Assadpoor, 2012). Polysaccharides extracted from Ulva fasciata (UFP) are anionically charged polysaccharides consisting of uronic acids, sulfate groups, and rare sugars, such as iduronic acid, which mostly found in mammalian glycosaminoglycans and required in the synthesis of heparin analogs with antithrombotic activities (Kovensky et al., 1999; Lahaye & Robic, 2007). On the basis of the peculiar chemical composition of UFP, the biomass can be a source of rare sugar precursors for the synthesis of fine chemicals. Besides monomers, some oligomers and polymers attract particular interest because of their effective bioactivity. Previous researches have reported that polysaccharides isolated from U. fasciata possess important pharmacological activities such as antioxidant activities (Chakraborty & Paulraj, 2010; Qi et al., 2005), anticoagulant activities (Sathivel, Raqhavendran, Srinivasan, & Devaki, 2008), and antitumor and immune modulation activities (Kaeffer, Benard, Lahaye, Blottiere, & Cherbut, 1999). One particular interesting feature of UFP is its ability to form gels in presence of borate and calcium ions, which shows the potential to be applied as food hydrocolloids (Robic, Gaillard, Sassi, Lerat, & Lahaye, 2009). UFP has amphiphilic properties due to its both protein moiety and carbohydrate fraction. The carbohydrate fraction imparts hydrophilic properties and contributes to emulsion stabilization by increasing viscosity and steric effects (Funami et al., 2011). The protein moiety bound to the carbohydrate

28

P. Shao et al. / Carbohydrate Polymers 135 (2016) 27–34

plays a key role in the emulsifying properties because it activates the oil/water interface by adsorbing favorably onto the surface of oil droplets (Akhtar, Dickinson, Mazoyer, & Lanendorff, 2002). Since U. fasciata is an abundant resource in the ocean, there is a great potential to utilize its polysaccharides as a thickening, stabilizing and emulsifying ingredient in food industry. Based on our previous work, the optimum UFP extraction conditions (Shao, Chen, Pei, & Sun, 2013), the rheological properties, moisture-preserving activity and structure characteristics (Shao, Qin, Han, & Sun, 2014; Shao, Shao, Han, Lv, & Sun, 2015) have been reported. Cinnamaldehyde is an aromatic ␣,␤-unsaturated aldehyde, and is the principal component of essential oil from some cinnamon species (Balaguer, Lopex-Carballo, Catala, Gavara, & HernandezMunoz, 2013; Wang, Wang, & Yang, 2009). Cinnamaldehyde has been shown to exert broad spectrum of antimicrobial activity including bacteria, yeasts, and molds (Otoni et al., 2014; Shen et al., 2015). Nevertheless, the direct application of essential oils in foods has limitations due to their strong flavor, high volatility, poor solubility in water, as well as toxicological and economic considerations when high concentration of essential oils are applied. In practice, essential oils are encapsulated in the microcapsules to improve their stability and bioactivity, where oil in water (O/W) emulsionbased system is an excellent candidate for the protection of the active ingredients and to mask the unfavorable odors and bitter tastes. The aim of the present work was to investigate the emulsifying properties of UFP in the cinnamaldehyde/water system and the factors that may influence its stability. Its emulsifying properties were also compared with some commercial gums such as gum Arabic and gum Ghatti, to evaluate the potential practical application in food industry. 2. Materials and methods 2.1. Materials and chemicals The U. fasciata was collected on the coast of Nanji Archipelago (Zhejiang province, China). The collected sample was washed with seawater to remove extraneous matters such as epiphytes and contaminations from other algae. Then the sample was again washed with deionized water and air dried. Finally, the sample was sealed in plastic bags and stored at room temperature. Gum Arabic (GA) and Gum Ghatti (GG) were both purchased from Sigma Chemicals Company (Tehran, Iran). Medium chain triglyceride (MCT) was obtained from KLK Oleo, Ltd. (Malaysia), which contains 58 wt.% C8 fatty acid and 42 wt.% C10 fatty acid, and has a relative density of 0.95 g/mL. Cinnamaldehyde (98%) was purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). The other chemicals used in the study were from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and were of analytical grade.

