Food Hydrocolloids Manuscript Draft Manuscript Number: FOODHYD-D-10-00280 Title: Stabilization of Water Droplets in Oil exclusively by gel formation

Article Type: Short Communication Keywords: Multiple emulsion; hydrocolloid; W/O-emulsion; orifice; High pressure emulsification; gelation Abstract: Highly fat containing food products, like mayonnaise, are common components of today's diets. But as obesity becomes a more and more recognized problem a need for fat-reduced products emerges. Multiple emulsions pose one alternative for fat-reduced products. Unlike other fat-reduction methods, where textural changes are compensated by addition of hydrocolloids to the continuous phase, it is assumed that no textural changes occur while reducing the fat content by substituting internal parts of the oil droplets with water droplets. Despite this advantage no food multiple emulsions are available on the European market. One main reason for that is the lack of emulsifiers that allow the production of these products, especially the production of W/O-emulsions applicable as inner emulsions in multiple emulsions. The lack is mainly caused by the strict laws regulating the type and amount of food additives within the European Union A process based on high-pressure emulsification with orifices was designed and tested to allow an exclusive application of gels as stabilization agents for W/O-emulsions. A proof of principal is given for pectin and gellan gum as gelling agents. Depending on the type of gel different droplets size distributions were achieved, at which pectin yielded smaller droplets sizes (< 10 m) at comparable processing conditions. As no emulsifiers are required it offers a food grade alternative for the processing of W/O-emulsions.

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Stabilization of Water Droplets in Oil exclusively by gel formation F. Wolf, F. Gmoser, H. P. Schuchmann Institute of Process Engineering in Life Sciences, Section of Food Process Engineering, KIT, 76131 Karlsruhe, Germany All correspondence sends to: Frederik Wolf Kaiserstr. 12 D-76131 Karlsruhe, Germany Tel..: +49 (0) 721 / 608-3614 Fax: +49 (0) 721 / 608-5967 E-mail: Frederik.Wolf@kit.edu Abstract: Highly fat containing food products, like mayonnaise, are common components of todays diets. But as obesity becomes a more and more recognized problem a need for fat-reduced products emerges. Multiple emulsions pose one alternative for fat-reduced products. Unlike other fat-reduction methods, where textural changes are compensated by addition of hydrocolloids to the continuous phase, it is assumed that no textural changes occur while reducing the fat content by substituting internal parts of the oil droplets with water droplets. Despite this advantage no food multiple emulsions are available on the European market. One main reason for that is the lack of emulsifiers that allow the production of these products, especially the production of W/O-emulsions applicable as inner emulsions in multiple emulsions. The lack is mainly caused by the strict laws regulating the type and amount of food additives within the European Union A process based on high-pressure emulsification with orifices was designed and tested to allow an exclusive application of gels as stabilization agents for W/O-emulsions. A proof of principal is given for pectin and gellan gum as gelling agents. Depending on the type of gel different droplets size distributions were achieved, at which pectin yielded smaller droplet sizes (< 10 m) at comparable processing conditions. As no emulsifiers are required it offers a food grade alternative for the processing of W/O-emulsions. Keywords: Multiple emulsion, hydrocolloid, W/O-emulsion, orifice, 1. Introduction Even so multiple emulsions have the potential to be applied in functional or fat-reduced foods (Muschiolik 2007a), one does not find food products employing multiple emulsions in European supermarkets. A common way of processing multiple emulsions is that in the first instance an inner, in food applications mostly water-in-oil (W/O) emulsion, is produced, which is then further dispersed in an outer aqueous phase yielding a water-in-oil-in-water (W/O/W) emulsion (Muschiolik 2007c, Muschiolik 2007d, Garti 1997). To ensure a pleasant sensory profile the oil droplets in oil-in-water (O/W) emulsions are typically in the range of 1-50 m (Singer 1996, Friberg, Larsson & Sjblom 2004). Assuming

