Beverage emulsions: Recent developments in

Food Hydrocolloids 42 (2014) 5e41

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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd

Review

Beverage emulsions: Recent developments in formulation, production, and applications Daniel T. Piorkowski, David Julian McClements* Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 May 2013 Accepted 9 July 2013 Available online 26 July 2013

Soft drinks are one of the most widely consumed and profitable beverages in the world. This review article focuses on the utilization of emulsion science and technology for the fabrication of soft drinks by the beverage industry. A brief overview of the various high and low energy methods available for preparing this type of beverage emulsions is given, as well as a discussion of the functional ingredients used to formulate these systems, including oil phases, emulsifiers, weighting agents, ripening inhibitors, and thickening agents. The influence of droplet characteristics on the physicochemical and sensory properties of beverage emulsions is reviewed, with special focus on their influence on product stability. Finally, we discuss recent developments in the soft drinks area, including fortification with vitamins, reduced calorie beverages, and “all-natural” products. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Beverages Soft drinks Nutraceuticals Flavors Emulsions Nanoemulsions

Contents 1. 2. 3.

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5.

6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 Emulsion science and technology in the beverage industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 Controlling droplet characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8 3.1. Droplet composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3.2. Droplet concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3.3. Droplet size distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3.4. Droplet charge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 3.5. Interfacial properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 3.6. Colloidal interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 Physicochemical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 4.1. Optical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 4.2. Rheology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 4.3. Molecular distribution and release characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Beverage emulsion shelf-life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13 5.1. Physical stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 5.1.1. Gravitational separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 5.1.2. Droplet aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 5.1.3. Ostwald ripening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 5.2. Chemical stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 5.3. Defining the end of shelf life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Beverage emulsion manufacture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18 6.1. High-energy approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 6.1.1. High pressure valve homogenizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 6.1.2. Microfluidizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 6.1.3. Ultrasonic homogenizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

* Corresponding author. Tel.: þ1 413 545 1019. E-mail address: [email protected] (D.J. McClements). 0268-005X/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodhyd.2013.07.009

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D.T. Piorkowski, D.J. McClements / Food Hydrocolloids 42 (2014) 5e41

6.2.

7.

8. 9.

10.

Low-energy approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 6.2.1. Spontaneous emulsification methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 6.2.2. Phase inversion methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Beverage emulsion ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 7.1. Oil phase components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 7.1.1. Flavor oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 7.1.2. Cloud oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 7.1.3. Nutraceutical lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 7.1.4. Fat soluble colorants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 7.2. Emulsifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 7.2.1. Mechanism of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 7.2.2. Factors influencing selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 7.2.3. Ingredient examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 7.3. Weighting agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 7.3.1. Mechanism of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 7.3.2. Factors influencing selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 7.3.3. Ingredient examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 7.4. Ripening inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 7.5. Thickening agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 7.5.1. Mechanism of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 7.5.2. Factors influencing selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 7.5.3. Ingredient examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 7.6. Sweeteners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 7.6.1. Mechanism of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 7.6.2. Factors influencing selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 7.6.3. Ingredient examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Commercial aspects of beverage emulsion formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Recent developments in beverage emulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 9.1. “All-natural” products and cleaner ingredient lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 9.2. Low-calorie and mid-calorie soft drinks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 9.3. Energy drinks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 9.4. Beverage concentrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 9.5. Fortification with vitamins, minerals, and nutraceuticals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 9.6. Bottled water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 9.7. New product innovation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

1. Introduction Globally, soft drinks are one of the most widely consumed and profitable beverages (Table 1). Cola is the top soft drink flavor currently consumed in the United States, with lemon-lime and orange being the second and third. All three of these soft drink flavors contain hydrophobic citrus compounds extracted from fruit peels. Soft drinks may also contain a variety of other hydrophobic components, such as clouding agents, weighting agents, nutraceuticals, oil-soluble vitamins, and oil-soluble antimicrobials. The non-polar character of flavor oils and other hydrophobic ingredients means that these ingredients cannot simply be dispersed directly into an aqueous phase e they would rapidly coalesce and separate through gravitational forces leading to a layer of oil on top of the product (Given, 2009). Instead they first have to be converted into a colloidal dispersion consisting of flavor molecules encapsulated within small Table 1 Global revenue from non-alcoholic beverage sales by major beverage manufacturing companies in 2010 (Beverage Executive. October 2011, “Worldwide 100”, pages 44e 48). Company

Annual sales (billions of dollars)

Coca-Cola Co. PepsiCo Kraft Foods Dr. Pepper Snapple Group

35.1 21.4 8.8 5.6

particles suspended within an aqueous medium, e.g., a microemulsion, nanoemulsion, or emulsion (McClements, 2011; McClements & Li, 2010). These colloidal delivery systems must be carefully designed to provide desirable physicochemical, sensory, and biological attributes to the final product. A number of desirable attributes of colloidal delivery systems suitable for application in beverage products are highlighted below (McClements, Decker, & Weiss, 2007; McClements & Li, 2010):  Composition: Ideally, the delivery systems should be fabricated entirely from “label friendly” food-grade ingredients that are economic and easy to handle.  Fabrication: Ideally, the delivery systems should be fabricated using robust, reliable and inexpensive manufacturing methods that are easily implemented.  Stability: The delivery systems should be designed to withstand all of the stresses that a product may experience during its production, storage, transport and utilization, such as temperature fluctuations, exposure to light and oxygen, exposure to mechanical forces (such as stirring, flow through a pipe, and vibrations), variations in aqueous phase composition (such as pH, ionic strength, buffer type, ingredient interactions), and exposure to microorganisms (such as yeasts, molds or bacteria).  Physicochemical and sensory properties: The delivery system should not adversely affect the optical properties, rheology, or

D.T. Piorkowski, D.J. McClements / Food Hydrocolloids 42 (2014) 5e41

Emulsion

Nanoemulsion

Microemulsion

• Thermodynamically unstable • d > 100 nm • Optically opaque

• Thermodynamically unstable • d < 100 nm • Transparent or slightly turbid

• Thermodynamically stable • d < 100 nm • Transparent or slightly turbid

Fig. 1. Schematic representation of different kinds of colloidal dispersions that can be used in the beverage industry: emulsions, nanoemulsions, and microemulsions.

flavor profile (aroma, taste, and mouthfeel) of the beverage product into which it is incorporated.  Biological activity: The delivery system should not adversely affect the biological activity of any encapsulated bioactive components, such as antimicrobials, vitamins, or nutraceuticals. This review article provides an overview of the current status of the design, formulation, and production of emulsion-based delivery systems suitable for utilization within the beverage industry. 2. Emulsion science and technology in the beverage industry Hydrophobic components (such as flavor oils, clouding agents, oil-soluble vitamins, and nutraceuticals) can be incorporated into a

