Engineering the Crystallization Behavior of Triacylglycerols Using High Behenic Acid Stabilizers

Ga Yae Kim

A Thesis presented to The University of Guelph

In partial fulfillment of requirements for the degree of Master of Science in Food Science

Guelph, Ontario, Canada

ABSTRACT

ENGINEERING THE CRYSTALLIZATION BEHAVIOR OF TRIACYLGLYCEROLS

USING HIGH BEHENIC ACID STABILIZERS

Ga Yae Kim Advisor:

University of Guelph, 2017 Dr. Alejandro G. Marangoni

High behenic acid stabilizer (HBS) was used to stabilize oils and accelerate the nucleation of fats.

HBS stabilized liquid oil more effectively when there were more high-melting triacylglycerols

(HMTs) (melting point > 30°C) in the oil to be stabilized and when cooled at a faster rate.

Maximum oil stabilization efficiency of HBS in peanut oil was correlated with the lowest Cg, the highest nucleation rate, and the highest G′. On the other hand, the effect of HBS on the nucleation rate of fats was inversely correlated to the amount of HMTs present in fats as the nucleation of fats with a high level of HMTs (> 67 %) was mostly controlled by the native

HMTs of fats. The mechanism of HBS on oil stabilization and nucleation enhancement was to co-crystallize with HMTs present in the “host” fat/oil, resulting in a formation of new fraction.

The molecular compatibility between HBS and the “host” fat/oil is prerequisite for the oil stabilization and the nucleation enhancing effect of HBS to be effective.

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ACKNOWLEDGEMENTS

First, I would like to thank Dr. Marangoni for his guidance, patience and support throughout my Master’s program. I could always count on him for direction and encouragement.

Our meetings were always constructive and full of innovative and creative ideas that inspired me to give my best. His advice and comments on my work helped me to improve my scientific writing and thinking. I am also thankful for the supportive environment that he fostered in the lab as well.

For their time, encouragement and guidance, I would like to thank my advisory committee, Dr. Lim and Dr. Rogers during my thesis. I would also like to thank the research associates in the lab for their support and insight. Thank you Dr. Fernanda Peyronel, Saeed

Mizaee Ghazani and Andrew Gravelle. I am very appreciative for all the help I got from you.

I was so fortunate to be part of my supportive lab community. Thank you Kristin, Pere,

Cendy, Brian, Braulio, Chloe, Ming, Reed, and all the visiting and summer students for keeping me sane and feeling welcomed. I couldn’t have asked for a better group of people. I truly wish you all the best.

Finally, the greatest thanks to my fiancé Paul, my family and friends for their continuous support, encouragement and unconditional love. I couldn’t have done this without you.

THANK YOU EVERYONE!!

TABLE OF CONTENTS

ABSTRACT ...... ii ACKNOWLEDGEMENTS ...... iii TABLE OF CONTENTS ...... iv LIST OF TABLES ...... vii LIST OF FIGURES ...... vii CHAPTER 1 Introduction...... 1 1.1 Objectives ...... 3 1.2 Hypotheses...... 3 References ...... 4 CHAPTER 2 Literature Review ...... 7 2.1 High Behenic Acid Stabilizer ...... 7 2.2 Stabilizing And Structuring Liquid Oil ...... 7 2.2.1 Liquid Oil Stabilization...... 8 2.2.2 Structuring Liquid Oil ...... 9 2.3 Controlling Fat Crystallization ...... 11 2.3.1 Cooling Rate ...... 11 2.3.2 Minor Components ...... 12 2.4 Conclusion ...... 16 References ...... 17 CHAPTER 3 Crystallization Behavior of High Behenic Acid Stabilizers in Liquid Oil ...... 22 3.1 Abstract ...... 22 3.2 Introduction ...... 22 3.3 Materials and Methods ...... 24 3.3.1 Materials ...... 24 3.3.2 Fatty Acid Composition ...... 24 3.3.3 Triacylglycerol Composition ...... 25 3.3.4 Critical Gelation Concentration Determination ...... 26 3.3.5 Nucleation Kinetics ...... 26 3.3.6 Solid Fat Content ...... 27 3.3.7 Thermal Behavior ...... 28

3.3.8 Polymorphism ...... 29 3.3.9 Small Deformation Rheology ...... 29 3.3.10 Polarized Light Microscopy ...... 29 3.3.11 Statistical Analysis ...... 30 3.4 Results and Discussion ...... 30 3.4.1 Fatty Acid Composition ...... 30 3.4.2 Triacylglycerol composition ...... 31

3.4.3 Critical Gelation Concentration Determination ...... 32 3.4.4 Nucleation Kinetics ...... 33 3.4.5 Solid Fat Content ...... 34 3.4.6 Thermal Behavior ...... 37 3.4.7 Polymorphism ...... 40 3.4.8 Small Deformation Rheology ...... 41 3.4.9 Polarized light microscopy ...... 42 3.5 Conclusions ...... 45 References ...... 47 CHAPTER 4 Engineering the Nucleation of Edible Fats Using a High Behenic Acid Stabilizer 49 4.1 Abstract ...... 49 4.2 Introduction ...... 49 4.3 Materials and Methods ...... 51 4.3.1 Materials and Sample Preparation ...... 51 4.3.2 Fatty Acid Composition ...... 52 4.3.3 Triacylglycerol Composition ...... 53 4.3.4 Nucleation Kinetics ...... 53 4.3.5 Solid Fat Content ...... 54 4.3.6 Thermal Behavior ...... 55 4.3.7 Polymorphism ...... 55 4.3.8 Polarized Light Microscopy ...... 56 4.3.9 Statistical Analysis ...... 56 4.4 Results and Discussion ...... 57 4.4.1 Fatty Acid Composition ...... 57 4.4.2 Triacylglycerol Composition ...... 58 4.4.3 Physical Properties of HBS-Fat Mixtures ...... 62 v

4.4.4 Interaction between HBS and Specific Triacylglycerols or a Mixture of Triacylglycerols ...... 75 4.5 Conclusions ...... 80 References ...... 81 CHAPTER 5 Conclusions and Future Work ...... 84

LIST OF TABLES

CHAPTER 3

Table 1 Fatty acid composition of high oleic safflower oil, sesame oil, peanut oil, and HBS. Shown are means and standard deviations of n=2 replicates...... 31 Table 2 Triacylglycerol composition of high oleic safflower oil, sesame oil, peanut oil, and HBS. Shown are means and standard deviations of n=2 replicates...... 32

Table 3 Cg of HBS (% w/w) for edible oils at different cooling rates...... 33 Table 4 DSC thermal data of 12 % HBS-oil mixtures. Shown are means and standard deviations of n=3 replicates...... 39 CHAPTER 4

Table 1 Fatty acid composition of milk fat, palm oil, palm kernel oil, palm stearin, high-melting milk fat fraction, and high behenic acid stabilizer. Shown are means and standard deviations of n=2 replicates...... 57 Table 2 Triacylglycerol composition of anhydrous milk fat, palm oil, palm kernel oil, palm stearin, and high-melting milk fat fraction. Shown are means and standard deviations of n=2 replicates...... 59

Table 3 Triacylglycerol composition of the high behenic acid stabilizer...... 60

LIST OF FIGURES

CHAPTER 3 Figure 1 Free energies of nucleation (ΔG) of 12 % HBS-oil mixtures as a function of temperature. Shown are means and standard deviations of n=6 replicates...... 34 Figure 2 SFC of 12 % HBS-oil mixtures as a function of (A and B) temperature (A: 3 °C/min, B: 0.6 °C/min) and (C) time at 20 °C. Shown are means and standard deviations of n=3 replicates...... 36 Figure 3 DSC crystallization curves of 12 % HBS-oil mixtures and neat HBS cooled at (A) 3 °C/min and (B) 0.6 °C/min...... 38 Figure 4 DSC melting curves of 12 % HBS-oil mixtures and neat HBS. All samples were heated at 5 °C/min...... 39

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Figure 5 Wide angle X-ray spectra of 12 % HBS-oil mixtures and neat HBS. (a)PeO- 3 °C/min (b) PeO-0.6 °C/min (c)HOSO-3 °C/min (d)HOSO-0.6 °C/min (e)SeO- 3 °C/min (f)SeO-0.6 °C/min (g)HBS-3 °C/min (h)HBS-0.6 °C/min...... 41 Figure 6 Amplitude sweep rheograms of 12%HBS-oil mixtures cooled at (A) 3°C/min and (B) 0.6°C/min...... 42 Figure 7 Polarized light micrographs of 12 % HBS-oil mixtures. The scale bar represents 100 um...... 44

CHAPTER 4 Figure 1 HPLC chromatograms showing the triacylglycerol profile of (A) high- melting milk fat fraction and (B) milk fat...... 61 Figure 2 Free energies of nucleation (ΔG) of (A) anhydrous milk fat (B) palm oil (C) palm kernel oil and (D) palm stearin and HBS mixtures as a function of temperature. Shown are means and standard deviations of n=6 replicates...... 63 Figure 3 Free energies of nucleation at 306 K as a function of (A) high-melting triacylglycerols (HMT) (% w/w) present in each fat and (B) high-melting triacylglycerols (% w/w). No significant correlations (ns) between ΔGn at 306 K and HMT for the 1.5 % HBS addition. Shown are means and standard deviations of n=6 replicates...... 64 Figure 4 SFC of (A) anhydrous milk fat (B) palm oil (C) palm kernel oil and (D) palm stearin as a function of time at 20°C. Shown are averages and standard deviations of n=3 replicates. The insets of panels A and B highlight the early stages of the crystal growth...... 66 Figure 5 Differential scanning calorimetric crystallization curves of (A) anhydrous milk fat (B) palm oil (C) palm kernel oil and (D) palm stearin and DSC melting curves of (E) anhydrous milk fat (F) palm oil (G) palm kernel oil and (H) palm stearin...... 68 Figure 6. Differential scanning calorimetric (A) crystallization curves and (B) melting curves of HBS-Palm oil mixtures...... 70 Figure 7 Pseudo two-component state diagram of HBS-Palm oil mixtures constructed using peak melting temperatures determined using differential scanning calorimetry (Figure 6B). Shown are means n=6 replicates...... 71 Figure 8 Wide angle X-ray spectra of (A) anhydrous milk fat, (B) palm oil, (C) palm kernel oil and (D) palm stearin. Numbers indicate the corresponding d-spacings for the reflection...... 72 Figure 9 Polarized light micrographs of HBS-fat mixtures. The scale bar represents 100 µm...... 74 viii

Figure 10 Phase diagrams of (A) tristearin (B) tripalmitin and (C) high-melting milk fat fraction and HBS mixtures. % HBS is the proportion to the total weight of the mixture. All HBS-TAG mixtures were prepared with a starting material which is a blend of TAG: HOAO at a 4:6 ratio. Shown are means and standard deviations of n=4 replicates...... 76 Figure 11 Small angle X-ray spectra of (A) tristearin (B) tripalmitin and (C) high- melting milk fat fraction and HBS mixtures. % HBS is the proportion to the total weight of the mixture. All HBS-TAG mixtures were prepared with a starting material which is a blend of TAG:HOAO at a 4:6 ratio. Numbers indicate the corresponding d-spacings for the reflection...... 77 Figure 12 Wide angle X-ray spectra of (A) tristearin (B) tripalmitin and (C) high- melting milk fat fraction and HBS mixtures. % HBS is the proportion to the total weight of the mixture. All HBS-TAG mixtures were prepared with a starting material which is a blend of TAG:HOAO at a 4:6 ratio. Numbers indicate the corresponding d-spacings for the reflection...... 78

CHAPTER 1 Introduction

Fat is one of the most widely used ingredients in food products. It appears as a solid at temperatures above 25°C, but it is comprised of fat crystals entrapping liquid oil. In fat-based products such as margarine, shortening, and nut/seed butter, fat is a main ingredient. Mechanical and organoleptic properties of fat-based products depend on the physical properties of fats

(Marangoni and Wesdorp 2012). For example, the βʹ polymorph and small, uniformly sized fat crystals are preferred in plastic fat products that are required to have desirable smoothness, mouthfeel and spreadability (Danthine and Deroanne 2003; deMan and deMan 1994). Physical properties of fats are determined by the crystallization process (Tan and Man 2002).

Triacylglycerols (TAGs), the main component of fats, crystallize differently depending on their chemical and molecular characteristics (Sato 2001; Sulaiman et al. 1997; Taylor 1994), the presence of additives, and processing conditions (Maleky et al. 2012; Metin and Hartel 2005;

Sato 2001).

There are many challenges that need to be addressed when manufacturing fat-based food products. In this study, we will focus on two main issues: oil separation and complex thermal behaviors of fats and oils. Many intensive studies have been conducted to minimize these issues

(Kellens 1993; Sulaiman et al. 1997). However, little is known about the mechanism behind the oil stabilizing and crystallization enhancing effect of stabilizers high in behenic acid (HBS).

HBSs are a blend of fully hydrogenated vegetable oils (FHVO) high in erucic acid (Sato 2001).

They are mostly manufactured and marketed as a stabilizer because they are great for increasing the oil stability of fat-based food products (Capanoglu and Boyacioglu 2008; Chien 2015;

Peyronel et al. 2016). Peyronel et al. (2016) reported its acceleratory effect on crystallization of palm mid fraction and palm olein.

Oil separation is a phenomenon where the liquid oil phase separates from the solid particles in the fat-matrix. It is considered a detrimental problem to some food products because it changes not only the organoleptic and mechanical properties but also decreases the shelf-life of the products as the exposure of oil promotes lipid oxidation. Many researchers and manufacturers have proposed strategies to prevent or delay oil separation in fat-based food products. For example, the effects of palm oil (PO) and FHVOs on oil stabilization in peanut butter have been reported (Aryana et al. 2003; Gills and Resurreccion 2000; Hinds et al. 1994).