kept overnight at room temperature. The precipitate was separated, washed twice with absolute ethanol, and finally lyophilized to obtain the crude polysaccharide. 2.2.2. Radial flow chromatography According to Shao et al. (2014), the crude polysaccharide was further purified by deproteinization using radial flow chromatography method. Briefly, the radial flow column (RFC) was prepared by packing with ion-exchange resin (A103S, Purolite International Ltd., USA). The saturated solution of the crude polysaccharide was loaded onto the RFC and treated as follows: sample volume of 100 mL, sample concentration of 10 mg/mL, sample flow rate of 2 mL/min, and elution flow rate of 40 mL/min. The eluent was concentrated by rotary evaporation under vacuum and lyophilized to obtain the deproteinized polysaccharide which was defined as UFP in this work. 2.3. Chemical composition analysis The protein content of the samples was determined from the nitrogen content, using the Kjeldhal method (AOAC 968.06). Additionally, total carbohydrate content of the samples was estimated using the phenol-sulphuric acid method (Dubois, Gilles, Hamilton, Rebers, & Smith, 1956). 2.4. Emulsion preparation The aqueous phase of UFP were prepared by dissolving known amounts of UFP in Milli-Q water using an overhead mixer (IKA Labortechnik, IKA works, Malaysia) for 2 min. The solutions were left overnight on a roller mixer, under continuous stirring at room temperature for complete hydration of the polysaccharide. An oil-in-water emulsion was prepared by mixing appropriate quantities of above UFP stock dispersion, cinnamaldehyde, MCT in an aqueous solution and adjusted to pH 5.0, followed by prehomogenization at 26,000 rpm for 3 min. The pre-emulsions were then passed through a high pressure homogenizer for one time at 75 MPa (Microfluidics M-110L, USA). Sodium azide solution (0.02%, v/v, 5 M) was added to the freshly homogenized emulsions as an antimicrobial agent. The final formulation of the emulsions was as follows: the cinnamaldehyde concentration and MCT concentration were kept at 10% and 4% (w/w), respectively, with various concentrations of UFP (0.1–4%, w/w). The pH of the final emulsions was checked again and fine-tuned to pH 5.0 if necessary. The emulsions stabilized by GA and GG were also prepared using the same method above. During the whole process the solutions were kept in an ice bath to prevent the potential high temperature oxidative degradation of cinnamaldehyde. 2.5. Evaluation of emulsion stability

2.2. Preparation of UFP 2.2.1. Extraction of polysaccharides The sample was ground into powders to pass through 80-mesh sieve. The powder was soaked with 95% ethanol (v/v) for 12 h and changed the solvent for three times to remove most lipids and pigments (Shao et al., 2014). The residue was dried in the air and extracted in distilled water in a ratio of 1:20 (w/v) at 100 ◦ C for 3 h. After extraction, the mixture was combined and centrifuged at 5000 rpm for 10 min at 4 ◦ C. The supernatant was collected and concentrated to 500 mL at 45 ◦ C by using a vacuum rotary evaporator (RE-2000, Yarong Biochemistry Instruments Ltd., China). Aqueous ethanol (95%, v/v) was added slowly to adjust the final ethanol concentration to 80%. The mixture was stirred vigorously and then

There are several methods to evaluate the long term stability of emulsions such as heating at elevated temperatures, centrifugation, shaking and stirring. In the present study, acceleration test at 60 ◦ C was adopted, as it could provide the most accurate prediction of emulsion stability (Al-Assaf, Phillips, Aoki, & Sasaki, 2007; Al-Assaf, Phillips, Williams, & du Plessis, 2007). Briefly, the prepared emulsions were sealed into 20 mL glass bottles with a small headspace to minimize evaporation and were left in an oven preset at 60 ± 0.1 ◦ C. Samples after storage for 0 day (freshly prepared emulsions), 3 days and 7 days, were taken for laser diffraction measurement (Malvern Instruments Ltd., Worcestershire, UK). Moreover, the droplet size of all samples was measured to make the experiment results more convincing.

P. Shao et al. / Carbohydrate Polymers 135 (2016) 27–34

2.6. Droplet size measurement

29

Doppler velocimetry (LVD) at 17◦ . The measurements of ␨-potential and conductivity were carried out immediately after emulsion preparation using a maintenance-free capillary cell and deionized water for diluting the sample. In order to avoid the presence of air bubbles, the filling was done using a 1 mL syringe. The capillary cell was filled with diluted emulsion (1:50) between 7 and 10 mm; then fully inserted into the module. The measurement of ␨-potential and conductivity was expressed in the units of mV and mS/cm, respectively. For data analysis, the measurements were reported as the average of three separate injections, with four readings made per injection.