7

variety of different colloidal delivery systems suitable for application within beverage products (McClements, 2012; McClements & Rao, 2011), with the most common being microemulsions, nanoemulsions, and emulsions (Fig. 1). Each of these colloidal dispersions has particular benefits and limitations for the encapsulation of hydrophobic compounds. Microemulsions are thermodynamically stable systems under a specific set of environmental conditions (e.g., composition and temperature), and are therefore easy to fabricate (often by simple mixing) and tend to have good long-term stability. Microemulsions typically contain very small particles (r < 25 nm) and therefore tend to be optically transparent, which is desirable for soft drinks that should be clear. On the other hand, the formation of microemulsions usually requires relatively high levels of synthetic surfactants and sometimes the use of cosurfactants/ cosolvents, which can be undesirable for cost, taste, and labeling reasons. Microemulsions may also become thermodynamically unstable if environmental conditions are altered (such as temperature or composition). Conventional emulsions (r > 100 nm) and nanoemulsions (r < 100 nm) are both thermodynamically unstable systems, and therefore tend to breakdown during storage through a variety of instability mechanisms (Fig. 2), such as gravitational separation, flocculation, coalescence and Ostwald ripening (McClements & Rao, 2011; McClements et al., 2007). Emulsion systems must therefore be carefully designed to inhibit these instability mechanisms and provide sufficient kinetic stability throughout the lifetime of the product. Emulsions usually contain larger droplets than microemulsions and therefore they scatter light more strongly and appear more turbid or cloudy. This is an advantage for soft drinks that are required to have a cloudy appearance, but a disadvantage for products where optical clarity is required. Nevertheless, recently it has been shown that emulsions with ultrafine droplets, often referred to as “nanoemulsions”, can be prepared that are optically transparent (McClements, 2012; McClements & Rao, 2011). A major advantage of emulsions and nanoemulsions is that the emulsifier-to-oil ratio required to formulate them is often much less than that required for microemulsions, and they can be formulated from all natural ingredients (such as proteins and polysaccharides) rather than synthetic surfactants (such as

Fig. 2. Schematic diagram of most common instability mechanisms that occur in food emulsions: creaming, sedimentation, flocculation, coalescence, Ostwald ripening and phase inversion.

8

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Tweens). In this article, we focus primarily on the utilization of emulsion systems (conventional emulsions and nanoemulsions) in the preparation of soft drinks but much of the material is also relevant to the formulation of microemulsions. It should be noted that the emulsions used in the beverage industry are typically divided into two groups: flavor emulsions and cloud emulsions. Flavor emulsions contain lipophilic compounds that are primarily present to provide taste and aroma to a beverage product (such as lemon, lime, or orange oils). On the other hand, cloud emulsions are used to provide specific optical properties to certain beverage products, i.e., to increase their turbidity (“cloudiness”). Cloud emulsions are typically prepared using an oil phase that is highly water-insoluble and that is not prone to chemical degradation, such as flavorless vegetable oils. In addition, the size of the droplets within cloud emulsions is designed so that they have dimensions where strong light scattering occurs, but are not too large to undergo gravitational separation (e.g., r ¼ 100e200 nm). Cloud emulsions are often added to beverages that only contain a relatively low percentage of juice and provide a desirable cloudy appearance that hides sedimentation and ringing. In this article, we will use the term “emulsion” to refer to both nanoemulsions and conventional emulsions because they have similar structures and properties. Generally, an emulsion consists of at least two immiscible liquids (usually oil and water), with one of the liquids being dispersed as small spherical droplets in the other (Dickinson, 1992a; Dickinson & Stainsby, 1982; Friberg, Larsson, & Sjoblom, 2004; McClements, 2005). In general, emulsions are classified according to the relative spatial organization of the oil and water phases. A system that contains oil droplets dispersed within water is called an oil-in-water (O/W) emulsion, whereas a system that contains water droplets dispersed in oil is called a water-in-oil (W/O) emulsion. It is possible to prepare more complex emulsion structures, e.g., oil-in-water-in-oil (O/W/O), water-in-oil-in-water (W/O/W) or oil-in-water-in-water (O/W/W) emulsions (Benichou, Aserin, & Garti, 2004; Garti & Bisperink, 1998; van der Graaf, Schroen, & Boom, 2005). Currently, almost all of the emulsions used in the beverage industry are of the O/W type, although there may be certain advantages to using other emulsion types for certain applications. For example, in principle it is possible to trap a hydrophilic bioactive component within the inner water phase of a W/O/W emulsion to protect it from chemical degradation or for taste masking. In practice, it is often difficult to formulate W/ O/W emulsions that have sufficient stability for commercial applications, although this is still an active area of research. Emulsions are thermodynamically unfavorable systems that tend to break down over time though a variety of physicochemical mechanisms, including gravitational separation (creaming and sedimentation), droplet aggregation (flocculation and coalescence) and droplet growth (Ostwald ripening) (Dickinson, 1992a; Friberg et al., 2004; McClements, 2005). It is possible to form emulsions that are kinetically stable for a reasonable period of time by including substances known as stabilizers, e.g., emulsifiers, weighting agents, ripening inhibitors, or texture modifiers. It is important to clearly distinguish the different physicochemical mechanisms involved in promoting emulsion stability for these different categories of stabilizers. Emulsifiers are surface-active molecules that adsorb to the surface of freshly formed droplets during homogenization, forming a protective layer that prevents the droplets from aggregating. Weighting agents are dense hydrophobic components added to low-density oils to prevent gravitational separation. Ripening inhibitors are water-insoluble components added to polar oils to prevent Ostwald ripening. Texture modifiers are substances used to increase the viscosity or gel aqueous solutions, thereby retarding or preventing droplet movement. A more detailed description of different types of stabilizers

that can be used in beverage emulsions is given in a later section. Selecting the most appropriate stabilizer(s) for a particular application is one of the most important factors determining the shelflife and physicochemical properties of beverage emulsions. 3. Controlling droplet characteristics The bulk physicochemical properties of beverage emulsions (such as optical properties, stability, rheology, molecular partitioning, and release characteristics) are largely determined by the properties of the droplets they contain (McClements, 2005), such as composition, concentration, size, and charge (Fig. 3). In this section, we discuss some of the most important droplet characteristics that can be controlled by beverage manufacturers in order to create products with specific desirable functional properties. 3.1. Droplet composition The composition of the oil phase has a major influence on the formation and stability of beverage emulsions, which has often been overlooked in academic research. Beverage emulsions may contain a variety of different hydrophobic components, including flavor oils, essential oils, triacylglycerol oils, oil-soluble vitamins, nutraceuticals, weighting agents, and ripening inhibitors. These components vary in their molecular characteristics (such as molecular weight, molecular conformation, and functional groups), which leads to changes in their physicochemical properties (such as polarity, water-solubility, density, viscosity, refractive index, physical state, and melting point). Many of these molecular and physicochemical properties have a major influence on the formation, stability, and functionality of emulsions. For example, oil viscosity influences the efficiency of droplet disruption during high energy homogenization e the closer the ratio of dispersed phase viscosity to continuous phase viscosity (hD/hC) is to unity, the more efficient is droplet disruption and the smaller is the particle size produced (Walstra, 1993, 2003). Oil density determines the rate of particle creaming or sedimentation within emulsions e the greater the density contrast between the droplets and surrounding fluid, the faster the rate of gravitational separation (McClements, 2005). Oil refractive index determines the efficiency of light scattering by droplets in emulsions e the greater the refractive index contrast between the droplets and surrounding fluid, the stronger the degree of light scattering and the more turbid the appearance (Chanamai & McClements, 2002). The watersolubility of an oil phase determines the stability of an emulsion to Ostwald ripening due to diffusion of oil molecules through the

Fig. 3. The particles in beverage emulsions can be designed to have different functional performances by varying their physicochemical and structural properties, such as size, composition, charge and interfacial properties.