In addition, mixtures of mono-, di-, and triacylglycerols, are used as stabilizers (Mccoy 1982;

Widlak 2002).

Because most natural fats and oils are complex, their crystallization and melting behaviors are difficult to control or optimize. A high content of diglycerides (DAG) in PO, for instance, is speculated to be one of the reasons that cause a slow crystallization (Goh and Timms 1985; Siew and Ng 1999). Slow crystallization often results in a long transformation time from the α to βʹ polymorph (De Clercq et al. 2012; Verstringe et al. 2014) and post-hardening problems during the storage of palm-based food products (Siew and Ng 1999). Another example is anhydrous milk fat (AMF) which has an appealing flavour and mouthfeel that make it an advantageous food ingredient (Kerr et al. 2011). Despite its ideal characteristics, its application is limited due to its wide melting range (about - 40 °C to + 40 °C). This wide melting range leads to several polymorphic forms which could change the physical properties of AMF (Lopez et al. 2001).

Therefore, there has been a myriad of research on controlling crystallization behavior of fats 2

using additives and minor components (reviewed in the next chapter) and optimizing processing conditions (Campos et al. 2002; Herrera and Hartel 2000; Herrera et al. 2015; Martini et al.

2002a, 2002b).

1.1 Objectives

 Study the crystallization behavior of HBS in liquid oil  Examine the effects of cooling rate and chemical composition of oils on the oil stabilization efficiency by HBS  Investigate the influence of HBS on fat crystallization  Determine the mechanism of HBS on crystallization enhancement

1.2 Hypotheses

 The addition of HBS to liquid oil will result in a gel formation  Crystallization behavior of HBS will be affected by the type of oil and a cooling rate  HBS crystals will entrap liquid oil, and consequently stabilize the oil phase  HBS will accelerate the crystallization of fats by forming a new compound as a result of co-crystallizing with a high-melting fraction of fats

References

Aryana KJ, Resurreccion AV, Chinnan MS, Beuchat LR (2003) Functionality of palm oil as a stabilizer in peanut butter. J Food Sci 68:1301-7 Campos R, Narine SS, Marangoni AG (2002) Effect of cooling rate on the structure and mechanical properties of milk fat and lard. Food Res Int 35:971-81 Capanoglu E, Boyacioglu D (2008) Improving the quality and shelf life of Turkish almond paste. J Food Qual 31:429–445 Chien Y (2015). Shelf life extension of seed butter made with sesame, sunflower and pumpkin seeds, Master's thesis, the Ohio State University, Columbus Danthine S, Deroanne C (2003). Blending of hydrogenated low-erucic acid rapeseed oil, low- erucic acid rapeseed oil, and hydrogenated palm oil or palm oil in the preparation of shortenings. J Am Oil Chem Soc 80:1069-75 De Clercq N, Danthine S, Nguyen MT, Gibon V, Dewettinck K (2012) Enzymatic interesterification of palm oil and fractions: monitoring the degree of interesterification using different methods. J Am Oil Chem Soc 89:219-29 deMan L, deMan JM (1994) Functionality of palm oil, palm oil products and palm kernel oil in margarine and shortening. Lipid Technol 6:5–10 Gills LA, Resurreccion AV (2000) Sensory and physical properties of peanut butter treated with palm oil and hydrogenated vegetable oil to prevent oil separation. J Food Sci 65:173-80 Goh EM, Timms RE (1985) Determination of mono- and diglycerides in palm oil, olein and stearin. J Am Oil Chem Soc 62:730-734 Herrera ML, Hartel RW (2000) Effect of processing conditions on crystallization kinetics of a milk fat model system. J Am Oil Chem Soc 77:1177–1188 Herrera ML, Rodríguez-Batiller MJ, Rincón-Cardona JA, Agudelo-Laverde LM, Martini S, Candal RJ (2015) Effect of cooling rate and temperature cycles on polymorphic behavior of sunflower oil stearins for applications as trans-fat alternatives in foods. Food Bioprocess Thecnol 8:1779-90 Hinds MJ, Chinnan MS, Beuchat LR (1994) Unhydrogenated palm oil as a stabilizer for peanut butter. J Food Sci 59:816-20 Kellens M (1993) New development in the fractionation of palm oil, proceedings of porim international palm oil congress. Palm oil research institute of malaysia, Bangi, Selangor, pp. 128–140

Kerr RM, Tombokan X, Ghosh S, Martini S (2011) Crystallization behavior of anhydrous milk fat− sunflower oil wax blends. J Agric Food Chem 59:2689-95 Lopez C, Lavigne F, Lesieur P, Bourgaux C, Ollivon M (2001) Thermal and structural behavior of milk fat. 1. Unstable species of anhydrous milk fat. J Dairy Sci 84:756-66 Maleky F, Acevedo NC, Marangoni AG (2012) Cooling rate and dilution affect the nanostructure and microstructure differently in model fats. Eur J Lipid Sci Technol 114:748-759 Marangoni AG, Wesdorp LH (2012) Structure and Properties of Fat Crystal. 2nd edn. CRC Press LLC, Boca Raton, pp. 16, 28-35 Martini S, Herrera ML, Hartel RW (2002a) Effect of cooling rateon crystallization behavior of milk fat fraction/sunflower oil blend. J Am Oil Chem Soc 11:1055–1062 Martini S, Herrera ML, Hartel RW (2002b) Effect of processing conditions on microstructure of milk fat fraction/sunflower oil blends. J Am Oil Chem Soc 11:1063–1068 Mccoy SA (1982) Peanut butter stabilizer (Patent US 4341814) Metin S, Hartel RW (2005) Crystallization of fats and oils. In: Shahidi F (ed) Bailey’s Industrial oil and fat products, 6th edn, vol 6. Wiley, London Peyronel F, Campos R, Marangoni AG (2016) Prevention of oil migration in palm mid fraction and palm olein using a stabilizer rich in behenic acid. Food Res Int 88:52-60 Sato K (2001) Crystallization behaviour of fats and lipids—a review. Chem Eng Sci 56:2255-65 Siew WL, Ng WL (1999) Influence of diglycerides on crystallisation of palm oil. J Sci Food Agric 79:722-6 Sulaiman MZ, Sulaiman NM, Kanagaratnam S (1997) Triacylglycerols responsible for the onset of nucleation during clouding of palm olein. J Am Oil Chem Soc 74:1553-8 Tan CP, Man YBC (2002) Differential scanning calorimetric analysis of palm oil , palm oil based products and coconut oil : effects of scanning rate variation. Food Chem 76:89–102 Taylor AM (1976) The crystallisation and dry fractionation of malaysian palm oil. Oleagineux 3:73–79 Van Niiekerk PJ, Burger A (1985) The estimation of the composition of edible oils mixtures. J Am Oil Chem Soc 62:531–538 Verstringe S, Danthine S, Blecker C, Dewettinck K (2014) Influence of a commercial monoacylglycerol on the crystallization mechanism of palm oil as compared to its pure constituents. Food Res Int 62:694-700

Widlak N (2002) Peanut butter stabilizer and method for manufacturing stabilized peanut butter (Patent US 6447833)

CHAPTER 2 Literature Review

2.1 High Behenic Acid Stabilizer

Fat stabilizers are widely used additives to yield desirable mechanical, functional and shelf-life properties of fat-based food products such as margarine, peanut butter, shortening and confectionery fats. Fully hydrogenated vegetable oils (FHVO) are often used as stabilizers. Some most commonly used FHVOs in the stabilizers are cottonseed oil (Gooding et al. 1973, Widlak

2002), peanut oil (Gooding et al. 1973), and rapeseed oil (Sanders 1964). They can be used by themselves or blended with other FHVO to maximize the effect.

Stabilizers high in behenic acid (HBS) are also a blend of FHVOs, mainly fully hydrogenated rapeseed, cotton seed and soybean oils. The ratio of FHVOs varies among different manufacturers depending on the application, so they have different TAG and fatty acid profiles.

The content of behenic acid ranges from 20 - 50 %, and other major fatty acids include stearic, palmitic and arachidic acids. Typical applications of HBS include the prevention of oil separation, promotion of a glossy surface and desirable texture, and acceleration of crystallization in peanut butter, margarines, spreads and fillings (Danisco emulsifiers 2016;

Palsgaard 2014).

2.2 Stabilizing And Structuring Liquid Oil

Fats are composed of fat crystals in a continuous liquid oil phase. Natural oils can be structured by directly blending them with different proportions of saturated fats such as FHVOs

(Nusantoro et al. 2013) or food grade gelators. Direct blending is a great alternative to fractionation, hydrogenation and interesterification because it is cheaper, simpler and 7

nondestructive (Hayati et al. 2009). FHVOs are often used to achieve similar physical characteristics and functionality as partially hydrogenated vegetable oils which were popular ingredients used in fat-based products before trans-fatty acids were found to have negative health effects (Marangoni and Garti 2011). They are also used to improve oil holding capacity and shelf-life of fat-based products that are susceptible to oil separation. The effects of FHVOs and other gelators on stabilizing and structuring liquid oil are reviewed in this section.

2.2.1 Liquid Oil Stabilization

The effects of FHVOs on improving oil stability and retarding oil separation in fat-based products have been widely studied over the last few decades. HBS, 1.25 % – 7 % (w/w), significantly reduced oil separation for blends of palm mid fraction and palm olein by forming a new fraction with high melting fractions of two fats, thus resulting in a denser crystal network with higher oil binding capacity (Peyronel et al. 2016). HBS effectively inhibited oil separation in almond paste and seed butters as well (Capanoglu and Boyacioglu 2008; Chien 2015). Lipid oxidation of almond paste was retarded as 0.5 % HBS improved oil separation during storage at

4 °C and 30 °C (Capanoglu and Boyacioglu 2008). The addition of 2 % and 3 % HBS in seed butters not only prevented oil separation but also improved hardness during storage of 28 days at

25 °C (Chien 2015). No oil separation was observed in peanut butter stabilized with 1.5 - 2.5 %

(w/w) of HBS during 153 days of storage time at 0 - 45 °C (Gills and Resurreccion 2000). The addition of 1 - 2 % (w/w) fully hydrogenated flixweed seed oil and HBS inhibited oil separation in peanut butter when stored at 4, 21, 40 °C for up to 24 weeks (Ahmadi and Shahmlr 2016). In both studies, HBS resulted in the highest level of hardness, and the hardness of peanut butter remained constant for a longer period of time compared to unstabilized peanut butter or other

treatments (Ahmadi and Shahmlr 2016; Gills and Resurreccion 2000). The incorporation of 2 %

(w/w) fully hydrogenated peanut oil, consisting of palmitic (47 %) and stearic (52 %) acids, in low fat butter spreads improved spreadability and stability against oil separation at 25 °C or

36 °C (Reddy et al. 1999).

2.2.2 Structuring Liquid Oil

Higaki et al. (2003, 2004) made β-fat gel by adding 1.5 - 4.0 % (w/w) fully hydrogenated rapeseed oil with a high content of behenic acid (FHRO-B) to low-melting fats, sal fat olein and cocoa butter, which are liquid at room temperature. A specific thermal treatment was necessary in order to form gels: rapid cooling (10 °C/min) to the crystallization temperature from 70 °C followed by heating to the final temperature where both temperatures were determined depending on the melting points of FHRO-B and low-melting fats. In addition to the thermal treatment, the α to β polymorphic transition was another important step prior to the gel formation.

The polymorphism as well as the size and morphology of FHRO-B crystals played a crucial role in gel formation.

A recent study has shown a potential application of the β-crystals of FHRO-B as an aerating agent in whipped oil without using any emulsifiers. The lamellar planes of FHRO-B crystals were arranged almost parallel to the air-oil surface, and these crystals encapsulated the air bubble

(Mishima et al. 2016). The transition of α to β polymorph, mentioned above, was also a prerequisite for the formation of the most functional organogel using FHRO-B. Fat crystals of the gel containing 2 % of FHRO-B and 98 % of low melting fats were small and uniformly sized and distributed. Small, evenly-distributed crystals lead to desirable texture, spreadability, and

smoothness of gel materials and plastic fat products (shortening, margarine, spread) (Marangoni and Garti 2011).

Food grade natural waxes have shown a great potential in structuring edible oils because they are efficient oil binders and emulsion stabilizers, readily available and relatively inexpensive

(only small amounts are needed) (Blake et al. 2014; Hwang et al. 2013; Hwang et al. 2015; Patel et al. 2013; Toro Vazquez et al. 2007). Patel et al. (2015) studied the gelation behavior of high oleic sunflower oil gel made by adding six natural waxes with different chemical compositions.

Critical gelation concentrations (Cg) of waxes ranged from 0.5 % to 7% depending on their chemical nature and the gelation temperatures. The strongest gel was obtained with carnauba wax (CRX), containing wax esters and the highest proportion of fatty alcohols. Gels formed with waxes containing a high proportion of lower melting fatty acids not only displayed a higher Cg but also showed a lower elastic modulus. Safflower oil was successfully made into gels using candelilla wax (CLX) (0.5 - 3 % w/w) (Morales-Rueda et al. 2009; Toro-Vazquez et al. 2007), and they were stable against phase separation for at least for three months. In later work, Toro-

Vazquez et al. (2009) were able to obtain CLX-high-oleic safflower oil gels with different thermomechanical properties by adding tripalmitin (0 – 1 % w/w) and changing the gelation temperature. The authors noted that the co-crystallization of tripalmitin and CLX resulted in gels with higher elastic modulus and yield stress and a narrower melting temperature range (36 °C –

38 °C). Dassanayake et al. (2009) studied physical properties of three waxes, CRX, CLX, and rice bran wax (RBW), in olive oil gels. Long needle-like RBX crystals were distributed in a more homogeneous fashion in liquid oil than the other two wax crystals. In addition, the Cg of RBW

was lower for olive oil gelation, and the rate of gel formation was higher for RBW-based gels than other gels.

