Retention of Flavor Compounds by Dispersed High-Amylose Maize Starch

Home , Menthone

The Pennsylvania State University

The Graduate School

Department of Food Science

RETENTION OF FLAVOR COMPOUNDS BY DISPERSED HIGH‐AMYLOSE MAIZE STARCH (HAMS) IN AN AQEUOUS MODEL SYSTEM

A Thesis in

Food Science

Lihe Yeo

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Master of Science

December 2009

The thesis of Lihe Yeo was reviewed and approved* by the following:

Donald B. Thompson Professor of Food Science Thesis Co-Advisor

Devin G. Peterson Associate Professor of Food Science Thesis Co-Advisor

Joshua D. Lambert Assistant Professor of Food Science

John D. Floros Professor of Food Science Head of the Department of Food Science

*Signatures are on file in the Graduate School.

Abstract

Flavor is one of the most important attributes considered when determining the acceptability and popularity of food products. The main objective of this study was to examine

how hydrophobicity and solubility of flavor compounds relate to retention of these flavor

compounds by dispersed native HAMS in an aqueous model system, for both single‐flavor and

binary‐flavor systems. The effects of time and native lipids on flavor retention by dispersed

HAMS and on precipitated starch yield were also examined. Ten cyclic compounds with similar molecular weights, but varying in water solubility and hydrophobicity, were studied. HAMS

(Hylon VII) (0.7% w/w, db) was dispersed at 160oC for 10 minutes in a high pressure vessel. After

cooling to 80oC, each flavor compound was added at a concentration 20% below its pre‐

determined solubility. The starch‐flavor mixtures were cooled and held for one day, one week,

or one month at room temperature. Flavor compounds in the headspace were subsequently

analyzed by static headspace GC. Flavor retention (%) by aqueous starch was calculated relative to water alone. Flavor retention for menthol (58%), menthone (57%) and thymol (29%) was significantly higher than for the other seven compounds (<5%). A plausible explanation for this behavior is that a combination of sufficient hydrophobicity and sufficient solubility, leading to sufficient concentration, is necessary to generate high flavor retention. In binary‐flavor systems, when limonene was added with menthone or thymol, an approximately 40‐70% increase in limonene retention was observed. The enhanced retention of limonene in the presence of either menthone or thymol might simply be due to the presence of pre‐formed single helices that are able to accommodate a limonene molecule when these molecules are otherwise

in insufficient concentration to generate stable single helices. No difference in flavor retention

was observed for native and lipid‐free starch dispersions. By X‐ray diffractometry of the dried precipitates at one week, B patterns were obtained for the flavor compounds (limonene,

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cymene, anethole, carvone) with low flavor retention (%) and low amount of flavor bound. V

patterns were observed for menthol, menthone, thymol, pulegone and terpinen‐4‐ol. Since the

flavor compounds were added into the starch dispersions in varied concentrations, a V pattern

precipitate would also be obtained if the amount of flavor bound was high, despite low flavor retention (%). The generation of V pattern would depend on whether the single helices would associate to form a precipitate and the packing of the helices. Single helical starch‐flavor complexes reduced the formation of starch double helices over time. Overall, this work suggests an improved basis for understanding the association of flavor compounds with starch, and the results may be applicable to starch‐containing foods.

Table of Contents List of Figures ……………………..……………………………………………………………………….…………………. vii List of Tables ……………………………………..……………………………………………………………….………….. viii Acknowledgements ………………………………..…………………………………………………………….……….. ix

Chapter 1 Review of The Literature 1 The big picture‐ Why flavor and starch? ………………….……………………………..………...……….. 1 2 Flavor …………………………………………………………………………………………………….….…….….……… 1 2.1 Flavor properties………………………..………………………..…………………….………..… 1 2.2 Flavor retention and release ……..…………………………………...………….…..…..… 3 3 Starch ………………………………..……………………….…………………………………………..…….…..…….… 3 3.1 Structure and composition ...……………………………..……………..…………..…….. 3 3.2 Phase transition ………….……………………………….…………….………………….…….…. 5 4 Starch‐flavor Interaction..………………………………….…………………………………..………...…….…. 6 4.1 Types of starch‐flavor interaction..………………………….….….…..……..………….. 6 4.1.1 Starch‐flavor Inclusion complexes 4.1.1.1 Structural characteristics 4.1.1.2 Formation for inclusion complex 4.1.1.3 Factors that influence inclusion complexation 4.1.2 Other types of starch flavor interaction 4.2 Methods to investigate starch‐flavor interaction…………………………….…….… 11 4.2.1 Precipitation method 4.2.2 Headspace method 4.3 The role of lipid in starch‐flavor interaction……………………………..….………… 15 4.4 Flavor retention by starch in a mixed‐flavor system …………………..….…..…. 16 4.5 Flavor retention by starch over time…………………..……………………..……….…… 17 5 Statement of the problem …………………………………………………..………………..………..….….…... 20 6 Goal…………………………………………………………………………….……………………………….…….….....… 21 7 Specific objectives……………..…………………………………………………………………….………….….…… 21 8 References …………..…………..…………………………………………………………………..…..……….….…… 22

Chapter 2 Materials and Methods 1 Materials …………………………………………..……………..………………………………………………...... ….. 26 1.1 Starches 1.2 Flavors 2 Methods ……………………….…………………………………………………………………………………..…….… 26 2.1 Preparation of lipid‐free starch………………………………………………………………. 26 2.2 Determination of lipid content ………………………………………………………………. 28 2.3 Octanol‐water partition coefficient analysis ……………………………………..…… 28 2.4 Determination of saturation point using headspace analysis …………………. 29 2.5 Starch‐flavor complex preparation ………………………………………………………… 29 2.6 Determination of flavor retention using headspace analysis………………..… 30 2.7 Determination of precipitation yield ……………………………………………………… 33 2.8 Wide angle X‐ray diffractometry……..……………………………………………………… 33 2.9 Statistical analysis ……………………………………………………………………………..….. 34 3 References ……………………………………………………………………………………………..………….………. 34

Chapter 3 Retention of Flavor Compounds by Native Starch Dispersions 1 Single‐flavor starch system ………………………………………………………………………………..…...….. 35 1.1 Results …………………………………………………………………………………………..…….. 35 1.1.1 Solubility and hydrophobicity of flavor compounds…….….……..... 35 1.1.2 Flavor retention by native starch dispersion...……………………..….. 40 1.1.3 Flavor retention by native starch dispersion over time.…..…..….. 40 1.2Discussion …………………………………………..…………………………………………..…..….. 40 1.1.4 Measurement of hydrophobicity and solubility of flavor compounds on flavor retention by native starch dispersion…………………………………………………………………………….….. 40 1.2.2 The relationship of hydrophobicity of flavor compounds and Their flavor retention by native starch dispersion ……………………. 45 2 Binary‐flavor starch system …….…..………………………………………………………………………...……... 53 2.1 Results ….……………….………………………….………..……………………………….……...….. 53 2.1.1 Flavor retention by native starch dispersion ……………….…..…..….. 53 2.2 Discussion..….……………….……………………………………………………………………..……. 53 2.2.1 Possible explanations of flavor retention by native starch dispersion…………………………………..……………………………………..….…. 53 3 Conclusion …….………………………………………….……………………………………………………….…………... 57 4 References …...………………………………………….……………………………………………………….…………... 57