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aqueous phase (Kabalnov, 2001; McClements, Henson, Popplewell, Decker, & Choi, 2012). Oil interfacial tension plays a number of important roles in determining emulsion formation and stability. First, the ease of droplet disruption during high energy homogenization decreases as the interfacial tension decreases (Walstra, 1993). Second, the rate of droplet coalescence increases as the interfacial tension decreases (Kabalnov & Wennerstrom, 1996). Third, the ability of emulsifiers to adhere to droplet surfaces decreases as the bare oil-water interfacial tension decreases (Chanamai, Horn, & McClements, 2002). Finally, the rate of droplet growth due to Ostwald ripening depends on the interfacial tension at the oil-water interface (Kabalnov, 2001). For flavor emulsions, it is important to control the type and concentration of the flavor molecules initially present in the oil phase. It is also important to be aware that the location of the flavor molecules within an emulsion is governed by their oil-water partition, which depends on carrier oil type (Choi, Decker, Henson, Popplewell, & McClements, 2009; Choi, Decker, Henson, Popplewell, & McClements, 2010b). The flavor profile of an emulsion may therefore change if the carrier oil type is altered, if the physical state of the carrier oil changes, or if an emulsion is diluted, since this will change the distribution of the flavor molecules in the oil, water and air (Choi et al., 2009; Choi et al., 2010b; Mei et al., 2010). It is important for beverage manufacturers to understand the composition of the oil phases used to formulate commercial products, and to understand how specific lipophilic components influence the formation, stability, and properties of final products. 3.2. Droplet concentration In general, the concentration of droplets in an emulsion influences its texture, stability, appearance, sensory attributes, and nutritional quality (McClements, 2005; McClements & Rao, 2011). Droplet concentration is usually characterized in terms of the dispersed phase volume fraction (f), which is the volume of emulsion droplets (VD) divided by the total volume of emulsion (VE): f ¼ VD/VE. Practically, it is often more convenient to express the droplet concentration in terms of the dispersed phase mass fraction (fm), which is the mass of emulsion droplets (mD) divided by the total mass of emulsion (mE): fm ¼ mD/mE. When the densities of the two phases are equal, the mass fraction is equivalent to the volume fraction. It is particularly important to convert the droplet concentration to the appropriate units when comparing experimental work with theoretical predictions. In beverage emulsions, controlling the droplet concentration is important for a number of reasons. Beverage emulsions are often prepared in a concentrated form (>10% oil) because this facilitates handling and transport, but they are highly diluted when they are introduced into the final product ( 90% of the droplets should be smaller than 800 nm. The precise criteria used will depend on the product being manufactured (especially whether it should be clear or opaque).

repulsion, such as globular proteins and ionic surfactants. On the other hand, electrostatic repulsion is less important in systems where the fat droplets are coated by emulsifiers that form thick interfacial layers that generate long range steric repulsion, such as polysaccharides (gum arabic and modified starch). For electrostatically-stabilized emulsions, the magnitude of the z-potential should be greater than about 20 mV to produce systems that are stable during long-term storage. For sterically-stabilized emulsions, the droplet charge may not be important in terms of their physical stability, but it may still be important in systems where chemical reactions occur within the oil droplets that are induced by water-soluble ionic species, such as oxidation of u-3 fatty acids by transition metals. 3.5. Interfacial properties

3.4. Droplet charge The droplets in most beverage emulsions have an electrical charge because of adsorption of ionic species to their surfaces, e.g., proteins, ionic polysaccharides, ionic surfactants, phospholipids, fatty acids, and some small ions (McClements, 2005). The electrical characteristics of a droplet surface depend on the type, concentration and organization of the ionized species present, as well as the ionic composition and physical properties of the surrounding aqueous phase. The electrical charge on the oil droplets in a beverage may be important for a number of reasons: it determines the stability of the droplets to aggregation due to its influence of the magnitude, range and sign of electrostatic interactions; it determines the interactions of droplets with other charged species in an emulsion e.g., ions (such as calcium or iron), or polyelectrolytes (such as proteins or polysaccharides); it influences how the droplets interact with electrically charged surfaces, such as storage vessels, bottles, cups, and the mouth; it influences the behavior of the droplets in an electrical field, which is important for measuring their charge using electrophoresis. The electrical characteristics of a droplet in an emulsion are usually characterized in terms of its surface charge density (s), electrical potential (J0), and/or z-potential (z) (Hunter, 1986). The surface charge density is the amount of electrical charge per unit surface area, which depends on the net number of ionized groups per unit interfacial area. The electrical potential is the amount of energy required to increase the surface charge density from zero to s. The electrical potential depends on the surface charge density, but also on the ionic composition of the surrounding medium due to electrostatic screening effects. At a fixed surface charge density, the electrical potential decreases with increasing ionic strength due to these effects. The zeta-potential (z) is the electrical potential at the “shear plane”, which is defined as the distance away from the droplet surface below which the counter-ions remain strongly attached to the droplet when it moves in an electrical field. Practically, the zpotential is a better representation of the electrical characteristics of an oil droplet because it inherently accounts for the adsorption of any counter ions or ionic species to the droplet surface. In addition, the z-potential is more convenient to measure than the surface charge density or electrical potential (Hunter, 1986). Typically, the electrical characteristics of the droplets in an emulsion are determined by measuring the z-potential versus pH under appropriate measurement conditions (such as ionic composition). Droplet aggregation is inhibited in many beverage emulsions by using ionic emulsifiers that adsorb to the droplet surfaces and prevent them from coming close together because of electrostatic repulsion (Dickinson, 1992b; Friberg et al., 2004; McClements, 2005). Electrostatic repulsion plays a major role in determining the aggregation stability of fat droplets coated by charged emulsifiers that only form thin layers that generate short range steric

The boundary between the oil and water phases in an emulsion consists of a narrow region (z1e50 nm thick) that surrounds each oil droplet, and contains a mixture of oil, water, and emulsifier molecules, as well as possibly other molecular species, such as mineral ions, polyelectrolytes, and polar lipids. The interfacial region makes up a significant fraction of the volume of a droplet when the droplet diameter is less than about 1 mm (McClements & Rao, 2011), and is therefore particularly important in beverage emulsions since they usually contain droplets considerably smaller than this size. The interfacial region can influence many important physicochemical and sensory properties of beverages emulsions, including their stability, rheology, mouthfeel, and flavor. For this reason, it is often important to have knowledge about the interfacial properties of the droplets in a beverage emulsion, and to establish the major factors that influence them. Some of the most important properties of the interfacial region are: composition; structural organization; thickness; rheology; interfacial tension; and charge. These properties are determined by the type, concentration and interactions of any surface-active species present, as well as by the events that occur before, during, and after emulsion formation, e.g., complexation, competitive adsorption, layer-bylayer formation (Dickinson, 2003). As mentioned earlier, the electrical charge on the droplet interface influences its interaction with other charged molecules, as well as its stability to aggregation. The thickness and rheology of the interfacial region influences the stability of emulsions to gravitational separation, coalescence and flocculation, and determines the rate at which molecules leave or enter the droplets (Dickinson, 2003; McClements, 2005). For example, the ability of interfacial coatings to prevent droplet flocculation is strongly influenced by their thickness. Beverage manufacturers should therefore be aware of the nature of the interfacial region surrounding the oil droplets in their products, and the fact that they may be able to manipulate its properties to improve product performance. 3.6. Colloidal interactions The attractive and repulsive colloidal interactions that operate between the oil droplets in beverage emulsions determine their stability to flocculation and coalescence, which in turn influences their creaming stability and rheology (Friberg et al., 2004; McClements, 2005). The colloidal interactions between two oil droplets can be described in terms of an interaction potential (w(h)), which is the energy required to bring two droplets from an infinite distance apart to a surface-to-surface separation of h (Fig. 4). The overall interaction potential is made up from contributions from various types of interactions, with the most important being van der Waals, steric, electrostatic, depletion, and hydrophobic interactions (Israelachvili, 2011; McClements, 2005). These individual

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11

Table 2 Summary of major colloidal interactions operating between oil droplets in beverage emulsions. It is assumed that the droplets are coated by the same type of emulsifier.