2.3 Controlling Fat Crystallization

Fat crystallization affects the physical properties of fat, consequently mechanical and functional characteristics of fat-based products. There is an ongoing challenge to control fat crystallization because changes in the physical state of a fat and modifications in fat crystallization behavior are directly related to the quality and stability of those products. One method of controlling fat crystallization is to modify determinant factors affecting crystallization behavior of fats. Some examples of determinant factors are chemical and molecular nature of

TAGs, the presence of minor components and processing conditions (cooling rate, crystallization temperature, shear, pressure, storage time etc.). In this section, cooling rate and minor components, are reviewed.

2.3.1 Cooling Rate

Many researchers have investigated the effects of cooling rate on crystallization behavior of fats. The effects vary depending on the chemical properties of fats and crystallization temperature. Upon slow cooling, TAGs normally experience a smaller driving force per unit time for crystallization, thus the rate of nucleation and crystallization decreases (Campos et al. 2002;

Marangoni et al. 2006a, 2006b; Herrera et al. 2015). Several studies have reported higher SFC values when milk fat and lard (Campos et al. 2002; deMan 1964; Herrera and Hartel 2000), butter (Julien et al. 1985) and sunflower oil stearins (Herrera 2015) were crystallized at higher cooling rates. In addition, TAGs have more time (longer induction time) to align themselves into

more stable polymorphic forms when they are cooled slowly (Campos et al. 2002; D’Souza et al.

1990; Maleky et al. 2012; Metin and Hartel 2005; ten Grotenhuis et al. 1999; Woodrow and deMan 1968). However, cooling rates have also been reported not to have an effect on the polymorphism of milk fat/sunflower oil (Martini et al. 2001), fully hydrogenated canola oil/canola oil (Maleky et al. 2012), and hydrogenated sunflower seed oil (Herrera 1994). These lipid systems required more time to crystallize and a longer induction time (Herrera 1994;

Martini et al. 2001). In these cases, cooling rates did not affect polymorphic forms, indicating polymorphism did not influence induction times. Slowly cooled samples required shorter induction time because the molecular organization already had taken place at temperatures higher than crystallization temperature, meaning less time was needed to nucleate at a specific crystallization temperature (Herrera 1994; Herrera et al. 2015; Martini et al. 2001)

Cooling rates affect crystalline microstructure and morphology of fat crystals. A fast cooling results in a higher number of fat crystals that are smaller and more uniformly sized compared to the ones cooled at a slower cooling rate. (deMan 1964; Campos et al. 2002; Herrera and Hartel

2000; Metin and Hartel 2005). When fat is being cooled slowly, TAGs form larger crystals and microstructures with a lower crystal volume fraction and a more heterogeneous spatial distribution of mass (Campos et al. 2002; Metin and Hartel 2005). These behaviors lead to the formation of softer fat (Campos et al. 2002)

2.3.2 Minor Components

Over the years, there has been a great interest in studying the effects of minor components on fat crystallization as they are commonly used in controlling crystallization. The amount of minor components used varies depending on that of major components and their applications. The 12

typical amount can range from less than 0.1 % to 10 % (w/w) (Smith et al. 2011). Minor components can be present endogenously or added/removed to/from “host” fats. They can be either endogenous to the fat or structurally different from fats. The similarity in the molecular structure between fats and minor components consequently result in different outcomes. The effects of minor components on crystallization of fat also depends on the type of minor components used, “host” fats, concentrations, the degree of undercooling and the cooling rate. In some cases, it is necessary to hinder nucleation or growth of crystals in order to prevent or impede polymorphic transitions, formation of large crystals, and recrystallization (Guth et al.

1989; Talbot et al. 2012; Widlak 1999). In other cases, acceleration of nucleation is preferred to strengthen crystal networks in natural fats with a broad range of crystallization rates between different TAGs (Yoshikawa et al. 2014). This section reviews publications on the effects of different minor components on fat crystallization, especially the fats studied in Chapter 3.

The effects of TAGs, DAG and monoacylglycerols (MAG) on the crystallization of PO and its blends and fractions have been extensively studied. Peyronel et al. (2016) examined the effect of HBS on crystallization of palm mid fraction and palm olein. The rate of crystallization and the mechanical strength were higher upon the addition of up to 7% (w/w) HBS. In addition, the fat crystal network was denser with smaller pores and crystals (Peyronel et al. 2016).

Sulaiman et al. (1997) identified three saturated TAGs, tripalmitin (PPP), dipalmitoyl- myristoylglycerol and dipalmitoyl-stearoyl-rac-glycerol, that are responsible for the formation of nuclei and an enhanced crystallization of palm olein. Basso et al. (2010) also reported that PPP in

PO promoted the formation of larger crystals and β-form and resulted in a higher solid fat content (SFC) and reduced induction time. The addition of MAGs led to an increased nuclei

number and earlier onset of crystallization, consequently accelerated crystal growth and smaller crystal formation were observed without affecting SFC in PO and PO-based fat (Basso et al.

2010; Daels et al. 2015; Fredrick et al. 2008; Verstringe et al. 2014; Wilson 1999). On the other hand, the addition of MAG inhibited crystallization of palm olein (Maruyama et al. 2016). The effect of DAG on PO crystallization varied depending on the degree of undercooling, the concentration, and the chemical composition of the DAG. Several studies have reported on an inhibitory effect of palm-based DAG, up to 10%, on PO crystallization and the polymorphic transition of βʹ to β (de Oliveira et al. 2014; Saberi et al. 2011; Siew and Ng 1999; Wassel and

Yong 2007). However, the addition of high concentrations of palm-based DAG, 30% and 50%

(w/w), accelerated the nucleation and crystallization rates of PO and PO-palm olein blends

(Saberi et al. 2011; Xu et al. 2016).

Milk fat is another natural fat that has been a common subject in studying the effects of minor components on crystallization. DAG-enriched milk fat displayed a greater induction time, thus slower crystallization process at low degrees of undercooling compared to milk fat (Wright et al. 2000; Wright and Marangoni 2002). The presence and absence of DAGs did not influence the dropping points, equilibrium SFC, polymorphism, or microstructure (Wright et al. 2000).

While Wright and Marangoni (2002) observed effects of the addition of 1% DAG on milk fat crystallization, Herrera et al. (1999) did not see the inhibition of nucleation due to DAG in milk fat. Thus, the effect of DAG is dependent on chemical and molecular composition of “host” fats and DAG, concentration of DAG and temperature (Foubert et al. 2004; Wright and Marangoni

2002). Simouneau and German (1996) replaced both saturated and monounsaturated long-chain

TAGs in milk fat with long-chain monounsaturated TAGs from cocoa butter, and it resulted in

enhanced co-crystallization and melting of the fractions. In addition, when long-chain monounsaturated TAGs were less than 30% of the total weight at 4°C, the texture of milk fat was firmer. MAGs and free fatty acids with different chemical compositions showed varying effects on milk fat crystallization. MAGs rich in stearic acid promoted interfacial heterogeneous nucleation, crystal growth and the α to βʹ polymorphic transition, while MAGs rich in oleic acid had no effect. MAGs rich in lauric acid had an intermediate effect even at higher concentrations

(Fredrick 2013). Long chain saturated fatty acids accelerated crystallization whereas short chain saturated and monounsaturated fatty acids had an opposite effect on milk fat crystallization

(Bayard et al. 2017).

Sucrose esters (SE), polyoxyethylene sorbitan monostearate (Tween 60) and glycerol either accelerated or delayed nucleation of high-melting milk fat fraction (HMF) (Cerdeira et al.

2003; Litwineneko et al. 2004). The differences in structures of SE and TAGs caused a delay in nucleation of HMF (Cerdeira et al. 2003). The addition of 0.1 % (w/w) Tween 60 effectively decreased the induction time of HMF compared to 0.5 % (w/w) Tween 60 while 0.1 % (w/w) glycerol hindered nucleation (Litwinenko et al. 2004). Another study on the effects of SE on fats reported that the acyl-acyl interaction between SE molecules and TAGs significantly hindered crystallization of fats (Widlak 1999). Consequently, the interactions between SE and TAGs retarded polymorphic transition and the growth of crystal sizes.

Award and Sato (2002) reported that sucrose oligoesters accelerated the crystallization rate and retarded crystal growth of palm kernel oil in O/W emulsion. The authors also noted that the effects were greater when the length of fatty acid chains of sucrose oligoesteres were longer.

Their results agreed with those from previous studies that the mechanism of these emulsifiers 15

was to act as heteronuclei by forming molecular aggregates at the oil-water interface (Katsuragi et al. 2001).

The addition of inorganic and organic additives that do not have similarities to TAGs (i.e., no hydrocarbon chains) effectively promoted crystallization of molten TAGs. They increased the crystallization temperature and promoted the formation of more stable polymorphic form

(Yoshikawa et al. 2014).

2.4 Conclusion

This chapter briefly reviewed the various methods of improving oil separation stability, structuring liquid oil, and controlling fat crystallization. FHVOs have been used as effective oil stabilizers and gelators. Many studies have reported the effects of cooling rate and minor components on the crystallization behavior of fat. These methods have been widely used/studied for many purposes, but they all have one common purpose which is to achieve desirable physical properties of fat, thus to improve the quality of fat-based products. The next two chapters present one more method of engineering the crystallization of TAGs, by using high behenic acid stabilizers as a gelator and a nucleation enhancer/inhibitor.

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CHAPTER 3 Crystallization Behavior and Oil Stabilization Efficiency of High Behenic

Acid Stabilizers in Liquid Oil

3.1 Abstract

The crystallization behavior and structure of mixtures of a high behenic acid stabilizer (HBS) in peanut oil (PeO), high oleic safflower oil (HOSO), and sesame oil (SeO) were studied to elucidate the mechanism behind liquid oil stabilization. Both chemical composition of the oil and cooling rate influenced crystallization behavior and structure of HBS. The critical gelation concentration of HBS ranged from 6.5 % for PeO gelation at 3 °C/min to 11 % for SeO gelation at 0.6 °C/min. The free energies of nucleation (ΔG) was the highest for 12 % HBS-SeO (142 kJ/mol) followed by HOSO (75.8 kJ/mol), and PeO mixture (15.9 kJ/mol). The 12 % HBS-PeO mixture displayed the highest storage modulus (G′) under both cooling rates studied. The oil stabilization effect was enhanced in PeO due to the presence of high-melting triacylglycerols with a melting point > 30 °C (HMT) and a low level of low-melting TAGs (LMT) (melting point

< -10 °C). In general, 12 % HBS-oil mixtures crystallized at a higher cooling rate exhibited higher SFC values, lower crystallization points, a predominance of the β′ polymorph, and uniformly sized small spherulites. Oil stabilization effect of HBS was greater with a faster cooling rate and the greater amount of HMT and a lower level of LMT in oils.

3.2 Introduction

Oil separation and migration in foods are major issues that need to be addressed to improve the overall quality and shelf life of food products such as nut butters and spreads. These phenomena take place when liquid oil separates from solid particles, resulting in two phases. The 22

addition of a stabilizer in a small dosage is one way of preventing these issues from occurring.

For example, the recommended amount of high behenic acid stabilizer (HBS) used in this study is 0.2 % - 2.0 % (w/w) of the total weight (Palsgaard 2014). Most commonly used stabilizers are

TAG-based and made up of fully hydrogenated vegetable oils (FHVO), rich in 16 - 18 carbon fatty acids, as well as oils that are high in erucic acid. Upon full hydrogenation, these become solid fats, rich in palmitic, stearic and behenic acids. Some of the most commonly used stabilizers are derived from cottonseed oil (Gooding et al. 1973, Widlak 2002), peanut oil

(Gooding et al. 1973), and rapeseed oil, particularly high erucic acid varieties (Sanders 1964).

Prevention of oil separation using HBS has been studied in various food matrices such as almond paste (Capanoglu and Boyacioglu 2008), seed butters (Chien 2015), and blends of palm mid fraction and palm olein (Peyronel et al. 2016). With the addition of 0.5 % HBS in almond paste stored at 4 °C and 30 °C, oil separation was significantly decreased, consequently retarding lipid oxidation (Capanoglu and Boyacioglu 2008). 2 % and 3 % HBS in the seed butter effectively prevented oil separation and increased hardness after 28 days of storage at 25 °C

(Chien 2015). Peyronel et al. (2016) have shown a significant improvement in oil binding in palm mid fraction and palm olein blends by adding from 1.25 % up to 7 % of HBS. The authors discovered a new fraction formed by the co-crystallization of HBS and the high melting fractions present in the blends through differential scanning calorimetry. They speculated that the creation of this new fraction, a complex between HBS and the HMF enhances nucleation and oil binding.

Although the ability of HBS to prevent oil separation has been well established, its crystallization behavior in liquid oil has not been reported. Moreover, the mechanism by which these HBS immobilize liquid oil at such low concentrations is not clear. Our hypothesis is that 23

HBS forms its own crystal networks that entrap liquid oil, thus preventing oiling out from solid phase. The objective of this work is to present a fundamental characterization of HBS when added to liquid oil and study the effects of chemical composition of oil and cooling rate on its crystallization behavior.

3.3 Materials and Methods

3.3.1 Materials

Palsgaard 6111, the high behenic acid stabilizer used in this experiment was a generous gift from Palsgaard (Palsgaard® , Juelsminde, Denmark). Three commercially available edible oils were used: organic fair trade virgin sesame oil (SeO) (Emile Noel, Pont Saint Esprit Cedex,

France), 100 % pure peanut oil (PeO) (No Name, Brampton, ON, Canada) and high oleic safflower oil (HOSO) (New Directions Aromatics, Mississauga, Ontario).