The freshly prepared and stored emulsions were analyzed for particle size and size distribution based on laser light scattering technique, using a Malvern Mastersizer MS 2000 (Malvern Instruments Ltd., Worcestershire, UK). A few drops of emulsion were added to a sample dispersion unit stirring at 2000 rpm until laser obscuration level of 10–20% was reached. The emulsion droplet size distribution was measured by fitting diffraction data to a general purpose model. Values of 1.45 and 0.001 were used as the refractive index and absorption index of oil phase, 1.33 and 0 as the dispersant deionized water, respectively. The pH of the dispersant water was adjusted to match the pH of the samples. The average size of emulsion droplets was characterized by surface weighted mean diameter D[3,2] and volume weighted mean diameter D[4,3], respectively. The D[3,2] was applied to freshly prepared emulsions, as it can best reflect emulsifying activities that are related to the amount of interfaces generated, whereas the D[4,3] was particularly used to monitor the stability of the emulsions during storage which is more sensitive to the development of large droplets. They were defined by the following equations. D[3, 2] =

 3 nd  i i2

All experiments were performed at least in duplicate, and analysis of all samples were run in triplicate and averaged. Statistical analysis was performed using the Statistical Analysis Systems (Origin Pro 8.5) software package. The results obtained were analyzed using one-way analysis of variance (ANOVA) for mean differences among the samples. P-values of <0.05 were considered to be statistically significant.

(1)

3. Results and discussions

D[4, 3] =

 4 nd  i i3

(2)

ni di

ni di

2.10. Statistical analysis

3.1. Particle size and size distribution of the fresh emulsions

where ni is the number of particles with diameter di . The droplet size population of the 10% O/W emulsions stabilized by UFP at different concentrations (i.e., 0.1%, 0.5%, 1%, 2%, 3%, 4%, wt/wt) was measured immediately after preparation and on subsequent days (3rd and 7th) under storage at 25 ◦ C and 60 ◦ C, respectively. The O/W emulsions stabilized with 3% of UFP, GA and GG were evaluated to compare the emulsifying properties. The size of the emulsion droplets was analyzed immediately after preparation and subsequent days (3rd and 7th) under storage at 60 ◦ C. 2.7. Rheological measurement Rheological properties of the fresh polysaccharide-stabilized emulsions were measured within 24 h after preparation at 25 ◦ C using a Haake RheoStress 6000 Rheometer (USA, Thermo Fisher Scientific) equipped with a cone and a parallel plate (P60 TiL, 60 mm in diameter and gap size of 1 mm). All samples were allowed to equilibrate at the measuring temperature for 10 min before the start of the test. Steady shear viscosity of various concentrations was determined at 25 ◦ C and measured at shear rate from 0.01 to 500 s−1 . 2.8. Visual phase separation Immediately after preparation, 15 mL of the emulsions was transferred into a transparent glass test tube (20 mm diameter and 70 mm height) and sealed with a plastic cap. The sample tubes were kept at 60 ◦ C and the movement of any creaming boundary was tracked with time for 7 days. Physical phase separation was monitored during this period.

The initial (day 0) particle size distribution and surface weighted mean diameter D[3,2] of the 10% O/W emulsions stabilized by UFP at different concentrations are shown in Fig. 1. The results indicated that UFP could form emulsions with monomodal droplet distributions and average droplet size (D[3,2]) less than 1.0 ␮m, using only 3% UFP concentration (Fig. 1A). There was a significant decrease in D[3,2] when the UFP concentration in the continuous aqueous phase increased from 0.1% to 3% (w/w) (Fig. 1B). At very low UFP concentration (≤0.5%), large oil droplets were formed as manifested by a broad droplet size distribution, which could be the result of droplet flocculation by macromolecular bridging. This is likely to be attributed to the insufficient amount of UFP needed to provide complete coverage to the initially formed droplets during homogenization (Robins, 2000). Consequently, the partially covered droplets coalesce with others until their surfaces become protected by a dense layer of molecules (Osano, Hosseini-Parvar, Matia-Merino, & Golding, 2014). Additionally, bridging flocculation may occur when a single biopolymer molecule adsorbs at the surface of more than one emulsion droplet. It thus acts as a polymeric link and promotes bridging flocculation (Bouyer, Mekhloufi, Rosilio, Grossiord, & Aqnely, 2012). At 1% concentration, UFP provided sufficient coverage of the droplets and resulted in the formation of small droplets with uniform size. When the UFP concentration was above 1%, the droplet size distribution had a significant decrease until the polysaccharide concentration reached 3%. However, further increase of UFP concentration (4%) resulted in a slight increase of the average particle size, which could probably due to the excess of non-adsorbed polysaccharide has increased the viscosity of the aqueous phase, and therefore reduced the efficiency of droplet disruption during homogenization (Huang, Kakuda, & Cui, 2001). Furthermore, bimodal distribution with two size ranges was not observed for the high concentration (>1%) of UFP-stabilized emulsion in this work.