Fig. 4. Schematic representation of the interaction potential between two emulsion droplets. The droplets may be stable, weakly flocculated, strongly flocculated, or coalesced depending on the attractive and repulsive forces between them.

interactions vary in their sign (attractive or repulsive), magnitude (weak to strong) and range (short to long). Each of the individual interactions usually has a simple dependence on surface-to-surface separation, but the sum of the interactions can exhibit a more complex dependence. For example, the interaction potential between two oil droplets coated by a layer of charged polymer molecules would have a number of maximum and minimum values at certain separations, such as short- and long-range energy barriers, and primary and secondary minima (Fig. 4). Generally, droplets tend to aggregate when attractive interactions dominate, but remain as individual entities when repulsive interactions dominate (McClements, 2005). It is particularly important for scientists working in the beverage industry to identify and understand the major colloidal interactions operating between the droplets in their particular product. This knowledge can then be used to establish the optimum approach for maintaining product stability during production, transport and storage. For example, if a beverage emulsion is stabilized by a protein-based emulsifier, then electrostatic repulsive interactions will play an important role in preventing droplet aggregation. In this situation, the system will be sensitive to environmental changes that reduce the magnitude and range of the electrostatic repulsion acting between droplets, such as altering the pH or adding salts (particularly multivalent counter-ions). On the other hand, if the beverage emulsion is stabilized by a polysaccharidebased emulsifier, then steric repulsive interactions will be most important for preventing droplet aggregation. In this case, the product will be much less sensitive to droplet aggregation when the pH or ionic strength is changed. In this latter case, emulsion stability depends on the thickness and hydrophilicity of the interfacial layer, which will depend on the molecular characteristics of the polysaccharide molecules. A summary of the major colloidal interactions in beverage emulsions is given in Table 2.

4. Physicochemical properties The physicochemical properties of beverage emulsions play an important role during the manufacturing process, as well as in determining the perceived quality attributes of the final product.

Interactions

Sign

Magnitude

Range

Factors affecting

Van der Waals Steric

Attractive

Intermediate

Intermediate

Repulsive

Strong

Short

Electrostatic

Repulsive

Depletion

Attractive

Strong-toweak Weak-tomedium

Long-toshort Short

Bridging

Attractive

Strong

Short

Hydrophobic

Attractive

Strong

Long

Always present Thickness and chemistry of interface pH and ionic strength Amount & type of non-adsorbed polymer Amount & type of adsorbing polymer Surface hydrophobicity

The most important physicochemical attributes of these systems are briefly discussed in this section. Some of these properties are more important in the concentrated form of the beverage, whereas others are more important in the diluted form. 4.1. Optical properties The first cue that a consumer uses to judge the quality or desirability of a finished beverage product is its visual appearance (provided it is packaged or poured into a transparent container, such as a bottle or cup). Each type of beverage product is expected to have a particular appearance depending on its nature, e.g., a dark brown cola, a cloudy orange juice, or a clear green lime juice. From a scientific viewpoint, emulsion appearance is categorized in terms of their opacity and color, which can be quantitatively described using tristimulus color coordinates, such as the L*a*b* system (McClements, 2005). In this color system, L* represents the lightness, and a* and b* are color coordinates: where þa* is the red direction, a* is the green direction; þb* is the yellow direction, b* is the blue direction; low L* is dark and high L* is light. The opacity of an emulsion can therefore by characterized by the lightness (L*), while the color intensity can be characterized by the chroma: C ¼ (a*2 þ b*2)1/2. The color intensity is usually inversely related to the lightness, so that the chroma decreases (fades) when the lightness increases. The optical properties of emulsions are mainly determined by the relative refractive index, concentration, and size distribution of the droplets they contain (Chanamai & McClements, 2002; Danviriyakul, McClements, Decker, Nawar, & Chinachoti, 2002; McClements, 2005). The lightness of an emulsion tends to increase with increasing refractive index contrast and increasing droplet concentration, and has a maximum value at a particular droplet size. This has important implications for the development of beverage products that should be either clear or opaque. In general, the lightness of emulsions increases steeply as the oil droplet concentration increases from about 0 to 5 wt%, but then increases more gradually at higher droplet concentrations (Fig. 5). As mentioned earlier, some beverages are expected to have optical clarity, whereas others are expected to be cloudy. Optimizing the initial particle size distribution of a beverage emulsion, as well as inhibiting any changes in the particle size during storage, is therefore a particularly important part of designing a commercial product with the desired optical properties. For clear products, the majority of droplets should be less than about 50 nm in diameter so that the light scattering is very weak (Wooster, Golding, & Sanguansri, 2008). The scattering efficiency of the individual oil droplets determines the maximum amount of oil phase that can be

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Fig. 5. Lipid droplets have a major impact on the texture, appearance and stability of emulsions.

incorporated into a clear beverage before it becomes noticeably cloudy. As a rule of thumb, a turbidity of 0.05 cm1 (at 600 nm) can be considered to be a rough cut-off point between transparent and cloudy products. For cloudy products, the majority of droplets should be between about 200 and 400 nm in diameter so that the light scattering is very strong (McClements, 2002). In this case, the scattering efficiency of the individual oil droplets will determine the minimum amount of a clouding emulsion required to reach a particular turbidity in the final product. 4.2. Rheology The rheological properties of beverage emulsions are also an important factor determining their manufacture and utilization. Most beverage emulsions are initially manufactured in a concentrated form, which is diluted appreciably during the production of the final beverage product. The droplet concentration in the beverage concentrate typically ranges from 3 to 30%, while that in the final product is typically 1) volatiles in the headspace and therefore reduces the perceived flavor profile (Fig. 6). This phenomenon is important to take into account when reformulating a beverage product so that it contains a different fat concentration, e.g., fortification with a bioactive lipid such as u-3 oils.

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these environmental stresses may promote emulsion instability through a variety of physicochemical mechanisms: loss of ingredient functionality (e.g., changes in solubility, surface activity, or stabilization capacity); acceleration of chemical degradation reactions (e.g., oxidation, polymerization, or hydrolysis); acceleration of physical instability mechanisms, (e.g., flocculation, coalescence or Ostwald ripening). In this section, a brief overview of some of the major instability mechanisms in beverage emulsions is given, and some suggestions for preventing them from occurring are provided.

3

Relative Flavor Intensity

2.5 2 Flavor Polarity

1.5

Non-polar

1

Polar

0.5 0 0

13

5.1. Physical stability

20 40 60 80 100 Oil Droplet Concentration (%)

Emulsions are thermodynamically unfavorable systems that tend to break down over time due to a variety of physicochemical mechanisms (Fig. 2), including gravitational separation, flocculation, coalescence and Ostwald ripening (Dickinson, 1992a; Friberg et al., 2004; McClements, 2005). All of these instability mechanisms lead to a change in the structural organization of the various components within the system, rather than in the type of molecules present. Nevertheless, changes in the chemical structure of active components can lead to changes in physical stability, and vice versa.

Fig. 6. Initial flavor intensity of emulsions with fixed flavor content is strongly dependent on oil droplet content.

