Rice Bran Wax Oleogel Water Holding Capacity and Its Effects on the Physical Properties of the Network

Home , Candelilla wax, Canola oil, Cocoa butter, Hazelnut, Microcrystalline wax, Mineral oil

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Erica Danielle Cramer

Graduate Program in Food Science and Technology

The Ohio State University

2016

Master's Examination Committee:

Professor Farnaz Maleky, Advisor

Professor Dennis Heldman, Advisor

Professor Yael Vodovotz

Copyrighted by

Erica Cramer

2016

Abstract

Over the past few years there has been much concern about the health effects of consuming saturated and trans fats. Therefore, the FDA revoked the GRAS status of partially hydrogenated oils in order to reduce trans fats in foods. The concern in the food industry is that it is not easy to replicate the structural attributes of foods provided by saturated and trans fats. Recently, studies introduced food-safe organogelators such as rice bran wax (RBW) to structure oil to mimic the behavior of solid fat. RBW oleogels have been tested as a saturated fat replacement in foods like ice cream and cheese.

However, the current research is focused on structuring pure lipid systems, and not much is known about structuring a mixture of oil and water. It would also be of interest to replace liquid oil with water and reduce some foods calories. Therefore, the objective of this project was to determine the water holding capacity of RBW oleogels, and to see how water and an emulsifier would affect the properties of the gel. Oleogel samples were made with RBW (10% w/w), glycerol monooleate (0 or 1.67%), and water (0, 5, 10 or 20

% w/w). Soybean oil made up the rest of the mixtures. Samples were prepared and stored at 5°C. Their water content, rheological properties and thermal behavior were tested over 27 days. They maintained their moisture content over the 27-day storage period. The storage and loss modulus, maximum force, and melting point of the gels remained consistent over the 27-day storage period. However, variations were seen between gels with different water contents. The addition of the emulsifier reduced these ii

variabilities. Microscope images showed that the wax had a needlelike structure and that the gels with higher water content had a higher frequency of larger water droplets. RBW oleogels can structure water and oil while maintaining the functional properties of a solid fat. These could be used to make foods with the same structural properties but reduced fat and calorie content.

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Acknowledgments

I would like to thank the Center for Advanced Processing and Packaging Studies and the

Ohio Agricultural Research and Development Center for their financial support on this project. I would like to thank Tanya Whitmar for her help with NMR, Steve Moeller for his help with TPA, and Norman St-Pierre for his help with statistics. I would like to thank Dr. Yael Vodovotz, Dr. Dennis Heldman, and Dr. Farnaz Maleky for their recommendations and their encouragement through this research project. Finally, I would like to thank Susan Cramer, Bryan Willett, and Nora Driscoll for all the amazing back-up support you provided me through my master’s program.

Vita

June 2010 ...... A. C. Reynolds High School

December 2013 ...... B.S. Food Science and Nutrition, The Ohio

State University

August 2014 to present ...... Graduate Research Associate, Department

of Food Science and Technology, The Ohio

State University

Fields of Study

Major Field: Food Science and Technology

Table of Contents

Abstract ...... ii

Acknowledgments...... iv

Vita ...... v

Fields of Study ...... v

Table of Contents ...... vi

List of Tables ...... viii

List of Figures ...... ix

Chapter 1: Introduction ...... 1

Chapter 2: Literature Review ...... 4

2. 1: Health impacts of fatty acids ...... 4

2.2: Methods for replacing trans and saturated fats in foods ...... 7

2.3: Oleogels...... 11

2.4: Recent advances of oleogels in foods ...... 18

2.5: Emulsions ...... 23

2.6: Measuring the properties of an emulsion ...... 27

Chapter 3: Materials and Methods ...... 34

3.1: Materials ...... 34

3.2: Oleogel production ...... 34

3.3: Moisture content...... 36

3.4: Nuclear magnetic resonance ...... 36

3.5: Melting properties ...... 37

3.6: Solid fat content ...... 37

3.7: Rheology ...... 38

3.8: Texture profile analysis ...... 38

3.9: Microstructure ...... 38

3.10: Statistical analysis ...... 39

Chapter 4: Results and Discussion ...... 41

4.1: Preliminary Study: Properties of oleogels made with 2% and 5% wax ...... 41

4.2: Moisture content of gels made with 10% RBW...... 45

4.3: Microstructure properties ...... 51

4.4: Physical properties ...... 66

Chapter 5: Conclusions ...... 85

References ...... 86

Appendix A: Data for low wax oleogel measurements ...... 94

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List of Tables

Table 1. Moisture content measured for samples after 1 day in storage. Samples A-D do not contain emulsifier, samples E-G do...... 48

Table 2. Solid fat content of gels without and with emulsifier, measured at 5°C...... 52

Table 3. Enthalpy of gels made with increasing water content...... 57

Table 4. G” in KPa of samples, comparing effects of water content and storage time, without and with emulsifier. Letters compare the effect of water content, while numerals compare storage time...... 80

Table 5. Tan delta of samples, comparing effects of water content and storage time, without and with emulsifier. Letters compare the effect of water content, while numerals compare storage time...... 80

Table 6. G* in KPa of samples, comparing effects of water content and storage time, without and with emulsifier. Letters compare the effect of water content, while numerals compare storage time...... 81

viii

List of Figures

Figure 1. Shape of a saturated and an unsaturated fatty acid. The unsaturated acid is bent, which contributes to its lower melting point and differing textural properties...... 6

Figure 2. A cryo-TEM micrograph of tubules formed by β-sitosterol and γ-oryzanol in a corn oil emulsion. Source: (Duffy and others)...... 14

Figure 3. The polyhedral structure of a protein oleogel formed via spray-drying of a β- lactoglobulin-stabilized water-in-oil emulsion. Source: (Romoscanu and Mezzenga

2005)...... 16

Figure 4. Set-up used for creating gels...... 35

Figure 5. Representative data for strain sweep measurements, using 5% RBW, 20% water, and emulsifier after 1 day in storage...... 42

Figure 6. G' of gels made with 2% wax and 0, 5, 10, and 20% added water content...... 43

Figure 7. G' of gels made with 5% wax and increasing water content...... 44

Figure 8. Measured moisture content of samples over 27 day storage period without emulsifier...... 47

Figure 9. NMR readings of rice bran wax oleogels made with varying water contents and emulsifier levels...... 50

Figure 10. Representative DSC graph showing 10% water...... 55

Figure 11. Peak melting point of gels made with increasing water content...... 55

Figure 12. Microscope images of gels made with different concentrations of water (0-

20%) without and with emulsifier, taken at 10x magnification...... 59

Figure 13. Microscope images of gels made with different concentrations of water (0-

20%) (a) without and (b) with emulsifier, taken at 10x magnification at 35° light angle 62

Figure 14. Gel made with 10% water, no emulsifier, cooled at 3°C per minute. After samples hit 67.5°C, the microscope was refocused to better observe the whole field of crystals...... 64

Figure 15. Example of TPA graph using 10% water after 27 days of storage...... 67

Figure 16. Force measurements compared by water content (a) without and (b) with emulsifier...... 69

Figure 17. Force measurements compared by storage time (c) without and (d) with emulsifier...... 70

Figure 18. Representative graph (5% w/E day 1) showing G’ and G” values measured over increasing strain...... 75

Figure 19. G’ of samples (a) without and (b) with emulsifier, comparing different water contents on the same day...... 76

Figure 20. G’ of samples (a) without and (b) with emulsifier, comparing storage time on the same water content...... 79

Figure 21. Yield point of RBW oleogels made with increasing water content and stored over a 27-day period, (a) without and (b) with emulsifier...... 82

Figure 22. Moisture content of samples made with (a) 2% and (b) 5% wax over 2 days storage...... 95

Figure 23. Tan delta of gels with (a) 2% wax without and (b) with emulsifier, and (c) 5% wax without and (d) with emulsifier...... 96

Chapter 1: Introduction

There are many favorite foods that are made with saturated and trans fats. These fats can make fluffy cakes and flaky pastries. They provide creaminess to ice cream and dairy products, and improve the texture of meat products. Unfortunately, there are also health risks associated with the consumption of saturated and trans fats. While research is still underway to understand the connection between dietary saturated fat and cardiovascular health, current dietary guidelines for Americans recommends reducing the consumption of saturated fats (USDA and DHHS 2015). Trans fats have a more established link to cardiovascular health risks (Mozaffarian 2006). In 2015, the Food and Drug

Administration declared that partially hydrogenated oils would no longer be considered

GRAS, meaning that this major source of trans fats will no longer be allowed to be used in foods after 2018 (FDA 2015). Therefore, new methods are needed to provide the desired structure to foods without these ingredients.

There are different methods currently used to replace saturated and trans fats. Replacing them in foods with water or sugar can compromise the structural or nutritional aspects of the foods. Starches and gums can provide some structure, but they may not offer the same mouthfeel properties, or they have off flavors. Protein products can offer some of the binding properties of fats, but they can be less effective and have off flavors. Lipid 1

modification techniques like interesterification can provide some of the structure and mouthfeel properties, but they may not reduce saturated fat content to the desired levels.

A new lipid modification technique has been gaining interest in the food industry. This technique results in oleogels, which are gels that bind edible oils. The gels are bound with small quantities of products called organogelators. Many different organogelators have been tested for use with edible oils, including sterols, proteins, cellulose derivatives, and waxes. A number of these products have been tested for use in foods. For example, ethylcellulose has been tested in frankfurters, and waxes have been used to structure oils for margarines, cookies, ice cream, and cheese.

One organogelator that has shown promise is rice bran wax (RBW). RBW is already approved for use in foods, particularly as protection for fruit (US FDA 2015b). It is a byproduct of rice manufacture: when white rice is separated from its bran, the bran can be processed for oil, which can be fractionated to obtain wax. Because rice is consumed in large quantities worldwide, it is fairly inexpensive to obtain RBW. Therefore, it would be desirable to food manufacturers. RBW can also gel oils at low quantities, with less than 3% w/w in oil to gel most oils (Dassanayake and others 2009). The amount of wax used in the total food product would also be fairly low. It has demonstrated effects in products including ice cream and cheeses (Zulim Botega 2013; Limbaugh 2015; Huang

2015).

The effects of RBW on gelling oils are established, but there is less information on how water would interact with this oleogel system. There is the question of how well the wax could stabilize a water-in-oil emulsion, which is a system seen in many food products.

Another way to look at the question would be to ask to what extent oil could be replaced with water in the gel. Not only would this help determine the limits of the emulsion, but this information could also be useful to create an oleogel with a lower overall fat content.

If oil can be replaced with water in the gel, then the same structural properties could be maintained with less overall fat and calories. The degree to which oil can be replaced with water in the gels can be referred to as the water holding capacity.

The objectives of this research determine the water holding capacity of RBW oleogels, and to quantify the physical properties of the water-added RBW oleogels. The extent to which oil could be replaced with water in a RBW gel was measured. Observations were made on how increasing the concentration of water would affect the physical and microstructural properties of the gel. In addition, an emulsifier was added to determine how that emulsifier could change the structural properties of the gel, and potentially enhance the interactions that occur between the oil/RBW and water.

Chapter 2: Literature Review

2. 1: Health impacts of fatty acids

Cardiovascular disease (CVD) is the leading cause of death in the United States (Centers for Disease Control 2015). Obviously, there is a desire to learn what steps can be taken to try to prevent death in this manner. There has been much research on the effect of dietary fat on the risk of CVD.

2.1.1: Saturated fat

There is debate on the impact that dietary intake of saturated fatty acids (SFA) has on

CVD risk. Epidemiological studies indicate that places where people consumer more dietary SFA show more incidence of CVD compared to places where SFA intake is reduced (Erkkila and others 2008). There is evidence that reducing SFA intake can decrease the ratio of total cholesterol to high-density lipoprotein (HDL) cholesterol (Jebb and others 2010). However, others studies question the impact of SFA on a variety of other markers for CVD (Mensink and others 2003, de Lorgeril and Salen 2012).

Potential links have also been found for SFA intake on insulin sensitivity, contributing to

Type II diabetes, and on risk for colorectal cancer (Pedersen and Kirkhus 2011).

However, more research is being done to confirm the validity of these links. Enough concern has been raised about the health effects of saturated fat that as the United States 4

Department of Agriculture (USDA) and the Department of Health and Human Services still recommend that Americans work to reduce their intake of dietary saturated fat

(USDA and DHHS 2015).