Chapter 4 Influence of Native Lipids on Flavor Retention and Precipitated Starch Yield 1 Effects of presence of native lipids on flavor retention…………………..…………………………….... 59 1.1 Results……………….………………………………….……………….…..……..…………..……….. 59 1.2 Discussion …………..….…………………………………………………..……..………….….…….. 59 2 Effects of presence of native lipids on precipitated starch yield over time without flavor addition…………………..………………………………………………………………………………………..….. 61 2.1 Results……………….………………………………….……………….…..……..…………..……….. 61 2.2 Discussion…………………………………………………………………………………………………. 62 3 Effects of flavor addition to native starch dispersion on precipitated starch yield over time…………..……………………………………………………………...…….……………..……..…….…….………… 62 3.1 Results…………………………………….………………………………………………………………... 62 3.2 Discussion ………………………………………………..…………………………………….…………. 68 4 Effects of flavor addition to lipid‐free starch dispersion on precipitated starch yield over time ……………………….………………………………………..…….……………..……..…………………..………… 70 4.1 Results………………………..……………….……………………………………………………………. 70 4.2 Discussion ………………………………………………..…………………………………….…….…… 70 5 Conclusion ….………………………………………….…………..…………………………………………………………. 72 6 References ….………………………………………….…………..…………………………………………………………. 73

Chapter 5 Suggested Future Works…..………………………..……………………….…..…………………..….. 74

Appendix Figures and Tables ……………………………………………………...... 76

List of Figures

Figure 1.1 The effect of amylose concentration and chain length on phase behavior in aqueous amylose solution .………………………………………………………………………….…. 7 Figure 1.2 A schematic diagram of a common method for preparation of starch‐flavor complexes………………………………………………………………………………………………………. 12 Figure 2.1 Starch sample in a 20 mL headspace vial ……………………………………………………….. 31 Figure 2.2 The cooling profile for starch‐flavor mixture ………………………………………………..... 32 Figure 3.1 Determination of saturation concentration for A) limonene, B) thymol .………... 36 Figure 3.2 The relationship between hydrophobicity (log Pow) and saturation concentration ……….………………………………………………………………………………….……. 38 Figure 3.3 Flavor retention by native starch dispersion for ten flavor compounds………..…. 39 Figure 3.4 Flavor retention by native starch dispersion at one day, one week or one month…………..…………………………………………………………………………………………. 41 Figure 3.5 The relationship between flavor retention, hydrophobicity (log Pow) and saturation concentration …….…………………………………………………………………………. 47 Figure 3.6 Flavor retention as a function of flavor added for A) menthone and B) thymol …………………………………………………………………………………………………….…. 50 Figure 3.7 Binding isotherms of menthone and thymol in starch………………………………….…. 51 Figure 3.8 Flavor retention by native starch dispersion in A) single‐flavor system, B) binary‐flavor system…………………………………………………………………………………… 54 Figure 3.9 Schematic of possible explanations to form menthone‐starch‐limonene complexes in a binary‐flavor system …………………………………………………………….… 55 Figure 4.1 Flavor retention by native and lipid‐free starch dispersions at day one …...... … 60 Figure 4.2 Starch yield for native and lipid‐free starch dispersions without flavor addition………………………………………………………………………………………………………….. 63 Figure 4.3 Starch yield for native starch dispersion with and without flavor addition at one day one week or one month …………………………………………………..………………... 65 Figure 4.4 Starch yield for lipid‐free starch dispersion with flavor addition at day one week or one month …………………..……………………………………………….……..……. 71 Figure A.1 Graphs to determine saturation concentration for flavor compounds..………..… 76 Figure A.2 Flavor retention by lipid‐free starch dispersion at one day, one week or one month……………………………………………………………………………………………………… 77 Figure A.3 Sample calculation of flavor retention by native starch dispersion relative to water …………………………………………………………………………………………………………. 78 Figure A.4 Bjerrum plots for the binding of menthone and thymol…………………………………… 79

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List of Tables

Table 1.1 Flavor retention by amylose for hexanal and nonenal in single‐flavor and binary‐flavor systems………………………………………………………………………………. 18 Table 2.1 Flavor and structural properties of ten cyclic flavor compounds…………………….. 32 Table 3.1 Saturation concentration, log Pow and amount of flavor added in starch for ten flavor compounds…..………………………………………………………………………………… 37 Table 3.2 Flavor retention by native starch dispersion at one day, one week or one month………………………………..……………………………………………………………………. 42 Table 3.3 Bound flavor by native starch dispersion at one day, one week, or one month……………………………………………………………….………………………………………….… 43 Table 3.4 Comparison of log P values by different methods…………………………………………… 46 Table 3.5 Flavor added, flavor concentration, flavor retention, bound flavor and free flavor for menthone and thymol in native starch dispersion ……………….………… 52 Table 4.1 Starch yield for native and lipid‐free starch dispersion with and without flavor addition at one day, one week or one month………….………………………………..…… 64 Table 4.2 The flavor bound, flavor retention and starch yield determined from the mixture of a flavor compound and native starch dispersion at one week are shown alongside with the XRD diffractogram.……………………………………..…….…… 67 Table A.1 Flavor retention by lipid‐free starch dispersion at one day, one week or one month……………………………………………………………………………………………………… 77 Table A.2 GC‐peak area of flavor compounds in the headspace of a water system and an aqueous dispersed starch system …………….……………………………….………………. 78 Table A.3 ANOVA output for showing the effects of three different factors (starch type, compound, time) on the response (flavor retention)……….....……….………………… 80 Table A.4 ANOVA output for showing the effects of three different factors (starch type, compound, time) on the response (starch yield) …………………………….……………… 80

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Acknowledgements

I thank Dr. Donald Thompson and Dr. Devin Peterson for their guidance in my pursuit of

academia excellence.

I also thank PMCA for their financial support through the Department of Food Science

endowed fellowship, the John W. Hess PMCA Graduate Confectionery Fellowship, and Eric

James who had assisted in organizing the PMCA confectionery internship.

Grateful acknowledgements are also extended to Dr. Joshua Lambert, for his efforts as my committee member, and Dr. Gregory Ziegler for the use of his high pressure vessel unit.

I especially thank my family, Josh, International Friendship House, colleagues and friends for their continuous encouragements throughout my Master.

Chapter 1

Review of The Literature

1 The big picture - Why flavor and starch?

Food is crucial to our biological and social life. We eat to nourish our bodies and satisfy emotional hunger. One of the main macronutrients in the human diet is carbohydrate. Dietary carbohydrates include mono- and oligosaccharides such as sugar, high fructose corn syrup and lactose, but the major dietary component is starch and non-starch polysaccharides (Stephen et al., 1995).

Most foods contain starch. In addition to providing calories, starch has also been used as a natural or modified ingredient for its functionality as a gelling agent, thickener, flavor encapsulation agent, texture enhancer, water binder and stabilizer in a wide range of food products (Thomas and Atwell, 1999). In addition to cost, availability and nutrition, the acceptability and popularity of these food products are driven by flavor.

The flavor properties of foods have been widely studied. One particular area of research is on how flavor compounds interact with the major food constituents. For example, evidence of interaction of starch, particularly amylose component, with flavor molecules has been extensively documented (Conde-Petit et al., 2006). The knowledge of starch-flavor interaction is important for providing a scientific basis to better control the flavor quality of food products during formulation, processing, storage and packaging.

2 Flavor

2.1 Flavor properties

The US society of Flavors Chemists defines flavor as the “sum total of those characteristics of any material taken in month, perceived principally by the sense of taste and 1 smell and also general senses of pain and tactile receptors in month, as perceived by brain”. A flavor can be a single chemical entity (rarely) or a blend of flavor compounds.

The quantitative measure of a flavor molecule’s hydrophilic or lipophilic nature is useful in predicting how flavor molecules interact with food components. Polarity, hydrophobicity and water solubility are commonly used for such quantitative measure.

A simple rule of thumb, “like dissolves like”, indicates that a solute will dissolve better in a solvent that has a similar polarity. Polarity is defined as the uneven distribution of electrons in a molecule that results when one atom is more electronegative than another (McMurry, 2000).

For example, menthone, which has a carbonyl group, is more polar than limonene that only consists of hydrocarbon. Polarity of a molecule is a continuum. Most organic compounds are miscible with water to limited extent, and thus are described as more or less polar and more or less hydrophobic.