The location of a constituent within a beverage emulsion is governed by its equilibrium partition coefficients (e.g., oil-water, oil-air, oil-interface) and its mass transport kinetics through the system (McClements, 2005). When a beverage emulsion is placed in the mouth there is a redistribution of flavor molecules, with some of the aroma compounds leaving the product and entering the nasal cavity. The rate at which flavor molecules leave the droplets in beverage emulsions is usually extremely quick ( 100 nm), whereas Brownian motion forces tend to dominate droplet movement in emulsions containing smaller droplets (McClements, 2011). Consequently, emulsions become more stable to creaming or sedimentation as the particle size decreases because the creaming velocity decreases (v f r2) and because Brownian motion effects increase. The above calculations assume that the particles in beverage emulsions are homogeneous spheres consisting entirely of oil phase. In practice, the particles in beverage emulsions actually have a coreeshell structure, consisting of an oil core and an interfacial shell. In this case, the overall particle radius is given by rparticle ¼ rcore þ d, and the overall particle density (rparticle) depends on the densities of the core (rC) and shell (rS) materials and the volume fraction of the shell (FS):

rparticle ¼ FS rS þ ð1  FS ÞrC

(4)

The shell layer usually has a higher density than the oil or aqueous phases, so that an increase in the volume fraction of the shell layer will tend to increase the overall particle density. This has important implications for preventing gravitational separation in beverage emulsions with small droplets sizes since it reduces the density contrast between the particles and aqueous phase. In addition, very small particles may actually sediment rather than cream if they contain sufficiently thick and dense emulsifier layers. Thus, it should be possible to produce density matched particles in beverage emulsions by controlling the oil core size and the thickness of the adsorbed emulsifier layer. The above discussion has highlighted a number of approaches that can be used to inhibit or prevent gravitational separation in beverage emulsions. First, gravitational separation can be prevented by matching the density of the dispersed (oil) and continuous (aqueous) phases. The density of the aqueous phase typically varies from about 1000e1050 kg m3, depending on the amount of sugars and other solutes present (Table 3). The density of most oil phases is less than this value, and therefore oil droplets will tend to move upwards. As already mentioned, the density of the coreeshell particles within a beverage emulsion can be matched to the surrounding aqueous phase by adding a weighting agent to the oil phase, or by controlling the thickness and density of the emulsifier

layer. Second, gravitational separation can be inhibited by reducing the size of the droplets in the emulsion, since the creaming velocity is proportional to the droplet size squared (Stoke’s Law). If the droplets are sufficiently small, then Brownian motion effects will dominate and the system will remain stable to creaming or sedimentation. Third, gravitational separation can be inhibited by increasing the viscosity of the aqueous phase, e.g., by adding thickening or gelling agents. This approach may not always be viable since it will also influence the texture and mouthfeel of the final product. Another approach some beverage manufacturers have used to mask the undesirable effects of creaming (“ringing”) on the appearance of a product is to design the packaging so as to obscure the effect, e.g., with appropriate placement of the labels or cap. 5.1.2. Droplet aggregation The aggregation state of the droplets in a beverage emulsion is important because it influences the stability of the product to gravitational separation. Changes in particle size during storage may also influence other important quality attributes of beverage products, such as their appearance (cloudiness or homogeneity). The tendency for droplet aggregation to occur in a beverage emulsion depends on the balance of attractive and repulsive forces operating between the droplets (see earlier). The nature of the colloidal interactions operating in a particular beverage emulsion depends on the physicochemical properties of the oil, water and interfacial phases (e.g., dielectric constant and refractive index), oil core characteristics (such as radius), interfacial shell characteristics (such as thickness, charge, packing, rheology and hydrophobicity), and the properties of the intervening fluid (such as pH, ionic strength, osmotic pressure, and temperature). To a first approximation the overall colloidal interactions between a pair of droplets in a beverage emulsion can be described by the sum of the van der Waals (wVDV), electrostatic (wE), and steric (wS) interactions (McClements, 2005):

wðhÞ ¼ wVDV ðhÞ þ wE ðhÞ þ wS ðhÞ

(5)

The van der Waals interactions are attractive, whereas the steric and electrostatic interactions are usually repulsive (Table 2). The van der Waals attraction operates between all kinds of droplets and would always cause aggregation if there were no opposing repulsive forces. The magnitude and range of the steric repulsion depend on the thickness and chemistry of the interfacial layer, whereas the magnitude and range of the electrostatic repulsion depend on the droplet charge (z-potential) and the ionic composition of the aqueous phase. To design a product that is stable to droplet aggregation one must assure that the repulsive interactions dominate the attractive interactions. This is usually achieved by using an emulsifier that generates repulsive interactions between the droplets. The emulsifiers used in the beverage industry typically stabilize the droplets against aggregation by generating steric and/ or electrostatic repulsive interactions. Emulsifiers that form relatively thick open interfaces (such as polysaccharides and non-ionic surfactants with large hydrophilic head-groups) can generate a steric repulsion that is sufficient strong and long range to overcome the attractive van der Waals interactions, and thereby stabilize the system against aggregation. Emulsifiers that form highly charged interfaces (such as proteins and ionic surfactants) can generate a strong electrostatic repulsion between droplets that prevent aggregation. However, emulsifiers that can only stabilize emulsions due to electrostatic interactions may be prone to instability when the pH or ionic strength is changed. Some emulsifiers use a combination of electrostatic and steric repulsion to stabilize the system, e.g., such as casein and whey proteins.

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The droplets in emulsions are in continual motion because of the effects of thermal energy, gravity, or applied mechanical forces, and as they move about they frequently collide with their neighbors. After a collision, emulsion droplets may either move apart or remain aggregated, depending on the relative magnitude of the attractive and repulsive interactions between them. Droplets aggregate when there is a primary or secondary minimum in the interaction potential that is sufficiently deep and accessible to the droplets (Fig. 4). The two major types of aggregation in beverage emulsions are flocculation and coalescence. 5.1.2.1. Flocculation. Droplet flocculation is the process whereby two or more droplets come together to form an aggregate in which the droplets retain their individual integrity (Fig. 2). Droplet flocculation is usually detrimental to beverage emulsion quality because it accelerates the rate of gravitational separation thereby reducing their shelf-life. Flocculation can also cause an appreciable increase in the viscosity of beverage emulsion concentrates, and may even lead to the formation of a gel. This may be undesirable since it would influence the transport, handling and dispersibility of the product. Flocculation may occur in beverage emulsions through a variety of different processes that either increase the attractive forces or decrease the repulsive forces operating between the droplets. The mechanism that is important in a particular emulsion depends largely on the nature of the emulsifier used and the solution conditions (e.g., pH, ion type and concentration, and functional ingredients). Reduced electrostatic repulsion: Electrostatically stabilized emulsions may flocculate when the electrostatic repulsion between the droplets is reduced. A number of physicochemical changes may cause this reduction in electrostatic repulsion (Israelachvili, 2011): (i) the pH is altered so that the net charge on the droplets is reduced; (ii) counter-ions bind to the surface of the droplets and reduce their charge (“charge neutralization”); (iii) the ionic strength of the aqueous phase is increased to screen the electrostatic interactions (“electrostatic screening”). Protein-coated oil droplets are particularly sensitive to flocculation due to reduction in the electrostatic repulsion between them when the pH or ionic composition is altered (Demetriades, Coupland, & McClements, 1997a; McClements, 2004). Increased depletion attraction: The presence of non-adsorbing colloidal entities in the continuous phase of an emulsion, such as biopolymers or surfactant micelles, generates an increase in the attractive force between the droplets due to an osmotic effect associated with the exclusion of the colloidal entities from a narrow region surrounding each droplet (Israelachvili, 2011). This attractive force increases as the concentration of colloidal entities increases, until eventually it becomes large enough to overcome the repulsive interactions between the droplets and causes them to flocculate. This type of droplet aggregation is usually referred to as depletion flocculation. The presence of relatively high concentrations of non-adsorbed biopolymer emulsifiers (gum arabic and modified starch) have been shown to induce depletion flocculation in model beverage emulsions (Chanamai & McClements, 2001). Depletion flocculation may also be promoted by other kinds of biopolymers that might be used in beverages, such as maltodextrin, pectin, xanthan gum, and carrageenan (Cao, Dickinson, & Wedlock, 1990; Cho & McClements, 2009; Gu, Decker, & McClements, 2004; Gunning, Hibberd, Howe, & Robins, 1988). Increased hydrophobic interactions: This type of interaction is important in emulsions that contain droplets that have some nonpolar regions exposed to the aqueous phase. A good example of this type of interaction is the effect of thermal processing on the flocculation stability of oil-in-water emulsions stabilized by globular proteins (Demetriades, Coupland, & McClements, 1997b). At room