3.3.2 Fatty Acid Composition

Materials and methods were obtained from Christie (1982). An Agilent 6890-series Gas

Chromatograph (GC) (Agilent Technologies, Inc., Wilmington, DE, USA) with 7683-series auto-sampler was used to investigate FA composition. 60 m × 0.22 mm internal diameter with

0.25 μm film thickness GC Agilent 6890 BPX70 column was used (SGE Inc., Austin, TX, USA).

The oven was programmed to increase from 110 °C to 230 °C (4 °C/min) and stay at 230 °C for

18 min. The maximum temperature for column was 260 °C. The injector was set at 250 °C and operating at 20.1 psi flow of 17.7 ml/min. An average velocity of the flow of helium, a carrier gas, was 25 cm/s. The detector was set at 255 °C with 450 ml/min air and 50ml/min helium flowing. The GC separation peaks were analyzed by using OpenLAB CDS (Agilent

Technologies) software. FA composition was determined by comparing peaks to internal standards and their corresponding signals.

3.3.3 Triacylglycerol Composition

TAG composition of three edible oils was determined by performing the chromatographic analyses with Waters Alliance model 2690 high performance liquid chromatograph (HPLC) with a refractive index detector Waters model 2410 (Waters, Milford, MA, USA). The chromatographic separation of the compounds in oils was achieved with a Waters xbridge C18 column with 4.6 mm × 250 mm internal diameter with 5μm particle size. Materials and sample preparation were obtained from Ghazani (2015). Instrument settings were as followed: isocratic flow (1ml/min), 40 °C (a sample and a column chamber and a detector), and a mobile phase containing acetone/acetonitrile 60/40 (v/v) (mixed in advanced). The data obtained were analyzed using Millenium32 (K&K Testing, LLC, Decatur, GA, USA). TAG composition was determined using the internal library for each TAG and its corresponding signal.

Due to a high melting point (Tm), HPLC was not a suitable method to determine the TAG composition of HBS. The same GC was used for determining the TAG composition of HBS, except it was equipped with a fused silica capillary column (DB-17HT, 30 m × 0.32 mm i.d. ×

0.15 μm film thickness, J&W Scientific, Folsom, CA, USA). 25 mg of HBS was dissolved in 1 mL chloroform and 2 μl of solution was injected in split ratio of 30:1. Helium was used as carrier gas and the flow rate was 2.0 ml/min. Both the injector and detector temperatures were maintained at 360 °C. The TAGs were separated based on the number of carbon atoms. The molar percentage of TAGs present in HBS was calculated using three equations where A, B and

C are the molar percentages of fatty acids A, B and C (Vander 1963). The molar percentage of

TAG containing only one fatty acid A is,

A3 %AAA = 10000 and that of TAG containing fatty acids A and B is,

3 × A2B %AAB = 10000 and that of TAG containing three different fatty acids A, B and C is,

6 × A × B × C %ABC = 10000

3.3.4 Critical Gelation Concentration Determination

The Cg for each sample was determined by placing the glass vial upside down for five seconds an hour after the sample preparation was done to check if the sample flows or not.

Samples were prepared by first putting 10 g of oil and different % (w/w) of HBS (ranging from

2 % to 11 % with every 0.5 % increment) in a 20 ml glass vial in an oven at 80 °C for 15 minutes to erase crystallization history. Vials were then cooled under two different cooling rates:

3 °C/min (Newtonian cooling) and 0.6 °C/min (stepwise cooling) rate to 20 °C in a static state.

3.3.5 Nucleation Kinetics

Nucleation kinetics of 12 % HBS-oil mixtures were studied by determining induction time using a cloud point analyzer and ultimately calculating the free energy of nucleation (ΔG) using

the Fisher-Turnbull equation. A Phase Transition Analyzer (Phase Technology, Richmond, BC,

Canada) was used to determine induction time at a specific undercooling temperature (the temperature was 5 °C to 10 °C lower than the highest melting component of the sample). 150 μl of melted 12 %HBS-oil mixtures was placed in the sample chamber using a pipette. Samples were first heated up to 70 °C for 1000 seconds then cooled at 50 °C/min, a cooling rate chosen to achieve isothermal crystallization, to undercooling temperatures. Five undercooling temperatures

5 °C to 10°C below the highest melting component of the mixture were chosen within the range of 3 °C for each sample. The data were obtained using the software LBT-466 (Phase

Technology). The induction times (τ) were determined by selecting the time value that corresponded to a deviation of 1 % from the starting points, the initial flat region of the time versus signal curve. For each HBS-fat mixture and its undercooling temperatures, the slope of a

2 linear regression curve of the plot ln(τTf) vs 1/(TfΔT ) was calculated where Tf is the undercooling temperature and ΔT=Tf - Tm (Tm is the melting point). The slope, m, was used to calculate ΔG by using the Fisher-Turnbull equation:

ΔG = m*k/ΔT2

2 where ΔT is created by using the same Tm but different Tf ranging from 20 °C to 50 °C and k is

Boltzman constant. Calculation and data analysis were done by using GraphPad Prism 5.0

(GraphPad Software, San Diego, CA, USA).

3.3.6 Solid Fat Content

SFC was determined using a Bruker mq20 Minispec Series PC 120 Nuclear Magnetic

Resonance (NMR) Spectrometer operating at 20 MHz and 0.47T (Bruker Optics, Milton, ON,

Canada). 12 % HBS-oil mixtures were transferred to an NMR glass tube, about 3 cm in height.

SFC values of oils in its liquid state were first measured as blank prior to sample measurements.

The SFCs of samples were determined serially after they were conditioned for 30 min at temperatures ranging from 20 °C to 80 °C, every 5 °C.

Isothermal crystallization behavior was determined by monitoring SFC as a function of time.

12 % HBS-oil mixtures were melted at 80 °C for 15 min to erase all crystal memory. Then the samples were placed in the water bath at 20 °C, and the SFC was measured every 30 seconds for the first 15 min, every minute for the following 15 min and every 10 min for the remaining 60 min followed by readings at intervals of 1 hour.

For SFC measurements, the Daily Check was performed every 24 h with three standard calibration samples supplied by Bruker. The collection and analysis of data from NMR analysis was done using the MiniSpec software V2.51 Rev 00/NT (Bruker Biospin, Gmbh). Values reported in this work are the average of three measurements.

3.3.7 Thermal Behavior

A Mettler-Toledo differential scanning calorimeter (DSC) (Mettler Toledo, Mississauga, ON,

Canada) was used to determine thermal behavior. 12 % HBS-oil mixtures ranged from 7 to 10 mg were weighed and hermetically sealed into an aluminum pan. Samples were heated at

10 °C/min to 80 °C and held for 15 min, cooled from 80 °C to 20 °C according to their cooling rate. They were then held at 20 °C for 15min and reheated to 80 °C at 5°C/min. Peak melting and crystallization points (Tm and Tc) and onset temperatures were calculated using STARe software

(Mettler Toledo). Exothermic peaks of HBS-oil mixtures cooled at 0.6 °C/min were manually integrated to determine the percentage of a small peak area.

3.3.8 Polymorphism

The polymorphism of fat samples was assessed by using a Rigaku Automated Powder X-Ray

Diffractometer (XRD) (Rigaku, Tokyo, Japan). The copper X-ray tube (wavelength of 1.54 Å ) operated at 40 kV and 44 mA. The measurement was at 0.5 °/min in the range 2θ = 1 ° - 35 ° at

20 ± 3 °C. Samples (12 % HBS-oil mixtures) were prepared by heating 10 g of oil with 12 %

(w/w) HBS at 80 °C for 15 min, cooled at 3 °C/min and 0.6 °C/min to 20 °C and stored for an hour prior to measurements. A sample was filled in a sample holder without being compressed.

The sample holder for this diffractometer was made from glass with 20 mm × 20 mm and 0.3 mm in depth. Polymorphic forms were determined using MDI Jade 9 (MDI, Livermore,

California USA).

3.3.9 Small Deformation Rheology

Rheological measurements were performed using a TA AR2000 rotational controlled stress rheometer (TA instruments, New Castle DE, USA) and a sandblasted plate (20 mm) with a 0.2 mm gap. Samples were heated at 80 °C for 15 min prior to crystallization which was taken at

3 °C/min and 0.6 °C/min to 20 °C then held for 20 min. An amplitude test was performed at

20 °C with the shear strain ranging from 0.001 % to 1000 % at an angular frequency of 1 rad/s.

3.3.10 Polarized Light Microscopy

The microstructure of 12 % HBS-oil mixtures were examined using a Leica DMRXA2 microscope (Leica, Wetzlar, Germany) equipped with a 20× objective lens. Polarized light

micrographs were taken using a ORCA-Flash2.8 Digital CMOS camera C11440 (Hamamatsu,

Hamamatsu, Shizuoka Pref., Japan). A 5 ul drop of each gel melt was placed on a preheated

(80 °C) glass microscope slide and covered with a preheated (80 °C) glass coverslip. Slides were heated at 80 °C for 15 min to erase crystallization history then cooled at 3 °C/min or 0.6 °C/min to 20 °C and held for 10 min prior to observations. The temperature and cooling rates were controlled using an LTS 350 heating and freezing stage operated by a TMS 93 temperature programmer (Linkam Scientific Instruments Ltd., Surrey, England). Liquid nitrogen and an electric element were used to cool down and heat up the stage, respectively. 7 micrographs were taken per slide, and two slides were prepared per sample. All samples were prepared in triplicate.

Micrographs were auto-contrasted using Photoshop CS5 (Adobe, San Jose, CA, USA).

3.3.11 Statistical Analysis

The average of triplicate measurements and their standard deviations were reported and used for all statistical analysis. A one way ANOVA and post hoc Turkey test (p<0.05) were performed using GraphPad Prism 5.0 (GraphPad Software).

3.4 Results and Discussion

3.4.1 Fatty Acid Composition

Table 1 shows the fatty acid compositions of oils used in this study and the HBS. All three oils were high in oleic acid (C18:1) with HOSO having the highest level out of three oils (79.5 %).

SeO had almost equal amount of oleic acid and linoleic acid (C18:2), and these two FA comprised of 83 % of oil. SeO showed a higher level of stearic acid (C18:0) (9.2 %) than other two oils. PeO was the only oil that contained long-chain fatty acids (LCFA) equal or longer than arachidic acid

(C20:0) (5.9 %) which were also found in HBS but at much higher levels. The two major fatty acids in HBS were stearic acid and behenic acid (C22:0), 41.9 % and 41.7 %, respectively.

Table 1 Fatty acid composition of high oleic safflower oil, sesame oil, peanut oil, and HBS. Shown are means and standard deviations of n=2 replicates.

FA (%wt.) HOSO SeO PeO HBS

Palmitic (C16:0) 4.7±0.1 9.2±0.0 9.3±0.5 4.9±0.5

Stearic (C18:0) 2.0±0.1 6.2±0.0 2.9±0.2 41.9±2.6

Oleic (C18:1) 79.5±0.0 41.6±0.3 57.1±2.2 0.7±0.5

Linoleic (C18:2) 13.1±0.0 41.4±0.1 23.3±1.2 Linolenic (C ) 0.6±0.0 1.3±0.1 18:3 Arachidic (C ) 1.3±0.1 10.0±0.2 20:0 Behenic (C ) 3.1±0.3 41.7±3.4 22:0 Lignoceric (C ) 1.5±0.2 0.8±0.2 24:0 Others 0.7±0.1 1.1±0.3 0.3±0.4

3.4.2 Triacylglycerol composition

The TAG composition of the oils was characterized using HPLC, and the results are shown in the Table 2. The most abundant TAG in both HOSO and PeO was OOO followed by OOL.

SeO contained OOL the most, 21.3 % followed by OLL (19.3 %) and OOO (16 %). The amount of high-melting TAGs (HMT) (TAGs with Tm > 30 °C) was the greatest in PeO (2.5 %), followed by SeO (1.3 %) and HOSO (0.0%). The level of low-melting TAGs (LMT) (TAGs with

Tm < -10 °C) was the highest for SeO and the lowest for PeO. Melting points of TAGs were determined using Triglyceride Property Calculator: An R Shiny App (Marangoni Research Lab,

Guelph, ON, Canada, http://www.crcfoodandhealth.com/services.php). In both SeO and PeO, there were more than 15 % of other components not identified, possibly MAG and DAG. The

TAG composition of HBS is presented in the Table 2. 31

Table 2 Triacylglycerol composition of high oleic safflower oil, sesame oil, peanut oil, and HBS. Shown are means and standard deviations of n=2 replicates.

TAG Melting TAG2 Melting HOSO SeO PeO HBS (% w/w) Point (°C) (% w/w) Point3 (°C) OLL -15.0 4.9±0.1 19.3±0.0 9.4±0.2 PPS 62.6 2.5 OOLn -13.1 1.1±0.1 PSS 64.4 7.7

OOL -9.5 18.9±0.5 21.3±0.0 17.9±0.2 BSP 66.1 21.2 PLL -3.2 3.3±0.1 SSB 70.7 19.9

POL 1.8 10.0±0.1 9.0±0.3 BSA 71.5 17.4

OOO 4.8 55.7±1.2 16.0±0.0 24.9±0.9 SSS 72.5 16.7 POO 18.5 9.6±0.2 7.4±0.1 9.9±0.2 BBS 73.5 14.6 SOO 23.5 3.9±0.1 4.4±0.1 3.6±0.1 TAG(CN)1 HBS OOB 23.6 0.9±0.0 50 3.0±1.3

POS 31.0 1.3±0.0 0.7±0.1 52 6.1±0.1

POP 37.2 1.2±0.0 54 1.1±0.3

PSS 64.4 0.6±0.0 56 13.8±0.1

Others 6.9±1.1 20.5±0.1 17.4±0.6 58 27.5±0.4 A Arachidic acid, L linoleic acid, Ln linolenic acid, O oleic 60 25.6±1.5 acid, P palmitic acid, S stearic acid, B behenic acid, CN carbon number 62 22.9±0.1 1 triaclyglycerol composition calculated based on the fatty acid composition assuming random distribution of fatty acids on glycerol molecule (Section 3.3. in Chapter 3) 2 triacylglycerol composition determined experimentally by high temperature gas-liquid chromatography

3.4.3 Critical Gelation Concentration Determination

Cg of HBS in the different oils and crystallized at slow and fast cooling rates are shown in

Table 3. More HBS was used to form gel with SeO than HOSO and PeO at both cooling rates.