2.9. Zeta potential measurements 3.2. Rheological characterization Zeta potential (␨) measurements of the emulsions were conducted on a Zetasizer Nano-ZS apparatus (Malvern Instruments, UK) equipped with an MPT-2 pH autotitrator. The apparatus has a 4 mW He/Ne laser emitting at 633 nm. Electrophoretic mobility of charged particles was measured by means of laser

Rheological properties of the cinnamaldehyde/water emulsions prepared by addition of 0.1%, 0.5%, 1%, 2%, 3% and 4% (w/w) UFP as emulsifying agent were investigated (Fig. 2). An increase in UFP content resulted in a systematic increase of apparent viscosity in

30

P. Shao et al. / Carbohydrate Polymers 135 (2016) 27–34

6 0.1% 0.5% 1% 2% 3% 4%

A

Volume (%)

12 10 8 6 4

B

5

D[3,2] (μ μm)

14

4 3 2

2 1 0 0.01

0.1

1

10

100

1000

10000

0

1

2

3

4

5

Concentration (%)

Partical Size (μ μm)

Fig. 1. Particle size distributions of 10% cinnamaldehyde O/W emulsions stabilized by UFP at different concentrations measured after prepared at room temperature. (A) Effect of UFP concentration (wt/wt) on the particle size distribution of the fresh emulsions. (B) Effect of UFP concentration (wt/wt) on the average particle size D[3,2] of the fresh emulsions.

0.1% 0.5% 1% 2% 3% 4%

0.10

Viscosity (Pas)

0.08 0.06 0.04 0.02 0.00

1

10 Shear Rate (s-1)

100

Fig. 2. Effect of UFP concentration (wt/wt) on the viscosity of 10% (wt/wt) cinnamaldehyde/water emulsions at 25 ◦ C.

-26

Zeta Potential (mv)

-28 -30 -32 -34 -36 -38 -40

0.10% 0.50%

1% 2% Concentration

3%

4%

Fig. 3. Effect of UFP concentration (wt/wt) on the ␰-potential of the emulsions.

the tested shear rate range (0.01–500 s−1 ). The viscosity property of UFP appears to have stabilized the emulsions, as confirmed by the absence of creaming in emulsions above 1% UFP concentration (Fig. 4). It is well known that most non-adsorbed polysaccharides provide increasing viscosity of the aqueous phase with increasing concentrations (Dickinson, 2009). Under these conditions, emulsions may become stable where the thickness of the continuous phase around the droplets restricted the movement of droplets in the system, consequently avoiding contact between the droplets and thereby preventing flocculation, coalescence and creaming.

Fig. 4. Phase separation of UFP-stabilized 10% (wt/wt) cinnamaldehyde/water emulsions after 30 days storage at 25 ◦ C.

Researchers also found that at higher hydrocolloid concentrations, creaming is inhibited due to the viscoelastic character of the interconnected regions of emulsion droplets that have become flocculated into a gel-like network (Garti, Slavin, & Aserin, 1999). However, an investigation indicated that viscosity was not the main factor affecting the emulsion stability (Huang et al., 2001). Factors such as electrostatic repulsions, protein content and varieties of polysaccharide would also have an influence on the emulsion stability. The UFP-stabilized emulsions also exhibited pseudoplastic behavior (Fig. 2), i.e., the curves show moderate to pronounced shear-thinning behavior, and a higher viscosity was observed at lower shear rates. It is probably the shear-thinning behavior of the emulsions at low UFP concentrations (<0.5%) was due to the breakage of bridged-droplets, achieving Newtonian profile at high shear rates. When higher amounts of UFP were used, a systematic increase in thinning behavior and viscosity occurred (Fig. 2). The shear-thinning behavior is probably the most common rheological behavior for emulsions. From their rheological parameter, it is possible to infer the stability of emulsions, their texture, and conditioning possibilities for future applications. 3.3. Zeta potential measurements Once an emulsion has been prepared, the main factor determining its stability is the strength of the repulsive interactions between