15

temperature, whey protein stabilized emulsions (pH 7) are stable to flocculation because of the large electrostatic repulsion between the droplets, but when they are heated above 70  C they become unstable. The globular proteins adsorbed to the surface of the droplets unfold above this temperature and expose non-polar amino acids that were originally located in their interior. Exposure of these non-polar amino acids increases the hydrophobic character of the droplet surface and therefore leads to flocculation because of the increased hydrophobic attraction between the droplets. Formation of biopolymer bridges: Many types of biopolymer promote flocculation by forming bridges between two or more droplets. Biopolymers may adsorb either directly to the bare oil surfaces of the droplets or to the adsorbed emulsifier molecules that form the interfacial layer. To be able to bind to the droplets there must be a sufficiently strong attractive interaction between segments of the biopolymer and the droplet surface. The most common types of interaction that operate in food emulsions are hydrophobic and electrostatic (Dickinson, 2003). For example, a positively charged biopolymer (such as chitosan) might adsorb to the surface of two negatively charged emulsion droplets causing them to flocculate (Ogawa, Decker, & McClements, 2003) or a negatively charged biopolymer (such as pectin, carrageenan or xanthan) might adsorb to the surface of two positively charged droplets causing them to flocculate (Dickinson, 2003; Guzey & McClements, 2006). The development of a suitable strategy to prevent droplet flocculation in a particular beverage emulsion therefore depends on identification of the physicochemical origin of flocculation in that system. In general, flocculation can be prevented by ensuring that the repulsive forces dominate the attractive forces, and that there are no additives that can promote bridging. 5.1.2.2. Coalescence. Coalescence is the process whereby two or more liquid droplets merge together to form a single larger droplet (Fig. 2). Coalescence causes emulsion droplets to cream or sediment more rapidly because of the increase in their particle size. In beverage emulsions, coalescence eventually leads to the formation of a layer of oil on top of the material, which is referred to as oiling off. This process is one of the main reasons for the shiny oily layers often seen on top of unstable beverage emulsions. The susceptibility of a beverage emulsion to droplet coalescence is highly dependent on the nature of the emulsifier used to stabilize the system, since this instability mechanism involves two or more droplets fusing together. In general, the susceptibility of oil droplets to coalescence is determined by the nature of the forces that act between the droplets (i.e. gravitational, colloidal, hydrodynamic and mechanical forces) and the resistance of the interfacial layer to rupture. The stability of emulsions to coalescence can be improved by preventing the droplets from coming into close proximity for extended periods, e.g., by preventing droplet flocculation, preventing the formation of a creamed layer, or having too high droplet concentrations (McClements, 2005). Alternatively, one can control the properties of the interfacial layer surrounding the oil droplets to make it more resistant to rupture, e.g., by selecting an appropriate emulsifier or other additives that alter surface properties. 5.1.3. Ostwald ripening This susceptibility of a beverage emulsion to Ostwald ripening (OR) is mainly determined by the solubility of the oil phase in the aqueous phase: the higher the solubility, the more unstable the emulsion. Oil phases with very low water-solubilities (such as the vegetable oils used in clouding emulsions) do not exhibit OR, but oil phases with relatively high water-solubilities (such as flavor or essential oils) may be highly unstable. Mechanistically, OR is the

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process whereby the size of the oil droplets in an oil-in-water emulsion increases over time due to diffusion of oil molecules from small to large droplets through the intervening aqueous phase (Kabalnov, 2001; Kabalnov & Shchukin, 1992). The driving force for this effect is the fact that the water-solubility of an oil contained within a spherical droplet increases as the radius of the droplet decreases, which means that there is a higher concentration of solubilized oil molecules in the aqueous phase surrounding a small droplet than surrounding a larger one (Kabalnov & Shchukin, 1992; McClements, 2005). The presence of this concentration gradient means that solubilized oil molecules tend to move from the immediate vicinity of smaller droplets to that of larger droplets. This leads to an increase in mean droplet size over time, which can be described by the following equation once steady state conditions have been achieved (Kabalnov & Shchukin, 1992):

5

0% corn oil 2.5% corn oil

Mean droplet diameter (µm)

16

5% corn oil

4

10% corn oil

3

2

1 32 aSN Dt dðtÞ  dð0Þ ¼ ut ¼ 9 3

3

(6)

Here, d(t) is the number-weighted mean droplet diameter at time t, d0 is the initial number-weighted mean droplet diameter, u is the Ostwald ripening rate, a ¼ 2gVm/RT, SN is the water-solubility of the oil phase in the aqueous phase, D is the translational diffusion coefficient of the oil molecules through the aqueous phase, Vm is the molar volume of the oil, g is the oil-water interfacial tension, R is the gas constant, and T is the absolute temperature. The most important factor determining the stability of a beverage emulsion to OR is the water-solubility of the oil phase (SN) (Weiss, Herrmann, & McClements, 1999). For this reason OR is not usually a problem for emulsions prepared using oils with a very low water-solubility, such as long chain triglycerides (e.g., corn, soy, sunflower, or fish oils). On the other hand, OR may occur rapidly for emulsions prepared using oils with an appreciable water-solubility, such as flavor oils and essential oils (Li, Le Maux, Xiao, & McClements, 2009; McClements et al., 2012; Wooster et al., 2008). OR can be retarded in these systems by adding a substance known as a ripening inhibitor. A ripening inhibitor is a nonpolar molecule that is soluble in the oil phase but insoluble in the water phase, e.g., a long chain triacylglycerol (such as corn oil). This type of molecule can inhibit OR by generating an entropy of mixing effect that counter-balances the curvature effects. Consider an oil-in-water emulsion that contains droplets comprised of two different lipid components: a water-insoluble component and a water-soluble component. The water-soluble molecules will diffuse from the small to the large droplets due to OR. Consequently, there will be a greater concentration of waterinsoluble molecules in the smaller droplets than in the larger droplets after OR occurs. Differences in the composition of emulsion droplets are thermodynamically unfavorable because of the entropy associated with mixing: it is more favorable to have the two lipids distributed evenly throughout all of the droplets, rather than to be located in particular droplets. Consequently, there is a thermodynamic driving force that operates in the opposite direction to the OR effect. The change in droplet size distribution with time then depends on the concentration and solubility of the two components within the oil droplets. This approach has previously been used to improve the stability of food-grade nanoemulsions, such as those containing short chain triglycerides, essential oils, and flavor oils (Li et al., 2009; McClements et al., 2012; Wooster et al., 2008). An example of this effect is shown in Fig. 7 which shows that droplet growth in orange oil-in-water emulsions during storage can be inhibited by adding a sufficiently high concentration of corn oil (the ripening inhibitor) (McClements et al., 2012). Orange oil (4-fold) has a relatively high solubility in water, and therefore is highly prone to OR, which leads to an appreciable