2.1.2: Trans fat

Trans fats have advantages in bakery products, which will be discussed later. They can also be found in small quantities in meat. The consumption of trans fats has been shown to raise low-density lipoprotein (LDL) levels, which is a marker for increased risk of

CVD. They also increase the ratio of total cholesterol to HDL cholesterol, another risk marker for CVD (Mozaffarian 2006). In 2015, the Food and Drug Administration (FDA) determined that partially hydrogenated oils (PHOs) are no longer Generally Recognized as Safe (GRAS) (FDA 2015). The loss of GRAS status means they may no longer be used in foods, and the food industry has been given three years to comply with the new regulations. Since PHOs are the primary source of trans fats in bakery goods and confections, there is a great need for products that will emulate the properties that those

PHOs provided.

2.1.3: Unsaturated fat

Historically, consumers have looked for “low-fat” or “reduced-fat” options on the market, but this approach ignores the fact that there are benefits to consuming unsaturated fats. Multiple studies have indicated that reducing saturated fat intake in favor of unsaturated fats can be beneficial in raising HDL cholesterol and improving the ratio of

total cholesterol to HDL (Mensink and others 2003, de Lorgeril and Salen 2012,

Mozaffarian and others 2010). Therefore, there is some interest in the food industry for reformulating products using unsaturated fats instead of saturated fats.

2.1.4: Role of saturated and trans fats

Saturated fatty acids are organic acids with long hydrocarbon tails. Those tails have only single bonds. In other words, every carbon on the tail has the maximum number of hydrogens: it is saturated with hydrogen. The structure of saturated and an unsaturated fatty acid can be seen in Figure 1.

Figure 1. Shape of a saturated and an unsaturated fatty acid. The unsaturated acid is bent, which contributes to its lower melting point and differing textural properties.

The structure of saturated fats influences properties such as the melting point of the fat, the crystalline structure, and the physical properties of the overall product. These properties impact different attributes of the foods that the fats are in. It provides texture to baked goods and dairy products (de Hoog and others 2011). It acts as a lubricant, which is useful for creating flaky pastries and aerating ice cream (van Aken 2007). The

lubrication effect improves the mouthfeel of the product. Fats can carry lipid-soluble flavor elements to the food. The fats can lubricate gluten chains, preventing long chains from forming and inducing the desired texture in baked goods. Trans fatty acids are fatty acids that have a double bond on the carbon tail that is arranged in a trans conformer.

Since the tail has a double bond, it has some more stability than its saturated counterpart.

When comparing fatty acids with the same carbon chain length, the saturated fat generally has the highest melting point, the trans fat has the second highest, and the cis fat has the lowest. Trans fats appear in foods in one of two ways. They can be a product of partially hydrogenated oils, or they may occur naturally in meats. The advantage of partially hydrogenating oils is that they can be customized so that specific properties can be achieved. These properties can influence the flakiness, hardness, volume, texture, and mouthfeel of bakery and other food products (Kodali 2014).

2.2: Methods for replacing trans and saturated fats in foods

Replacing saturated and trans fats in foods is challenging. Those fats provide specific functionalities to the foods that they are in, and any replacements must match those functionalities in foods.

2.2.1: Carbohydrate-based methods

There are different methods that can be used to mimic the structure of saturated fats is foods. Initially, some manufacturers may reformulate their products by reducing total fat content and filling the bulk with water or sugar. Meat products that replace fat with

water are often more rubbery than the full-fat product, which is undesirable to consumers

(Berry and Leddy 1984). Bakery products that replace fat with sugar lose the nutritional benefits for which they might have been aiming (Atkinson 2011).

Starches, fibers, and gums often get used to try to replicate the properties of saturated fats in food. Starches form gels when heated in the presence of water, and these gels can trap water to make a moister product. Potato starch has been used in ground beef and frankfurters to improve the texture properties of those products (Berry and Wergin 1993,

Carbello and others 1995). Starches have been tested in bakery products, but they bind water that may need to evaporate for a fluffy product, so they can lead to an undesirable texture (Sudha and others 2007). In ice cream, the final product is often harder than the one made with full fat (Underdown 2011). Different fibers have been tested, as they can also bind water in foods. Oat fibers have been used in ground meat products, and while they absorbed the water in the cooked product, there was difficulty in binding in the raw mix (Barbut 2011). Oat fiber, pea fiber, and starches were tested in bologna, resulting in a product that was grainier and less juicy than the full-fat product (Claus and Hunt 1991).

Gums like xanthan, locust bean, guar, and carrageenan have been tested in a variety of food applications. They can improve the mouthfeel of certain products as they bind water, but they often melt at high temperatures in a way that is undesirable in the final product. Xanthan gum was found to be a superior gum to use in frankfurters when compared to carrageenan, locust bean gum, and low methoxy pectin due to its resistance to temperature effects (Wallingford and Labuza 1983). However, it was also found that

alginate and xanthan gums do not offer the gel strength to the frankfurter emulsion that fat offers (Whiting 1984). Carrageenan was tested in conjunction with oat fiber in frankfurters, and while the product held water, the texture was not as accepted by consumers as was the full fat product (Cofrades and others 2000). Gums have also been tested for their use in certain bakery products. They help maintain the moisture content of the dough and allow it to be processed easily (Atkinson 2011).

2.2.2: Protein-based methods

Protein products have also been considered for replacing the functionality of saturated fats in foods. Egg white was used in frankfurters, where it had better mouthfeel and texture results than a starch-based product, but it did not help the binding properties of the frankfurter (Carbello and others 1995). Soy protein was also tested for its properties in frankfurters. The end product was less hard and fracturable than the full fat product

(Claus and Hunt 1991). Whey-based products have been tested for use in ice cream, but they can throw off the flavor of the final product (Welty and others 2001).

2.2.2: Emulsifier-based methods

Certain products known for their emulsifying properties have been tested for their ability to reduce the overall fat content of food products. In bakery products, emulsifiers can reduce the size of the fat droplets that surround gluten chains, so that less fat is required to achieve the same shortening effect (Atkinson 2011). Mono- and diglycerides are a popular choice for this type of application, and lecithin has been used as well.

Emulsifiers have been used in ice cream products as well, although the end product can have a less desirable texture than the full-fat product (Underdown 2011).

2.2.4: Fat-based methods

Different techniques have been used regarding fat for reducing saturated fat content in foods. Simply replacing a saturated fat with an unsaturated alternative can yield undesirable properties in the final food product. Frankfurters using oil as a substitute for solid fat are harder and more rubbery (Barbut 2011). Bakery products run the risk of oil migration, where oil may seep out of the food and into the surroundings, which is an undesirable quality attribute (Atkinson 2011). In addition, they may not provide the lubrication and leavening effects that a solid fat would provide (de Cindio 2011).

Originally, the partial hydrogenation of oils was used to obtain products with less saturated fat that would still have the properties of a regular saturated product. However, as information about the health effects of the resulting trans fats came to light, this method was no longer feasible for use in the food industry. Bakery products depend on solid fat to have the right texture, hardness, and mouthfeel properties. Proper fat addition also makes sure the product will expand during baking, but still be soft after that process

(de Cindio 2011). Structured water-in-oil emulsions have been used to try to replicate the effects of butter, which is high in saturated fat. These products can have significantly less fat than butter, but still maintain some of the physical and rheological properties of the original product. Another method for structuring liquid oils is interesterification.

Lipids in foods are mostly triglycerides, which consist of three fatty acids on a glycerol

backbone. With interesterification, fatty acids are rearranged on the glycerol backbone in order to change the properties of the final product. The fatty acid composition may be consistent, but if saturated fatty acids are arranged closer together than in the original oil, it can alter the melting point and other physical properties of the fat (Criado and others

2006). A third technique for increasing unsaturated fat relative to saturated fat is to mix fats together. There are a number of butter and oil mixtures available on the market, advertised as spreadable butters. There are potential applications here for customizing the properties of a fat that can be used in different applications.

2.3: Oleogels

A recent technique that has shown promise as an alternative to saturated fats in foods has been the development of oleogels. To create an oleogel, small quantities of certain additives are added to edible oils. These additives, known as organogelators, impart specific qualities to the oil that it would not have otherwise. The organogelators generally form a network that provides structure to the gel. These gels are thermoreversible, so that after they are formed they can be remelted and cooled as needed. They are semisolid, and the strength of the gel is usually related to the concentration of the organogelators. The melting point of the gels depends on the organogelator, and it depends on the concentration of the organogelator used. When used in food products, the oleogels offer a fatty acid profile that resembles the liquid oil that was used. However, the semisolid nature of the gel has attributes closer to a saturated or trans fat product. Therefore, these gels can provide the physical structures to food that

are usually provided by the saturated or trans fats, but they can potentially provide the health effects associated with unsaturated fats. The concentration of organogelator needed to gel an oil is often very small, around 1% w/w (Marangoni 2012). The concentration of the organogelator in the final food product is therefore much smaller.

Some organogelators have already been approved for use in foods in specific applications or concentrations, while others await GRAS status. For the organogelators that currently have GRAS status, the small concentrations should be within the guidelines that would be allowed for those products. Research into the actual health effects of oleogels is in progress.

Different organogelators use different mechanisms to structure the liquid oil. Some organogelators self-assemble into small particles that can structure gels. Some organogelators form networks that can trap the liquid oil in a matrix. Wax-based organogelators can form crystals that interact with the liquid oil that shape the gel.

2.3.1: Self-Assembling Organogelators

Some organogelators self-assemble into fibers that can be used to structure gels. 12-

Hydroxystearic acid (HSA) is a long chain organic acid with a hydroxyl group on the twelfth carbon in the chain. It has been used for lubricating grease, as well as a technique for cleaning oil spills (Tamura and Ichkawa 1997). It can do this because it forms a fairly solid gel at a concentration as low as 1% w/w (Marangoni 2012). The reason why 12-

HSA works is because it forms crystals along a specific dimension, forming a stacked v-

shape with a bend at a 117° angle (Kuwahara and others 1996). When these crystals reach a certain concentration, they start to self-assemble into a fiber network. This network can then use capillary force to trap the solvent that it is in, and it imparts solid- like properties to the resulting gel. 12-HSA is not currently approved for use in food, but its potential as an organogelator has stimulated interest in determining if that approval can be obtained (Marangoni 2012).

Phytosterols, which occur naturally in plants, have shown promise as organogelators.

This interest is compounded by their potential to reduce LDL cholesterol (Katan and others 2003). The most common phytosterols that have been reported for use are β- sitosterol and γ-oryzanol (Zinic and others 2005). It has been found that a combination of

40% β-sitosterol and 60% γ-oryzanol is ideal for creating tubules that provide structure to the liquid oil (Chen and Terentjev 2009). An example of a phytosterol gel can be seen in

Figure 2.

Figure 2. A cryo-TEM micrograph of tubules formed by β-sitosterol and γ-oryzanol in a corn oil emulsion. Source: (Duffy and others).

Ceramides are compounds that have also been shown to gel oils. They are a type of lipid that contains the amino alcohol sphingosine. They can be found in the intercellular spaces in the epidermis, which influences the permeability of the epidermis (Imokawa and others 1991). They also act as chemical messengers relating to cell division and cell death (Selzner and others 2001). Rogers and others (2009) used synthetically formed ceramides as well as those synthesized from milk and egg products to test in vegetable oil. It was found that the shorter the chain on the ceramide, the greater the gelation effect; with C-2 chains, only 2% w/w was needed for a gel to form, but for longer chains, at least 5% w/w was needed for gelation. The ceramides structured the gel by crystallizing in a hexagonal arrangement (Raudenkolb and others 2003). The pure ceramide samples, which contained only one chain length, formed fibrillar or needle-like structures (Rogers and others 2009). However, the samples that contained a mix of chain lengths, or those obtained from the milk and egg products, yielded spherical crystals. 14

The change in crystal structure could be an explanation for why a higher concentration of those compounds was required for gelation.

