Influence of Interfacial Composition on Droplets Flocculation of Oil-in- Water Emulsions Stabilized By Garden Cress Seed Gum

1-Arezoo Fazeli 2- Masoud Najaf Najafi Young Researchers Club Institute of Scientific Quchan Branch, Islamic Azad University Quchan, Applied Higher-Education Jihad-e-Agriculture Iran Mashad, Iran [email protected] [email protected]

3- Ali Mohamadi Sani 4-Arash Koocheki Department of Food Science and Technology Department of Food Science and Technology Quchan Branch, Islamic Azad University Ferdowsi University of Mashhad Quchan, Iran Mashhad, Iran [email protected] [email protected]

Abstract—Both keeping emulsion droplets away from I. INTRODUCTION instability and using natural components are important factors in making colloidal systems. Directly interaction between oil and water is not Garden cress seed gum as a novel natural thermodynamically favorable because of hydrocolloid source used in 0, 0.5 and 1% hydrophobic effects [1]. So we need a third party concentrations to determine the gum efficiency on which may have surface activity or electrical droplets interface and its ability to avoid flocculation. charge. Surface active molecules have both For this purpose surface and interfacial tensions, hydrophilic and lipophilic regions distributed along creaming index and microscopic images studied. their backbones. In emulsion science an interface is 0 Hydrocolloid solutions kept for 24 h at 4 C to attain a narrow region that separates the two phases maximum hydration. Surface and interfacial tensions commonly oil and water [2]. Bulk physicochemical measured at 250C after reaching to their equilibrium time. Storage time considered to be 28 days for all properties of emulsions, such as their ease of treatments. The result showed surface and interfacial formation, stability and texture are governed by the tensions parameters decreased by increasing gum nature of the interface and therefore it is important concentration. Surface tension was least for 1% to understand its composition [3]. Flocculation concentration but it was not much less than 0.5% occurs when droplets smash to each other but, they level. In the control sample (without gum), emulsions keep their initial membrane. The magnitude of droplets flocculated immediately and consequently flocculation depends on colloidal interaction which phase separation occurred at the first day. At the relative to thickness and electrical charge of 0.5% concentration hydrocolloids associated at the interfacial layer in turn [1,4]. interface and made a stable emulsion. Partial flocculation happened at this level but no In recent years many studies done on plant gravitational separation observed. Emulsions made seeds to find a good source of hydrocolloids being by 1% gum concentration were completely stable able to substitute instead of synthetic ones. during storage. At this level, the interface was Lepidium perfoliatum [5,6], mesquite [7], tamarind saturated of hydrocolloids and the extra amount [8], Jatropha curcas [9], okra [10], fenugreek stayed at media. Non-adsorbed hydrocolloids [11,12] are some examples. Although these increased solution viscosity and also produced polysaccharide are able to form stable emulsions repulsion forces with adsorbed ones. It assumes that but, there is still some debate about the molecular Garden cress seed gum can prevent origin of their surface activity. dropletaggregation by thickening effect and electrostatic repulsion rather than interfacial Garden cress seeds are bitter and find activities. applications in a wide range of biological functions and diseases such as leprosy, skin diseases and as a Keywords-interfacial properties; garden cress; diuretic [13,14]. The seeds contain a large amount emulsion stability; surface activity; flocculation of mucilaginous substances and a gum of high