Hyrophobicity means lacking affinity for water. Hydrophobic molecules result in affinity between water molecules being stronger than the affinity between water and solute molecules themselves (Goss and Schwarzenbach, 2003). A useful measure of the hydrophobicity of a substance is given by its distribution ratio between water and a standard water-immiscible organic solvent. Octanol-water partition is commonly used, and the partition coefficient is expressed as log Pow. In other words, log Pow is a measure of differential affinity of a compound for octanol and water.

Water solubility refers to the maximum concentration of a solute that can be dissolved in water. It is dictated by the balance of intermolecular forces between the water and the flavor molecule, and the entropy change associated with solvation. Both hydrophobicity and water solubility are properties of flavor compounds that might be of relevance to understand starch- flavor interaction.

2.2 Flavor release and retention

The flavor quality is, in part, determined by the release of flavors from the food when eating, and the perception of the released flavors by the olfactory and gustatory system (Taylor,

2002). The term “flavor release” in a broad sense includes the release of flavor from a food during manufacture, storage, preparation or eating (Reineccius, 2006). However, today, fIavor release usually refers to the dynamic process by which flavor is liberated from the food matrix to the gas phase during eating and mastication of food (Reineccius, 2006). The main driving force for the movement of flavor compounds from one phase to another is the difference in flavor concentration. The rate of the diffusion is determined by concentration gradients and the mass transfer coefficients of the flavor compounds in each of the phases.

Flavor release is influenced by the nature and concentration of flavor molecules present in food due to interactions with different food components such as starch, lipid and protein

(Bakker, 1995). Flavor molecules that are retained in the food matrix during consumption will not be perceived. Therefore, retention of flavor compounds is required for flavor release. The retention of flavor compounds in a food can be assessed by the equilibrium distribution of flavor compounds between the food matrix and the gas phase.

3 Starch

3.1 Structure and composition

Starch is the primary carbohydrate in cereal grains, as a form of stored energy in the seed (Watson, 2003). Primary sources of starch in the food we consume everyday are rice, wheat, corn (maize), potato, cassava, and sago. Corn originated in Mexico 7,000 years ago and spread throughout the Americas. The U.S. is currently the largest producer of corn, producing

3 about 40% of the world’s total (FAOSTAT, 2004). Regardless of the botanical source, starch is built from monosaccharide D-glucose and is the most abundant storage glucan in the world.

Starch occurs naturally in the form of granules with alternating amorphous and semi- crystalline growth rings. When viewed under polarized light, the granules appear birefringent.

Starch is a mixture of amylose, a predominantly linear polymer of α-1,4-glycopyranosyl units, and amylopectin, a highly branched polymer of α-1,4-linked glucan chains with α-1,6-linked branched points. Normal maize starch has about 25% amylose and 75% amylopectin (Thomas and Atwell, 1999).

In general, maize starches with >40% amylose can be considered as HAMS (Shannon and

Garwood, 1984). The high amylose content of HAMS is caused by ae mutant which results in the lack of starch-branching enzyme IIb (SBEIIb) (Boyer and Preiss, 1981). It was shown that amylose from HAMS has smaller molecular size (690-740 DP) and shorter constituent chains (CL 215-255) than from normal maize starch (930-990 DP, CL 295-335) (Takeda et al., 1988; Takeda et al.,

1989). The amylopectin from HAMS is also different from the normal starch, being less branched with longer inner and outer chains than normal maize amylopectin (Takeda et al., 1993; Klucinec and Thompson, 2002).

HAMS was shown to have greater iodine binding capacity than normal starches (Gerard et al., 2001). The high amylose content and the presence of more longer chains in HAMS might relate to its higher capacity interact with iodine, lipids, flavor and other small organic molecules.

HAMS’s structural characteristics lead to unique starch behavior and applications. HAMS is commonly used in the confectionery industry to produce rapid set for gum candies and hence improve productivity. Adding HAMS can also help to retain moisture in the food system and provide a film formation for further thermal processing such as reducing oil retention in batters and breading (Thomas and Atwell, 1999).

Other constituents in maize starch are proteins (~0.3%), lipids (~1%), moisture (~12%) and ash (~0.5%). The total content of internal lipids of cereal starches ranges from 0.6% to 1.5%

(w/w) (Morrison, 1988). They are composed exclusively of free fatty acids (FFAs) and lysophospholipids, which both are the monoacyl lipids. HAMS tends to have higher lipid content, probably associated with the higher long chain α-1,4-glucan content (South et al., 1991). Such lipids readily associate with amylose, which forms a single helix with the hydrocarbon chain in the core. The hydrocarbon chain of fatty acid resides in the helical cavity of amylose, but not the polar group of the fatty acid due to steric hindrance (Godet et al., 1993; Morrison et al., 1993).

By X-ray diffraction, the aggregate of the ordered association of these single helices can yield V- type crystalline pattern. It has been suggested that such V-types complexes can also be formed with lipids in vivo (Morrison et al., 1993; Morgan et al., 1995).

3.2 Phase transition

Starch is almost never in a thermodynamic equilibrium but rather exists in a metastable state. The final structure of starch such as starch spherulites, gel or glass, heavily depend on hydrothermal history and water content. In aqueous starch dispersion, the starch molecules exist in random coils (Ring et al., 1985). When starch is cooked in excess water, starch granules are disrupted and gelatinization occurs. Irreversible changes that accompany this process include granular swelling, loss of birefringence, leaching of amylose and native crystalline melting (Buleon and Colonna, 2007). The extent of these changes varies according to numerous parameters such as moisture content, heating temperature, time and shear force.

Gelatinized and solubilized starch polymers tend to reassociate after heating. This reassociation process of starch chains in more ordered structure is called retrogradation. During retrogradation, double-helical association can promote gelation and/or precipitation under

5 appropriate conditions. The amylose component in the starch crystallizes faster than the amylopectin component (Miles et al., 1985). The long term effect of the crystallization of amylopectin has been associated with changes in texture (Gudmundsson and Eliasson, 1990).

During retrogradation, amylose, which is a predominantly linear, may form longer double helices (40-70 glucose unit) than amylopectin, which is highly branched (Leloup et al., 1991).

The structure of starch during retrogradation is influenced by the chain length distribution, amylose-amylopectin ratio, concentration, and also cooling conditions (Figure 1.1)

(Gidley and Bulpin, 1987; Gudmundsson and Eliasson, 1990). The kinetics of gelation appears to be governed by the interaction of junction zones to form an inter-connected network.

Precipitation is the phase separation of the crystalline aggregates of double helices from the solution. Gidley and Bulpin (1987) observed that amylose with DP <100 precipitated readily for

<5% amylose concentration. Above DP 100 and >1% amylose concentration, gelation was observed, and more readily at higher cooling rate 15oC/min rather than 3oC/min.

4 Starch-flavor interaction

4.1 Types of starch-flavor interaction

4.1.1 Starch-flavor inclusion complexes

4.1.1.1 Structural characteristics

One kind of binding of flavor compounds to starch, particularly the amylose portion, is called inclusion complexation. In addition to flavor compounds, a variety of other ligands such as alcohol, iodine and monoacyl lipid, are also shown to form inclusion complexes with amylose

(Kuge and Takeo, 1968, Conde-Petit et al., 2006; Godet et al., 1993, Osman-Ismail and Solms,

1973, Tapanapunnitikul et al., 2008).

Figure 1.1 The effect of amylose concentration and chain length on phase behavior in aqueous amylose solution (Gidley and Bulpin, 1987).

In contrast to the formation of double helices during retrogradation, in the presence of suitable ligands, the amylose can adopt a single left-handed helix conformation to form inclusion complex. The helical cavity the starch is considered hydrophobic (Godet et al., 1993) and can accommodate guest molecules with sufficient hydrophobicity. There is also evidence suggesting that larger ligand which is too large to fit in the cavity might reside in the inter-space between the helices (Helbert and Chanzy, 1994; Nuessli et al., 2003; Biais et al., 2006).