Lecithin is an emulsifier that is generally removed during the degumming step of oil processing. Therefore, it is reasonable to assume that lecithin could structure oil to form an oleogel (Shchipunov 2001). Unpurified lecithin cannot gellate an oil in any concentration, so other techniques are needed. Lecithin has been shown to gellate other organic solvents if it is purified to about 95% phosphatidcholine, so it has the potential to gel an edible oil under the same circumstances (Scartazzini and Luisi 1988). Lecithin can work in conjunction with sorbitan esters like sorbitan tri-stearate (STS). It has been shown that when lecithin is combined with STS in a ratio of 2:3 or in 3:2 that it can form a gel, using as little as 4% structurant (Pernetti and others 2007).

2.3.2: Network-Forming Organogelators

Certain organogelators function by forming a network that can trap oil. Proteins have been used to form such networks. Protein-stabilized emulsions have been created and dried as a technique to create an organogelator. Mezzenga (2011) made an emulsion consisting of paraffin oil or olive oil mixed with protein-rich water, using β-lactoglobulin as the protein. The proteins were then cross-linked by either thermal or chemical means.

The emulsion was then dried to 0.25% water content, leaving proteins cross-linked on the surface of the oil droplets. The cross-linked proteins provided the structure for the gel.

The resulting dried gel was reversible and rehydratable. However, gels made with

unrefined olive oil were less reversible, perhaps due to the impurities in the oil. The proteins were found to create a polyhedron structure (Romoscanu and Mezzenga 2006).

The rheological properties of these gels indicated that the protein structure had foam-like properties. An example gel can be seen in Figure 3.

Figure 3. The polyhedral structure of a protein oleogel formed via spray-drying of a β- lactoglobulin-stabilized water-in-oil emulsion. Source: (Romoscanu and Mezzenga 2005).

So far, only one polymer product has been found to be an effective organogelator. Most carbohydrates are water-soluble, so they have limited interactions in hydrophobic environments. Ethylcellulose has been demonstrated as effective in edible oils (Dey and others 2011, Laredo and others 2011). It is synthesized from cellulose. Ethylcellulose is typically dissolved in oil by heating it above its glass transition point, around 125-130°C.

Because of this high temperature, research has been done to investigate the effects of oil oxidation on the structure of the gels (Gravelle and others 2012). In general, the higher the molecular weight of the ethylcellulose, the stronger the gel. There is also a 16

relationship between the number of double bonds in the oil and the strength of the gel.

Flaxseed oil gels have a higher elastic modulus than those made with canola oil (Zetzl and others 2012). Ethylcellulose creates a coral-like structure with many small pockets that trap the oil. The resulting gels are very firm, especially at higher temperatures.

Ethylcellulose has been tested in different foods, which will be discussed later.

2.3.3: Wax-Based Organogelators

Wax is an ester that is formed by the combination of a long-chained alcohol and a fatty acid. Waxes tend to have long hydrocarbon chains, which result in fairly stable structures. A number of different waxes have been tested as organogelators, and different waxes offer different properties. Yilmaz and Ogutcu (2014) have used beeswax and sunflower wax to gel olive oil as a method for creating a spreadable olive oil product.

Candelilla was comes from a Yerba evergreen shrub, and it can gel an oil with 2% w/w concentration. If the sample is held at less than 5°C, it can gel at an even lower percentage (Morales-Rueda and others 2009). If the sample is heated and cooled repeatedly, it will show the same melting point and energy each time, demonstrating the thermoreversability of this product (Toro-Vazquez and others 2007). Like with all wax organogelators, a higher concentration will lead to a higher melting point of the oleogel.

Carnauba wax comes from a Brazilian palm plant (Copernicia prunifera), and it is often used for polishing and high-value applications. At least 4% w/w (Dassanayake and others 2009) is needed to gel oil.

Rice bran wax has shown great potential as an organogelator. It comes from dewaxing rice bran oil, which is a side product that results from polishing rice. This wax is found abundantly in Asia, which contributes to its inexpensiveness (Wolfmeier and others

2005). It has GRAS status and is currently approved for use as a coating for candy and fruit (US FDA 2015b). The properties of RBW as an organogelator have been studied extensively by Dassanayake and others (2009). They found that 0.5% w/w was enough to gel oil, which is much lower than the amounts needed for candelilla and carnauba wax.

The crystals formed by RBW in an oleogel are long and needle-like, whereas those or candelilla and carnauba wax are more sphere-shaped (Dassanayake and others 2009).

The needle-like crystals contribute to the structuring abilities of the wax, because they enable more liquid oil to be trapped in the structure. When comparing waxes at 6% w/w concentration, RBW oleogels were harder than those made with candelilla or carnauba wax. Studies of the melting properties indicate that the higher the concentration of wax in the gel, the more the melting properties resemble those of the pure wax. Because

RBW is easy to obtain, inexpensive, and useful in small quantities, it has been tested in several food products as a replacement for the functionality of saturated fat. These properties also led RBW to be the focus of this research study.

2.4: Recent advances of oleogels in foods

There are many products that can be used as organogelators, but it is important to determine if they can actually be used in foods.

2.4.1: Frankfurters

In the meat industry, saturated fats provide plasticity to comminuted meat products, such as frankfurters. Youssef and Barbut (2009) found that frankfurters made with canola oil had higher hardness values than products made with beef fat, which resulted in a more rubbery product that would not appeal to consumers. Ethylcellulose was used to gel canola oil (10% ethylcellulose in oil) to be used as the fat in a new frankfurter formulation (Zetzl and others 2012). It was found that the oleogels produced hardness and chewiness values comparable to the frankfurters made with beef fat, while canola oil values were higher. In addition, fat globule sizes were more similar between the beef fat and the oleogel, while those from canola oil were much smaller. The ethylcellulose stabilized the canola oil enough to prevent fat globules from breaking down during processing. This study shows that ethylcellulose could be used to create a frankfurter product with a fatty acid profile closer to that of canola oil.

2.4.2: Bakery Goods

Saturated fat has a shortening effect in baked goods. These effects include desired tenderness and texture, mouthfeel, lubrication of gluten and starch, incorporation of air, and improved shelf life (Ghotra and others 2002). There is an interest in reducing saturated fat and trans fat in baked goods, even though they do provide these structural benefits. Patel and others (2014) used methylcellulose and xanthan gum to gel sunflower oil for cakes that were compared to cakes made with margarine and shortening. It was found that the cake batters made with oleogel provided a batter with rheological

properties that matched the margarine and shortening batter than the cake batter made with straight oil. The cake also showed hardness values similar to margarine after it was stored, while the cake made with oil was much harder. Jang and others (2015) made cookies using candelilla mixed with canola oil at 3 and 6%. When compared to cookies made with shortening, the two wax concentrations resulted in cookies that were less firm, and they spread more during the baking process. However, the fatty acid profile yielded

90-92% unsaturated fat, while the shortening cookies had about 47% unsaturated fat.

Consumer acceptability of these cookies was not studied, but Yilmaz and Ogutcu (2015) did another study that covers consumer acceptance of cookies made with oleogels. They made gels using 5% beeswax or sunflower wax mixed with hazelnut oil. The cookies made with shortening had higher hardness and lower fractionability than the ones made with the oleogels. A sensory panel noted the differences between the cookies made with the shortening versus those with the oleogels. However, in a hedonistic test, they rated the cookies with the oleogels as being more preferable than the ones made with shortening. Therefore, oleogels do have the potential to be used as acceptable substitutes for shortening in baked goods, and they can be used to create a fatty acid profile that will be more desirable to consumers.

2.4.3: Margarine

Margarine is a product often used as a substitute for butter. It is required to contain at least 80% fat (US FDA 2015a). Other commercial spreads can be made with lower fat contents, and they will often have different spreadabilities. Margarines are traditionally

made with hydrogenated oils, but with concerns about trans fats from the hydrogenation process, other methods have been researched. Oleogels could be used to simulate the structural properties found in margarine and other spreads. Beeswax and sunflower wax were combined with olive oil to produce gels that were compared to commercial spreads

(Yilmaz and Ogutcu 2014). It was found that the gelled olive oil showed hardness and stickiness values comparable to the spread with 7% beeswax or with 3% sunflower wax.

Hardness and stickiness relate to the effect experienced by the consumer who spreads the product before consumption. Hwang and others (2013) used different waxes to gel soybean oil to produce an actual margarine substitute. It was found that sunflower wax produced the firmest margarine that was comparable to the commercial products, while

RBW produced a softer margarine. The melting points of the products with the waxes were higher than those of the commercial spreads due to the melting point of the waxes.

More work would be needed to determine if the products could pass consumer standards.

2.4.4: Ice Cream

Another dairy product that could benefit from oleogels is ice cream. Ice cream utilizes milkfat, whose triglycerides have a broad melting range between -40 and 40°C, to crystalize and form a network around the air bubbles that get whipped into the product, which helps stabilize the ice cream structure (Goff 1997). The stabilization effect is influenced by the presence of saturated fats in the milkfat. The use of unsaturated fats in ice cream could produce a product that appeals to consumers for its potential health benefits. Zulim-Botega and others (2013) worked on developing an ice cream

formulation that used RBW to replace the solid fat normally found in ice cream. RBW and high-oleic sunflower oil were combined (10% wax in oil), then added to the ice cream mix before the freezing procedure. Ice cream that uses straight oil as a substitute for solid fat melts much faster than that made with milkfat. Ice cream made with RBW and other emulsifiers melted more slowly than those made with just oil, although milkfat still had the slowest melting rate. RBW was shown to be superior to candelilla wax and carnauba wax for this purpose. The RBW produced larger fat droplets in the finished product, closer in size to those from the milkfat. This may contribute the reduced melting rate of the wax-based formulation.

2.4.5: Cheese

Cheese is another dairy product that depends on saturated fats for structure and texture properties. Recent work in our labs has been done to develop processed cheese and cream cheese products that use oleogels to replace the functionality of saturated fats in the product. For the processed cheese, soybean oil was gelled by RBW or sunflower oil, then combined with Swiss cheese for a product that had less saturated fat. The concentration of wax influenced the hardness, oil-binding capacity, and melting properties of the processed cheese (Huang 2015). For the cream cheese, RBW or ethylcellulose were mixed with soybean oil to produce a cream cheese product that was compared to the commercial products. It was found that the oleogel cream cheese products had about 25% less saturated fat than the commercial products. The hardness, stickiness, and rheological properties of the oleogel cream cheeses were comparable to

the commercial products made with full saturated fat (Limbaugh 2015). Consumer acceptance of these products is still under study. These products show that oleogels have the potential to develop cheeses that will have a fatty acid profile that will be more desirable to consumers.

2.5: Emulsions

2.5.1: Emulsions in foods

Oil and water are naturally immiscible. Attempts to combine the two result in a layer of oil on top of a layer of water. The reason for this separation is that water is a polar molecule, whereas oils are composed of nonpolar molecules that tend to avoid the dipole movements of water. In order to mix oil and water, emulsifiers need to be added. Many emulsifiers will have two parts: one part that is hydrophilic, which allows it to interact with water, and the other is hydrophobic, allowing it to interact with the oil. These emulsifiers often end up at the interface between the oil and water phases, helping the emulsion keep its structure.

There are two main types of emulsions. For an oil-in-water (o/w) emulsion, oil droplets are dispersed through the continuous water phase. For a water-in-oil (w/o) emulsion, the water droplets are dispersed through the oil phase. Both types of emulsions are common in foods. Milk, mayonnaise, and many salad dressings are o/w emulsions. Butter, cheese, and margarine are w/o emulsions.

When the oil phase of the emulsion is high in saturated fat, the resulting product can have semi-solid or gel-like properties. Butter is high in saturated fats, lending to its solid state at ambient temperatures and thus is usefulness in baking. In trying to reduce saturated fat, different structurants are needed to replicate the original structure. Oleogels can play a role in building that structure.

2.5.2: Emulsions in oleogels

There have been a number of methods tried to use organogelators to provide structure to an emulsion. Bot and others (2011) used β-sisosterol and γ-oryzanol to form tubules in sunflower oil that would stabilize an emulsion. They added preheated water at 10, 30, and 60% w/w. The result was a Pickering emulsion, stabilized by the solid particles that adhered to the surface of the water droplet. It was found that adding water to a 16% sterol oleogel would disrupt the formation of the tubules, while in a 32% sterol oleogel, two types of tubules formed. The water droplets were trapped between the fibers formed by the tubules, and in the course of a week, the droplets grew in size. The tubule structure grew more complicated in that time period.