P. Shao et al. / Carbohydrate Polymers 135 (2016) 27–34

60

1% UFP-fresh 1% UFP-25°C3d 1% UFP-25°C7d 1% UFP-60°C3d 1% UFP-60°C7d

A

40 30 20

2% UFP-fresh 2% UFP-25°C3d 2% UFP-25°C7d 2% UFP-60°C3d 2% UFP-60°C7d

B

50

Volume (%)

Volume (%)

50

60

40 30 20 10

10 0 0.01

0.1

1

10

100

1000

0 0.01

10000

0.1

3% UPF-fresh 3% UFP-25°C3d 3% UFP-25°C7d 3% UFP-60°C3d 3% UFP-60°C7d

C

60

40 30 20 10 0 0.01

10

100

1000

10000

4% UFP-fresh 4% UFP-25°C3d 4% UFP-25°C7d 4% UFP-60°C3d 4% UFP-60°C7d

D

50

Volume (%)

Volume (%)

50

1

Particai Size (µ µm)

Partical Size (µ µm) 60

31

40 30 20 10

0.1

1

10

100

1000

Partical Size (μ μ m)

10000

0 0.01

0.1

1

10

100

1000

10000

Partical Size (μ μ m)

Fig. 5. Change in droplet size distribution of different concentration of UFP-stabilized emulsions during storage at room temperature and 60 ◦ C ((A) 1% UFP, (B) 2% UFP, (C) 3% UFP, and (D) 4% UFP).

Table 1 Comparison of the content of protein and carbohydrate and the average droplet size of the emulsions stabilized by 3% concentration gums. Gum

GA GG UFP

Protein content (%)

2.6 3.3 1.2

Total carbohydrate content (%)

82 86 89

Average droplet size (␮m) D[3,2]

D[4,3]

1.726 ± 0.045 0.851 ± 0.013 0.975 ± 0.001

5.496 ± 0.003 0.961 ± 0.012 1.640 ± 0.036

Fig. 6. Comparison of the particle size distributions (A) and the viscosity (B) of the freshly prepared emulsions with different gums at 3% concentration.

32

P. Shao et al. / Carbohydrate Polymers 135 (2016) 27–34

30

GA-fresh GA-60°C3d GA-60°C7d

A

40

B

GG-Fresh GG-60°C3d GG-60°C7d

30 Volume (%)

Volume (%)

20

10

20

10

0.01

0.1

1

10

100

1000

10000

Partical Size (μm)

40

C

0.01

0.1

1

10

100

1000

10000

Partical Size ( μm)

UFP-fresh UFP-60°C3d UFP-60°C7d

Volume (%)

30

20

10

0.01

0.1

1

10

100

1000

10000

Partical Size (μm) Fig. 7. Comparison of the particle size of different gum emulsions at 3% concentration during storage at 60 ◦ C ((A) GA, (B) GG, and (C) UFP).

the surfaces of closely approaching droplets. As shown in Fig. 3, ␨-potential values of the UFP-stabilized cinnamaldehyde/water emulsions varied from −26.30 to −38.37 mV, and were depended on the UFP concentrations. This may be attributed to the increase of UFP concentration leading to the increase of negative surface charge of emulsion droplets and consequently negatively charged ␨-potential (repulsive forces) which stabilize the emulsion system (Dickinson, 2009). In fact, research has indicated that UFP is basically an anionic polysaccharide consisting of 35% uronic acid and 19% sulfate with two major repeating aldobiuronic acids designated as type A: ulvanobiuronic acid 3-sulfate (A3S ) and type B: ulvanobi-uronic acid 3-sulfate (B3S ) (Lahaye & Robic, 2007). The negatively charged polysaccharide (i.e., UFP) can enhance the electrostatic repulsive forces among emulsion droplets. Similarly, the negatively charged emulsion droplets repel each other and retard the aggregation and flocculation.

3.4. Visual phase separation To estimate the stability of different UFP-stabilized emulsions upon storage, phase separation of emulsions stabilized with various UFP concentrations was recorded after storing quiescently for 30 days. The results indicated that the higher the concentration of UFP, the lower the possibility of the phase separation of emulsions (Fig. 4). The emulsions stabilized by lower UFP concentrations (≤1%) showed phase separation after 30 days, but this did not occur in the emulsions containing >1.0% (w/w) UFP. As discussed above, the higher creaming rate of the lower UFP emulsions could be attributed to their larger particle size and wider size distribution.