Like starch-lipid inclusion compelxes, the crystalline packing of the starch-flavor single helices is also characterized as V pattern by wide-angle X-ray diffraction. Three types of V polymorphs, with 6, 7 or 8 glucosyl units per turn have been proposed, suggesting that the amylose cavity can be tailored based on the size and shape of the ligand (Biais et al., 2006).

4.1.1.2 Formation of inclusion complexes

Menthone has been shown to form inclusion complex with starch readily (Kuge and

Takeo, 1968). Equation 1 describes an equilibrium equation in which menthone is added to an aqueous starch system to form starch-menthone inclusion complexes.

Starch (aq) + Menthone (aq) Starch-Menthone (aq) ----- Equation 1

At equilibrium, inclusion complexation can also be viewed as host-guest binding assembly measured by association constant Ka.

Ka = [Starch-Menthone]/[Starch][Menthone]

Ka is mathematically related to ∆G:

∆G = -RT ln Ka

where R=gas constant (K/mol), T= temperature (oK)

Ka is a measure of binding affinity of flavor compounds to starch. It was found to vary greatly among different flavor compounds (Rutschmann and Solms, 1990a; Wulff, et al., 2005).

In order to form inclusion complexes, the reaction must be accompanied by a decrease in Gibbs free energy (∆G).

∆G = ∆H - T ∆S

Prior to the formation of starch-flavor inclusion complexes, the water molecules that interact with flavor compounds or the hydrophobic surfaces of the interior of the amylose cavity are much more organized than they are in bulk water. When the flavor compounds replace the water molecules, the previously imposed organization of water molecules is less. The release of structured hydration water contributes to the positive entropy change and negative change in free energy. Since the natural tendency is always toward the lower energy state, inclusion complexes are formed.

Under favorable conditions, the single helical amylose-flavor complexes may initiate nucleation events and crystallize. Due to the heterogeneous structure of starch molecules, the interaction with flavor molecules may produce partly crystalline, partly random coil configurations, giving rise to phase separation and precipitation (Heinemann et al., 2001).

Amylose-flavor complexes have been extensively studied for a long time by means of precipitation. Kuge and Takeo (1968) examined the ability of >100 organic compounds to form precipitate, concluded that the ability of ligands to form precipitate varies. In this study, the initial cooling temperatures for formation of starch-flavor complexes were shown to be different among the ligands. In a similar study, Osman-Ismail and Solms (1973) concluded that some minimum concentration for each flavor compound was required to initiate the formation of aggregated helices and lead to precipitation.

4.1.1.3 Factors that influence the formation of starch-flavor compound inclusion complexes

The formation of an inclusion complex is known to be influenced by the structure of starch. The chain length of amylose was shown to affect the inclusion complexes with monoacyl lipids. Amylose having DP <20 seems unable to form these inclusion complexes. Stability of complexes increased with longer amylose chain length (Godet et al., 1995). By analogy to amylose-lipid complexes, amylose-flavor complexes may exhibit similar binding behavior. Starch with high amylose content might be advantageous in forming inclusion complexes.

Another factor that affects inclusion formation is the physical properties of the flavor compounds. Various physical properties of the flavor compounds such as molecular size, solubility, shape, volatility, and hydrophobicity were shown to affect the interaction of starch with flavor compounds. The functional group of the flavor compounds, which contributes to these physical properties, also affects the formation of inclusion complexes. Most alcohols were shown have better ability than aldehydes and ketones to form complexes (Kuge and Takeo

(1968). The same group concluded that it seems impossible to predict the complexing ability solely by solubility, and speculated that hydrophobicity of flavor compounds might affect complexing ability as well. Arvisenet et al. (2002a) studied the complexing behavior of isoamyl acetate, ethyl hexanoate and linalool with amylose. Their data showed that linalool and ethyl hexanoate, but not isoamyl acetate, formed inclusion complexes with amylose. The authors suggested that the different behaviors were probably caused by their differences in solubility.

Tapanapunnitikul et al. (2008) used terpenes, limonene, cymene, menthone and menthol to make inclusion complexes with HAMS. In this group, the flavor compounds with higher water solubility gave higher starch yields and flavor retention. The results of how solubility and hydrophobicity affect flavor retention in starch are unclear. The problem might be due to too small a number of flavor compounds (3-4 flavor compounds) or the presence of other confounding variables such as molecular size and shape. For linear compounds, there is some

10 evidence that flavor compounds with higher molecular weight corresponds to higher retention of these compounds in cyclodextrin (Goubet et al., 1998). For non-linear flavor compounds, as their molecular size increase, those bulky flavor compounds might not be included in the cavity due to stearic hindrance (Goubet et al., 1998).

4.1.2 Other types of starch-flavor interaction

Another mode of starch-flavor interaction is sorption, which usually refers to the interaction of flavor molecules with materials at low water content. One example is the sorption of flavor molecules on the surface of the starch granules or powder made from extruded starch

(Boutboul et al., 2002; Hau et al., 1998). It was shown that the starch samples with higher surface area and more pores exhibited better flavor retention.

After gelatinization or dispersion, many starch-based products, such as snack foods and cereals, are further processed to reduce moisture content. In many of these products, starch can exist in the glassy state. Due to the low mobility and diffusion rate in the glassy state, flavors can be retained (Boutboul et al., 2002). The application of glassy carbohydrate mixtures has been exploited in flavor encapsulation technology.

4.2 Methods to investigate starch-flavor interaction

4.2.1 Precipitation method

Numerous studies have been conducted on starch-flavor inclusion complexes prepared by the mean of precipitation (Kuge and Takeo, 1968; Osman-Ismail and Solms, 1973; Arvisenet et al., 2002a; Arvisenet et al., 2002b; Tapanapunnitikul et al.,2008). The strategy for preparing these starch-flavor complexes is presented in Figure 1.2.

Starch, amylose Heat to 120-200oC, 30-60 min

Starch dispersion Add flavor compound (0.03-1000 mmol/mol glucose), 25-80oC

Shake or stand 20s-1hr, rt-80oC

Starch-flavor mixture

Cool down

Store for 24-48hrs

Centrifuge and discard supernatant Precipitate

Figure 1.2 A schematic diagram of a common method for preparation of starch complexes.

The three main steps in this method are: 1) molecular dispersion of starch 2) formation of starch-flavor complexes 3) precipitation of starch-flavor complexes. The starch variety and conditions for preparation differ among the studies reported in the literature. Potato starch or tapioca starch (having low level of internal lipids compared to other botanical sources) was used by some to prevent monoacyl lipids from competing with the flavors (Kuge and Takeo, 1968;

Osman-Ismail and Solms, 1973; Arvisenet et al., 2002a; Arvisenet et al., 2002b; Heinemann et al., 2001; Rutschmann and Solms, 1990a). Commercial amylose and HAMS were also used because there is strong evidence showing that linear chains of amylose can readily form inclusion complex with monoacyl lipids, iodine and flavor compounds (Tapanapunnitikul et al.,

2008, Wulff, et al., 2005). Due to the structural differences, higher temperature is required to disperse HAMS than normal starches. It was suggested that initial heating to >160oC resulted in a molecular dispersion of amylose crystals in HAMS (Boltz and Thompson, 1999).

The starch concentration used in the above studies ranged from 0.5% to 3% to prevent gelation. To induce precipitation, a large amount of flavor ranging from 500 to 30000μL/L was typically used. The temperature when flavor was added varied from room temperature to 80oC.

Adding flavors at higher temperature might minimize the extent of retrogradation (double helices) prior to the formation of inclusion complex (single helices)(Tapanapunnitikul et al.,

2008). After adding flavor, the starch sample is cooled to promote association of helices and lead to precipitation.