Ogutcu and others (2015) used beeswax in olive oil to build emulsions that were compared to commercial spreads. Their goal was to build an emulsion that would stabilize at room temperature. Tween 20 and Tween 80, or polysorbate, were added as emulsifiers, and xanthan gum was also added to boost stability. The firmness and stickiness values of the emulsions remained stable during the 90 days of storage. The

beeswax formed fine crystals that gathered at the surface of water droplets, stabilizing the system. Those crystals remained stable during the 90-day storage period. The solid fat content (SFC) was only affected by the content of the beeswax, showing that the composition of the lipids was not affected by the combining of the two lipid sources.

Beeswax made up 3.75-4.5% of the sample, and the SFC was within that range.

Toro-Vazquez (2013) examined how candelilla wax and monoglycerides could be used to stabilize emulsions. They used a high pressure homogenizer to build the emulsion, and controlled the temperature of the final product so that it could solidify before the emulsion broke. The samples were still stable after one month of storage at 5°C. The monoglycerides built a crystal network that trapped the droplets of the emulsion. As the concentration of the monoglycerides increased, more microplatelets were formed. When the concentration of the candelilla wax increased, the gels became more solid.

Beri and others (2013) studied carnauba wax as a way to stabilize emulsions with an eye towards moisturizing lipsticks. Carnauba and microcrystalline wax were added to castor oil, and several emulsifiers were added for stability. The samples were emulsified, then frozen to allow for crystallization. It was found that the samples made with polyglycerol polyricinoleate, and emulsifier, had the smallest water droplets. The other samples had larger water droplets with greater variability in droplet size. The carnauba wax contributed to crosslinking that strengthened the product, while the microcrystalline wax made small irregular crystals that decreased overall strength. The rheological properties,

like elastic modulus and viscous modulus, decreased with the addition of water, but the extent was affected by the concentration of wax.

Shellac was also used to create stable emulsions without the use of additional emulsifiers

(Patel and others 2013). Emulsions were prepared by using shellac in rapeseed oil as the oleogel, and 20% water. The emulsions were cooled to room temperature before studying. Shellac can gel oil at 2% concentration. Increasing the concentration of shellac in the gel increases the firmness of the resulting emulsion. The emulsions were stable after weeks of storage, with neither water nor oil trying to separate from the structure.

Shellac can weakly interact with water, so the stability of the emulsion was a combination of the physical entrapment of the water and the influence of shellac crystals gathering at the interface of the water.

Some organogelators lose their functionality in the presence of water, and some have altered properties. Some organogelators form crystals that adhere at the interface of water droplets, and some trap the water in the bulk of the structure. To predict how RBW would interact in a given food system, it would be of interest to know how the presence of water might change the behavior of the wax. It would also be useful to determine for how long the structure will remain stable. Emulsifiers build the strength of emulsions by interacting with both the oil and water phases, and it can provide structure that the wax alone might not offer.

2.6: Measuring the properties of an emulsion

There are a number of techniques that can be used to determine the structure of an emulsion and how it reacts to physical changes.

2.6.1: Microstructure

Microscopes are useful for determining the shape and size of crystals that make up the structure of a lipid. One technique that is especially useful for this purpose is polarized light microscopy (PLM). This technique uses light waves that are directed by a polarizer to hit the sample at a specific angle. The alignment of the light allows the crystalline structure of fats to be observed. Fat crystals are birefringent and tend to organize along specific axes. Regular light would hit the sample in all directions, showing just a generic structure. Directing the angle of the light allows the pattern along the axes to be revealed.

To accurately determine the melting and crystallization properties of a lipid structure, differential scanning calorimetry (DSC) might by used. This technique uses a machine that measures the energy flowing in or out of a sample relative to a standard during changes in temperature. Two identical pans are placed in a cell of the DSC unit: one holding the sample of interest, and one empty. The cell changes temperature at a rate determined by the experimenter, and the heat flow is measured. The difference between the heat flow of the sample pan relative to the empty pan is the data of interest. The result is a graph that shows the heat flow over a temperature range. During a heating cycle, the graph will have dips that indicate where components are melting. For example,

in a fat that contains different fatty acids and crystal structures, there will be dips related to the melting point of the various fatty acids, and potentially dips related to α and β forms of the fat crystals. Cocoa butter is an example of a fat that would show this trend

(Ali and Dimick 1994). During a cooling cycle, there are peaks that show where the crystals form, and where the sample changes phases from a liquid to a solid. The temperature of crystallization is generally a bit lower than the melting temperature. The enthalpy of the melting or crystallization reaction can also be measured by calculating the area under the curve where the reaction takes place. Some lipids change in their crystal structure during aging. This change could reveal itself as a change in the melting properties over storage time.

2.6.2: Solid fat content

Solid fat content (SFC) is an indication of the extent to which the fat within a sample has crystallized into a solid, as opposed to being in a more liquid state. The SFC affects many rheological and texture properties of the fat sample. It is temperature dependent: samples at a higher temperature will generally have a lower SFC, as certain triglycerides within the sample have a lower melting point than others. This change can be observed from a practical standpoint in that butters and shortenings stored at a lower temperature will be harder than those stored at a higher temperature. Solid fat content is measured using NMR. The official method for SFC determination involves using a 20 MHz NMR unit that sends a 90° pulse through the sample and taking readings at two different time points. The first time point is soon after the pulse, and the readings include

measurements from protons in the solid and liquid portions of the sample. At the second time point, the protons in the solid portion of the sample should have returned to their original orientation, while the protons in the liquid portion would still be reorienting and giving off a signal.

2.6.3: Water holding capacity

In an emulsion, the water and oil portions tend to try to avoid each other, resulting in the separation of the two components, or the breaking of the emulsion. One method to determine the stability of the emulsion is to determine how well it is holding on to its water over time. In addition to the microscopy information, which would provide visual input as to where the water is in relation to the emulsion, the moisture content of the sample could indicate if water has evaporated from the sample during its time in storage.

There are several methods that can be used to determine the moisture content of a sample.

One method involves drying the sample. The initial mass of the sample is taken, and then the sample is heated for a period of time to allow the moisture to evaporate off. The final mass of the sample is measured, and the change between the initial and final masses is considered the moisture content. There are several ovens specifically designed to take these measurements, and to track the change of the mass during the heating time. The challenges associated with this method are based on the fact that the sample is heated to drive off water. If the sample is heated too far, it could burn or lose volatiles. If this happens, then the moisture content of the product would be reported at a higher value than the actual content. Another challenge would be if the water is somehow tightly

bound within the sample. If this happens, the water would not evaporate during the heating time, and the reported moisture content would be lower than the actual value.

Based on the challenges of the gravimetric method, it would be helpful to have a technique that measures the water content of a sample directly. One method for doing this involves nuclear magnetic resonance (NMR). This technique uses a series of magnetic pulses to agitate specific atoms within the sample. The behavior of those atoms gives an indication as to make-up of the sample. In 1H NMR, the atom of interest is hydrogen. A magnetic field passes through the sample to align the hydrogens so that they will spin in the same direction. The magnetic field then briefly changes directions at a

90° angle, causing the hydrogens to realign their spin in that direction. The time it takes the hydrogens to return to their original spin direction is measured. The time it takes is related to what the hydrogen is doing in the sample, and how it is interacting with other hydrogens in the sample. For example, hydrogens that are part of a long hydrocarbon tail are going to return to their original spin faster than those that are part of a water molecule

(Merlic 1997). This is because the hydrocarbon tail hydrogens are influenced strongly by their neighboring hydrogens, whereas the ones in the water are more free to move. The behaviors of hydrogens in certain formations have predictable behaviors that can be used to predict the overall structure of the sample. If the behavior of water hydrogens is distinct from the behaviors of other hydrogens in the sample, it is simple to estimate the concentration of water within the sample. The concentration is related to the proportion of the water to the rest of the signal in the output data.

2.6.4: Physical properties

There are different ways to measure the reaction of an emulsion to changes in physical force. One of them is through rheology. Rheology measures the changes experience by a sample during shearing. An example would be spreading butter on bread. At a certain level of force, the butter acts like a solid, and no changes occur. However, as the force applied increases, it reaches a point where the butter starts to flow more like a liquid, and this is the point where the butter can actually be spread. Of course, these changes are also temperature dependent: warm butter is spread more easily than cold butter straight from the fridge. A product like a gel is considered to have two components acting in reaction to the spreading force: a solid-like component, and a liquid-like component. The solid-like components of the gel are measured as the G’ of the sample, while the liquid- like components are G”. If G’ is greater than G”, then the gel is behaving more as a solid than as a liquid. When the values crossover, then the sample has broken down and is now flowing as a solid. Tan delta is used to describe one interaction between the solid and liquid-like behaviors of the gel. Tan delta is defined as the ratio of G”/G’. If the value is greater than 1, then the sample behaves more like a liquid than like a solid. If the value is less than 1, then it behaves more like a solid. The complex modulus (G*) can also give some insight as to the interaction between the solid and liquid-like properties of the gels.

Rheology measurements tend to start with a strain sweep, where the sample undergoes increasing strain at a constant frequency of oscillation. During the test, the sample is put

under a probe that oscillates to cause strain in the sample. The distance that this oscillation occurs in a given amount of time increases during the duration of the test.

Initially, the strain does not cause significant changes to the sample. At this point, the sample is behaving like a solid, so if any deformations occur during the test, they soon return to the original state. This is the elastic property of the gel. Eventually, the strain reaches a level where the deformations become more permanent, and the gel starts to break down. As long as the deformations can be resisted, the gel is considered to be in its linear viscoelastic region (LVR). This region is useful for determining measurements for other rheologic tests.

Rheology is a popular technique used for measuring oleogels. Oleogels are structured by the organogelators to have solid-like properties, but the presence of liquid oil will contribute to the liquid-like portions of the gel. The addition of water should change the rheologic properties of the gel. The higher the water content, the more liquid-like the gel should behave. However, if the organogelator is interacting with the water, it could cause the gel to somehow seize up and behave more as a solid.

Another method for measuring the textural properties of the gels is through texture profile analysis (TPA). This technique is generally designed to simulate mastication, in that a certain amount of force is applied to the food product, and the amount of force applied to compress a product will give insight into the hardness and other properties of the product. TPA provides information about the hardness, stickiness, resilience,

gumminess, chewiness, and springiness of the product. To do this, a probe compresses the gel sample to a specific distance. A certain amount of force is required to achieve this compression. This force is the hardness. If the sample breaks before the highest hardness is reach, the force at which the sample breaks is the maximum force of the sample. As the probe returns to its original position, the sample may cling to the probe, giving a negative force reading. This negative force gets measured as the stickiness or adhesiveness of the sample. The resilience of the sample relates to how much the sample tries to return to its original height while being compressed, while springiness indicates how much the sample returned to its original height after the first compression. Oleogels are specifically designed to act like solid fat, so comparing the hardness or maximum force of an oleogel to a solid fat sample could indicate how successful such an attempt was. More importantly, when TPA is applied to foods that incorporate oleogels, it can indicate how successful the organogelator was at achieving a match to the full-saturated fat sample.

Chapter 3: Materials and Methods

3.1: Materials

Rice bran wax (RBW) pastilles were obtained from Koster Keunen (Watertown, CT).

Glycerol monooleate (GMO) was obtained from Hallstar (Chicago, IL). Soybean oil came from Kroger (Cincinnati, OH).

3.2: Oleogel production

Samples were prepared in 200g batches. The samples contained 2, 5, or 10% RBW, 0 or

1.67% GMO, and 0, 5, 10, or 20% deionized water. Previous research has demonstrated that RBW is effective at gelling oil at low concentrations (Dassanayake and others 2009), and it can be used in food products (Limbaugh and others 2015, Huang and others 2015).

GMO has been used to enhance the stability of food products made with RBW oleogels when used at this concentration (Zulim and others 2013). The remainder of the sample consisted of soybean oil (68.33-90%), which is used in many commercial products

(USDA 2012).

First, oil and GMO were weighed and mixed by hand. They were then placed in the cell of a heater attached to a hot water bath (Figure 4). The water bath was preheated to 85°C and maintained that temperature during the mixing process. The RBW was also added to 34

the cell. The cell was covered with aluminum foil. The sample was then mixed with at

300 RPM for 15 minutes, using a Caframo stand mixer (Georgian Bluffs, Ontario,

Canada) and a mix head with three small blades.

Figure 4. Set-up used for creating gels.