molecular weight has been identified [15,16]. The tests were carried out in triplicate at ambient macromolecular component of the gum has a temperature. molecular weight of 540 kDa, and is nearly as rigid as xanthan with regard to chain conformation [17]. C. Microscopic Observation Also, it was measured that the gum powder To find out the main mechanism which is contains 77 % carbohydrate, 2.45 % protein and responsible for droplets instability we conducted 1.85 % fat [15]. microscopic observation. Every emulsion diluted There is no published information about how with 1 g/L SDS and stirred to reach desired Garden cress seed gum can affect the interface concentrations (1:1000 SDS). Afterwards, a small composition, flocculation amount and in drop of these mixtures were placed onto glass consequence the emulsions long term stability slides covered with slips and observed by optical against gravitational separation. This study aimed microscope (Lx-400 Labomed, USA) equipped to determine the properties of interface composition with 9 mp (TSTD Tucsen, China) digital camera at and its effects on droplets flocculation by using × 10 magnifications. All samples were observed Garden cress seed gum as a surface active immediately after preparation and 28 days after polysaccharide. being kept at 4 0C. One droplet of the 0.5 % emulsion monitor for 3 hours to understand the II. MATERIAL AND METHOD phenomena involved on droplets instability. Images Analytical grade chemicals were provided by took every hour with different magnifications. Merck (Germany). Garden cress seed gum was extracted based on the method presented by Karazhiyan et al. [18]. Commercial corn oil D. Creaming Measurement purchased from local markets in Mashad. Fifteen ml of each fresh emulsion was transferred into test tubes, tightly covered to A. Emulsion Preparation prevent air penetration, and then stored at 4 0C for The dispersions were prepared by dissolving 0, 28 days. Creaming index was measured every 0.5 and 1 g of gum powder into 80 g distilled water seven days by the method presented by Sciarini while stirring with IKA-RCT basic (Germany) [20]. The measurement was performed in triplicate magnetic stirrer for 30 min. Sodium azide in 0.01 samples. The extent of creaming was determined as % proportion added to all dispersions to avoid a creaming index: bacterial growth. Afterward, the solutions kept at 4 0 HS C overnight to attain complete hydration. The final CI(%) = × 100 pH of all dispersion adjusted to be neutral by using HE 1 molar hydrochloric acid and sodium hydroxide. Corn oil in water emulsions were prepared by The height of the serum layer (HS), the total height adding 20 ml corn oil into dispersions while stirring of the emulsion (HE) by a magnetic stirrer for 10 min. Homogenization III. RESULT AND DISCUSSION done with a laboratory rotor stator homogenizer A. Surface and Interfacial tensions (Ultra Turrax T-25, IKA Instruments, Germany) at 20000 rpm for 5 min at room temperature. Surface and interfacial tensions give useful information about emulsifying properties of a B. Surface and Interfacial Tensions Measurement component and stability of droplets against aggregation and Ostwald ripening [19]. The surface Wilhelmy plate and Du Nouy ring method of and interfacial tension activities of Garden cress Kruss K100 tensiometer (Germany) used to seed gum solutions presented in Fig. 1. According measure surface and interfacial tensions to this Figure, adding gum decreased surface and respectively. The equilibrium time for interfacial tensions significantly. Although there measurements obtained to be 30 min. To minimize were more hydrocolloids available at the 1 % level the movement and to maximize the steady but the 0.5 % concentration was more effective in condition the sample holder fixed at the hairbreadth reducing both surface and interfacial tension. At the to sensitive force measuring device. The force 1 % level, gum concentration was more than the exerted on the plate when goes upward shows interface requirement while at 0.5 % it has been surface tension while the force acting on the ring enough to cover newly formed oil droplets. Spatial when the vessel holding is lowered indicates oil- prevention at higher concentration hampered gum interface [19]. The ring caught at the air–water hydrocolloid to cover oil droplets, so additional interface was progressively and carefully covered hydrocolloid molecules in the media led to gelation with oil, and the interfacial tension was and viscosity enhancement and we could not continuously recorded. The reported surface measure logical interface value. Huang et al. (2001) tension and interfacial tension numeral are mean compared physical properties of 14 kinds of gums values of 15 and 3 measurements respectively. All with 0.5 % concentration in 40 % canola oil-in-