To quantify the flavor molecules, the starch-flavor complex precipitates are usually dispersed in sodium hydroxide for flavor extraction and subsequently analyzed by gas chromatography (GC). Further characterization of amylose-flavor complex precipitate has been performed using differential scanning calorimetry (DSC), X-ray diffraction, and iodometric

13 titration (Heinemann et al., 2001; Nuessli et al., 2003; Heinemann et al., 2004; Tapanapunnitikul et al., 2008)

Precipitation is a convenient screening method to measure the ability of flavor compounds to form insoluble inclusion complexes with starch. However, in order to induce precipitation, flavor concentration used is several magnitudes larger than normally used in food products (0.01 - 500μL/L) (Flavor-Base, Georgia, USA). Another limitation for this method is that only the crystalline phase of inclusion complexes is studied.

4.2.2 Headspace method

Both static and dynamic HS-GC (headspace-gas chromatography) analyses have been used to study starch-flavor interactions in food. For static HS-GC, the equilibrium partition coefficient, Kgp (“gp” refers to gas-product), describes the thermodynamic distribution of volatilities between the gas phase and product phase in liquid systems, is expressed as:

K gp = Cg/Cp

The partition coefficient Kgp is the ratio of Cg, the concentration of flavor compound in the in the headspace gas, and Cp, the concentration of flavor compound in the product (Kolb & Ettre, 2006).

Prior to static headspace analysis, the sample is equilibrated and an aliquot of the headspace gas is transferred directly into a gas chromatography (GC) column.

The static headspace analysis has been used to measure flavor retention in starch extruded at low water content (Boutboul et al., 2002), starch gels (Arvisenet et al., 2002ab;

Lafarge et al., 2008), native starch (Hau et al., 1996), and model food systems (Philippe et al.,

2003). The flavor retention is generally calculated as the partition coefficient in the product-gas phase relative to that of in the water-gas phase, from the following equation:

Flavor retention = ( Kgw – Kgp ) /Kgw = 1- Kgp/Kgw

The dynamic headspace method is a process by which volatiles are continuously extracted from the matrix. For static conditions, partitioning phenomena are considered; while for dynamic conditions, mass transport phenomena are considered in addition to partitioning phenomena. In the mouth, mass transport is affected by viscosity and eating behavior. The rate of the diffusion is determined by the flavor concentration gradients and the mass transfer coefficients of the flavor compounds in each of the phases. Since the equilibrium conditions are impossible to achieve during eating, the dynamic headspace method has also been useful to study the flavor release from food (Decourcelle, et al., 2004; Pozo-Bayon et al., 2008). The disadvantage for the dynamic headspace method is that the data produced might have larger variation than those performed under equilibrium conditions (Kolb and Ettre, 2006).

Nevertheless, both static and dynamic headspace methods require little sample preparation, and provide useful information that is of relevance to flavor perception.

4.3 The role of lipids in starch-flavor interaction

Most flavor compounds tend to be lipophilic, but their lipophilicity can vary greatly. In a system in which water is the continuous phase, lipophilic compounds tend to be driven into the air. When lipid is added in the system to form an emulsion, the flavor compounds will tend to interact preferentially with the lipid phase, decreasing the headspace concentration. Therefore, by changing the fat content, one would expect changes in the composition, intensity of flavor compounds and rate of flavor release (Plug and Haring, 1993).

In food technology, monoglycerides might be added to the starch-based matrix to improve rheological quality and stability of food products. For example, monoglycerides were used to retard staling in bread making (Boyle, 1997), and to improve freeze-thaw stability

(Mercier et al., 1980). The long aliphatic chains of monoglycerides are able to form inclusion

15 complexes with amylose, preventing retrogradation that is associated with staling. Similarly, the aliphatic chains from free fatty acids and lysophospholipids present in the native maize starch can also form single helices with amylose (Morrison, 1988).

The effect of monoacyl lipid on the formation of starch-flavor complexes has been investigated and contradictory conclusions have been obtained. In ternary systems comprising starch, menthone and monostearate, addition of monostearate was shown in one study to reduce the retention of menthone in starch (Rutschmann and Solms, 1990a). In another study,

Tapanapunnitikul et al. (2008) used HAMS, with and without native lipid, to make inclusion complexes with two pairs of terpenes with high and low water solubility. The data showed that the presence of native lipid improved the flavor retention for terpenes with little water solubility. This data contradicted the general idea in which defatted starch or potato starch (low monoacyl lipid content) was commonly used to eliminate the interference of lipids during complexation (Osman-Ismail and Solm, 1973; Rutschmann and Solms, 1990ab; Heinemann et al.,

2003). Little literature investigating the effects of native starch lipids on flavor retention in starch is available.

4.4 Flavor retention by starch in a multi-flavor system

By analogy to the influence of monoacyl lipids in interfering with flavor retention, the presence of other lipophilic flavor compounds might also affect flavor retention in a starch- based system. In a starch system, when flavor compounds are present in a mixture, rather than individually, some flavors might be driven to the air more readily while others are preferentially retained in the matrix. The net balance of interactions between the starch and the flavor molecules ultimately could influence flavor perception.

Rutschmann and Solms (1990b) studied the precipitate formed by a mixture of potato starch, decanal and menthone. They found that decanal was favored over menthone to be retained in the starch. As the decanal concentration increased, the menthone retention decreased.

In another study, Wulff et al. (2005) studied the retention of hexanal and nonenal by amylose. In a single flavor system containing 5 mmol of hexanal or nonenal, the retention by amylose was 5.5% and 6.8% respectively (Table 1.1). When hexenal and nonenal were added together (a total of 10 mmol), their retention decreased to 2.0% and 2.9% respectively. When they were added together but with their flavor concentration lowered by 5 times (a total of

2mmol), the percent retention of nonenal was 2.5 times higher than that of hexanal. A higher retention of nonenal than hexanal in the binary-flavor system might be due to the higher association constant (Ka) for the formation of inclusion complexes with nonenal (210 L/mol) compared to that of hexanal (36 L/mol) (Wulff et al., 2005). The competition between nonenal and hexanal appeared to be stronger at lower flavor concentration.

In summary, in a multi-flavor system, retention of one flavor compound by starch might affect the retention of another. The basis for such behavior has not been elucidated.

4.5 Flavor retention by starch over time

Crystalline starch-flavor spherulites and starch-flavor complexes showed little degradation during storage. In the study by Wulff et al. (2005), very little flavor loss or oxidative degradation (<3%) was observed in the freeze-dried amylose-flavor complexes for hexanal, 2- nonenal, 1-octen-3-ol and guaiacol after one year of storage under “dry conditions” at room temperature. Tapanapunnitikul et al. (2008) found no changes in the retention of thymol, menthone and cymene by a precipitate, but limonene retention decreased by 40% after 20

Table 1.1 Flavor retention by amylose for hexanal and nonenal in single-flavor and binary-flavor systems Added flavor (mmol/g amylose) Flavor retention (%) Hexanal Nonenal Hexanal Nonenal 5 0 5.5 - 0 5 - 6.8 5 5 2.0 2.9 1 1 0.4 1.0 Data were obtained from Wulff et al., 2005.

18 weeks at room temperature. In the same study, menthone-starch complexes showed the highest stability even at 50oC (Tapanapunnitikul et al., 2008).

Most storage studies have been performed on starch-flavor precipitates. There is no or little literature of how time influences flavor retention in an aqueous starch system. Structural change in an aqueous system can be more dynamic than in a dry form due to higher mobility and rearrangement of starch molecules. During storage, retrogradation might occur and the changes in starch structure might interfere with flavor retention, or vice versa.