During this time, water was preheated to 100°C. The mixing speed was then increased to

1000 RPM. The water was weighed, so that it was about 80°C when it was added to the water. This water was added to the cell with the oil mixture. The sample was mixed for three more minutes, then poured into the final container required for the appropriate test method (glass slides, storage tubs, NMR tubes, etc.). The sample was then immediately

transferred to a refrigerator at 5°C. Samples were stored in the refrigerator until the time for testing.

3.3: Moisture content

A Mettler Toledo moisture analyzer (MJ33, Columbus, OH) was used to verify the moisture content of the gels and to measure the change in water content during the 27- day storage period. The device used infrared waves to heat the sample, and it measured the change in the mass of the sample during the heating period. An aluminum pan suitable for the unit was used for each sample, and 0.900-1.00 grams of sample was weighed into the pan. The sample was then heated at 115°C for 60 minutes. Moisture content readings were taken from the display screen. Three samples were tested for each variable, and the test was performed in triplicate.

3.4: Nuclear magnetic resonance

The moisture contents were verified using nuclear magnetic resonance (NMR). After mixing, the hot sample was placed into 5mm glass NMR tubes. The tubes were stored at

5°C until they were measured. Measurements were taken using a 600 MHz unit (Bruker

BioSpin AG, Billerica, MA) at 25°C. Data were analyzed using TopSpin 3.5 software

(Bruker, Billerica, MA). The area of the peak associated with water and the total area under the NMR curve were calculated using the TopSpin software. The water content was calculated as seen in Equation 1.

Equations 1:

Moisture content = [area under water peak]/[total area under curve]

3.5: Melting properties

The melting properties of the gels were tested using a differential scanning calorimeter

(DSC). The DSC unit (DSC Q2000 Texas Instruments, New Castle, DE) was calibrated with indium, and samples were tested in T-zero aluminum pans. The samples were first equilibrated at 5°C for 1 minute, then heated to 85°C at a rate of 1°C per minute. The peak melting point and enthalpy were determined using TA Universal Analysis 2000 software (Version 4.5a, TA Instruments, New Castle, DE).

3.6: Solid fat content

Solid fat content (SFC) was measured based on the AOCS Official Method Cd 1b-93.

After the samples were finished mixing, they were poured into 8mm NMR tubes and placed in 5°C storage. The samples were kept at 5°C until they were measured. The SFC measurement was done using a Bruker PC/20 series Minispec NMR Analyzer, using a procedure preprogrammed in the unit. Three replicates from each gel sample were measured, and the test was performed in duplicate.

3.7: Rheology

Samples for rheology testing were prepared in plastic containers for cooling and storage.

The containers were 10cm in diameter and filled to 1 cm in height. Rheological testing was done with an Anton-Paar MCR 302 rheometer (Graz, Austria) using a 20mm sandblasted parallel plate probe and a sandblasted plate, with a geometry gap of 2mm..

About 1g of each sample was spread on the plate. Strain sweep tests used a strain from

0.001-10% at a frequency of 10 rad/sec. Tests were performed after 1, 3, 7, and 27 days in storage. There were 4-5 repetitions performed, and the experiment was replicated 3 times.

3.8: Texture profile analysis

Samples were prepared using a set of plastic molds 20mm in diameter and 12mm in height. The samples were stored at 5°C until the test was performed. Analysis was done with a TAXT2 analyzer (Stable Microsystems Ltd, Surray, UK) using a 25mm Plexiglass probe, and data were analyzed with Exponent software (version 6,1,5,0, Texture

Technologies, Hamilton, MA). The sample was compressed to 40% of its original height during a 2-cycle compression. The probe head moved at 1mm/second at all stages of the test, and there was a 5 second pause between strokes. There were 6 replicates performed for each test, and the test was done in duplicate.

3.9: Microstructure

Polarized light microscopy (PLM) was used to study the samples’ microstructure. The samples were examined with an AxioCam MRc 5 (Zeiss, Thornwood, NY). To look at the final structure of the gels, slides and coverslips were preheated to 85°C. After mixing, a few drops of the sample were placed on the slide. The coverslip was quickly applied, and the slides were placed in 5°C storage. The samples were imaged after 1 day of storage. The slides were placed on a scanning stage and photographed using 10x magnification. Fat crystals are anisotropic, which means that under polarized light, they scatter the light specifically based on the direction of the crystal. This results in birefringence, and the fat crystals will appear light against a dark background (Marangoni

2005). Water droplets cannot be observed with this technique, so the light microscope settings were used to observe them.

The gels were also analyzed to observe crystal growth behavior during the cooling process. To do this, samples were viewed using a temperature controlled stage (Linkam

Scientific Instruments, Tadworth, UK). The stage and the blank slides were heated to

75°C. As soon as the sample was done mixing, a few drops were applied to the slide, and a coverslip was added. The slide was then cooled on the stage at 5°C per minute to 5°C.

Images were taken as the sample cooled.

3.10: Statistical analysis

Each of the analytical tests were performed on all samples at least in duplicate. The effects of water content and storage time on the samples were analyzed using 1-way

ANOVA, with Tukey HSD post hoc. The effects of the emulsifier were tested using t- test, since it was a pair-wise comparison. The samples were analyzed at 95% confidence interval. The software used was JMP 11.2.0 (2013 SAS Institute Inc.).

Chapter 4: Results and Discussion

4.1: Preliminary Study: Properties of oleogels made with 2% and 5% wax

Rice bran wax (RBW) can structure soybean oil at a low concentration (Dassanayaki and others 2009). Preliminary tests were conducted with gel samples made using 2% and 5% wax w/w.

4.1.1: Rheological properties of low wax RBW oleogels

Rheology is the study of how materials flow, or the structural changes that occur when some small amount of force is applied. When materials are sheared, they display different properties depending on their content and the amount of shear applied. The rheology of liquids often focuses on how they might flow through pipes, but gels will have very different rheological behaviors. A practical application of gel rheology is how a material can be spread, like margarine on toast. There is a certain amount of force needed to spread the margarine, and it will depend on the temperature, crystalline structure, and composition of the ingredients. If the amount of shear force is low, the margarine will resist being spread, and nothing will change. Once a critical shear force is reached, the spreading will occur quite easily. Butter, which has a different composition and crystalline properties, will display different reactions to the same amount of shear force. Rheology helps to quantify those differences. 41

Figure 5 shows a representative graph obtained from the rheology measurements. It is based on a 5% wax sample.

2.50E+06

2.00E+06

1.50E+06 Pa 1.00E+06 G' G" 5.00E+05

0.00E+00 0.005 0.05 0.5 5 Strain (%)

Figure 5. Representative data for strain sweep measurements, using 5% RBW, 20% water, and emulsifier after 1 day in storage.

The flat part at the beginning of the sweep is the linear viscoelastic region (LVR). At this point, the gel can recover its original form when the strain on it relaxes The values in the

LVR region were used to compare the effects of water content on the structural properties of the gels.

The G’ value gives information regarding the solid-like properties of the gel. It indicates how firm the gels are. Figure 6 shows the properties of gels made with 2% wax.

0.70 0.60 A 0.50 a B b 0.40 B b 0% c

0.30 C 5% G' (MPa) G' 0.20 10% 0.10 20% 0.00 1 2 Time (days)

0.80 B b B a 0.70 A A a a 0.60 0.50 0% 0.40 5%wE

G' (MPa) G' 0.30 10%wE 0.20 0.10 20%wE 0.00 1 2 Time (days)

Figure 6. G' of gels made with 2% wax and 0, 5, 10, and 20% added water content.

At 2% wax, the G’ of the samples decreases with the addition of 5% water. While 5 and

10% water are similar, the 20% water sample shows another decrease. All of the samples had higher G’ values when the emulsifier was added. This observation suggests that the emulsifier could be stabilizing the gel network. It may be stabilizing water droplets in the 43

gels, preventing them from coalescing before the gel finishes cooling (Chen and

Dickinson 1999). However, some loss of structure is still seen with 20% water. No loss of structure was observed over the 3-day storage period. Figure 7 shows the G’ values of the waxes made with 5% wax.

3.00

2.50 A a a AB a 2.00 B 0% 1.50 5% G' (MPa) G' 1.00 b C 10% 0.50 20%

0.00 1 3 Time (days)

3.50 A A 3.00 A A a a a a 2.50 2.00 0%

1.50 5%wE G' (MPa) G' 1.00 10%wE 0.50 20%wE 0.00 1 3 Time (days)

Figure 7. G' of gels made with 5% wax and increasing water content. 44

At 5% wax, there are no significant differences between the control sample and the ones with 5 and 10% water. When 20% water is added, the structure is not as strong, resulting in a lower G’. This trend is not seen in the samples that contain the emulsifier. The emulsifier has an effect on stabilizing the structure of the gels, so that they remain firm even with the addition of 20% water. The gels made with 5% wax are more effective than 2% wax at maintaining their structure when oil is replaced with water. However, loss of structure is still seen at higher water concentration.

At lower wax concentrations, oil can be replaced with water to the same extent as a gel made with 10% wax. However, the addition of the water disrupted the gel system such that gels with a higher water content would not have the same functionality as gels made without the water (Toro-Vazquez and others 2013, Patel and others 2013, Beri and others

2013).. By 10% wax, that functionality was present, which could be seen through the physical properties of the gels. Since the 2 and 5% wax gels lost their structural properties at higher water concentrations, further investigations were curtailed. The remainder of this study focuses on gels made with 10% wax.

4.2: Moisture content of gels made with 10% RBW

There are some benefits to replacing the oil in an oleogel with water. From a nutrition standpoint, when oil is replaced with water, the total fat content and total calorie content of the gels decreases. This property can be useful in foods to create products that have

the same functional properties, but with a reduced fat or calorie content. In addition, manufacturers may prefer to use water instead of oil in processing as a cost reduction step.

4.2.1: Water holding capacity of RBW oleogels

Preliminary testing was done to determine the extent to which the oil in the gels could be replaced with water. While emulsions were easily prepared with 5 and 10% water, the cooling rate became critical in preparing the samples with 20% water. The 20% samples had larger water droplets that would try to separate after compression during rheological and TPA testing. When 30% water was added, the water coalesced before the sample was able to cool. Therefore, phase separation occurred. Since the system was primarily composed of oil and water, the two components would try to separate between the end of the mixing time and the crystallization point. Based on these observations, 20% water was the maximum amount used in all subsequent testing.

4.2.2: Moisture analyzer method for determining water content

The moisture content of the samples was calculated based on the NMR values. The values that were obtained were compared with the values from the moisture analyzer, which used evaporation to measure moisture content. Since the values matched, the moisture analyzer was determined to be an accurate method to use for determining the water content of the gels. It also ensured that any discrepancies in between the water added to the gel and the water measured within the gels would be due to evaporation

during processing or storage. Further tests were used to measure evaporation during storage.

I I I I 20

15 1 3 10 a a a a 7

Moisture Moisture Content % 27 5 A A A A

0 5% 10% 20% Water Content

Figure 8. Measured moisture content of samples over 27 day storage period without emulsifier.

Figure 8 demonstrates the moisture contents of the samples measured during storage time. Based on the moisture content analyzer, the water content of the gels did not change significantly over the 27 day test period. There was no significant difference noticed between the moisture contents of the samples with the emulsifier and those without. It was thought that the presence of the emulsifier would enhance the formation of small water droplets (Zulim and others 2013), and allow for separation that would prevent the droplets from coalescing during storage (Chen and Dickinson 1999).

One of the major concerns for these samples is whether they would come out of emulsion during storage. This change would present itself as syneresis, a process where water separates from the gel network and pools on the surface of the sample. This phenomenon can be observed in products like yogurt, where a small amount of water can be observed on the surface of the food. In addition, water droplets inside the sample could begin to coalesce, making it easier for the water phase to separate from the continuous lipid phase.

During the 27 day storage period, no excess water was observed in the containers holding the samples.

Table 1 shows the amount of water measured after 1 day in storage. The measured water content was slightly lower than the amount initially added to the sample. The 5% gel samples showed values around 3.8% water, the 10% had 8.6%, and the 20% samples were around 19%.

Table 1. Moisture content measured for samples after 1 day in storage. Samples A-D do not contain emulsifier, samples E-G do.