water emulsions [21]. They found that all the gums flocculation [19]. In higher level (1%) due to reduced surface and interfacial tensions except viscosity effect and repulsion interaction of high carrageenan and xanthan because of the mentioned charge biopolymers, droplets moved slower and reason. Bouyer et al. (2011) investigations on therefore they clashed too tardy. interfacial properties of gum arabic indicated this According to Fig. 4, at the first two hours the gum needed time to diffuse towards the interface, droplets arrangement changed from open to adsorption, and rearrange itself [22]. Unlike to this compact and they started to share their membranes. research, no change was observed for Garden seed Flocculation occurred because the interfacial layer during the measuring time. was not hydrocolloid rich, so there were not It deduced that Garden cress seed gum could enough repulsion forces between oil droplets. Also, not prevent droplets instability just by interfacial Garden cress seed gum has rigid chain effects. It might be related to the protein sector of conformation [18] which created pores and built these seeds and its surface activity too. Also Ellis et attractive forces between droplets. At the third hour al. reported surface activity of guar gum can be a droplets films started to ruptured, coalescence and result of the high number of galactos branches and made larger droplets. When interfacial layer is not either protein content [23]. Youssef et al. (2009) viscoelastic it cannot resist against rupture [19] like purified fenugreek (treated with phenol) to produce the evaluated interface that ruptured with a little non-protein species. They found out by reducing pressure from other molecules. protein content from 3.74 % in raw fenugreek to 0.16 % in purified one, surface activity decreased significantly [24].

70 Surface tension

60 Interfacial tension

) 50

1 - a) 40

30

Tensions (mN m (mN Tensions 20

10

0 b) 0 0.5 1 Gum concentration (%)

Figure 1. Surface and interfacial activities of Garden cress seed gum solutions (0, 0.5, and 1%)

B. Microscopy Observation c) Micrograph displayed the phenomenon Figure 2. Microscopic images of fresh emulsions made of, a) involved in emulsions instability and of course determined emulsions microstructure (Figs. 2 and no gum, b) 0.5% and c) 1% of Garden cress seed gum 3). At control samples after removing stress, droplets smashed to each other and fused rapidly (Fig. 2a). As it was expected, low viscosity and lack of repulsion forces influenced droplets movement at these emulsions. By adding 0.5 % gum the movement of droplets reduced. Electrostatically repulsion forces were the main reason that retarded droplets instability at this level. McClements indicated that large diameter, low a) interfacial tension and increase in contact area between flattened droplets can change extremity of

100 0% 90 0.50% 80 1% 70 60 50 40 30

b) (%) Index Creaming 20 10 0 0 7 14 21 28 Time (day) Figure 5. Creaming stability of Garden cress seed gum at different concentration after 28 days kept at 40C c) Fig. 5 shows creaming stability of emulsions Figure 3. Microscopic images of emulsions made of, a) no gum, during storage time. The control sample segregated b) 0.5% and c) 1% of Garden cress seed gum after 28 days being into three phases; oil phase, cream layer and kept at 40C opaque fluid after 24 h kept in 4 0C (Fig. 6a). Oil droplets were observed from test tube wall in 0.5 % ration. Although flocculation and consequently × 40-First hour coalescence happened in this level, but no creaming saw after 28 days. At 1 % concentration emulsions were completely stable against gravitational instability. Repulsion forces between adsorbed and non-adsorbed hydrocolloid and also between covered droplets were stronger than attractive forces among them at this proportion. This repulsion forces precluded intermolecular interactions and prevented droplets aggregation. Georgiadis et al. (2012) had the same experience × 40-Second hour about emulsions prepared by polysaccharide which extracted from orchid roots [28]. Although the Garden cress seed gum could not completely prevented droplets flocculation just by interfacial effect but its hydrophilicity nature relayed this ability. Garden cress seed gum has rigid conformation which minimizes its flexibility [18]. So the non-adsorbed hydrocolloids made networks which trapped oil droplets, immobilized × ퟏퟎퟎ-Third hour and prevented the Brownian motion. Dokic, Krstonic, Nikolic and also Yang and Jiang (2012) explained thickening effect by using Stocks law [25,26]. These scientists studied the stabilizing properties of modify starch. According to tentative work done by Kang et al. (2012), if inhibition of the oil-water interface cannot overcome droplets aggregation, they would begin to coalescence [27].