The process of retrogradation is of great interest to the food industry because it affects the sensory properties of the starch-based food products. It is well-known that monoglycerides and polar lipids could retard retrogradation of starches by minimizing the formation of amylose- lipid complex single helices (Gudmundsson and Eliasson, 1990). In the presence of flavor compounds, starch-flavor single helices can be formed. The formation of these helices leads to the interest of whether flavor addition could retard retrogradation as well. However, a negative aspect of the formation of these singles helices is that the flavor compounds might be strongly retained by the starch and may not be available for perception.

Food products are commonly stored at ambient conditions over a period of time.

Understanding flavor retention in starch model systems and its influence on starch retrogradation might be useful in controlling food quality and shelf life.

5 Statement of problem

Based on the current evidence indicating that the solubility and hydrophobicity might influence starch-flavor interaction (Kuge and Takeo, 1968; Tapanapunnitikul et al., 2008), this research is designed to investigate the effects of these parameters on starch-flavor interactions.

Prior to this work, the results of how hydrophobicity and solubility affect flavor retention in starch have been inconclusive due to the presence of confounding variables such as molecular size and shape. In the current study, ten cyclic compounds with similar molecular size were chosen to minimize such confounding effects.

Tapanapunnitikul et al., (2008) concluded that the presence of a native lipid improved flavor retention by precipitated starch, particularly for terpenes of low water solubility. This new finding emphasizes the possible significance of native lipid in affecting its interaction with flavor compounds having different solubility. Understanding the effect of lipids on starch-flavor interaction and retrogradation may provide strategies to improve flavor quality as well as shelf- life in starch-based products. The influence of starch-flavor interaction on retrogradation in an aqueous starch system has not yet been documented.

Unlike the precipitation method, the static headspace method can measure flavor retention that resulted from the formation of either soluble or insoluble starch-flavor complexes.

In addition to that, flavor concentration high enough to induce precipitation is not necessary for the headspace method.

6 Overall goal of the research

To understand the interaction of high-amylose maize starch (HAMS) with flavor compounds in an aqueous system.

7 Specific objectives

1 To examine how hydrophobicity and solubility of flavor compounds relate to retention of the

flavor compounds by dispersed HAMS, for both single-flavor and binary-flavor systems

2 To determine the effect of time on flavor retention by HAMS

3 To determine the effect of flavor addition on precipitated starch yield over time

4 To determine the effect of native lipids on flavor retention by HAMS and on precipitated

starch yield over time

8 References

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Chapter 2

Materials and Methods

1 Materials

1.1 Starches. Commercial high-amylose maize starch (HAMS) Hylon VII was obtained from National Starch and Chemical Company, Bridgewater, NJ. It is referred to as native HAMS.

The lipid-free starch was prepared from the native starch using Method 2.2.1. The moisture contents of native and lipid-free starch were 12.04 ± 0.07% and 7.64 ± 0.33%, respectively

(AACC method 44-15A). The lipid contents were 1.11 ± 0.25 and 0.02 ± 0.01 wt% (db), respectively (Method 2.2.2).

1.2 Flavors. 10 cyclic flavor compounds with a series of solubility and hydrophobicity

(Table 2.1) were purchased from Sigma-Aldrich, St. Louis, MO. The 10 compounds were: (R)-(+)- limonene (Sigma Aldrich, purity 97%), p-cymene (Aldrich, purity 99%), trans-anethole (Aldrich, purity 99%), menthone (Fluka, purity > 97%), menthol (Sigma-Aldrich, 99%), thymol (Sigma-

Aldrich, 99.5%), (R)-(-)-carvone (Aldrich, purity 98%), pulegone (Aldrich, purity 85%), terpinen-4- ol (Fluka, purity > 95%), and guaiacol (Sigma).

2 Methods

2.1 Preparation of lipid-free starch. Lipid-free HAMS was prepared using Klucinec and

Thompson’s method (1998). 10 g of native HAMS was dispersed with 200 mL of dimethyl sulfoxide/water (90:10) mixture in a polypropylene centrifuge tube. The sample was heated in a boiling water bath with constant stirring for 3 h. Following dispersion, 160 mL of ethanol (95%, v/v in water) was added into 40 mL of dispersion. The mixture was agitated until no clumps remained. The mixture was then centrifuged at 6,500 x g for 10 min. The supernatant was

Table 2.1 Flavor and structural properties of ten cyclic flavor compounds.

27 discarded and the pellet was washed by dispersing in 50 mL of ethanol followed by recentrifugation (6,500 x g). The washing step was repeated once with 50 mL of ethanol and once with 50 mL of acetone. The precipitate was dried at 40oC overnight in a hot air oven.

2.2 Determination of lipid content. The amount of lipid in the native and lipid-free

HAMS was determined by gas chromatography, after conversion of fatty acids to fatty acid methyl esters (Godet et al., 1995). In a screw cap test tube, 100 mg of starch was mixed with 5 mL of MeOH/H2SO4 solution (98/2, v/v). 150 μl of heptanoic acid in toluene (1 mg/ml) was added as an internal standard. The mixture was placed in a 80oC water bath for 90 min for methylation. The mixture was then extracted with 5 mL of hexane at room temperature or 1 h.

The extract was dried with anhydrous NaSO4. Gas chromatography (GC) was performed with an

Agilent 6890 (Agilent Technologies, Palo Alto, CA) utilizing a flame ionization detector equipped with a DB-5 ((5%-Phenyl)-methylpolysiloxane) capillary column (30 m x 0.25 mm i.d. with a 0.30

μm film thickness), and a Combi-Pal autosampler (CTC Analytics, Carrboro, NC). The operating conditions were as follows: sample was injected in split mode (5:1); inlet temperature was

200oC, detector was 250oC, oven program was 150oC for 2 min, then increased at 10oC/min from

150°C to 200oC, then increased at 3oC/min to 250oC and held for 3 min.

2.3 Octanol-water partition coefficient analysis. The log Pow values for all flavor compounds were determined by a shake flask method as previously described by Griffin and others (1999) with a slight modification. Both octanol and water were mutually saturated for 24 h prior to use. For those compounds suspected to have log P >3 (based on estimated Clog P data, Table 3.2), a ratio of 2 mL of octanol to 200 mL water was used. For those compounds suspected to have log P <3, a ratio of 5 mL of octanol to 200 mL water was used. A known amount of flavor (~40mg) was weighed and added into octanol and mixed well. Either a 2 mL or

5 mL fraction of this octanol-flavor mixture was added into 200 mL water for log P analysis. The

28 octanol-water-flavor mixture was inverted 100 times in 5 min. The samples were then centrifuged for 30 min at 4000 rpm to destroy the emulsion. Prior to GC injection, the octanol layer was diluted 1:1 with MeOH containing 200 μL/L methyl hexanoate as the internal standard. GC analysis was conducted with the same instrumental setup as outlined in Section

2.2 with following modifications. The operating conditions were as follows: 1 μL sample was injected in split mode (50:1); inlet temperature was 200oC, detector was 250oC, oven program was 40oC for 2 min, then increased at 10oC/min from 40°C to 200oC, and held at 200oC for 2 min.

2.4 Determination of saturation point using headspace analysis. A series of known amounts of flavor were added to 20 mL screw-thread headspace vials (MicroLiter Analytical

Supplies, Inc, Suwanee, GA, USA), each containing 10 mL of water. All vials were capped with

PTFE/Silicone screw thread caps. The samples were equilibrated at 35oC for 30 min in the agitator prior to injection. GC analysis was conducted with the same instrumental setup and oven conditions as outlined in Section 2.3, except 1 mL of gas phase was sampled and the split ratio was changed to 1:20. The saturation point was determined as the point which the GC peak area reaches a maximum as the flavor concentration increased.