Label Input Water Content (%) Measured Moisture Content (%) A 0.00 -- B 5.00 3.76±0.27 C 10.0 8.58±0.41 D 20.0 19.2±0.44 E 5.00 4.02±0.12 F 10.0 8.55±0.23 G 20.00 19.2±0.74

The likely reason for this discrepancy is due to evaporation during processing. The initial method used for creating the gels yielded low moisture contents. When steps were added to reduce the likelihood of evaporation in the samples, the final moisture contents increased. In addition, while the mixing cell was covered with aluminum foil, a small amount of steam was observed escaping from the hole that allowed the mix head into the cell. The moisture content values were validated using other methods to determine that the total amount of water in the sample was being measured.

Work on emulsions made with candelilla wax and monoglycerides in sunflower oil by

Toro-Vazquez and others (2013) showed a similar level of stability. They made emulsions with 20% water and 2-3% candelilla wax. They observed no water separating from the system after one month in 5°C storage. Patel and others (2013) found that preparing gels with shellac and rapeseed oil had similar results. They measured the size of the water droplets in their samples every 6 weeks for 18 weeks, and found no significant changes in the size. This indicates that the water was not coalescing, and the water was not leaving the sample. No phase separation was observed over this time.

Therefore, rice bran wax is consistent with other wax-based oleogels in maintaining the stability of the emulsion over time.

4.2.2: Nuclear magnetic resonance measurements of RBW oleogels

There were three possible explanations for difference in water content from expected values: water bound in the gel that did not evaporate, water lost during sample

preparation, or water lost during storage. A method was sought to determine if there was

any tightly-bound water within the gel. The method to verify the water content of the

gels was by using nuclear magnetic resonance (NMR). This method can show the total

amount of water in the gels based on the interaction of the magnetic field with the protons

in the water molecules. It would be helpful in identifying water that is tightly bound in

the system, which would not be measured by evaporation-based methods. To ensure the

accuracy of the moisture analyzer values, NMR was used to check the moisture content.

The peaks can be seen in Figure 9.

Fatty acid chains Water peak Water peak Esters & alcohols

20% w/E 20% w/E

20% 20%

10% w/E 10% w/E

10% 10%

5% w/E 5% w/E

5% 5%

0% 0%

Figure 9. NMR readings of rice bran wax oleogels made with varying water contents and emulsifier levels.

There were three major components in the samples that would show peaks in the NMR graph: the soybean oil, the wax, and the water. Barison and others (2009) gathered NMR data on soybean oil to determine the fatty acid composition. Bakota and others (2013) did a similar process on rice bran wax. The peaks from their studies correspond to the ones seen in Figure 9. The peaks represent the fatty acid chains and the esters and alcohols that made up the bulk of the gels. The only additional peak is the one at 4.8ppm, which corresponds to water (Merlic 1997). There was a trend as the added water content increased for a decrease in the resolution in the peaks. The peaks for the fatty acid chains were less prominent as moisture content increased. This change indicated that the structure of the gels was more amorphous as the moisture content increased.

4.3: Microstructure properties

4.3.1: Solid fat content

Solid fat content (SFC) indicates how much of the fat in the sample has crystalized into a solid form at a given temperature (Marquez and others 2013). This behavior impacts many properties of the fat product (Leung and others 1985). A product that has a high

SFC may have a higher melting point, which can be observed with DSC (Walker and

Bosin 1971). It would show greater crystal formations when viewed under PLM.

Properties like rheology and hardness are greatly influenced by the SFC of the sample, because the motion of solid particles past each other is what is being measured.

Therefore, SFC is a critical measurement to examine.

There are two components in the RBW oleogels that can affect SFC. The wax itself melts at around 78°C, while the melting point for soybean oil is around -3°C. However, certain fatty acids within the oil will start crystallizing below 5°C (O’Brien 2009). Since the gel samples were tested after storage at 5°C, a small amount of solid fat from the soybean oil was expected. The RBW was expected to provide most of the solid fat, and since the amount of RBW was the same in all samples, the solid fat from the wax was expected to be about the same. The SFC of the samples can be seen in Table 2.

Table 2. Solid fat content of gels without and with emulsifier, measured at 5°C.

Label Solid Fat Content (%) Label Solid Fat Content (%) (with emulsifier) A 7.38±0.46A,a A 7.38±0.46A,a B 7.55±0.34A,a E 7.51±0.34A,a C 7.78±0.27A,a F 7.94±0.54A,a D 8.26±0.47B,a G 8.42±0.51B,a

The SFC of the gels was around 7.5%. The trend shows that as water content increased, the SFC of the gels was consistent. However, only the 20% gel samples were significantly different from the other samples. At the higher water contents, the ratio of wax to oil in the gel increased. This may have caused the wax to dissolve less easily in the oil, yielding the higher SFC. The presence of the emulsifier had no significant effect on SFC.

The soybean oil contributed a small amount to SFC, since a small amount of fatty acids in the oil can solidify in 5°C storage. Hayati and others (2009) reported that the SFC of 52

soybean oil at 5°C was around 0.16%. Therefore, the contribution to the total SFC of the gels should be minimal. The major component structuring the gels is the wax. Since the gels were made with 10% wax, it was predicted that the SFC would be around 10%.

However, Blake and others (2014) found that gels made with 10% rice bran wax would have about 7% SFC when measured at 20°C. They attributed the difference in SFC contents to components in the wax that dissolved in the oil. Since they were dissolved in the oil, they did not crystallize and contribute to the SFC of the gels.

Toro-Vazquez and others (2013) made gels using candelilla wax and monoglycerides, and they tested the effects of adding 20% water to the gels. They found that the gels made with 20% water had SFC values about 20% less than the comparable ones made with just oil and wax. They made their gels by preparing the oleogel separately, then melting down the oleogel to mix it with water. Therefore, they attributed the loss of SFC to the reduced amount of oleogel in the sample. In this study, the wax content remained the same, while only the oil was replaced with water. Because of that, the ratio of wax to oil increased as the water content increased. This may have allowed solid crystals to form more easily, thus increasing the SFC.

4.3.2: Differential scanning calorimetry

The SFC of a fat product can have an impact on the melting point and the amount of energy needed to melt the fat (Walker and Boxin1971). Differential scanning calorimetry

(DSC) is a technique that can be used to examine the melting and crystallization

properties of a sample, as well as identify changes in the crystallization patterns of the sample (Foubert and others 2008). In a fat sample, different fats melt at different temperatures. The melting point of fats is influenced by a number of factors, including the fatty acid content, the form of the crystal, and the arrangement of fatty acids on the glycerol backbone.

Rice bran wax is primarily composed of fatty acids with esters ranging from 16-32 carbon numbers, and fatty alcohols with carbon numbers ranging from 24 to 38

(Dassanayake and others 2009). The highest percentage of esters comes from carbon chains with 22 and 24 carbons. Rice bran wax contains a single class of aliphatic fatty acids that have been esterified into fatty alcohols. Since the variety of fatty acids in the wax is fairly small, rice bran wax has a sharp melting pattern, with a peak melting point at 78°C (Dassanayake and others 2009). Dassanayake (2009) tested rice bran wax mixed with canola and soybean oils in different concentrations. It was found that as the concentration of rice bran wax increased, the melting peak became more pronounced, and the peak temperature wax closer to that of the pure wax. At lower wax concentrations, melting occurred more gradually, and the peak was at a lower temperature (Dassanayake and others 2009).

Figure 10 shows a representative graph generated from the DSC data. Even with the additional water, all the graphs showed one peak at the melting point that was influenced by the wax.

0 -0.02 -0.04 -0.06 -0.08 Heat (W/g) flow Heat 73.5 -0.1 0 10 20 30 40 50 60 70 80 90 100 Temperature (°C)

Figure 10. Representative DSC graph showing 10% water.

The peak melting temperature occurs at the lowest peak in the figure. Then enthalpy is calculated based on the area of the dip in the figure. Figure 11 shows the peak melting temperature of the gels taken after 1 and 7 days in storage.

75.5 75 A A 74.5 a a a A A 74 a a a A A a 73.5 A 73 72.5 1 day 72

Temperature(oC) 7 days 71.5 71 70.5 0% 5% 10% 20% 5% w/E 10% 20% w/E w/E Water Content

Figure 11. Peak melting point of gels made with increasing water content.

In this study, the melting point of rice bran wax oleogels mixed with different concentrations of water was measured. It was found that as the water contents of the gels increased, there was no significant change in the melting point (Figure 11). This changes could be due to the higher wax-to-oil ratio in the gel portion, which would cause the gels to melt at a slightly higher temperature. However, the variations between the gels were not statistically significant. The peak was around 73.5°C.

Storage time can have an effect on fat crystals, as crystals grow or shift to more stable forms in storage. This change can be observed using DSC techniques with shifts in melting points that occur where specific crystals formed. However, there was no change observed in the melting properties of the samples during the storage time.

The effect of an emulsifier on the melting point of the gels was tested. The samples made with the emulsifier were not significantly different from the one without the emulsifier.

Enthalpy is an indication of how much energy was used while the reaction occurred.

Melting is an endothermic reaction, which means that the sample absorbs energy from the environment during the process. This can be seen graphically by the dip in the graph where melting occurs. Enthalpy can be calculated by finding the area of the dip in the graph, the part where the energy flowing through the system varies from the baseline amount of energy. Enthalpy results can be seen in Table 3.

Table 3. Enthalpy of gels made with increasing water content.

Label Day 1 Day 7 A 16.7±2.7 16.4±0.9 B 15.9±0.3 18.6±3.4 C 12.1±1.2 16.6±3.8 D 18.0±0.3 16.7±0.1 E 18.3±0.1 16.5±1.8 F 18.5±1.3 16.9±0.5 G 17.3±1.7 16.6±0.5

It was found that the enthalpy of the gels was consistent as the water content increased.

Enthalpy provides insight into the SFC properties of the gels, and the types of fat crystals in the sample. The SFC values were similar across increasing water contents, seen in

Table 2. Therefore, the amount of energy required to melt the solid fat was similar, as seen in Table 3. The values were consistent over the time period measured, which means that the SFC of sample did not change much during the storage time. The specific types of fat crystals in the gels were not observed in this study. The addition of the emulsifier seems to have reduced the variations seen between samples. The emulsifier seems to have an effect on stabilizing the gel system.

4.3.3: Polarized light microscopy

Polarized light microscopy (PLM) is a technique that uses light filtered to a certain angle in order to highlight different parts of the image. Fats samples can have two parts: the solid part, made with crystal structures, and a liquid portion, from any fatty acids with a low melting point (Marquez and others 2013). Fat crystals are birefringent, which means that when light bounces off the crystals at a specific angle, they will stand out against the

liquid portions (Marangoni 2005). In the rice bran wax oleogels, the solid fat crystals will stand out from the soybean oil that surrounds it. Since the wax crystals solidify quickly at high temperatures, any crystals formed from fatty acids in the soybean oil will probably grow off the structure provided by the wax (Zhang and others 2012).

The structure of the wax-oil matrix may be sturdy enough to prevent water droplets from coalescing on its own. The interactions between the wax-oil matrix and the water droplets will be further observed using PLM.

Figure 12 shows the gels after one day in storage. The white portions are the wax crystals, while the black is either liquid oil or water.

100µm

Without emulsifier With emulsifier

Figure 12. Microscope images of gels made with different concentrations of water (0-20%) without and with emulsifier, taken at 10x magnification.

The wax crystals are long and needle-like, which can be observed in all the images in

Figure 12. According to Dassanayake and others (2009), this structure can explain why rice bran wax is such an effective organogelator, especially at low concentrations. The needle-like crystals form the matrix that traps the liquid oil. They tested rice bran wax, carnauba wax, and candelilla wax, and found that while rice bran wax had needlelike structures, the other two waxes formed more spherulitic structures that were not conducive to structuring the oil. Consequently, rice bran wax could gel oil at a lower concentration than the other two waxes.

In Figure 12 D (at 20%), there are dark shapes with wax around them. These dark spots have been circled to highlight them. These dark spots could be water droplets. The wax formed around the water droplets, but it did not cross the line of the drop. This indicates that the wax is not actively interacting with the water. The emulsion is stabilized by the wax cooling quickly enough to trap the water droplets in a crystalline shell. Hodge and

Rousseau (2003) noticed that gels made with water, mineral oil, and paraffin wax made more stable emulsions when the wax was crystalized after the emulsion was made. They attributed this stabilization to the rapid formation of the crystal network that trapped water droplets during cooling. Toro-Vazquez and others (2013) also noticed this behavior when making gels with candelilla wax, safflower oil, and water.