Figure 4. Monitoring images of droplets behavior at present of 0.5% hydrocolloid gum

C. Creaming Stability

REFERENCES [1] W. Norde: Colloids and Interfaces in Life Sciences, Marcel Dekker, New York (2003). [2] E. Dickinson, T.V. Vliet, and P. Walstra: Studying food colloids: Past, present and future. In: Food Colloids, Biopolymers and Materials, E. Dickinson and T.V. Vliet (Eds.), Royal Society of Chemistry, Cambridge, UK, (2003) pp. 391-400. [3] D.G. Dalgleish, Adsorption of protein and the stability of emulsions, Trends Food Sci. Technol. 8 (1997) 1–6. [4] E. Dickinson, Hydrocolloids at interfaces and influence on the properties of dispersed systems, Food Hydrocoll. 17 (2003) 25-39. [5] M. Soleimanpour, A. Koocheki, and R. Kadkhodaee, Influence of main emulsion components on the physical properties of corn oil a) b) in water emulsion: Effect of oil volume fraction, whey protein concentrate and Lepidium perfoliatum seed gum, Food Hydrocoll. (in press). [6] M. Soleimanpour, A. Koocheki, and R. Kadkhodaee, Effect of Lepidium perfoliatum seed gum addition on whey protein concentrate stabilized emulsions stored at cold and ambient temperature, Food Hydrocoll. 30 (2013) 292-301. [7] Y.L.L. Francoa, C.I.C. Montanoa, K.G.M. Robinsona, J.L. Mendozaa, and L.E.R. Ozunab, Physicochemical characterization and functional properties of galactomannans from mesquite seeds (Prosopis spp.), Food Hydrocoll. 30 (2013) 656- 660. [8] A.M. Avachat, K.N. Gujar, and K.V. Wagh, Development and evaluation of tamarind seed xyloglucan-based mucoadhesive buccal films of rizatriptan benzoate, Carbohydrate Polym. 91 (2013) 537-542. c) [9] Z.Z.B. Abidnia, N.S.M., Shamsudina, N. Madehia, and S. Sobria, Optimisation of a method to extract Figure 6. Emulsions macroscopic observation after 28 days kept the active coagulant agent from Jatropha curcas 0 seeds for use in turbidity removal, Industrial Crops at 4 C, a) no gum, b) 0.5%, c) 1% and Prod. 41 (2013) 319-323. [10] M.S. Alamri, A.A. Mohamed, and S. Hussain, Effects of alkaline-soluble okra gum on rheological and thermal properties of systems with wheat or IV. CONCLUSIONS corn starch, Food Hydrocoll. 30 (2013) 541-551. This investigation did on the ability of Garden [11] M. Kasrana, S.W. Cuib, and H.D. Goffa, Covalent cress seed gum to associate at the oil-water attachment of fenugreek gum to soy whey protein interface. Results showed this gum had not isolate through natural Maillard reaction for significant surface activity and the minor effect improved emulsion stability, Food Hydrocoll. 30 (2013) 552-558. may belong to its protein content. Amount and rate of flocculation was directly relative to surface and [12] M. Kasrana, S.W. Cuib, and H.D. Goffa, Emulsifying properties of soy whey protein interfacial activity of this hydrocolloid but other isolate–fenugreek gum conjugates in oil-in-water reasons might be effective too. Control sample emulsion model system, Food Hydrocoll. 30 (2013) could not retain its monotonous form even for 1 691-697. hour and immediately started to segregate. At the [13] G. H. Amin: Medicinal plants of Iran (1st Ed.). 0.5 % concentration, less hydrocolloid existed at Tehran University Publication, Tehran (2005) pp. the interface while at 1 % it was saturated of 106 (in Persian). hydrocolloid, so droplets were more stable against [14] M.K. Avachat, and A.G. Dhamne, Method of flocculation at the latter concentration. Both 0.5 manufacturing of husk from Lepidium sativum and 1 % emulsions withstood toward gravitational seeds. The Patent Cooperation Treaty (PCT), WO 02/102856A1 (2002). separation after being kept for 28 days at 4 0C. It is clear that Garden cress seed gum has little surface [15] H. Karazhiyan, Extraction optimization and physical properties of cress seed hydrocolloid using and interfacial activities but mostly can act as a response surface methodology, PhD Thesis, thickener. Ferdowsi University of Mashhad, Iran (2008).