0 0

5 10 Storage Time (day)

15

Fig. 7. Droplet growth due to Ostwald ripening of orange oil emulsions can be inhibited by adding a corn oil, which acts as a ripening inhibitor (McClements et al., 2012).

increase in mean droplet size during storage. On the other hand, corn oil has a very low solubility in water, and therefore it can retard OR if it is incorporated into the oil phase prior to homogenization. These results show that incorporating  10% corn oil into the oil phase was sufficient to inhibit OR in these systems (Fig. 7). OR may also be retarded by adding certain kinds of weighting agents (such as ester gums) since these substances also have a very low water solubility and therefore act as ripening inhibitors (Lim et al., 2011). 5.2. Chemical stability A number of lipophilic compounds that may be present in beverage emulsions can undergo chemical degradation during storage, which leads to a loss of color, flavor and/or nutrients. A few representative examples of chemical degradation of lipophilic components in oil-in-water emulsions are given below. Citrus degradation. Several mechanisms lead to the chemical decomposition of citrus flavor components (such as citral, d-limonene, and citronellal), including oxidation, hydrolytic reactions, the formation of terpene alcohols, and the cyclization of terpene aldehydes (Clark, Powell, & Radford, 1977; Kimura, Iwata, & Nishimura, 1982; Kimura, Nishimura, Iwata, & Mizutani, 1983a, 1983b). Acid-catalyzed decomposition and oxidation reactions change the desirable flavor profile of citrus oils by reducing the concentration of desirable flavor components and increasing the concentration of undesirable flavor components (Tan, 2004; Ueno, Masuda, & Ho, 2004). The beverage industry would therefore like to identify effective strategies for preventing these undesirable chemical degradation reactions. There has been a great deal of research on establishing the major factors that influence the chemical degradation of citral because this is one of the most important flavor compounds found in commercial beverages. The degradation rate of citral in aqueous solutions has been shown to increase with decreasing pH (Choi et al., 2009) (Fig. 8). Most commercial beverages have acidic aqueous phases and are therefore highly susceptible to flavor loss during storage due to this acid-catalyzed mechanism. The

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Citral Concentration (%)

0.05

0.04

0.03 pH 7 pH 6 pH 5 pH 4 pH 3

0.02

0.01 0

1

2 3 4 5 Storage Time (days)

6

7

Fig. 8. Influence of pH on the chemical degradation rate of citral in aqueous solutions: the degradation rate increases with decreasing pH (Choi et al., 2009).

chemical stability of citral has been shown to be much higher when it is located within an oil phase than in an aqueous phase (Choi et al., 2009). Consequently, the chemical degradation of citral in beverage emulsions can be improved by ensuring that the citral molecules are located primarily in an oil phase rather than in the aqueous phase. Indeed, studies have shown that citral stability can be improved by increasing the oil droplet concentration (Choi et al., 2009) or by adding surfactant micelles to the aqueous phase (Choi et al., 2010b), although these strategies may not be practical for most commercial products. It was proposed that citral stability may be improved by encapsulating it within solid lipid particles rather than within liquid oil droplets, however the opposite was found to be true experimentally, which was attributed to the expulsion of the citral molecules into the aqueous phase after droplet crystallization (Mei et al., 2010). Addition of various kinds of natural antioxidants to flavor oil emulsions has also been shown to improve the stability of citral to chemical degradation (Yang, Tian, Ho, & Huang, 2011). The oil droplets in beverage emulsions are surrounded by a coating of emulsifier molecules, and so it may be possible to improve the stability of the citral molecules within them by engineering the properties of the interfacial layer (Decker & McClements, 2001; Given, 2009). Indeed, studies have shown that citral degradation was faster in flavor oil droplets coated by an anionic surfactant than those coated by a non-ionic or cationic surfactant, which was attributed to differences in the accumulation of catalytic protons near the droplet surfaces (Choi, Decker, Henson, Popplewell, & McClements, 2010a). A high local concentration of protons is believed to accelerate the citral degradation mechanism at the droplet surfaces. Coating flavor oil droplets with a cationic biopolymer layers has also been shown to improve the stability of citral to chemical degradation (Djordjevic, Cercaci, Alamed, McClements, & Decker, 2007, 2008; Yang, Tian, Ho, & Huang, 2012). Polyunsaturated lipid degradation. There has been great interest in the beverage industry in fortifying products with u-3 lipids (such as flax, fish and algal oils) since these lipids have been claimed to have health benefits and are currently under-consumed by the general population. Nevertheless, there are many technical difficulties associated will incorporating these lipids into beverage products due to their high susceptibility to oxidation. Lipid oxidation affects the quality of emulsion-based products, influencing

17

their flavor, odor, and nutritive value (Frankel, Satué-Gracia, Meyer, & German, 2002). The oxidation of polyunsaturated lipids is a highly complex series of chemical reactions that is initiated when a lipid interacts with an oxygen reactive species, and proceeds through molecular cleavage and oxygen addition reactions to the formation of a wide variety of volatile compounds (McClements & Decker, 2000; Waraho, McClements, & Decker, 2011). The rate at which oxidation takes place is dependent on several factors: the molecular structure of the lipids; storage conditions; the presence of pro-oxidants and antioxidants; and the structural organization of the system. Based on this knowledge a variety of strategies have been developed to inhibit or prevent lipid oxidation in emulsified products: addition of oil-soluble and water-soluble antioxidants; chelation of pro-oxidant transition metals; engineering the interface to prevent pro-oxidants from coming into close proximity to lipid substrates; controlling environmental conditions, such as exposure to heat, oxygen, or light. Carotenoid degradation. Carotenoids are natural compounds found in many fruits and vegetables that are may be used in foods as colorants or nutraceuticals because of their potential health benefits (Mayne, 1996; Ryan, O’Connell, O’Sullivan, Aherne, & O’Brien, 2008). One of the major factors currently limiting the incorporation of carotenoids into many food and beverage products is their high susceptibility to chemical degradation. In particular, carotenoids have a conjugated polyunsaturated hydrocarbon chain that makes them highly prone to autoxidation (Boon, McClements, Weiss, & Decker, 2009). A number of factors have previously been shown to promote the oxidation of carotenoids, including highly acidic environments (Konovalov & Kispert, 1999), light (Mortensen & Skibsted, 1996), heat (Mader, 1964), singlet oxygen (Krinsky, 1998), transition metals (Gao & Kispert, 2003; Williams et al., 2001), and free radicals (Liebler & McClure, 1996; Woodall, Lee, Weesie, Jackson, & Britton, 1997). Once carotenoid degradation has been initiated a number of secondary reaction products may form, including epoxides, endoperoxides, apocarotenals and apocarotenones (Gao & Kispert, 2003; Woodall et al., 1997; Yamauchi, Miyake, Inoue, & Kato, 1993). The chemical degradation of carotenoids leads to color fading, and may reduce their beneficial health properties. Recent studies have examined the influence of interfacial properties (i.e., emulsifier type), storage conditions (i.e., pH, ionic strength, and temperature) and antioxidant addition (i.e., vitamin E, Coenzyme Q10, EDTA and ascorbic acid) on the chemical degradation of b-carotene encapsulated within oil-in-water nanoemulsions (Qian, Decker, Xiao, & McClements, 2012). The rate of b-carotene degradation was found to increase with decreasing pH and increasing temperature, was faster for a nonionic surfactant (Tween 20) than for a protein (b-lactoglobulin), and decreased with increasing antioxidant addition to either the oil or aqueous phase. 5.3. Defining the end of shelf life The end of the shelf life of a product can be defined as the time when it becomes unacceptable to consumers, which depends on the rate of the various physical and chemical instability mechanisms occurring. A product may become unacceptable when a ring of oil droplets is visible at the top of the bottle, when the flavor components decompose/oxidize and create an unacceptable flavor profile, when the color changes beyond an acceptable level, or when the product is microbiologically unsafe to consume. A beverage manufacture should establish quantitative criteria that can be used to establish the end of the shelf life of their particular product. They should then develop a systematic testing scheme that can be used to predict the shelf life of products.