The crystals seen in Figure 12 are also consistent in size across water contents and emulsifier content. The consistency in crystal size may also give some insight to the

properties observed with the DSC. The crystals observed with PLM were similar in size and distribution, so it is likely that the melting properties of the gel would be just as similar.

It was observed that the crystals were similar in size with or without emulsifier, which explains why no significant differences have been observed between the gels made with and without the emulsifier at the same storage time and water content. The emulsifier was expected to have some effect on water droplets in the gels. The emulsifier has a glycerol head, which is more polar, and the oleic tail, which is less polar. The head will form bonds with the water in the samples, while the tail would bond more with the oil.

These interactions would enhance the structure of the emulsion, helping it to last longer.

Glycerol monooleate tends to adhere to the droplet interface, enhacing, which helps to stabilize the emulsion (Chen and Dickinson 1999). However, no difference was observed in the samples.

To better observe the water droplets in the samples, pictures were taken at a different light angle. Figure 13 shows the samples at another light angle and 10x magnification.

100µm

Without emulsifier With emulsifier

Figure 13. Microscope images of gels made with different concentrations of water (0- 20%) (a) without and (b) with emulsifier, taken at 10x magnification at 35° light angle

The water droplets stand out much more clearly in these images. The control sample

(0%) shows only the crystal network, which are the darker strands, and the surrounding oil. At 5% water, a few water droplets appear. Larger droplets appear with greater frequency as the water content increase to 10 and 20%. The images at 50x magnification also reveal many small water droplets. It is likely that there are more small water droplets that cannot be seen with the level of magnification offered by the PLM (Toro-

Vazquez and others 2013). These small water droplets are more evenly dispersed through the sample. The large water droplets may be the result of coalescence that occurs during the cooling process.

PLM images were also taken during the cooling process of the gels. Attempts were made to capture images during the crystallization process. Figure 14 shows representative images of cooling during the process.

100µm

68°C

100µm

Figure 14. Gel made with 10% water, no emulsifier, cooled at 3°C per minute. After samples hit 67.5°C, the microscope was refocused to better observe the whole field of crystals.

It can be seen that once the crystallization temperature was reached (~69°C), the crystals formed very rapidly. The few degrees of change indicates about 3 minutes of crystal growth. Cooling was not carried out at a controlled rate for rheology samples. They were placed in cold storage as rapidly as possible in an attempt to have a high cooling rate. The actual rate at which the gels cooled in the fridge is not completely comparable to the rate used with the PLM, but the PLM can provide some insight as to how it may have happened.

Patel and others (2013) made gels using rapeseed oil and up to 6% shellac, and they added 20% water to the gels. They observed their gels after 18 weeks in storage and found no change in the size of the water droplets. The shellac is very effective at structuring the oil so that the matrix does not allow the coalescence of water droplets during storage (Patel 2013).

Toro-Vazquez and others (2013) made gels using candelilla wax and 20% water. They observed no aggregation of crystals around the water droplets, indicating that the emulsification stability of the gels was probably due to the entrapment of the water droplets in the rapidly cooled gel structure. They described their systems as emulsion- filled gels, where the structure of the matrix would have the greatest influence on the physical properties of the gel.

4.4: Physical properties

4.4.1: Texture profile analysis

Texture profile analysis (TPA) is a technique that is used to measure the attributes of a product that can be sensed by a person eating the product (Friedman and others 1963).

For example, hardness can correlate to the amount of force required to bite into a product.

TPA differs from rheology in that it is a large deformation measure, while rheology is a small deformation technique (van Vliet 2014). Rheology is beneficial for measuring the small changes that take place within the structure when exposed to stress. TPA helps measure the changes that occur in the structure as it is broken down.

In a double compression test, the TPA unit measures the amount of force that the probe experiences as a function of time during the compression process. The probe compresses the sample, withdraws, rests, compresses a second time, and withdraws again. Figure 15 shows a typical graph that results from this test on the oleogel samples.

3000 Maximum force 2500

2000

1500

1000 Force (g) Force 500

0 0 5 10 15 20 25 30 -500 Time (s)

Figure 15. Example of TPA graph using 10% water after 27 days of storage.

There are several points in this graph to consider. The first peak is considered as the maximum force of the sample. When the probe first touches the sample, it encounters a great deal of resistance to being compressed, but the sample is finally broken. The compression continues, and the sample displays a certain degree of resistance to continual compression. The gel is trying to maintain its structure in the presence of continued force. The sample then with draws, and a negative force area can be seen.

This area relates to adhesiveness, which is how much the sample tries to adhere to the probe. In testing adhesiveness, great care must be taken to ensure consistency between the amounts of sample adhering to the probe. For example, the sample could break in half and part of the sample could cling to the probe. There is no way to ensure that the amount of sample adhering to the probe is the same. Care was taken in the methodology to prevent this kind of clinging from happening. However, this test was not specific enough to measure adhesiveness accurately. The probe then rests for a few seconds, then 67

continues with the second compression. This compression shows much lower values, since the sample has already broken down to some extent. The different points on the graph can be used to measure different attributes of the sample. The first attribute to consider is the maximum force of the samples. Figure 16 shows the maximum force of the samples compared based on water content.

3500 II 3000 B i AA B a a a a II i i C i 2500 I I 2000 A

1500 B Force(g) C 1000 D 500 0 1 day 3 days 7 days 27 days Time (days) (a) 3500

3000 b A A ab ab i A a I I i ii i-ii 2500 B I I 2000 A

1500 E Force (g) Force 1000 F G 500

0 1 day 3 days 7 days 27 days Time (days) (b) Figure 16. Force measurements compared by water content (a) without and (b) with emulsifier.

It was found that the water content of the samples had no trend that effected the maximum force values of the samples. There were some small variations between the maximum force values of the samples; however, these variations were not consistent over testing, and would not likely be detected by a consumer. The samples with the emulsifier

had maximum force values that were not significantly different from those taken on the same day at the same water content level.

Figure 17 shows the maximum force values of the samples compared over time in storage.

3500 i 3000 a I i AB A B a a a II II i 2500 C III i 2000 1 day

1500 3 days Force (g) Force 1000 7 days 27 days 500

0 A B C D Sample (a) 3500

3000 AB A a a II i i i B a b II i 2500 C I I 2000 1 day

1500 3 days Force (g) Force 1000 7 days 27 days 500

0 A E F G Sample (b) Figure 17. Force measurements compared by storage time (c) without and (d) with emulsifier.

Samples with the same water content did not have a trend in the changes in their maximum force over storage time. The samples with the emulsifier added showed less variability over time and across water contents, compared to the samples without the emulsifier. However, there were no significant differences in the maximum force values noted between the samples with the emulsifiers and those without.

It was expected that increasing the water content of the samples would decrease the hardness of the sample. Increasing water content would mean that there is less oleogel available to structure the overall emulsion. It seems that the 10% wax was sufficient to structure the system, despite the increase in water content. It was also expected that adding the emulsifier might increase the maximum force values, since it could prevent water droplets from coalescing and allow more fat crystals to form, strengthening the network of the gel. However, no effect was noted.

In general, increasing the amount of organogelator in the oil will increase the hardness of the sample (Toro-Vazquez 2013, Beri and others2013, Hwang and others 2013). For waxes, this is due to the higher number of crystals in the oil that can provide structure to the gel (Hwang and others 2012). This study focused on one concentration of wax in the system. However, as the water concentration increased, the ratio of wax to oil increased slightly. No effect on the hardness value was seen. This could tie in to the solid fat content of the gel.

Hwang and others (2013) made margarine samples that were 80% oleogel and 20% aqueous mixture that contained a number of ingredients that are common to margarine.

They found that rice bran wax was not effective at creating a firm margarine, which may have been due to the emulsifier that they used. They found a correlation between wax content and firmness. Toro-Vazquez and others (2013) used 2-3% candelilla wax and monoglycerides to form an emulsion with 20% water. They found that their emulsified gels had lower firmness values than their emulsions. The gels they made would correspond to the 0% and 20% gel samples used in this study. This study found no change between 0% and 20% water content using 10% rice bran wax, showing that the

10% wax is effective at maintaining the gel structure. Beri and others (2013) used 10-

40% water in their organogels made with castor oils and different waxes. They calculated Young’s modulus for their samples, which is an indication of how much the sample acts like an elastic solid (Norton and others 2011). They found that adding the water decreased the modulus and the point of fracture for their samples made with 5% paraffin and 10% microcrystalline wax. However, once the gels had water in them, increasing the water content from 10-40% had no effect. When they investigated other waxes and emulsifiers, they found that generally, as water content increased, the modulus and point of fracture decreased. The extent depended on the wax and emulsifier that were used.

Storage time can have an effect on the hardness values of fats. The changes in crystal form discussed in the rheology section can also have an effect on the hardness of fats. As the fats sit in storage, the crystals change shape into more stable forms. This process is called maturation (Ronholt and others 2012).

When the oleogels are placed in 5°C storage, they immediately form a number of needle- like crystals that structure the oil, which was seen under microscopy. Since changes in the crystals are minimal during storage time, there is not any particular change observed in the hardness of the samples. It has been noticed that pure soybean oil can start to solidify into a gel after a few days in 5°C storage. This is due to the presence of small- chain fatty acids in the sample that can solidify at a temperature higher than the melting point of the oil, which is about -3°C. These fatty acids could solidify in the gels after a few days of storage. If this was happening, it was not observed in the maximum force measurements of these gels.

Ogutcu and others (2015) made margarine-type samples using beeswax and virgin olive oil. They measured the firmness of their samples over a 90-day storage period. For all their emulsified samples, they found no trend in the firmness values over the storage period. Each sample showed a certain degree of variability from day to day, but it was within the same level for each type of emulsion. This variability was also seen in the samples in this study. That variability may be associated more with errors within the

sample or within the machine, as opposed to some property inherent to the nature of the emulsion.

Other attributes that can be measured with TPA include resilience, cohesion, springiness, and gumminess. These attributes were similar to the maximum force in that there was no trend seen in the changes from water content, storage time, or emulsifier content.

4.4.2: Rheology

Hardness measurements require large scale deformation. However, the differences between the samples do not provide information about the internal structure of the gels.

Therefore, it was of interest to investigate more minor effects in the gels’ structures. The movement of the solid particles within the gel could be different based on the water contents (Brummer 2006), and fine tune measurements were needed to measure these differences. This was one appeal for rheology, which is a small scale deformation technique.

The gel samples had similar patterns for the G’ and G”, which can be seen in Figure 18.

The initial part of the graph, where the increasing strain is having a minimal effect on the

G’, is called the linear viscoelastic (LVR) region (Brummer 2006). When the strain reaches a certain point, the gel starts to break and the G’ decreases sharply. The G” shows a similar pattern to the G’.

1.00E+06

1.00E+05

Pa G' 1.00E+04 G"

1.00E+03 0.001 0.01 0.1 1 10 Strain (%)

Figure 18. Representative graph (5% w/E day 1) showing G’ and G” values measured over increasing strain.

The data for all the gels at all the time points follows the same pattern as was seen in

Figure 18. In addition, the values for G’ and G” were very close for all samples. In order to further investigate the effects of water content and time on the gels, a data point in the

LVR region was examined. The samples were compared at a strain of 0.005% for analysis. The point was selected using the computer software.

The data were compared for different water contents, on different days, in Figure 19.

600 a 500 A A a AB I i a B a I ii 400 B I I i-ii i-ii A 300

B G' (KPa) G' 200 C 100 D

0 1 day 3 days 7 days 27 days Time (days)

600

500 A a a AB a I i B I 400 B a I I i i i A 300

E G' (KPa) G' 200 F 100 G

0 1 day 3 days 7 days 27 days Time (days)

Figure 19. G’ of samples (a) without and (b) with emulsifier, comparing different water contents on the same day.