that multiple emulsions yield a comparable sensory profile at comparable droplet-sizes, the inner droplets must be significantly smaller than 1-50 m. The water droplets should be about 10-100 times smaller than the enveloping oil droplets (Vladisavljevic & Schubert 2001), hence droplet sizes not exceeding 5 m. To stabilize small droplets, emulsifiers must be employed that adsorb fast at the interface and stabilize the formed droplets. For application in multiple emulsions the stabilization furthermore need to ensure that the inner droplets are stable against mechanical and thermal stresses during the second emulsification step (processing of the multiple emulsion). One main reason for the absence of multiple food emulsions is the lack of emulsifiers that allow the production of these products, especially the production of W/O-emulsions applicable as inner emulsions in multiple emulsions. The lack is caused by the strict laws regulating the type and amount of food additives within the European Union (in Germany represented by Zusatzstoff-Zulassungsverordnung ZZulV, 29. January 1998 - (BGBl I S. 231)). The W/O-emulsifiers Polyglycerol Polyricinoleate (PGPR) (Muschiolik 2007b, Dickinson, Evison, Owusu & Williams 1994, Benichou, Aserin & Garti 2007, Surh, Vladisavljevic, Mun & McClements 2007, Mun, Choi, Rho, Kang, Park & Kim 2010) and Phosphatidylcholine (PC)-depleted phospholipids (Muschiolik 2007b, Akhtar & Dickinson 2001) are often used in literature to produce W/O-emulsions and yield good results. The former only works at high concentrations which are too high for legal application, while the latter can be employed quantum satis but is sensitive to changes of the recipe and thus hard to handle (Weiss, Scherze & Muschiolik 2005, Knoth, Scherze & Muschiolik 2005a, Knoth, Scherze & Muschiolik 2005b). Both are representative for stabilization characteristics of the available food-grade W/O-emulsifiers. There are so far no emulsifiers available capable of fulfilling the task to stabilize W/O-emulsions in a satisfying and legal way, so that there are no food products based on multiple emulsions commercially available. Thus, a substitute for these emulsifiers in food applications to allow the processing of W/Oemulsions for application in multiple emulsions is essential. The approach of this study is to stabilize emulsions with gels instead of emulsifiers (see Fig. 1). As they are capable of forming rigid droplets and are food grade substances they fulfill the basic requirements as alternative stabilization system. Gels are already employed for partial stabilization of emulsions. (Weiss, Scherze & Muschiolik 2005) for example used alginate gels to form a rigid layer around W/O-droplets in an aqueous matrix and thus enable a targeted release. Works of (Surh, Vladisavljevic, Mun & McClements 2007) showed further that gels can be employed in the aqueous phase of W/Oemulsions that were further employed in the processing of multiple emulsions. But all emulsions from their study contained PGPR and only additionally a gel. Works of (Erni, Cramer, Marti, Windhab & Fischer 2009, Walther, Cramer, Tiemeyer, Hamberg, Fischer, Windhab & Hermansson 2005) show that it is possible to form particular, non-spherical systems stabilized exclusively by gelation of the emulsion droplets. As it is undesirable that already gelled droplets are broken up, to prevent plugging of processing machinery and ensure defined, spherical structures, the gel formation must take place after the droplet break-up. These restrictions can only be met if the droplets are broken up without the presence of a stabilizing system whatsoever and are stabilized after break-up. Furthermore it is necessary that the droplets are stabilized as fast as possible before coalescence processes take place. As gel formation, in case of the presented study by complex formation with Ca2+-ions, is triggered by lowering temperature below a critical value (Harris

1990), the process needs to be capable of realizing sufficiently fast cooling rates and the gel formation kinetics need to be fast (Erni, Cramer, Marti, Windhab & Fischer 2009). This study gives a proof of principle that stabilization of water droplets in an oily matrix exclusively by gel formation is possible employing a process based on high pressure emulsification with orifices. 2. Materials & Methods 2.1. Preparation of emulsions 2.1.1. Materials The gelling agents employed in this study were pectin of type Classic AU 708 kindly provided by Herbstreith & Fox KG, Neuenbrg, Germany and gellan gum purchased from AppliChem GmbH, Darmstadt, Germany. CaCl2 was acquired from Carl Roth GmbH + Co. KG, Karlsruhe, Germany. The vegetable oil (mixture of canola and sunflower oil) was purchased from Floreal Haagen GmbH, Saarbrcken, Germany. To stabilize the reference samples the W/O-emulsifier PGPR (PGPR 90) was employed which was kindly provided by Danisco A/S, Denmark. 2.1.2. Preparation of pre-emulsion For all samples a pre-emulsion was prepared with demineralized water, the gelling agent and CaCl2 as the aqueous phase and plain vegetable oil as the oily phase. The aqueous phase was stirred with a propeller stirrer at 500 rpm for 1 h to ensure complete solution of the gelling agent and CaCl2 .The preparation for the gelling agents pectin and gellan gum differed in employed recipe and preparation temperatur. 3 % Pectin was dissolved into water at 80 C. At this temperature 0.3 % CaCl2 was added to avoid premature gelation. In case of gellan gum 1 % was dissolved at 50 C. At this temperature 0.1 % CaCl2 was added to again avoid premature gelation. As a reference W/O-emulsions were prepared and stabilized by 5 % PGPR in the oily phase. For these samples plain demineralized water was used as the aqueous phase. To avoid coalescence and to show the general feasibility of the process, all samples and the reference had a disperse phase content of 4 %. The pre-emulsions were prepared by slowly adding the aqueous phase to the oily phase while stirring with a propeller stirrer at 500 rpm. The pre-emulsions were at all times tempered to 80 C (pectin) or 50 C (gellan gum) respectively.