[16] S.M.A. Razavi, R. Farhoosh, and A. Bostan, [23] P.R. Ellis, Q. Wang, P. Rayment, Y. Ren, and S.B. Functional properties of hydrocolloid extract of Ross-Murphy: Guar gum: agricultural and some domestic Iranian seeds, Research project botanical aspects, physicochemical and nutritional No.1475, Unpublished report, Ferdowsi University properties, and its use in the development of of Mashhad, Iran (2007). functional foods. In: Handbook of dietary fiber: An applied approach, S. Sungoo and M.L. Dreher [17] H. Karazhiyan, S. M. A. Razavi, G.O. Phillips, Y. (Eds.), Marcel Dekker, New York (2001). Fang, S. Al-Assaf, and K. Nishinari, Rheological properties of Lepidium sativum seed extract as a [24] M.K. Youssef, Q. Wang, S.W. Cui, and S. Barbut, function of concentration, temperature and time, Purification and partial physicochemical Food Hydrocoll. 23 (2009) 2062-2068. characteristics of protein free fenugreek gums, Food Hydrocoll. 23 (2009), 1-5. [18] H. Karazhiyan, S. M. A. Razavi, and G.O. Phillips, Extraction optimization of a hydrocolloid extract [25] L. Dokic, V. Krstonosic, and I. Nikolic, from cress seed (Lepidium sativum) using response Physicochemical characteristics and stability of oil- surface methodology, Food Hydrocoll. 25 (2011) in-water emulsions stabilized by OSA starch, Food 915-920. Hydrocoll. 29 (2012) 185-192. [19] D.J. McClements: Food emulsions: principles, [26] J.S. Yang, B. Jiang, W. He, and Y.M. Xia, practice, and techniques, CRC Press, Boca Raton, Hydrophobically modified alginate for emulsion of FL (2005). oil in water, Carbohydrate Polym. 87 (2012) 1503- 1506. [20] L.S. Sciarini, F. Moldonado, P.D. Ribotta, G.T. Perez, and A.E. Leon, Chemical composition and [27] W. Kang, L. Guo, H. Fan, L. Men, and Y. Li, functional properties of Gleditsia triacanthos gum, Flocculation, coalescence and migration of Food Hydrocoll. 23 (2009) 306-313. dispersed phase droplets and oil– water separation in heavy oil emulsion, J of Petroleum Sci. and Eng. [21] X. Huang, Y. Kakuda, and W. Cui, Hydrocolloids 81 (2012) 177-181. in emulsions particle size distribution and interfacial activity, Food Hydrocoll. 15 (2001) 533- [28] N. Georgiadis, C. Ritzoulis, E. Charchari, C. 542. Koukiotis, C. Tsioptsias, and C. Vasiliadou, Isolation, characterization and emulsion stabilizing [22] E. Bouyer, G. Mekhloufi, I. Le Potier, T.d.F. de properties of polysaccharides form orchid roots Kerdaniel, J.L. Grossiord, V. Rosiliol, and F. (salep), Food Hydrocoll. 28 (2012) 68-74. Agnely, Stabilization mechanism of oil-in-water emulsions by β-lactoglobulin and gum Arabic, J of Colloid and Interface Sci. 354 (2011) 467–477.