The differences in the water samples are most significant at 5 and 10% water, but this trend is not the same every day. The water in these gels may be mixed fully, such that the water content is not having an effect on the structural properties of the gel. The fat crystals in the gel seem to be having a greater effect on the firmness of the gels than the

water content. The water does not seem to be significantly interrupting the structure of the fat crystals. If they had disrupted the structure, the G’ might be lower. The values for the 20% water samples do tend to be on the higher side, compared to those from the other samples. This difference may be due to the fact that the water is not as well incorporated into the gel as it is for the other systems. The 20% gels were the most difficult to form, because if there was too much time before they started cooling, the water droplets would try to coagulate, and the water would separate from the rest of the sample. If the water droplets are taking up less of the sample, the place where the wax crystals are would have a higher ratio of wax to oil, which would increase the G’. In trying to place the gel on the rheometer, there was a risk with the 20% samples that the water would try to squeeze out from the samples. This indicates that at 20% water, the formation of the emulsion was dependent on the structure of the cooled wax crystals, and that the water was not interacting with the wax-oil matrix. The addition of the emulsifier seems to reduce this trend. This may be due to the emulsifier interacting with the water droplet interface, preventing them from coalescing into larger droplets before cooling. The SFC of the samples could also provide some insight into this rheological effect. It was noted that the samples with 20% water had a significantly higher SFC than those with lower water contents. An increase in SFC has been correlated with an increase in G’ (Deman and

Beers 1987).

Since they are the same size, it can explain why the differences between the rheological properties and the maximum force values were so small. Rheology depends on the way

the crystals move against each other when the sample is being sheared. If the crystals are the same size regardless of water content and emulsifier content, then they would react in a consistent manner when exposed to shear force. Maximum force testing is based on the effect of compressing the total structure, so minimal changes in crystal size may not affect the overall hardness of the sample.

Figure 20 shows the pattern of G’ over time. It was predicted that during storage, the fat crystals might grow or change form in such a way that would alter the structure of the gel. The storage time did not have a significant effect on the gels. The changes can result in an increase in G’ values as the sample starts exhibiting more solid-like behaviors.

600 i 500 A A i a i i A I 400 A abab b I I I 1 day 300

3 days G' (KPa) G' 200 7 days 100 27 days

0 A B C D Water Content

600

500 A A a i I i i A A I 400 ab ab b I I i b 1 day 300

3 days G' (KPa) G' 200 7 days 100 27 days

0 A E F G Water Content

Figure 20. G’ of samples (a) without and (b) with emulsifier, comparing storage time on the same water content.

Similar trends are seen when looking at the G” of the samples. Table 4 shows these trends. It can be seen that increasing the water content of the samples yields no significant effect on G” values. The same can be said for the effects of storage time.

Table 4. G” in KPa of samples, comparing effects of water content and storage time, without and with emulsifier. Letters compare the effect of water content, while numerals compare storage time.

G” (KPa) Sample Day 1 Day 3 Day 7 Day 27 A 140.3±7.0A,I 150.2±12.3 A,I 120.6±9.3 A,I 109.5±6.3 AB,I B 136.3±8.3 AB,I 118.8±7.6 A,I-II 118.2±8.9 AB,I-II 111.1±10.3 B,II C 111.2±7.6 B,I 122.8±9.6 A,I 113.9±10.8 A,I 110.7±7.2 AB,I D 158.4±13.4 A,I 155.8±9.9 A,I 134.5±6.1 A,I 135.7±9.1 A,I E 111.8±7.4 ab,I 110.1±5.3 a,I 128.3±11.7 a,I 107.3±6.9 A,I F 125.0±8.7 b,I 121.1±10.3 a,I 108.9±5.8 a,I 105.3±7.5 A,I G 135.6±8.2 a,I 138.7±11.9 a,I 116.7±8.3 a,I 138.8±6.7 A,I

Tan delta is the ratio of G” to G’. Since the G’ and G” have similar differences in values, it makes sense that tan delta for the gels is almost the same across water contents and storage times. Table 5 demonstrates this trend.

Table 5. Tan delta of samples, comparing effects of water content and storage time, without and with emulsifier. Letters compare the effect of water content, while numerals compare storage time.

Tan delta Sample Day 1 Day 3 Day 7 Day 27 A 0.311±0.005 A,I 0.329±0.003 A,I 0.331±0.005 A,I 0.322±0.006 A,I B 0.310±0.004 A,I 0.320±0.004 A,I 0.327±0.004 A,I 0.321±0.004 A,I C 0.314±0.004 A,I 0.313±0.004 A,I 0.331±0.004 A,I 0.330±0.008 A,I D 0.321±0.003 A,I 0.317±0.004 A,I 0.320±0.003 A,I 0.325±0.004 A,I E 0.325±0.004 a,I 0.324±0.004 a,I-II 0.331±0.008 a,I-II 0.320±0.004 a,II F 0.321±0.003 a,I 0.319±0.004 a,I 0.333±0.004 a,I 0.321±0.004 a,I G 0.337±0.005 a,I 0.332±0.006 a,I 0.338±0.003 a,I 0.335±0.005 a,I

The average tan delta value is around 0.324. This indicates that the gels behaved much more as a solid than it did as a liquid. Since the tan delta was so consistent, it indicates that whatever the hardness values of the gels are, the internal structure is consistent through all the gels.

The G* of the samples showed a similar pattern to the G’ and G”, which can be seen in Table 6.

Table 6. G* in KPa of samples, comparing effects of water content and storage time, without and with emulsifier. Letters compare the effect of water content, while numerals compare storage time.

G* (KPa) Sample Day 1 Day 3 Day 7 Day 27 A 471.2±20.1 A,I 401.0±43.3 A,I 382.6±27.8 A,I 359.6±22.6 AB,I B 422.3±26.5 AB,I 357.7±26.0 A,I-II 337.0±30.1 A,I-II 285.9±48.9 B,II C 370.1±23.8 B,I 378.6±32.4 A,I 276.5±29.9 A,I 328.9±32.4 AB,I D 412.3±35.8 A,I 470.1±34.9 A,I 441.1±19.1 A,I 440.5±31.2 A,I E 360.7±22.0 ab,I 358.2±18.5 a,I-II 343.6±4.76 a,I-II 327.6±32.1 a,II F 372.5±22.2 b,I 382.0±41.5 a,I 310.2±32.8 a,I 344.9±25.7 a,I G 390.7±43.1 a,I 399.1±49.9 a,I 321.6±28.1 a,I 405.3±36.1 a,I

The G* values of the gels were very similar to the G’ values. This indicates that the gels’ behaviors were more like solids than they were like liquids. This trend backs up the data seen in the tan delta figures.

The yield point of the gels is the point at which the gels would break from the increasing strain. This point was calculated using the 0.2% method, which is standard for yield stress measurements. The yield stress occurs at the point where the stress deviates by

0.2% of that which was seen in the LVR. The yield point data can be seen in Figure 21.

0.035 A A a I a I I i i i 0.03 A I i A a a 0.025

0.02 A

0.015 B C 0.01 Yield Point (% Strain) D 0.005

0 Day 1 Day 3 Day 7 Day 27 Storage Time (Days) (a)

0.035 a i A I i 0.03 A A A a a I I i a I i 0.025

0.02 A

0.015 E F 0.01 Yield Yield PointStrain) (% G 0.005

0 Day 1 Day 3 Day 7 Day 27 Storage Time (Days) (b)

Figure 21. Yield point of RBW oleogels made with increasing water content and stored over a 27-day period, (a) without and (b) with emulsifier.

The yield point of the gels was expected to decrease as the water content increased, because the water was expected to disrupt the gel network. However, there was no significant difference observed between samples made with increasing amounts of water.

In addition, there were no significant differences observed with the increase in storage time, or with changes in emulsion content. These data show that the structure and behavior of the oleogels were very consistent when 10% RBW was used.

Many studies for rheology have shown that the higher the concentration of organogelator, the larger the G’. Beri and others (2013) observed the effects of adding water to their wax-stabilized emulsions. They used 5% paraffin 10% microcrystalline wax. They found that as the water content of their samples increased, the G’ decreased. They attributed this change to the crystal network being less developed as more water droplets disrupted the system. In addition, as the water content increased, the phase angle of their gels decreased. Phase angle is another technique for comparing the G’ and G” of the gels, similar to tan delta. A decrease in phase angle means that the gels were starting to behave more like liquids than like solids. The needlelike crystals of the rice bran wax may have stabilized the gels to prevent the changes that were observed in the samples with paraffin and other waxes. In all the gels, the G’ remained above G” for their tests, indicating that the gels acted more as solids than as liquids.

Patel and others (2013) used shellac and rapeseed oil to make emulsions. Their gels used up to 6% shellac, and the emulsion contained 20% water. While only one concentration of water was used, they found that the emulsified gels had lower G’ values than those 83

made without water. In addition, increasing the shellac content resulted in an increase in

G’.

Chapter 5: Conclusions

It was found that 2% RBW was not a sufficient concentration to maintain the structure of the oleogels as the added water content increased. With 5% RBW, the structure could only be maintained if emulsifier was added. By 10% RBW, the wax concentration was sufficient to maintain structure as water content increased. The samples were able to hold 20% w/w water without losing any over 27 days in storage, so the water holding capacity was consistent through storage. Samples with higher water content had larger water droplets that appeared more frequently through the samples. However, the increase in water content had a minimal effect on the physical and thermal properties of the gels.

Where those differences occurred, the addition of emulsifier reduced the variability that was seen between those samples.

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Appendix A: Data for low wax oleogel measurements

The first issue to understand with the gels is whether they are capable of holding their initial water content during storage. The moisture content analyzer was the first method used to determine the actual amount of moisture lost from the samples during storage.

This method uses infrared heating to dry the sample, while maintaining a record of the loss of mass during the testing period. Figure 22 shows the moisture contents of these gel samples after 1 and 3 days.

20 18 16 14 12 10 1 8 3 6

Moisture Content (%) Content Moisture 4 2 0 5we 10we 20we

20 18 16 14 12 10 1 8 3 6

Moisture Content (%) Content Moisture 4 2 0 5we 10we 20we

Figure 22. Moisture content of samples made with (a) 2% and (b) 5% wax over 2 days storage.

The gels were tested at two time points. There was some loss relative to the amount of water initially added to the sample due to evaporation during processing. However, no moisture was lost during the storage time. There was no loss of water from the samples

with either 2 or 5% wax. That low concentration was enough to trap the water droplets in the gel matrix.

Tan delta is the ratio of G” to G’. While G’ refers to the solid-like properties of the gel,

G” represents the liquid behaviors. When tan delta is 1, then the gel is behaving equally as a solid and a liquid. If tan delta is less than 1, the solid-like properties are more dominant. Figure 23 shows the tan delta values for the gels.

0.50 0.45 B a 0.40 A A A a a a 0.35 0.30 0% 0.25

0.20 5% Tan delta Tan 0.15 10% 0.10 20% 0.05 0.00 1 3 Time (days)

Figure 23. Tan delta of gels with (a) 2% wax without and (b) with emulsifier, and (c) 5% wax without and (d) with emulsifier. (Continued)

Figure 23. Continued

0.50 0.45 A A A a a a 0.40 A a 0.35 0.30 0% 0.25

0.20 5%wE Tan delta Tan 0.15 10%wE 0.10 20%wE 0.05 0.00 1 3 Time (days)

0.50

0.45 A b 0.40 0.35 A 0.30 A a a a A 0% 0.25

0.20 5% Tan delta Tan 0.15 10% 0.10 20% 0.05 0.00 1 3 Time (days)

(Continued)

Figure 23. Continued

0.50 0.45 0.40 b b 0.35 B b B B 0.30 A a 0% 0.25

0.20 5%wE Tan delta Tan 0.15 10%wE 0.10 20%wE 0.05 0.00 1 3 Time (days)

For 2% wax, there were no significant differences in tan delta observed between gels with increasing water contents. The gels were rather soft due to the low wax content, so the additional water content is not affecting the degree to which the sample behaves like a solid or liquid. This trend was true for samples with and without the emulsifier. The gels made with 5% wax were also not influenced by the addition of water to the system, until

20% water. For 0 to 10% water, the tan delta is very consistent, indicating that the addition of water is not significantly affecting the properties of the system. When 20% water is added, the tan delta is increased significantly, indicating that the gel would behave much more as a liquid. The addition of the emulsifier mitigates the effects of the

20% water. This change may be due to interactions between the emulsifier and the water droplets; the emulsifier is helping keep the water droplets in place so that they do not move in response to increased strain. The resistance to strain is more solid-like behavior.

The samples made with the 2% gels all have higher tan delta values than the samples made with 5% wax. Since there is less wax to structure the oil, the gels are behaving more as liquids.