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6. Beverage emulsion manufacture Beverage emulsions are usually prepared using a two-step process: a beverage emulsion concentrate (3e30 wt% oil) is prepared, which is then diluted extensively to create the finished product ( 1 the optimum curvature is concave (favoring W/O systems). For non-ionic surfactants, the head group is relatively large compared to the tail group (p < 1) at temperatures below the PIT and so O/W emulsions are favored. Upon heating, the head group becomes progressively dehydrated and so the packing parameter increases. At the phase inversion temperature (PIT), the head group and tail group have similar sizes (p ¼ 1) and so liquid crystals or microemulsions are formed. Above the PIT, the head group is relatively small compared to the tail group (p > 1) and so

22

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Fig. 13. Schematic diagram of formulation-composition map for a typical surfactant-oil-water system. This map can be used to understand the formation of emulsions and nanoemulsions suing various phase inversion methods.

W/O emulsions are favored. The relative solubility of nonsurfactants in the oil and water phases also changes with temperature because of head group dehydration, which has also been used to interpret the PIT phenomenon (Anton & Vandamme, 2009; Anton et al., 2007). At low temperatures, the head group is highly hydrated and so the surfactant tends to be more soluble in water. As the temperature is raised and the head group becomes progressively dehydrated and the solubility of the surfactant in water decreases, while its solubility in oil increases. At a particular temperature (zPIT), the solubility of the surfactant in the oil and water phases is approximately equal. At higher temperatures, the surfactant becomes more soluble in the oil phase than in the water phase. Thus at high temperatures the surfactant is mainly located in the oil phase, and when the system is cooled it has a tendency to move into the aqueous phase. Thus, oil droplets are formed by a mechanism similar to spontaneous emulsification. The PIT method is relatively straightforward to implement (Fig. 10). A mixture of surfactant, oil, and water (SOW) is initially heated up to a temperature around or slightly above the PIT, which leads to the formation of a microemulsion or liquid crystalline phase. The SOW system is then quench cooled to a temperature well below the PIT with continuous stirring, which leads to the spontaneous formation of an oil-in-water emulsion or nanoemulsion (Anton & Vandamme, 2009). An example of the phase behavior of a SOW mixture consisting of a non-ionic surfactant (13% Tween 80), a flavor oil (10 wt% lemon oil), and water (77%) upon heating and cooling is shown in Fig. 14 (Rao & McClements, 2010). Initially, the surfactant, oil and water were blended together to form a coarse O/W emulsion that was optically opaque. Upon heating the system becomes transparent when the PIT is reached, and then becomes opaque when heated above the PIT due to formation of a W/O emulsion. Upon cooling, the system goes from turbid to transparent indicating that a nanoemulsion was formed (d ¼ 45 nm). One of the limitations of the phase inversion temperature method is that the emulsions produced are often highly prone to

droplet coalescence when they are stored at temperatures approaching the PIT. This could be a problem in food and beverage applications that require some form of thermal treatment (such as pasteurization, sterilization or cooking) or that are stored at elevated temperatures (e.g., in warm or hot climates). Recently, we developed an approach to overcome this problem by forming emulsions using a non-ionic surfactant with a relatively low PIT, and then diluting the resulting emulsions in a solution containing another surfactant with a high PIT (Rao & McClements, 2010). This second surfactant partially displaces some of the original surfactant

Fig. 14. Change in turbidity when a coarse lemon oil emulsion (10% lemon oil, 13% Tween 80, 77% water) is heated and cooled. A very fine transparent nanoemulsion is formed when the system is heated, and then cooled below its phase inversion temperature. (Rao & McClements, 2010).

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23

from the droplet surfaces, thereby altering the optimum curvature and PIT of the surfactant monolayer, as well as increasing the repulsive interactions between the droplets. This approach could be used in the food and beverage industry to form very fine droplets using the PIT method, and then stabilizing them by diluting them in a different surfactant solution. The PIT method can only be used for certain combinations of oils and surfactants. The PIT must be within a practical range that can be implemented within the food industry (e.g. somewhere between 40 and 90  C) e if it is too high then it will not be possible to create emulsions, and if it is too low the emulsions formed will be highly unstable to droplet growth. The PIT tends to increase with increasing molecular weight and hydrophobicity of oil molecules, which means that for many triacylglycerol oils the PIT is too high to practically reach. On the other hand, flavor oils seem to be amenable to emulsification using the PIT method. Nevertheless, it is still important to select an appropriate type and amount of surfactant to ensure the PIT is in the correct range. 6.2.2.2. Emulsion inversion point (EIP) method. The emulsion inversion point (EIP) method can also be implemented very easily (Fig. 10). It simply involves titrating increasing amounts of an aqueous phase into an organic phase to induce a catastrophic phase inversion from a W/O to an O/W system. Initially, an organic phase is prepared that contains oil and a hydrophilic surfactant. The aqueous phase is then slowly titrated into this organic phase with constant stirring. As the amount of water added increases the system converts from a W/O emulsion, to an O/W/O multiple emulsion, to an O/W emulsion (Jahanzad, Crombie, Innes, & Sajjadi, 2009; Sajjadi, 2006). The formation of these multiple emulsions requires that the hydrophilic surfactant is initially located in the oil phase. At relatively low surfactant concentrations the formation of multiple emulsions is suppressed and only relatively large oil droplets are produced in the final emulsion, which are similar in size to those that would be produced if the surfactant had been dissolved in the water phase prior to homogenization (Jahanzad et al., 2009). At relatively high surfactant concentrations, multiple emulsions are formed during the titration process, and the final oil droplet size within the O/W emulsions is determined by the size of the inner oil droplets in the intermediate O/W/O emulsions. The value of the critical concentration where phase inversion occurs, as well as the size of the oil droplets produced, depends on process variables, such as the stirring speed, the rate of water addition, and the emulsifier concentration (Thakur et al., 2008). The emulsifiers used in catastrophic phase inversion are usually limited to small molecule surfactants that can stabilize both W/O emulsions (at least over the short term) and O/W emulsions (over the long term). We have recently used the EIP method to produce emulsions with relatively small droplet diameters (90%), whereas the major constituents in 10 lemon oil were monoterpenes (z35%), sesquiterpenes (z14%) and oxygenates (z33%). The concentration of both polar and non-polar components increased with increasing oil fold, while intermediate polar components decreased. The density, interfacial tension, viscosity, and refractive index of the lemon oils increased as the oil fold increased (i.e., 1