2.5 Starch-flavor complex preparation. The procedure was based on the method of

Tapanapunnitikul et al. (2008). HAMS (0.7 w/w, db) was weighed in a beaker and was brought to

100 g with distilled water. After mixing for 15 min using a stirring bar, the starch-water mixture was transferred to a pressure vessel (Auto Engineers, PA, USA). The pressure vessel had a bolt closure and was connected to an electric band heater (Watlow, St. Louis, USA). The pressure vessel was heated to 160oC and held for 10 min. The heating element was then switched off. The vessel was immediately sprayed with water for 1 min and allowed to sit for an additional 5 min to avoid splashing of hot dispersion during dispensing. 200 μl of sodium azide solution (10% w/v) was added to the starch dispersion to prevent bacterial growth. To a 10 mL volumetric cylinder,

29 a 10 mL aliquot of hot starch dispersion was measured and transferred to 20 mL headspace vials

(Figure 2.1). The headspace vials were equilibrated in an 80oC water bath for 15 min. Each flavor compound was added into the headspace vial at 80oC at a concentration 20% below its predetermined saturation point. The mixture was vortexed immediately and placed in the 80oC water bath for 1h. Each headspace vial was calculated to contain 67.5 ± 0.5 mg of starch (db).

The headspace vials containing the starch-flavor mixture were cooled slowly by placing them in a screw cap jar (14.0 x 15.6 cm) at room temperature. Each jar contained 5 headspace vials submerged in 600 mL of water with an initial temperature of 80oC. The samples were cooled to room temperature over 6 hours (Figure 2.2). They were stored for 1 day, 1 week or 1 month at room temperature. The samples were subsequently analyzed indirectly by static headspace GC

(Figure 2.1).

2.6 Determination of flavor retention using headspace analysis. The samples were equilibrated at 35oC for 30 min in the agitator prior to injection. GC analysis was conducted with the same instrumental setup and oven condition as outlined in Section 2.4. Flavor retention (%) by aqueous starch was calculated relative to water alone.

Flavor retention ( %) = (HS starch- HS water ) / HS water * 100

HS refers to the peak area of a flavor compound in the headspace. This calculation is valid based on the assumption that the partition coefficients of a flavor compound between gas and aqueous phases are not influenced by the presence of starch in the aqueous phase (see

Table A.2, Figure A.3 in Appendix). In the binary-flavor systems, flavor retention (%) was calculated relative to water added with two flavor compounds, instead of one flavor compound.

Figure 2.1 Starch-flavor mixture in a 20 mL headspace vial.

90 80

C) 70 o 60 50 40 30 20 Temperature ( Temperature 10 0 0 1 2 3 4 5 6

Time (hour)

Figure 2.2 The cooling profile of the starch-flavor mixture. Mean values were plotted, n=2.

2.7 Determination of precipitation yield. After storing for 1 day, 1 week or 1 month, the sample was centrifuged at 1000 x g for 10 min. The supernatant was collected and diluted

100 times with deionized water prior to performing the phenol-sulfuric acid assay. The total carbohydrate in the precipitate (as glucose content), was determined indirectly using the method of Dubios et al. (1956). 1 mL of supernatant was added to a 25 mL glass test tube. 1 mL of 5% phenol solution was added to the sample. After vortexing, 5 mL of concentrated sulfuric acid was added quickly and the sample mixture was vortexed immediately. The sample was cooled for 20 min at room temperature. Absorbance was read at 490nm using a spectrophotometer (UltraSpec 3000; Pharmacia Biotech; Cambridge, England). The amount of total carbohydrate was determined from a standard curve constructed using glucose standards in the range of 0-100 μg/mL. A correction factor of 0.9 was used to convert mass of glucose to mass of starch. The factor was calculated by dividing the molecular weight of glucose less one molecule of water (180-18) by the molecular weight of gluclose (180). The % precipitated starch yield is calculated as:

[Total carbohydrate of added starch – Total carbohydrate in supernatant]*100

Total carbohydrate of added starch

2.8 Wide angle X-ray diffractometry (XRD). The analyses were conducted using a desktop X-Ray diffractometer (MiniFlex II) and MDI Jade8 Software for data collection. The samples were equilibrated at water activity 0.58 prior to analysis. The samples were analyzed between 40 and 300 2θ at a step size of 0.02o, a scan speed of 2.0sec/step, at a tension of 40kV, and a current of 45mA.

2.9 Statistical analysis. Flavor retention values and precipitated starch yields were evaluated by three-way ANOVA. The effects of three factors (flavor compounds, time, and types of starch) on the response of flavor retention were tested. Significant effects were determined using Tukey’s pair-wise comparisons. The significance level was set a 0.05. Statistical analyses were performed using Minitab 13.0 (Minitab, Inc., State College, PA, USA).

3 References

Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., and Smith, F. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350-356.

Godet, M.C., Colonna, T.P., and Buleon, A. 1995. Inclusion/exclusion of fatty acids in amylose complexes as a function of the fatty acid chain length. Int. J. Macromol. 17:405-408.

Griffin, S., Wyllie, S.G., and Markham, J. 1999. Determination of octanol-water partition coefficient for terpenoids using reversed-phase high-performance liquid chromatography. J. Chromotogr. A. 864: 221-228.

Klucinec, J.D., and Thompson, D.B. 1999. Amylose and amylopectin interact in retrogradation of dispersed high-amylose starches. Cereal Chem. 76:282-291.

Tapanapunnitikul, O., Chaiseri, S., Peterson, D.G., and Thompson, D.B. 2008. Water solubility of flavor compounds influences formation of flavor inclusion complexes from dispersed high- amylose maize starch. J. Agric. Food Chem. 56:220-226.

Chapter 3

Retention of Flavor Compounds by Native Starch Dispersions

1 Single-flavor starch system

1.1 Results

1.1.1 Solubility and hydrophobicity of flavor compounds

Water solubility refers to the saturated concentration or maximum concentration of a solute that can be dissolved in water. In this study, the water solubility was determined by GC- headspace analysis as the concentration at the point which the headspace concentration achieved a maximum with increasing concentration. For example, as shown in Figure 3.1, the saturation concentrations of limonene and thymol were determined to be 45 and 1000 μL/L, respectively. The saturation concentrations and saturation graphs for the other 8 compounds can be found in Table 3.1 and Figure A.1 (Appendix), respectively.

Hydrophobicity values as determined by the logarithm of octanol-water partition coefficient (log Pow) are also shown for the ten model flavor compounds in Table 3.1. The compounds are listed in the order of increasing saturation concentration. In general, as the saturation concentration in water increased, the log P decreased (Figure 3.2). The relationship appeared not perfectly linear. For example, thymol and menthol have similar log Pow values but the former was twice as soluble in water.

1.1.2 Flavor retention by native starch dispersion

Figure 3.3 and Table 3.2 show the flavor retention (%) determined after one day for the ten compounds. The flavor retention of menthol (58%), menthone (57%) and thymol (29%) were significantly higher than the rest of the flavor compounds (<5%).

Figure 3.1 Determination of saturation concentration for A) limonene and, B) thymol, n=2.

Table 3.1 Saturation concentration, log Pow and amount of flavor added in starch (μL/L) for the ten flavor compounds.

a Mean values with standard deviation, n=3.

Figure 3.2 The relationship between hydrophobicity (log Pow) ( ) and saturation concentration (μL/L) ( ) for nine flavor compounds. Guaicol, having a log Pow of 1.39 and a saturation concentration of 18000 μL/L is not included in this figure due to the scale.

65% 55% 45% 35% 25% 15% Flavor retention (%) retention Flavor 5% -5%

Figure 3.3 Flavor retention (%) by native starch dispersion for ten flavor compounds. Mean values are expressed with standard errors, n=3.