The differences in Cg were greater when the mixtures were cooled at a faster rate. I speculate that the presence of HMT PeO contributed to the crystallization of the mixture. Although SeO has more HMTs than HOSO, it also has a greater amount of LMT which hindered the crystallization of the mixture. In general, a higher cooling rate during crystallization leads to a lower Cg for all

systems. For SeO, faster cooling reduced the amount of HBS by 1 % whereas for PeO and

HOSO the amount was reduced by 2.5 %.

Table 3 Cg of HBS (% w/w) for edible oils at different cooling rates Cooling rate 3 °C/min 0.6 °C/min Oil Type PeO 6.5 % 10.0 % HOSO 8.0 % 10.5 % SeO 10.0 % 11.0 %

3.4.4 Nucleation Kinetics

The free energies of nucleation (ΔG) of 12 % HBS in oil from 48 °C to 51 °C are shown in

Figure 1. PeO-HBS gel had the lowest ΔG of 15.9 kJ/mol while the SeO-HBS had the highest

G  n RT value of 142 kJ/mol. The rate of nucleation (vn) is a function of ΔGn,  ~ e , thus a higher ΔG translates to a lower rate of nucleation. A higher rate of nucleation would result in a larger number of smaller crystallites being formed. This could explain the trends observed for Cg, where PeO with the lowest ΔG also had the lowest Cg. Further evidence for this is given below.

Figure 1 Free energies of nucleation (ΔG) of 12 % HBS-oil mixtures as a function of temperature. Shown are means and standard deviations of n=6 replicates.

3.4.5 Solid Fat Content

SFC vs. temperature and SFC vs. time at 20 °C plots are shown in Figure 2. Most of 12 %

HBS in oil mixtures crystalized at 20 °C, as SFC values at the temperature were near 12 %. A sharp decrease in the SFC occurred above 50 °C for all mixtures in Figure 2-A where the HBS started melting. However, gels cooled at 0.6 °C/min displayed significantly lower SFC values

(p<0.05) from 20 °C to 50 °C compared to those of gels cooled at 3 °C/min. The higher SFC in rapidly cooled mixtures was probably due to enhanced mixed crystal formation between TAGs in a metastable polymorphic form, which would allow for a greater variety of TAG molecules to participate in crystalline solids formation. Previous studies reported higher SFC values in rapidly cooled fats (Julien et al. 1985; Campos et al. 2002; deMan 1964; Herrera and Hartel 2000).

When fats are rapidly cooled, more liquid oil is absorbed to crystals, resulting in less liquid oil present in fats (Julien et al. 1985).

The effect of differences chemical composition of oils on SFC values was reflected in the

SFC curves. Greater amounts of HMT in SeO and PeO resulted in the SFC being significantly higher (p < 0.05) than HOSO at 20 °C for both melting (mixtures cooled at 3 °C/min) and crystallization.

Figure 2 SFC of 12 % HBS-oil mixtures as a function of (A and B) temperature (A: 3 °C/min, B: 0.6 °C/min) and (C) time at 20 °C. Shown are means and standard deviations of n=3 replicates. 36

3.4.6 Thermal Behavior

Figure 3 and 4 show crystallization and melting thermograms of 12 % HBS-oil mixtures, as well as neat HBS. A summary of DSC thermal data is presented in Table 4. Neat HBS showed a sharp exothermic peak under both cooling rates, indicating the narrow distribution of TAGs. In

12 % HBS-oil mixtures, the overall HBS melting behaviour was not affected by the cooling rate or type of oil. The onset temperature for crystallization point was higher when the mixtures were cooled at a slower rate. Crystallization starts at a higher temperature upon slow cooling because

TAGs have more time to form crystals during the cooling process (Metin and Hartel 2005).

Sharp crystallization peaks were observed at ~ 41 °C when the mixtures were cooled at

3 °C/min (Figure 3). Upon slower cooling, two exothermic peaks were observed: the first exothermic peak (at higher temperature) was sharper and taller than the peak observed at lower temperatures. High and low melting components tend to be more easily separated upon very slow cooling (Kawamura 1981). Two groups of TAGs in HBS were categorized based on their melting points. Three TAGs containing palmitic acid, PPS (Tm = 62.6 °C), PSS (Tm = 64.4 °C), and BSP (Tm = 66.0 °C), account for 31.4 % (w/w) of all TAGs, and they are relatively lower melting components compared to rest of the TAGs which above 70 °C. The percentage of small exothermic peak area for PeO was the lowest (23 % ± 0.01) followed by SeO (32.56 % ± 0.06) and HOSO (35.83 % ± 0.01). Perhaps HBS and HMTs from PeO formed new fraction which caused parts of small exothermic peak to shift towards higher temperatures resulting in a bigger first exothermic peak. Same trend was observed when HBS was added to palm mid fraction and

palm oil (Peyronel et al. 2016).

Figure 3 DSC crystallization curves of 12 % HBS-oil mixtures and neat HBS cooled at (A) 3 °C/min and (B) 0.6 °C/min.

Figure 4 DSC melting curves of 12 % HBS-oil mixtures and neat HBS. All samples were heated at 5 °C/min.

Table 4 DSC thermal data of 12 % HBS-oil mixtures. Shown are means and standard deviations of n=3 replicates.

Sample Crystallization point (°C) Melting point (°C) Onset1 Peak1 Onset2 Peak2 Onset Peak PeO-3 °C/min 41.6±1.0 40.8±0.9 - - 46.5±3.3 54.8±1.1 HOSO-3 °C/min 42.8±1.2 41.4±1.3 - - 47.3±1.5 55.6±1.0 SeO-3 °C/min 41.7±0.8 41.1±1.0 - - 47.3±1.5 55.1±0.9 PeO-0.6 °C/min 43.9±1.0 42.2±0.9 34.6±0.0 30.5±0.1 44.3±0.6 54.6±0.8 HOSO-0.6 °C/min 44.7±0.9 43.4±1.2 34.6±0.7 32.4±0.2 46.5±1.1 55.5±1.2 SeO-0.6 °C/min 44.3±1.1 43.0±1.0 33.6±1.9 31.1±0.5 46.3±1.5 55.2±1.2 1 Onset and peak for the first exothermic peak at higher temperature

2 Onset and peak for the later exothermic peak

3.4.7 Polymorphism

Powder X-ray diffraction spectra of 12 % HBS in oil and neat HBS are shown in Figure5.

Neat HBS displayed α polymorphism when crystallized both at 3 °C/min and 0.6 °C/min. It is not uncommon for very high melting TAGs to form α crystals, and a similar result has been previously reported (Ahmadi et al. 2008). Longer fatty acid chains require more time to assemble themselves into more stable polymorphs, and because HBS crystalizes very rapidly, a rapid increase in viscosity during crystallization of neat HBS causes a mass transfer limitation that prevents the metastable form from transforming to more stable crystal forms.

The transition from α to β and/or β′ did take place for 12 % HBS in oil. When HBS was diluted in oil, its TAGs were able to pack more efficiently leading to the formation of the more stable polymorphic form (Rousseau et al. 2005). A slower cooling rate led to the formation of more stable polymorphic form (β) as expected (Campos et al. 2002; Metin and Hartel 2005).

3.4.8 Small Deformation Rheology

Figure 6 presents amplitude sweep rheograms of 12 % HBS-oil mixtures. For all mixtures, G′ was larger than a loss modulus (G″), and G′ dominated the viscoelasticity of the system, confirming the gel-like behavior. The order of G′ was SeO < HOSO < PeO which is the opposite of Cg and ΔG of the mixtures. Cooling rate did not affect G′ and G″. Mixtures cooled at

0.6 °C/min displayed overshoots where both G′ and G″ slightly increased before a second deformation.

Figure 6 Amplitude sweep rheograms of 12%HBS-oil mixtures cooled at (A) 3°C/min and (B) 0.6°C/min.

An overshoot is observed when a system resists further deformation until it yields at a higher shear strain. Van den Tempel proposed the existence of two types of bonds in fats. “Primary” bonds are stronger while “secondary” bonds are weaker and probably break or stretch at a lower strain causing early deformation (Van den Tempel, 1961). It is likely that gels cooled at a slower rate had more time to form primary bonds which can store more energy than secondary bonds.

This would explain the overshoot observed in slowly cooled samples at large deformations, after secondary bonds have already yielded.

3.4.9 Polarized light microscopy

Microstructures of 12 % HBS-oil mixtures cooled at different cooling rates are shown in

Figure 7. Faster cooling rate yielded a higher crystal volume fraction comprised of smaller spherulites uniform in size (averaged ~45 um). Size of spherulites in mixtures cooled at 42

0.6 °C/min ranged from 10 um to 100 um. The relationship between the morphology of fat crystals and a cooling rate is well-established in the literature (Campos et al. 2002; Herrera and

Hartel 2000; Maleky et al. 2012; Metin and Hartel 2005; Toro-vazquez and Marangoni 2004). As mentioned earlier, when fat is cooled slowly, crystal growth will predominate over nucleation events and thus TAGs would have more time to form more organized crystal networks containing larger crystals ( Campos et al. 2002; Herrera and Hartel 2000).

Figure 7 Polarized light micrographs of 12 % HBS-oil mixtures. The scale bar represents 100 um.

It is most likely that the presence of HMTs in PeO led to the highest oil stabilization efficiency by HBS. Maximum structuring efficiency of HBS in PeO correlated with the lowest

Cg, the highest nucleation rate, and the highest G′. It has been reported that the efficiency in the use of emulsifiers/stabilizers increases when the host matrix had similar fatty acid profiles as the stabilizer (Garbolino et al. 2005; Smith and Povey 1997). In addition, PeO was the only oil that suggested this as the possible mechanism of HBS mediated structural stabilization. When 12 %

HBS in HOSO and SeO was crystallized at 0.6 °C/min, the position of the smaller exothermic peak representing palmitic acid containing TAGs remained the same while parts of it shifted towards higher temperatures in PeO. This result suggests a formation of new fraction by HBS and high melting fraction of PeO. Peyronel et al. (2016) reported a newly formed solid species with the addition of the stabilizer rich in stearic acid (51.65 %) and behenic acid (32 %) into blends of palm mid fraction and palm oil. It would be seen that optimal oil stabilization by this family of high behenic acid stabilizers depends on the presence of “complementary” TAGs in the oil being stabilized. This is somewhat worrying since the new generation high oleic oils lack many of these high melting point TAGs and would thus be more prone to oil separation. In this case, the HBS would not be as efficient in stabilizing high oleic acid nut butter oils.

3.5 Conclusions

The crystallization behavior of HBS was influenced by the type of oil and cooling rate. PeO-

HBS gel was formed with the addition of lowest amount of HBS, and it showed the lowest ΔG and highest G′ under both cooling rates among the three oils used. In fatty acid and TAG composition analyses, PeO contained the highest level of HMT and the lowest level of LMT. A slower cooling rate led to lower nucleation rates of 12 % HBS-oil mixtures. Consequently, a 45

lower nucleation rate led to higher Cg, lower SFC values (p < 0.05), higher onset temperature for crystallization, the presence of larger spherulites, and fewer spherulites. TAGs in 12 % HBS-oil mixtures had sufficient time to arrange themselves into more stable and thermodynamically preferred polymorph (β) when cooled slowly (0.6 °C/min).

The range of Cg of HBS, 6.5 % to 11 % (w/w) was a function of the oil and cooling rate used.

This level is very high considering the recommended dosage is from 0.2 % to 2.0 % (w/w) in food products such as nut butters. HBS stabilizes liquid oil more effectively when there are more

HMTs in the oil to be stabilized and when cooled under a faster rate. Faster cooling is also preferred to form oil gels at lower stabilizer concentrations, and with desirable physical properties such as higher levels of the β′ polymorph and smaller crystals. I believe that the stabilizer can form crystal networks by itself, but its effects on oil stabilization are enhanced when it interacts with certain TAGs, particularly TAGs with Tm > final crystallization temperature. Further work needs to be carried out on the interaction between these high behenic acid stabilizers and HMTs in specific oils and fats.