Creaming of emulsions under still conditions can be considered as the contribution of two factors, one is the Brownian movement and the other is the movement of the droplets under the gravitational field. During the migration process, droplets descend to the lower part of the tube, and droplets collide between each other. During this period the stability against coalescence is mainly governed by the particle interfacial film resistance. Coalescence probability increases when fluctuations become so important to form holes that can pass from one droplet to another. According to Stoke’s law, the creaming stability of emulsion can be improved by reducing the droplet size, increasing viscosity of the continuous phase, or minimizing density differences between the droplets and the continuous phase (Depree & Savage, 2001). As discussed previously, the viscosity of the emulsions was increased with increasing UFP concentrations (Fig. 2). In addition, the adsorption of polysaccharide at the emulsion droplets surface increased the negative surface charge, and resulted in stable droplets against aggregation and flocculation. Both factors may have contributed to the improved stability in higher UFP emulsions. 3.5. Emulsion stability during acceleration test To further estimate the storage stability of the cinnamaldehyde/water emulsions prepared with 1%, 2%, 3%, and 4% UFP as emulsifying agent, changes in particle size and size distribution during storage at 25 ◦ C and 60 ◦ C for 7 days were determined (Fig. 5). The data showed that there were significant changes in particle size and size distribution for most of the emulsions except the emulsion stabilized with 3% UFP. Although as shown in Fig. 5C and D, the changes in particle size and size distribution of the emulsion

P. Shao et al. / Carbohydrate Polymers 135 (2016) 27–34

33

stabilized by 3% UFP are similar to 4% UFP. Under lower polysaccharide concentrations (≤1%), droplet flocculation causes creaming instability because the increase in effective size of the particles (which promotes creaming) more than compensates for the increase in continuous phase viscosity (which retards creaming) (Fig. 4). This arises when pairs of particle surfaces approach within a distance less than the mean diameter of the free polymer molecule in aqueous solution. The exclusion of the polymer from the intervening gap is associated with an attractive inter-particle force, which is resulted from the tendency of solvent to flow out from the gap under the influence of the local osmotic pressure gradient to form big droplets.

emulsions for at least 30 days. The viscoelasticity of UFP stabilized emulsions appeared to be dependent on its concentration. Additionally, the stability of the emulsions may be attributed to the UFP’s both protein and polysaccharide fractions and non-adsorbed polysaccharide in the aqueous phase. A possible mechanism of UFP on the stabilization of the O/W emulsion could be the adsorption of UFP at the oil-water interfaces, providing a steric (mechanical) barrier against droplet coalescence. When the formulation and process conditions are carefully controlled, the emulsions showed higher stability by the UFP than those by gum Arabic and gum Ghatti. This study may have provided useful information on utilization of UFP as a natural emulsifying and stabilizing ingredient in food industry.

3.6. Comparison the emulsifying property of UFP with other commercial gums

Acknowledgments

The emulsifying properties of the different gums at a fixed concentration of 3% (wt/wt), in terms of particle size (D[3,2] and D[4,3]) as shown in Table 1, revealed that both UFP and GG produced the emulsion droplets with monomodal distribution (Fig. 6A). The UFP with only 1.2% protein level (wt/wt) formed emulsion droplets of D[3,2] value of 0.975 ␮m, which was similar to the D[3,2] value of the commercial GG gum with a relatively high protein level of 3.3%. However, GG exhibited better emulsifying properties than UFP as a smaller proportion of big droplets were formed (D[4,3], 0.961 ␮m) in GC emulsions compared to UFP (D[4,3], 1.64 ␮m) emulsions. Furthermore, the emulsions produced by another commercial gum of GA (2.6% protein) exhibited a biomodal type size distribution, with a droplet size value of D[3,2] 1.726 ␮m, which was larger than those of UFP and GG emulsions. The results suggested that the emulsifying properties of these gums could not only be attributed to their protein fractions, but also to their particular different structural features. In addition, the emulsions prepared with different gums at 3% concentration had different viscosities (Fig. 6B). The UFP-stabilized emulsion had the highest viscosity amongst the emulsions tested, which may partly explain its good stability during storage. The emulsions stabilized by 3% GA exhibited a very low viscosity. Research has pointed out that GA exhibited high water solubility and low viscosity in comparison to other polysaccharides of similar molecular weights (Sanchez, Renard, Robert, Schmitt, & Lefebvre, 2002). The flow behavior of GA is considered to be Newtonian at concentrations as high as 50% (w/v) at shear rates >100 s−1 (Mothé & Rao, 1999). Our observation supported above findings and suggested that the rheological behavior of the emulsions appeared to be mainly influenced by the rheological behavior of the emulsifying agent (gums) used. In the case of UFP, the non-adsorbed UFP may have dominated the rheological properties. In terms of stability during storage, the GA stabilized emulsions appeared to be the least stable, as observed by the coalescence and dramatic growth in average droplet size (D[3,2]) during storage (Fig. 7A). GG produced the most stable emulsion, in terms of the monomodal particle size distribution (Fig. 7B). In the GA stabilized emulsions, the droplet size changed a lot over time, probably due to its big particle sizes (>1 ␮m) and the low viscosity of the aqueous phase, which was insufficient to prevent creaming. Huang et al. (2001) reported that it required 12.5% GA (containing 2% protein) to produce stable 40% (wt/wt) O/W emulsions. This suggested that UFP could be a better emulsifying agent than GA, because only a small amount (3%) is required to emulsify and stabilize the system (Fig. 7C). 4. Conclusions In this work, 3% (wt/wt) UFP was observed to form small emulsion droplets (<1.0 mm) and stabilize the cinnamaldehyde/water