2.1.3. Preparation of W/O-emulsion All samples were prepared with the experimental setup depicted in Fig. 2, which is based on a high pressure emulsification process employing an orifice. The pre-emulsions were added to feed bin 1 and there further stirred with a propeller stirrer at 200 rpm to avoid sedimentation. Depending on the recipe the whole plant to the orifice was heated to the corresponding temperature (80 C for pectin, 50 C for gellan gum, no heating for PGPR). Into feed bin 2 cold oil (0 C) was added in case of the samples stabilized with gelling agents and regular oil for the reference sample. The pre-emulsion was pumped through the orifice by means of a one piston high

pressure pump from Microfluidics Corp., Newton MA, USA of type M-110Y. The orifice was mounted on the pump instead of the original disruption unit. The homogenizing pressures were varied between 50 and 1000 bar. The orifices diameters were d= 0.8 mm (pectin) and 0.2 mm (gellan gum), respectively, a length of 2 mm and an exit diameter D = 1.6 mm. At a distance of 15 mm after the orifice the oil from feed bin 2 was added by means of a Mohnopump of type VARMECA37M by Erich Netzsch GmbH & Co. Holding KG, Selb, Germany at a feed rate of 1.9 l/min. Here again the pipe diameter is increased to 5 mm to avoid backflow. Due to the feed the disperse phase content of the emulsion is reduced to 2 %. Samples from the formed emulsions were taken after feeding of the cold oil. 2.2. Measurement of droplet size distribution To classify the resulting emulsions the particle size distribution was taken as a measure. Because of the targeted size < 5 m and the expected droplet sizes between 100 nm and 100 m, static laser light scattering (LS 230 with PIDS technology) by Beckman Coulter Inc., Brea, USA was chosen as method to determine the droplet size distribution of the samples. The LS 230 is able to detect droplet sizes from 0.04 to 2300 m. The emulsions were measured immediately after preparation. All samples were diluted with the same vegetable oil as used for the preparation of the emulsions. All emulsions were prepared three times and each sample measured three times. The presented results display the averaged values and standard deviations of all measurements. 3. Results and discussion 3.1. Results 3.1.1. Pectin In Fig. 3 the results for pectin are depicted. The cumulative volume distribution Q3 is plotted against the droplet size x. This mode of representation was chosen as the target is to form small droplets holding as much volume as possible of the employed water. In the depicted plot the amount of small droplets containing a certain percentage of the total water volume can be read of the ordinate. It can be seen that the results for the pectin stabilized and the PGPR stabilized reference emulsions yield very comparable droplet size distributions and standard deviations. Both show a bimodal droplet size distribution with most droplets at a size range between 2 and 10 m, thus still a bit larger then required. The amount of water in small droplets in the area below 1 m is low, it only sums up to about 10 %. For droplets below 5 m it sums up to about 60 % of the volume. 3.1.2. Gellan Gum In Fig. 4 the results for gellan gum are depicted. The droplets are larger than the droplets of the PGPR stabilized reference emulsion, unlike the results from pectin. An attempt was made to yield more small droplets by increasing the energy input (increase of pressure difference). For low energy inputs three-modal distributions can be observed. For higher energy inputs a, to the reference comparable, bimodal distribution can be observed. Nonetheless are the larger droplets always larger than in the reference. The amount and size of the small droplets below 1 m is comparable. But the majority of droplets are not between 2 and 10 m, but rather between 5 and 200 m. It can be seen that an increase of specific energy inserted into the system leads to a reduction of the droplet size fraction of large droplets. Like pectin the standard deviation is very broad. But never the less a clear tendency can be observed. For the first increases in pressure more large droplets

are broken up. Between 600 and 1000 bar the changes in droplet sizes are only minor. 4. Discussion The results show that with the proposed process the stabilization by temperature-triggered gelation successfully was triggered after the break-up in the orifice. The gelling agents were liquid until the point of feed. The temperature reduction by the fed cold oil seems to be sufficient to lower the temperature below the critical temperature of around 30 C for gellan gum and 55 C for pectin, thus triggering a complex formation with Ca2+ ions leading to stable gel droplets stabilizing a W/O-emulsion. But obviously differ the results for the two gelling agents as they yield different droplet size distributions. The reasons - insufficient droplet break-up or insufficient gel formation due to aggregation or coalescence - are not determined yet, but subject to current studies. 5. Conclusions These results pose a proof of principal for the exclusive application of gels as stabilization agent for W/O-emulsions processed in a high-pressure homogenizer employing orifices. As no emulsifiers are required it offers a food grade alternative for the processing of W/Oemulsions. A process was suggested in which this principle can be applied. It was found that the gel type is a parameter of importance. With pectin 60 % of the water volume could be stabilized in droplets of sizes below 5 m. Droplet stabilized with gellan gum showed a strong dependency on the employed specific energy input, but even at 1000 bar droplets sizes were still too large. Reducing the amount of remaining large droplets is subject to current studies. The gel formation kinetics in for this process relevant time scales, the impact of the cooling feed, the break-up mechanism of droplets in orifices need to be understood to evaluate their impact on the resulting droplet size distribution.

Acknowledgement Financial support for this research from Deutsche Forschungs Gesellschaft is gratefully acknowledged. Figure caption: Figure 1: Principal structure of W/O/W-emulsion with/without gelled water droplets Figure 2: Experimental setup for preparation of W/O-emulsions exclusively with gel Figure 3: Measured droplet size distribution of W/O-emulsions stabilized with pectin compared with a PGPR stabilized reference Figure 4: Measured droplet size distribution of W/O-emulsions stabilized with gellan gum compared with a PGPR stabilized reference

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