References

Christie WW (1982) A simple procedure for rapid transmethylation of glycerolipids and cholesteryl esters. J Lipid Res 23: 1072-1075 Campos R, Narine S., Marangoni AG. (2002) Effect of cooling rate on the structure and mechanical properties of milk fat and lard. Food Res Int 35:971–981 Capanoglu E, Boyacioglu D (2008) Improving the quality and shelf life of Turkish almond paste. J Food Qual 31:429–445 Chien Y (2015). Shelf life extension of seed butter made with sesame, sunflower and pumpkin seeds, Master's thesis, the Ohio State University, Columbus deMan JM (1964). Effect of cooling procedures on consistency, crystal structure and solid fat content of milk fat. Dairy Ind 29:244–246 Garbolino C, Bartoccini M, Flöter E (2005) The influence of emulsifiers on the crystallisation behaviour of a palm oil-based blend. Eur J Lipid Sci Technol 107:616–626 Ghazani SM (2015) HPLC Protocol to detect the % of TAGs in edible fats. Food and Soft Material Laboratory. University of Guelph Gooding CD, Melnick D, Parker W (1973 Oct 16) Peanut butter stabilizer. United States patent US 3,766,226 Herrera ML, Hartel RW (2000) Effect of processing conditions on crystallization kinetics of a milk fat model system. J Am Oil Chem Soc 77:1177–1188 Julien JP, Nadeau JP, Dumais R (1985) Dairy science and technology (Principles and applications) .La Fondation de technologie laitière de Québec, Quebec Kawamura K (1981) The DSC thermal analysis of crystallization behavior in high erucic acid rapeseed oil. J Am Oil Chem Soc 58:826–829 Maleky F, Acevedo NC, Marangoni AG (2012) Cooling rate and dilution affect the nanostructure and microstructure differently in model fats. Eur J Lipid Sci Technol 114:748–759 Metin S, Hartel RW (2005) Crystallization of Fats and Oils. Bailey's industrial oil and fat products Narine SS, Marangoni AG (1999) Microscopic and rheological studies of fat crystal networks. J Cryst Growth 198:1315–1319

Palsgaard (2014) Palsgaard® 6111 Product profile. Palsgaard®. Juelsminde, Denmark

Peyronel F, Campos R, Marangoni AG (2016) Prevention of oil migration in palm mid fraction and palm olein using a stabilizer rich in behenic acid. Food Res Int 88:52-60

Rousseau D, Hodge SM, Nickerson MT, Paulson AT (2005) Regulating the β′→β polymorphic transition in food fats. J Am Oil Chem Soc 82:7–12 Smith PR, Povey MJW (1997) The effect of partial glycerides on trilaurin crystallization. J Am Oil Chem Soc 74:169–171 Toro-vazquez JF, Marangoni AG (2004) Effects of crystalline microstructure on oil migration in a semisolid fat matrix. Cryst Growth Des 4:731-736 Vander RJ (1963) The Determination of Glyceride Structure I. J Am Oil Chem Soc 40:242–247 Widlak N, Hartel RW, Narine S, editors (2001) Crystallization and solidification properties of lipids. 1st ed. Champaign (IL): The American Oil Chemists Society. pp. 53-78

CHAPTER 4 Engineering the Nucleation of Edible Fats Using a High Behenic Acid

Stabilizer

4.1 Abstract

A stabilizer high in behenic acid (HBS) was used to control nucleation of edible fats. The addition of HBS led to an enhanced nucleation of anhydrous milk fat (AMF) and palm oil (PO) which had lower levels of high-melting triacylglycerols (HMTs) (melting point > 30 °C, relative to the crystallization temperature) compared to other fats. With the addition of 1.5 % HBS, there was an increase in crystallization onset temperatures and density of the microstructure in these two fats. Further studies were conducted to investigate the interactions between HBS and specific homogeneous triacylglycerols (TAGs) or a mixture of triacylglycerols. HBS displayed solid-state incompatibility (eutectic behavior) with tripalmitin and tristearin, whereas it displayed compatibility (monotectic partial solid solution formation) behavior when mixed with the high- melting milk fat fraction (HMF). This suggests two mechanisms for nucleation enhancement of

HBS. One mechanism would involve surface nucleation on top of pre-formed TAG surfaces, for tripalmitin and tristearin, while the other mechanism would involve additionally co- crystallization with the nucleating agent, for the case of HMF.

4.2 Introduction

Edible fats are widely used in confectionery products and fat-based food products such as margarines and shortening. They are used to enhance flavor and properties like mouthfeel, spreadability, and texture (Marangoni and Wesdorp 2012). These organoleptic characteristics of foods highly depend on the crystallization behavior of fats. For example, a faster nucleation rate 49

leads to formation of smaller and denser crystals, thus an adequate functionality and improved texture of final products (Ghotra et al. 2002). Another example is post hardening caused by a slow crystallization behavior of palm oil which tends to cause a grainy mouthfeel texture after manufacturing (Omar et al. 2015). Controlling the crystallization behavior of edible fats continues to be a technological challenge for the food industry. This has been exacerbated lately due to consumer and public health demands for removal of partially hydrogenated oils and excessively saturated oils. Moreover, new pressures have arisen to minimize the use of palm oil due to sustainability issues. A consequence of these pressures is that the amount of crystalline structuring material has been drastically reduced and changed in nature. Therefore, it is quite pressing to find strategies to enhance the nucleation behavior of fats and control crystal growth to achieve maximal structuring using minimal amounts of crystalline material.

The influence of additives and minor components on crystallization of fats has been extensively reviewed (Patel and Dewettinck 2015; Ribeiro et al. 2015; Smith et al. 2011; Talbot et al. 2012;). Some of the works that have not been covered in these reviews are: MAGs, DAGs, and polyglycerol esters in palm oil, palm olein, palm-based margarine fat, non-hydrogenated fat not based on palm and fat blend containing 75 % coconut oil and 25 % sunflower oil (Daels et al.

2015; de Oliveira et al. 2014; Maruyama 2016; Rizzo et al. 2015; Verstringe et al. 2014; Xu et al.

2016), free fatty acids and their esters in milk fat (Bayard et al. 2017), sorbitan monooleate in soybean-based interesterified fat (Ming and Gonçalves 2015), lecithins in commercial fats used in margarine and cocoa butter (Daels et al. 2015; Miyasaki et al. 2016; Rigolle et al. 2015), sorbitan monostearate and monooleate in cocoa butter (Masuchi et al. 2014), to name a few.

However, only a few studies have been conducted on the effects of stabilizers on nucleation and

crystallization behavior of edible fats. Peyronel et al. (2016) have reported an increase on the rate of crystallization in palm mid fraction and palm olein blends by adding from 1.25 % up to 7 % of

HBS. The authors speculated that HBS enhances nucleation by forming a new fraction which is the result of interaction between HBS and high melting fractions in the blends.

The purpose of this chapter was to determine whether HBS can be used to control the nucleation behavior of edible fats. And if HBS has any affects, our interest was to elucidate its mechanism of action by studying its interaction with homogeneous TAGs (tristearin and tripalmitin) and a mixture of TAGs in HMF.

4.3 Materials and Methods

4.3.1 Materials and Sample Preparation

Palsgaard 6111, the high behenic acid stabilizer used in this experiment was a generous gift from Palsgaard (Palsgaard, Juelsminde, Denmark). Palm kernel oil (PKO) was obtained from

Palsgaard (Palsgaard). Palm oil (PO) and palm stearin (PS) were from Jomalina (Jomalina Sdn.

Bhd. Klang, Selangor, Malaysia). Anhydrous milk fat (AMF) was provided by Kraft (Kraft

Foods Group, Inc., Northfield, IL, USA). Fat samples for all the analyses were prepared first by heating up 10 g of fat with 0, 0.2, or 1.5 % (w/w) HBS in a 20 ml glass vial at 80 °C for 20 min, then stored in an incubator (20 °C) with a lid closed for a week prior to analyses.

Tristearin (SSS) and tripalmitin (PPP) were purchased from Sigma-Aldrich (Sigma

Aldrich, St. Louis, MO, USA). HMF was fractioned in the lab. 200 g of AMF was mixed with

800 ml ethyl acetate in a glass bottle with a lid. The bottle was stored at 5 °C for 2 h, gently

swirled every 20 min. The mixture was vacuum filtered to obtain crystallized HMF. HMF crystals were spread on a tray and left overnight in a fume hood to evaporate solvent residue.

SSS, PPP, and HMF were diluted in high oleic algal oil (HOAO) at a ratio of 4:6 (w/w)

TAG:HOAO prior to making HBS-TAG mixtures. HBS was added to a TAG/HOAO blend at 5 %

(w/w) then with 10 % increments starting from 10 % to 100 % HBS. Once HBS-TAG mixtures were prepared in a 20 ml glass vial, they were heated at 100 °C for 20 min and stored at 20 °C in an incubator with a lid closed for two weeks.

4.3.2 Fatty Acid Composition

Materials and method were obtained from Christie (1982). An Agilent 6890-series Gas

Chromatograph (GC) (Agilent Technologies, Inc., Wilmington, DE, USA) with 7683-series auto-sampler was used to investigate FA composition. 60 m × 0.22 mm internal diameter with

0.25 μm film thickness GC Agilent 6890 BPX70 column was used (SGE Inc., Austin, TX, USA).

The oven was programmed to increase from 110 °C to 230 °C (4 °C/min) and stay at 230 °C for

18 min. The maximum temperature for column was 260 °C. The injector was set at 250 °C and operating at 20.1 psi flow of 17.7 ml/min. Helium, a carrier gas, flew at an average velocity of 25 cm/s. The detector was set at 255 °C with 450 ml/min air and 50 ml/min helium flowing rates.

Chromatograms were analyzed by using OpenLAB CDS (Agilent Technologies) software. FA composition was determined by comparing peaks to internal standards and their corresponding signals.

4.3.3 Triacylglycerol Composition

30 mg of neat fat was weighed and placed in a 2 ml high performance liquid chromatography

(HPLC) vial. 600 μl chloroform and 1 ml 60:40 HPLC-grade aceton:acetonitrile solution were added to dissolve the sample. TAG composition of HMF was determined by performing the chromatographic analyses with Waters Alliance model 2690 high performance liquid chromatograph with a refractive index detector Waters model 2410 (Waters, Milford, MA, USA).

The chromatographic separation of the compounds in HMF was achieved with a Waters xbridge

C18 with 4.6 mm × 250 mm internal diameter with 5 μm particle size. Instrument settings were as followed: isocratic flow (1 ml/min), 40 °C (a sample and a column chamber and a detector), and a mobile phase containing acetone/acetonitrile 60/40 (v/v) (mixed in advanced). The data obtained were analyzed using Millenium32 (K&K Testing, LLC, Decatur, GA, USA). TAG composition was determined using the internal library for each TAG and its corresponding signal

(Ghazani 2015). TAG profile of HBS was obtained from Chapter 3.

4.3.4 Nucleation Kinetics

Nucleation kinetics of HBS-fat mixtures was studied by determining induction time using a cloud point analyzer and ultimately calculating ΔG using the Fisher-Turnbull equation. A Phase

Transition Analyzer (Phase Technology, Richmond, BC, Canada) was used to determine induction time at a specific undercooling temperature (the temperature was 5 °C to 10 °C lower than the highest melting component of the sample). 150 μl of melted HBS-fat mixtures was placed in the sample chamber using a pipette. Samples were first heated up to 70 °C for 1000 seconds then cooled at 50 °C/min, a cooling rate chosen to achieve isothermal crystallization, to undercooling temperatures. Five undercooling temperatures 5 °C to 10 °C below the highest

melting component of the mixture were chosen within the range of 3 °C for each sample. The data were obtained using the software LBT-466 (Phase Technology). The induction times (τ) were determined by selecting the time value that corresponded to a deviation of 1 % from the starting points, the initial flat region of the time versus signal curve. For each HBS-fat mixture and its undercooling temperatures, the slope of a linear regression curve of the plot ln(τTf) vs

2 1/(TfΔT ) was calculated where Tf is the undercooling temperature and ΔT=Tf - Tm (Tm is the melting point). The slope, m, was used to calculate ΔG by using the Fisher-Turnbull equation:

ΔG = m*k/ΔT2

2 where ΔT is created by using the same Tm but different Tf ranging from 20 °C to 50 °C and k is

Boltzman constant. Calculation and data analysis were done by using GraphPad Prism 5.0

(GraphPad Software, San Diego, CA, USA).

4.3.5 Solid Fat Content

SFC was determined using a Bruker mq20 Minispec Series PC 120 NMR Spectrometer operating at 20 MHz and 0.47 T (Bruker Optics, Milton, ON, Canada). Isothermal crystallization behavior was determined by monitoring SFC as a function of time. Samples were melted at

80 °C for 15 min to erase all crystal memory. HBS-fat mixtures were transferred to an NMR glass tube, about 3 cm in height. Those glass tubes were placed in the water bath at 20 °C, and the SFC values were measured every 4 min for the first 14 min, every 10 min for the following

30 min, every hour for the following 3 h and every 2 h for the remaining 2 h followed by two readings after one and three weeks. For SFC measurements, the Daily Check was performed every 24 h with three standard calibration samples supplied by Bruker. The collection and

analysis of data from NMR analysis was done using the MiniSpec software V2.51 Rev 00/NT

(Bruker Biospin, Gmbh). Values reported in this work are the average of three measurements.

4.3.6 Thermal Behavior

4.3.6.1 Thermal behavior of HBS-Fat mixtures

A Mettler-Toledo differential scanning calorimeter (DSC) (Mettler Toledo, Mississauga, ON,

Canada) was used to determine thermal behavior. Fat samples ranged from 7 to 10 mg were weighed and hermetically sealed into a pan made from aluminum. Samples were held at 20 °C for 15 min and heated to 80 °C at 5 °C/min then held for 15 min followed by cooling to -20 °C at

-5 °C/min. Tm, Tc, and onset temperatures were calculated using STARe software (Mettler

Toledo).