This work was supported by National Natural Science Foundation of China (No. 31301560, 31571833). We also thank Dr. Fang from International Institute of Agri-Food Security, Curtin University, Australia for his valuable suggestions. We confirm that there is no conflict of interest. References Akhtar, M., Dickinson, E., Mazoyer, J., & Lanendorff, V. (2002). Emulsion stabilizing properties of depolymerized pectin. Food Hydrocolloids, 16(3), 249–256. Al-Assaf, S., Phillips, G. O., Aoki, H., & Sasaki, Y. (2007). Characterization and properties of Acacia senegal (L.) Willd. var. senegal with enhanced properties (Acacia (sen) SUPER GUMTM ): Part 1—Controlled maturation of Acacia senegal var. senegal to increase viscoelasticity, produce a hydrogel form and convert a poor into a good emulsifier. Food Hydrocolloids, 21(3), 319–328. Al-Assaf, S., Phillips, G. O., Williams, P. A., & du Plessis, T. (2007). Application of ionizing radiations to produce new polysaccharides and proteins with enhanced functionality. Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms, 265(1), 37–43. Balaguer, M. P., Lopex-Carballo, G., Catala, R., Gavara, R., & Hernandez-Munoz, P. (2013). Antifungal properties of gliadin films incorporating cinnamaldehyde and application in active food packaging of bread and cheese spread foodstuffs. International Journal of Food Microbiology, 166(3), 369–377. Bouyer, E., Mekhloufi, G., Rosilio, V., Grossiord, J. L., & Aqnely, F. (2012). Proteins, polysaccharides, and their complexes used as stabilizers for emulsions: Alternatives to synthetic surfactants in the pharmaceutical field? International Journal of Pharmaceutics, 436(1–2), 359–378. Chakraborty, K., & Paulraj, R. (2010). Sesquiterpenoids with free-radical-scavenging properties from marine macroalga Ulva fasciata Delile. Food Chemistry, 122(1), 31–41. Chivero, P., Gohtani, S., Yoshii, H., & Nakamura, A. (2015). Effect of xanthan and guar gums on the formation and stability of soy soluble polysaccharide oil-in-water emulsions. Food Research International, 70, 7–14. Choy, A. L., May, B. K., & Small, D. M. (2012). The effects of acetylated potato starch and sodium carboxymethyl cellulose on the quality of instant fried noodles. Food Hydrocolloids, 26(1), 2–8. Depree, J. A., & Savage, G. P. (2001). Physical and flavour stability of mayonnaise. Trends in Food Science & Technology, 12(5–6), 157–163. Dickinson, E. (2003). Hydrocolloids at interfaces and the influence on the properties of dispersed systems. Food Hydrocolloids, 17(1), 25–39. Dickinson, E. (2009). Hydrocolloids as emulsifiers and emulsion stabilizers. Food Hydrocolloids, 23(6), 1473–1482. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28(3), 350–356. Funami, T. (2011). Next target for food hydrocolloid studies: Texture design of foods using hydrocolloid technology. Food Hydrocolloids, 25(8), 1904–1914. Funami, T., Nakauma, M., Ishihara, S., Tanaka, R., Inoue, T., & Phillips, G. O. (2011). Structural modifications of sugar beet pectin and the relationship of structure to functionality. Food Hydrocolloids, 25(2), 221–229. Garti, N., Slavin, Y., & Aserin, A. (1999). Surface and emulsification properties of a new gum extracted from Portulaca oleracea L. Food Hydrocolloids, 13(2), 145–155. Huang, X., Kakuda, Y., & Cui, W. (2001). Hydrocolloids in emulsions: Particle size distribution and interfacial activity. Food Hydrocolloids, 15(4–6), 533–542. Jafari, S. M., Beheshti, P., & Assadpoor, E. (2012). Rheological behavior and stability of d-limonene emulsions made by a novel hydrocolloid (Angum gum) compared with Arabic gum. Journal of Food Engineering, 109(1), 1–8. Kaeffer, B., Benard, C., Lahaye, M., Blottiere, M., & Cherbut, C. (2015). Biological properties of Ulvan, a new source of green seaweed sulfated polysaccharides, on cultured normal and cancerous colonic epithelial cells. Planta Medica, 65(6), 527–531. Kovensky, J., Duchaussoy, P., Bono, F., Salmivirta, M., Sizun, P., Herbert, J. M., et al. (1999). A synthetic heparan sulfate pentasaccharide, exclusively containing