4.3.6.2 Thermal behavior of HBS-SSS, PPP, and HMF mixtures

Measurements were carried out using a TA Instruments DSC model Q200 (TA

Instruments, New Castle, DE, USA). HBS-TAG mixtures ranging from 5 mg to 7 mg were placed in hermetically sealed alodined aluminum pans. They were heated to 100 °C at 5 °C/min and held for 20 min then cooled to -5 °C at -5 °C/min. Data were analyzed using Universal

Analysis (TA Instruments)

4.3.7 Polymorphism

The polymorphism of fat samples and HBS-TAG mixtures were assessed by Rigaku

Automated Powder X-Ray Diffractometer (XRD) (Rigaku, Tokyo, Japan). The copper X-ray tube (wavelength of 1.54 Å ) was operated at 40 kV and 44 mA. The measurement was at

0.5 °/min in the range 2θ = 1 ° - 35 ° at 20 ± 3 °C. Fat samples were filled in a sample holder 55

using a flat spatula without being compressed. The sample holder for this diffractometer was made from glass with 20 mm × 20 mm and 0.3 mm in depth. For HBS-TAG mixtures, the same sample preparation (section 3.1) procedure was followed but on a sample holder instead of a vial.

Once samples were prepared for HBS-TAG mixtures, sample holders were placed in a container at 20 °C for two weeks. Polymorphic forms were determined using MDI Jade 9 (MDI,

Livermore, California USA).

4.3.8 Polarized Light Microscopy

The microstructures of fat samples were examined using a Leica DMRXA2 microscope

(Leica, Wetzlar, Germany) equipped with a 20× objective lens. Polarized light micrographs were taken using a ORCA-Flash2.8 Digital CMOS camera C11440 (Hamamatsu, Hamamatsu,

Shizuoka Pref., Japan). A 5 ul drop of each mixture melt was placed on a preheated (80 °C) glass microscope slide and covered with a preheated (80 °C) glass coverslip. Slides were stored at

20 °C for a week prior to observations. Seven microgpahs were taken per slide, and two slides were prepared per sample. All samples were prepared in triplicate. Micrographs were auto- contrasted using Photoshop CS5 (Adobe, San Jose, CA, USA).

4.3.9 Statistical Analysis

The average of n=3 replicates and their standard deviations were reported and used for all statistical analysis. A one-way ANOVA and post hoc Tukey test (p < 0.05) were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA).

4.4 Results and Discussion

4.4.1 Fatty Acid Composition

Table 1 shows fatty acid compositions of fats and HBS. HBS was mostly composed of long chain saturated fatty acids (>14 carbon atoms), and two major ones were stearic acid

(41.9 %) and behenic acid (41.7 %). In general, all fats, except for PKO, had a high content of long chain saturated fatty acids, palmitic acid in particular. AMF and HMF had a greater content of stearic acid compared to PO and its fractions. Both AMF and PKO contained medium-chain saturated fatty acids with 8-12 carbon atoms. HMF had a higher content of palmitic acid and stearic acid and a lower content of unsaturated fatty acids and medium and short-chain saturated fatty acids than AMF.

Table 1 Fatty acid composition of anhydrous milk fat, palm oil, palm kernel oil, palm stearin, high-melting milk fat fraction, and high behenic acid stabilizer. Shown are means and standard deviations of n=2 replicates.

FA ( %wt.) AMF PO PKO PS HMF HBS Caprylic (C8:0) 1.0±0.1 3.9±0.3 Capric (C10:0) 2.9±0.03 3.7±0.2 Lauric (C12:0) 3.8±0.01 48.8±1.3 2.0±0.02 Myristic (C14:0) 11.8±0.03 1.3±0.2 17.1±0.1 1.3±0.02 14.2±0.2 Myristoleic 3.7±0.02 (C14:1) Palmitic (C16:0) 30.8±0.2 46.6±0.2 8.7±0.4 58.8±0.4 46.0±0.4 4.9±0.5 Palmitoleic 1.5±0.0 3.1±0.02 (C16:1) Stearic (C18:0) 12.6±0.1 4.5±0.01 2.2±0.2 4.7±0.04 27.8±0.1 41.9±2.6 Oleic (C18:1) 27.5±0.4 38.0±0.2 13.4±1.0 29.0±0.2 7.6±0.3 0.7±0.5 Linoleic (C18:2) 2.5±0.0 9.7±0.1 2.4±0.2 6.2±0.1 Arachidic 10.0±0.2 (C20:0) Behenic (C22:0) 41.7±3.4 Lignoceric 0.8±0.2 (C24:0)

4.4.2 Triacylglycerol Composition

TAG compositions of the fats used in this study are shown in Table 2. TAGs have been arranged in order of increasing melting point. HBS was composed of TAGs with 50 or more carbon atoms (Table 3). The content of HMTs (Tm > 30°C) was PKO > PS > PO > AMF (Table

2).

HPLC chromatograms of HMF and AMF are presented in Figure 1. Major peaks were identified with their corresponding TAGs. As expected, HMF had a lower proportion of low- melting TAGs and hardly any diacylglycerols or low melting TAGs compared to AMF (Figure

1-B).

Table 2 Triacylglycerol composition of anhydrous milk fat, palm oil, palm kernel oil, palm stearin, and high-melting milk fat fraction. Shown are means and standard deviations of n=2 replicates.

TAG ( % Melting AMF PO PKO PS HMF w/w) point1 (°C) OLL -15.0 1.50±0.2 2.6±0.1 OLO -9.5 1.5±0.2 1.2±0.1 2.9±0.1 3.3±2.6 PLL -3.2 2.6±0.1 1.5±0.02 1.2±0.3 PLO 1.8 1.8±0.1 4.9±0.4 9.3±0.2 OOO 4.8 3.0±0.1 3.1±0.1 0.9±0.1 2.0±0.1 LaOO 5.1 3.5±0.1 LaLaO 10.3 4.72±0.3 MOO 12.8 1.5±0.1 LaOM 16.8 4.6±0.1 POO 18.5 3.6±0.1 18.7±0.02 1.2±0.1 13.0±0.2 LaOP 20.5 3.7±0.1 PPL 23.3 2.6±0.2 7.17±0.1 1.4±0.1 SOO 23.5 3.5±0.1 3.3±0.03 0.5±0.1 1.5±0.2 CLaLa 24.3 1.8±0.04 2.8±0.1 MOP 27.0 3.0±0.2 1.9±0.03 CaCaLa2 30.0 3.8±0.4 5.8±0.1 POS2 31.0 4.1±0.1 3.4±0.1 0.3±0.01 6.6±0.2 6.7±0.1 MLP2 33.5 2.7±0.03 CaLaLa2 34.1 2.5±0.4 8.6±0.1 POP2 37.2 3.9±0.04 23.0±0.2 1.5±0.01 36.4±1.6 7.1±0.1 LaLaM2 42.3 16.3±0.02 SOS2 44.5 3.3±0.4 2.2±0.3 0.8±0.1 LaLaP2 45.6 8.2±0.4 LaLaLa2 45.7 0.8±0.1 19.4±0.02 LaPM2 49.4 5.0±0.01 PPM2 53.3 2.1±0.04 14.1±0.1 LaPP2 54.4 0.8±0.1 MMM2 57.1 2.1±0.04 PPS2 62.6 1.6±0.6 2.9±0.1 2.9±0.0 16.8±0.04 PSS2 64.4 1.6±0.5 2.5±0.2 0.3±0.01 8.2±0.1 PPP2 65.9 2.6±0.2 3.3±0.2 0.9±0.02 14.7±0.2 18.8±0.1 SSS2 72.5 2.7±0.2 1.69±0.0 C-Caprylic; Ca-Capric; La-Lauric; M-Myristic; P-Palmitic; S-Stearic; O-Oleic; L-Linoleic 1 Determined using Triglyceride Property Calculator: An R Shiny App (Marangoni Research Lab, Guelph, ON, Canada, http://www.crcfoodandhealth.com/services.php) 59

2 High melting triacylglycerols with Tm > 30°C

Table 3 Triacylglycerol composition of the high behenic acid stabilizer (HBS)

TAG ( % w/w)1 Melting point3 HBS TAG( % w/w)2 HBS (°C) PPS 62.6 2.5 CN50 3.0±1.3 PSS 64.4 7.7 CN52 6.1±0.1 BSP 66.1 21.2 CN54 1.1±0.3 SSS 72.5 16.7 CN56 13.8±0.1 SSB 70.7 19.9 CN58 27.5±0.4 BSA 71.5 17.4 CN60 25.6±1.5 BBS 73.5 14.6 CN62 22.9±0.1 P-Palmitic; S-Stearic, B-Behenic, A-Archidic 1 triaclyglycerol composition calculated based on the determined fatty acid composition assuming random distribution of fatty acids on glycerol molecule (Section 3.3. in Chapter 3) 2 triacylglycerol composition determined experimentally by high temperature gas-liquid chromatography 3 Determined using Triglyceride Property Calculator: An R Shiny App (Marangoni Research Lab, Guelph, ON, Canada, http://www.crcfoodandhealth.com/services.php)

Figure 1 HPLC chromatograms showing the triacylglycerol profile of (A) high-melting milk fat fraction and (B) milk fat. La-Lauric; M-Myristic; P-Palmitic; S-Stearic; O-Oleic; L-Linoleic

4.4.3 Physical Properties of HBS-Fat Mixtures

4.4.3.1 Nucleation Kinetics

The free energies of nucleation (ΔG) of HBS-fat mixtures from 29 °C to 33 °C are shown in Figure 2. The addition of 1.5 % HBS significantly and progressively decreased ΔG in AMF and PO (P<0.05). The induction time of PO was significantly reduced when a hard fat rich in stearic and behenic acid was added (de Oliveria et al. 2015). HBS acted as a nucleation inhibitor or enhancer in PKO depending on the concentration used. The addition of 0.2 % HBS increased the ΔG while addition of 1.5 % HBS led to a decrease in ΔG. The nucleation of PS was not significantly influenced (p < 0.05) by the addition of 1.5 % HBS, but the 0.2 %HBS-PS mixture showed a significantly lower ΔG compared to neat PS.

In general, the greater the HMTs in fats, the lower ΔG (Figure 3A). The stabilizer lowered ΔG in a more pronounced fashion when the level of such HMTs was relatively lower. At higher HMT contents, the stabilizer was less effective, since the ΔG behavior was mostly controlled by the native HMT content of the fat (Figure 3B). There were significant correlations between ΔG at 33 °C (306 K) and HMT content for mixtures with 0 % and 0.2 %

HBS added but not for 1.5 % HBS. The ΔG values were too low with the addition of 1.5 % HBS.

Our results suggest that the efficacy of this stabilizer will be highly dependent on the amount of

HMTs present in the fat mixture at a particular temperature, since the concept of an HMT is relative to the temperature used to crystallize the material. This needs to be taken into account in the practical application of this technology.

Figure 2 Free energies of nucleation (ΔG) of (A) anhydrous milk fat (B) palm oil (C) palm kernel oil and (D) palm stearin and HBS mixtures as a function of temperature. Shown are means and standard deviations of n=6 replicates.

Figure 3 Free energies of nucleation at 306 K as a function of (A) high-melting triacylglycerols (HMT) (% w/w) present in each fat and (B) high-melting triacylglycerols (% w/w). No significant correlations (ns) between ΔG at 306 K and HMT for the 1.5 % HBS addition. Shown are means and standard deviations of n=6 replicates.

4.4.3.2 Crystallization curves and solid fat content

Crystal growth curves (SFC increases) for HBS-fat mixtures as a function of time at

20 °C are shown in Figure 4. AMF and PO showed similar trends in SFC profiles: a rapid increase in the SFC until a week of storage at 20 °C. These two fats display a slow crystallization behavior. SFC values of HBS-PKO mixtures reached a plateau of about 40 % after 60 min. A sharp increase in SFC for HBS-PS mixtures was observed until it reached an SFC of about 45 % at after 20 min followed by a gradual increase to 62 % after one week.

All fats used in this study had a high SFC, ranging from 20 % to 60 %, and the addition of up to 1.5 % of HBS did not have significant impact on the final equilibrium SFC (p > 0.05).

However, inset graphs (Figs. 4A & C) show that the rate of crystal growth in the early stages of crystallization was higher when the 1.5 % of HBS was added in AMF and PKO. These results correspond to the trend seen in ΔG.

Figure 4 SFC of (A) anhydrous milk fat (B) palm oil (C) palm kernel oil and (D) palm stearin as a function of time at 20°C. Shown are averages and standard deviations of n=3 replicates. The insets of panels A and B highlight the early stages of the crystal growth.

4.4.3.3 Thermal behavior

Figure 5 shows crystallization and melting curves of HBS-fat mixtures. Two contiguous peaks, a small peak at higher temperatures and a large peak at lower temperatures were observed in the crystallization curves of AMF and PO mixtures. The high-temperature peak of PO was associated with a high melting, or “stearin”, fraction while the low-temperature peak was

associated with a low melting, or “olein”, fraction (Braipson-danthine and Gibon 2007; Peyronel et al. 2016; Tan and Man 2002). The high-temperature peak became broader and its peak temperature increased as more HBS was added. Similar tendencies were observed in previous studies when a low concentration (< 3 % w/w) of hard fat with trisaturated TAGs were added to

PO (de Olivia et al 2015; Verstringe et al. 2013). Authors found that these additives promoted the crystallization of HMTs in PO while they did not affect the crystallization of lower melting

TAGs (partly or fully unsaturated TAGs). The crystallization curves of PKO mixtures were composed of two overlapping peaks when 0 % - 0.5 % HBS was added. Upon further addition of

HBS, however, peaks superimposed and became sharper. Crystallization curves of PS were not affected by addition of HBS.

The melting curve of neat AMF displayed a broad melting peak which became sharper with increases in added HBS. Little changes were evident in the melting curves of PO and PKO mixtures. Two distinct peaks were observed in the melting curves of 0 % and 0.2 % HBS-PS mixtures. They started to overlap and eventually superimposed with an increase in the amount of

HBS added. It is likely that the peak at the lower temperature was due to the olein fraction while the other peak was due to the stearin fraction (Tan and Che Man 2000; Tan and Man 2002).

Figure 5 Differential scanning calorimetric crystallization curves of (A) anhydrous milk fat (B) palm oil (C) palm kernel oil and (D) palm stearin and DSC melting curves of (E) anhydrous milk fat (F) palm oil (G) palm kernel oil and (H) palm stearin.

Further DSC analysis was carried out on PO to better define the effect of HBS on its thermal behavior. As previously discussed, two peaks were visible before HBS was added

(Figure 6A). The high-temperature peak became broader and split into two peaks as a result of the addition of > 5 % HBS. de Olivia et al. (2015) and Verstringe et al. (2013) observed a segregation of trisaturaed TAGs from additives and HMT from PO upon the addition of > 3 % additives. Starting from 10 % HBS-PO, the crystallization peak caused at ~ 40 °C became taller and shifted to higher temperatures as more HBS was added. The low-temperature peak of PO stayed intact until 60 % or more HBS was added to PO. Similar results were reported by

Peyronel et al. (2016). Upon heating of 0 % HBS-PO, a broad melting peak (I) and a small peak

(II) were observed (Figure 6B). Peak II disappeared when 7 % or more HBS was added. Peak III appeared with the addition of 3 % HBS and gradually increased in size until 10 % of HBS was added. With the addition of 20 % HBS, part of it eventually superimposed with peak I while the rest formed peak IV which is also a shoulder peak to peak V. Peaks I and III disappeared when

50 % or more HBS was added. Peak IV became sharper and its peak temperature decreased about 4 °C as more HBS was added.

Figure 6 Differential scanning calorimetric (A) crystallization curves and (B) melting curves of HBS-Palm oil mixtures. * 40 %-100 % HBS data modified to fit in graphs

4.4.3.4 Multicomponent state diagram

Changes in the melting points of the four peaks from HBS-PO mixtures (Figure 6B) as a function PO:HBS ratios are summarized as a state diagram in Figure 7. PO showed two melting points, and PO with > 60 % HBS showed one melting point. PO with intermediate HBS proportions displayed two or more melting points. The first state transition took a place when 3 %

- 20 % HBS was added. Two other state transitions were observed at higher temperatures with

the addition of 10 % - 50 % HBS. The formation of new fractions between higher melting TAGs from PO and HBS led to multiple state transitions.

Figure 7 Pseudo two-component state diagram of HBS-Palm oil mixtures constructed using peak melting temperatures determined using differential scanning calorimetry (Figure 6B). Shown are means n=6 replicates.

4.4.3.5 Polymorphism

The X-ray diffraction patterns of HBS-fat mixtures are presented in Figure 8. Samples containing 0 % and 0.2 % HBS-AMF displayed βʹ polymorphism, whereas 1.5 % HBS-AMF displayed β and βʹ polymorphism, as indicated by a strong reflection at 4.6 Å and weak reflections at 3.8 Å and 4.2 Å . The same trend was observed in the 1.5 %-PKO mixture except that the 4.6 Å reflection representing the β form was weaker. This suggests that the addition of

1.5 % HBS to AMF and PKO promoted the transformation of some of the βʹ crystals into the

more stable β form. In PO and PS, higher HBS proportions caused stronger reflections at 3.8 Å and 4.2 Å . Both PO and PS have a high content of palmitic acid which naturally crystallizes into the βʹ form, and results in a more stable βʹ polymorph (Szydłowska-Czerniak et al. 2005). This, in turn, suggests that the tendency of palmitic acid to crystallize into the βʹ form is greater than the effect of HBS on the polymorphism of PO and PS.

Figure 8 Wide angle X-ray spectra of (A) anhydrous milk fat, (B) palm oil, (C) palm kernel oil and (D) palm stearin. Numbers indicate the corresponding d-spacings for the reflection.

4.4.3.6 Polarized light microscopy

Microstructures of HBS-fat mixtures crystallized for a week at 20 °C are shown in

Figure 9. AMF and PKO mixtures containing 1.5 % HBS showed a denser microstructure with needle-shaped crystals between spherulites. A similar observation was made in blends of palm mid fraction and palm oil containing a similar stabilizer (Peyronel et al. 2016). There were too many of these crystals for them to only represent HBS crystals, since only 1.5 % of the mixture was HBS. I speculate that these needle-like crystals are not just HBS but of HBS co-crystallized with a particular TAG or TAG family in the fats studied.

Figure 9 Polarized light micrographs of HBS-fat mixtures. The scale bar represents 100 µm.

4.4.4 Interaction between HBS and Specific Triacylglycerols or a Mixture of

Triacylglycerols

In this section, we examined how HBS interacts with specific TAGs and a mixture of

TAGs to elucidate its mechanism behind the nucleation enhancement effect. Melting points of

HBS-TAG mixtures were measured using DSC, and plotted against HBS % (w/w) (Figure 10).

By relating phase behaviors with the polymorphism of HBS-TAG mixtures, we propose two different mechanisms of HBS in this section.

TAG-HOAO blends (4:6 w/w) were found in the β polymorphic form and neat HBS was in the α polymorphic form (Figure 11). Homogenous TAGs pack into a bilayer structure and form stable β polymorphs (Kodali et al. 1987). The β polymorph is formed when milk fat with a higher content of high-melting TAGs crystallizes (Tzompa-Sosa et al. 2016). Both SSS and PPP showed a similar trend in the phase diagrams (Figure 10), small angle (Figure 11) and wide angle

X-ray spectra (Figure 12). A eutectic point was identified between HBS and both SSS/HOAO (~

40 % HBS) and PPP/HOAO (~ 15 % HBS). This would suggest a solid-state incompatibility between these substances. These concentrations were in accordance with the maximum amount of HBS added before excess HBS crystals (α-polymorph) were observed in wide angle X-ray spectra. We propose that HBS crystallizes first and serves as a surface upon which TAGs like

SSS and PPP would crystallize. A surface nucleation of these TAGs on top of HBS could possibly be an epitaxial process of deposition of SSS and PPP on the surface of HBS TAGs.

Figure 11 Small angle X-ray spectra of (A) tristearin (B) tripalmitin and (C) high-melting milk fat fraction and HBS mixtures. % HBS is the proportion to the total weight of the mixture. All HBS-TAG mixtures were prepared with a starting material which is a blend of TAG:HOAO at a 4:6 ratio. Numbers indicate the corresponding d-spacings for the reflection. * 90 % and 100% HBS data modified to fit in graphs

Figure 12 Wide angle X-ray spectra of (A) tristearin (B) tripalmitin and (C) high-melting milk fat fraction and HBS mixtures. % HBS is the proportion to the total weight of the mixture. All HBS-TAG mixtures were prepared with a starting material which is a blend of TAG:HOAO with a 4:6 ratio. Numbers indicate the corresponding d-spacings for the reflection. * 100 % HBS data modified to fit in graphs

However, for the case of HMF, a different behavior was observed (Figure 10C). Here the pattern is clearly monotectic, suggesting a solid solution of HBS in HMF and a high degree of structural complementarity, and thus compatibility between these families of molecules. I therefore postulate that the presence of mixed HMTs in HMF could be responsible for the observed solid state compatibility. HBS TAGs co-crystallized with HMF TAGs, thus creating a new crystals phase with a higher melting point and resulting in an enhanced nucleation rate. It is possible that the pronounced effect of HBS on ΔG of AMF (Figure 3A) is due to this effect.

In this study, two mechanisms responsible for the observed nucleation enhancement were identified. HBS formed a complex with TAGs present in HMF while it acted as a heterogeneous seed in SSS and PPP. Molecular compatibility allowing a partial solid solution formation should enhance the activity of these stabilizers over merely providing a surface to nucleate on. These aspects should be taken into consideration when designing such stabilizers. Compatibility can be assessed in a relatively straightforward fashion using calorimetry and multicomponent state diagrams.

4.5 Conclusions

Different amounts of HBS were added to AMF, PO, PKO, and PS to control their nucleation behavior. The nucleation enhancing effect of HBS was greater when the level of HMTs in fats was lower. However, this effect will depend on the particular temperature conditions used since the definition of HMTs used in this study is relative to the crystallization temperature. The addition of up to 1.5 % HBS did not influence the final equilibrium SFC of fats, but the addition of 1.5 % HBS in AMF and PKO significantly increased the SFC during the first 4 and 60 min, respectively. Upon the addition of 1.5 % HBS, crystallization points of the high-melting fraction of AMF and PO increased about 8 °C and 5 °C, respectively. Needle-like crystals were observed between fat crystals under polarized light microscopy, making the microstructure denser. Based on the phase diagrams and X-ray spectra, we determined that HBS acted differently with pure

TAGs than when it is in a mixture of HMTs. With SSS and PPP, HBS crystallizes first, providing a surface for nucleation of SSS and PPP. For the case of HMF, however, HBS TAGs co-crystallized with HMF TAGs, suggesting the formation of a partial solid solution. The formation of this fraction due to molecular compatibility was required for the observed enhanced nucleation observed in HMF.

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CHAPTER 5 Conclusions and Future Works

In this work, the crystallization behavior of HBS in liquid oils and the effect of cooling rate on HBS crystallization were first examined. Both the cooling rate and chemical properties of oil affected the crystallization behavior of HBS and its physical properties. PeO was the only oil among three oils studied that contained fatty acids with 20-24 carbon atoms (5.9 % w/w) and middle and TAGs with Tm > 30°C such as POS, POP, and PSS (2.5 % w/w). The interaction between HBS and high melting fraction of PeO enhanced the crystallization of HBS and the oil stabilization efficiency. PeO required the lowest Cg (6.5 % HBS w/w) when the mixture was cooled at 3 °C/min. The free energies of nucleation (i.e. the energy barrier to overcome for the nucleation to happen) were also the lowest among three oils. In addition, 12 % HBS-PeO mixture showed the highest Gʹ. Results showed that, a faster cooling rate (3 °C/min vs.

0.6 °C/min) facilitated the crystallization of HBS independent of the type of oil used. The Cg values were 1 % - 2.5 % (w/w) lower upon faster cooling. The fast cooled 12 % HBS-oil mixtures showed higher SFC values at 20 °C and a faster melting of crystalline upon heating.

When the mixtures were cooled at a faster rate, the crystallization of HBS started at lower temperatures. A fast cooling promoted the prevalence of the βʹ polymorph and the formation of a larger number of smaller crystals. In conclusion, the presence of HMTs (relative to the crystallization temperature) and a faster cooling rate accelerated the crystallization of HBS as well as the stabilization of liquid oil.

The effects of HBS on the crystallization of edible fats depended on the concentration and the type of “host” fat. The nucleation rate of AMF and PO increased as more HBS was added. However, HBS had a varying effect on the nucleation rate of PKO and PS. The addition 84

of 0.2 % HBS significantly hindered the nucleation of PKO while 1.5 % HBS had the opposite effect. For PS, the addition of 1.5 % did not affect the nucleation rate but 0.2 % accelerated the nucleation. There was a strong correlation between ΔG and the proportion of HMT (relative to the crystallization temperature) (% w/w) in fats. Both AMF and PO had smaller proportions of

HMTs compared to PO and PS, and the effect of HBS on nucleation rate was the greatest in those two fats. The addition of 1.5 % HBS increased the initial crystallization rate of AMF and

PKO although the final equilibrium SFC of fats were not affected by HBS. The crystallization temperature of AMF and PO increased, and their crystallization peaks became broader. Upon the addition of 1.5 % HBS, β polymorphs were formed in AMF and PKO while reflections corresponding to the β′ form became stronger in PO and PS. Additionally, under the polarized light microscope, needle-like crystals were observed between fat crystals, resulting in a denser microstructure. The multicomponent state diagram of HBS-PO mixtures showed multiple state transitions when the mixtures contained 3 % - 50 % HBS.

The interaction between HBS and specific TAGs or a mixture of TAGs was further studied. The eutectic phase was formed between SSS and PPP with HBS. The minimum HBS concentration until excess HBS crystals separated was 40 % for SSS and 15 % for PPP. Above this concentration, melting points increased and α crystals from neat HBS were detected along with β crystals from neat TAGs. These results suggest that there was a solid-state incompatibility between HBS and these homogeneous HMTs. On the other hand, the monotectic phase was formed in the mixtures for HMF and HBS. This phase behavior suggests that partial solid solution was formed between HMF and HBS. HBS TAGs and HMF TAGs co-crystallized, forming a new fraction with a higher melting point, and thus accelerating the nucleation.

Liquid oils were successfully structured and stabilized using HBS. Fast cooled oil gels displayed some of the desirable physical properties of plastic fat such as β′ polymorph and small crystals. This finding provides the potential to make oil gels that can be incorporated into fat- based food products using HBS. However, maintaining the β′ form and oil stability during the storage time are critical to preserve the desirable mechanical and physical properties of fat-based food products. A further research can be done to study the stability of β′ polymorph and oil phase in HBS-oil mixtures.

Regarding the crystallization enhancing effect of HBS, there are many methods that can be utilized to maximize the effect. It will be interesting to investigate the synergistic effect of

HBS and other methods on controlling fat crystallization. Fats with a variety of TAGs with different melting points and chemical moieties can be fractionated to produce a fraction enriched with HMTs. In addition, other minor components such as emulsifiers can be added along with

HBS to accelerate the crystallization.

To conclude, HBS was used to control crystallization of TAGs in fats and oils. Its gel forming and oil stabilizing ability were improved with the presence of HMTs in oils and a faster cooling rate. HBS also accelerated the nucleation of fats with relatively low contents of HMTs

(25 % < HMTs < 50 %) (TAGs with Tm > final crystallization temperature). Two mechanisms of

HBS on nucleation enhancement were identified: acting as a heterogeneous seed and forming a partial solid solution. In order to maximize the effect, it is important that the “host” fat is molecularly compatible with HBS for the formation of a partial solid solution.