34

P. Shao et al. / Carbohydrate Polymers 135 (2016) 27–34

l-iduronic acid, displays higher affinity for FGF-2 than its d-glucuronic acid-containing isomers. Bioorganic & Medicinal Chemistry, 7(8), 1567–1580. Lahaye, M., & Robic, A. (2007). Structure and functional properties of Ulvan, a polysaccharide from green seaweeds. Biomacromolecules, 8(6), 1765–1774. Mothé, C. G., & Rao, M. A. (1999). Rheological behavior of aqueous dispersions of cashew gum and gum Arabic: Effect of concentration and blending. Food Hydrocolloids, 13(6), 501–506. Osano, J. P., Hosseini-Parvar, S. H., Matia-Merino, L., & Golding, M. (2014). Emulsifying properties of a novel polysaccharide extracted from basil seed (Ocimum bacilicum L.): effect of polysaccharide and protein content. Food Hydrocolloids, 37(0), 40–48. Otoni, C. G., Moura, M. R., Aouada, F. A., Camolloto, G. P., Cruz, R. S., Lorevice, M. V., et al. (2014). Antimicrobial and physical–mechanical properties of pectin/papaya puree/cinnamaldehyde nanoemulsion edible composite films. Food Hydrocolloids, 41, 188–194. Qi, H., Zhang, Q., Zhao, T., Chen, R., Zhang, H., Niu, X., et al. (2005). Antioxidant activity of different sulfate content derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta) in vitro. International Journal of Biological Macromolecules, 37(4), 195–199. Robic, A., Gaillard, C., Sassi, J. F., Lerat, Y., & Lahaye, M. (2009). Ultrastructure of Ulvan: A polysaccharide from green seaweeds. Biopolymers, 91(8), 652–664. Robins, M. M. (2000). Emulsions—Creaming phenomena. Current Opinion in Colloid & Interface Science, 5(5–6), 265–272.

Sanchez, C., Renard, D., Robert, P., Schmitt, C., & Lefebvre, J. (2002). Structure and rheological properties of acacia gum dispersions. Food Hydrocolloids, 16(3), 257–267. Sathivel, A., Raqhavendran, H. R., Srinivasan, P., & Devaki, T. (2008). Anti-peroxidative and anti-hyperlipidemic nature of Ulva lactuca crude polysaccharide on d-galactosamine induced hepatitis in rats. Food and Chemical Toxicology, 46(10), 3262–3267. Shao, P., Chen, M., Pei, Y., & Sun, P. (2013). In intro antioxidant activities of different sulfated polysaccharides from chlorophytan seaweeds Ulva fasciata. International Journal of Biological Macromolecules, 59, 295–300. Shao, P., Qin, M., Han, L., & Sun, P. (2014). Rheology and characteristics of sulfated polysaccharides from chlorophytan seaweeds Ulva fasciata. Carbohydrate Polymers, 113, 365–372. Shao, P., Shao, J., Han, L., Lv, L., & Sun, P. (2015). Separation, preliminary characterization, and moisture-preserving activity of polysaccharides from Ulva fasciata. International Journal of Biological Macromolecules, 72, 924–930. Shen, S., Zhang, T., Yuan, Y., Lin, S., Xu, J., & Ye, H. (2015). Effects of cinnamaldehyde on Escherichia coli and Staphylococcus aureus membrane. Food Control, 47, 196–202. Wang, R., Wang, R., & Yang, B. (2009). Extraction of essential oils from five cinnamon leaves and identification of their volatile compound compositions. Innovative Food Science & Emerging Technologies, 10(2), 289–292.