1.1.3 Flavor retention by native starch dispersion over time

Since the preliminary F test (three-way ANOVA) showed a significant effect of flavor compounds and time but not starch type (Appendix Table A.3), only flavor compounds and time for native starch dispersion are shown in Table 3.2. For menthol and thymol, flavor retention

(%) did not increase significantly over one month (Figure 3.4, Table 3.2). The menthone retention increased significantly from 58% at one day to 72% at one month. When calculated as amount of bound flavor (μL), menthol, menthone and thymol also had higher values than the rest (Table 3.3).

No significant increase in flavor retention over time was observed for the remaining seven flavor compounds (Figure 3.4, Table 3.2). Due to the large standard deviation observed among these values, it is plausible to say that these values cannot be distinguished from “zero” (Table

3.2).

Despite low flavor retention (%), the amounts of bound flavor (μL) for carvone, pulegone, terpinen-4-ol and guaicaol were higher than that of limonene, cymene and anethole (Table 3.3).

1.2 Discussion

1.2.1 Measurement of hydrophobicity and solubility of flavor compounds on flavor retention by native starch dispersion

By far the most common technique to measure hydrophobicity have been obtained by the shake-flask method, whereby a solute is shaken between octanol and water until the equilibrium is obtained (Leo et al., 1971). In the literature, most log Pow data have been generated for the pharmaceutical industry and environmental chemistry (Sangster, 1997). The experimentally determined log Pow data for flavor compounds are not as common (Piraprez et al., 1998).

85%

75%

65%

55%

45%

35%

25%

15%

Flavor retention (%) retention Flavor 5%

-5%

-15%

Figure 3.4 Flavor retention (%) by native starch dispersion at one day ( ), one week ( ) or one month ( ). Mean values are expressed with standard errors, n=3.

Table 3.2 Flavor retention (% ) by native starch dispersion at one day, one week or one month. Flavor 1 day 1 week 1 month Compounds Flavor Retention a (%) Limonene 3 ± 7 Ca 6 ± 2 Ca 3 ± 15 Ca Cymene 3 ± 11 Ca 2 ± 6 Ca -5 ± 13 Ca Anethole 4 ± 3 Ca 0 ± 4 Ca 8 ± 6 Ca Menthol 58 ± 4 Aa 65 ± 4 Aa 72 ± 2 Aa Menthone 57 ± 2 Ab 67 ± 0 Ab 74 ± 2 Aa Thymol 29 ± 2 Ba 32 ± 0 Ba 48 ± 5 Ba Carvone 3 ± 3 Ca 9 ± 8 Ca 5 ± 4 Ca Pulegone 3 ± 1 Ca 4 ± 3 Ca 17 ± 4 Ca Terpinen-4-ol -2 ± 4 Ca 11 ± 2 Ca 10 ± 2 Ca Guaiacol 1 ± 6 Ca 6 ± 0 Ca 3 ± 2 Ca aMean values with standard deviations, n=3. Statistical analysis by three-way ANOVA (flavor compounds, starch type, time) was performed. Preliminary F test showed significant effect of flavor compounds and time on flavor retention, but not starch type. Only flavor compounds and time for native starch dispersion are shown in this table. Significance was determined using Tukey’s pair wise comparison. Means in the same column (across flavor compounds) with the same capital letter are not significantly different at P>0.05. Means in the same row (across time) with the same small letter are not significantly different at P>0.05.

Table 3.3 Bound flavor (μL/L) by native starch dispersion at one day, one week or one month. Flavor 1 day 1 week 1 month Compounds Bound flavor a (μL/L) Limonene 1 ± 3 2 ± 1 1 ± 6 Cymene 1 ± 6 1± 3 -2 ± 6 Anethole 2 ± 2 0 ± 2 5 ± 4 Menthol 209 ± 14 234 ± 13 259 ± 7 Menthone 228 ± 8 269 ± 2 295 ± 9 Thymol 246 ± 15 275 ± 4 406 ± 38 Carvone 28 ± 33 89 ± 73 50 ± 38 Pulegone 25 ± 14 42 ± 33 169 ± 35 Terpinen-4-ol -46 ± 99 284 ± 51 255 ± 54 Guaiacol 149 ± 1045 1096 ± 55 604 ± 271 aMean values with standard deviations, n=3. The units are expressed as amount of flavor compounds (μL) in native starch dispersion (L). 10 mL of native starch dispersion was used and the dispersion contained 0.7% (db) of starch. Bound flavor = Flavor retention(%)*Flavor concentration (μL/L). Flavor concentration was calculated as 80% of the saturated concentration for each flavor compound.

In the current study, the reported for Pow menthol, thymol and carvone were 2.73, 2.71, and 2.49 respectively, as compared to the values of 3.23, 3.30 and 2.52 in another study (Ruelle and Kesselring, 1998). The log Pow for limonene and menthone were 3.15 and 2.80 respectively in this study, and 4.35 and 2.83 in literature (Gunning et al., 2000). Despite the discrepancies among the log Pow values from different studies, the trend was similar within the same study.

The shake-flask method has been reported to be time consuming. It is also limited to compounds with a log Pow between -2 and 4 owing to the required precision and sensitivity of the analytical technique (Leo et al., 1971). The average standard deviation of log Pow has been reported to be 0.18; however when data of different workers were combined, the standard deviation increased to 0.7 (Shiu and Mackay, 1986). By measuring the log Pow values of the ten compounds in this study, the values were relatively meaningful, even if they might not be absolutely accurate. Inconsistencies in the literature values have been ascribed mainly to experimental error and techniques used. Changes in the experimental procedure with respect to shaking times, centrifugation steps to clarify emulsion, the ratio of octanol and water, or the presence of small amount of impurities, are expected to contribute to the variation in Pow values reported (Leo et al., 1971).

For convenience, many computer software models have been developed to estimate log

Pow (Sangster, 1997). These models are usually based on an additive-constitutive procedure which the contributions of various atoms, groups and structural information are summed up to estimate log Pow values (Sangster, 1997). In one of these methods, the Crippen log P, log P is calculated based on atom contribution methods. By a second method, Clog P, log P is calculated based on a fragmental analysis – in which a solute structure is dissected into chemically- meaningful fragments (Mannhold and Waterbeemd, 2001). Both Crippen log P and Clog P data can be obtained from CambridgeSoft ChemDraw Ultra 8.0 (Massachusetts, USA).

A comparison of experimental and predicted data is shown in Table 3.4. Experimental and predicted data are in agreement (<0.5 unit difference) except thymol and carvone. In general, the ordering from most hydrophobic to least hydrophobic was in agreement for the predicted and the experimental data.

Reasonable agreement was found between the solubility data in the current study and the literature. In the current study, the solubility for limonene, cymene, menthol, menthone, and thymol were 40, 50, 450, 500 and 1000 μL/L, respectively. In other studies, the solubility for these compounds was 11 (Jouenne and Crouzet, 2000), 51, 464, 560, and 690 μL/L, respectively

(Lun et al, 1997). The slight differences might be due to the different methods used to determine water solubility. In the literature, a direct method to measure solubility has been commonly used. In this method, a saturated solution of an excess amount of flavor compound and water were mixed and equilibrated. The aqueous phase is then injected to HPLC or GC to determine the flavor concentration (Phillippe et al., 2003). In current study, an indirect measurement of solubility was obtained using the static headspace method. This study is more rapid, and the amount of flavor required is appreciably lower than the mutual solubility method

(Tanemura et al., 1998).

1.2.2 The relationship of hydrophobicity and solubility of flavor compounds to their flavor retention by native starch dispersions

The relationship among flavor retention (%), hydrophobicity and saturation concentration in a dispersed starch-flavor model is summarized in Figure 3.5. Previous studies have attributed low flavor retention to either too low solubility or too low hydrophobicity

(Arvisenet et al., 2002, Tapanapunnitikul et al., 2008). Based on the data in Figure 3.5, my interpretation is that both the concentration of the flavor added to the sample and its

Table 3.4 Comparison of log P values by different methods. Experimental data Estimated data Flavor Octanol-water partition b b compounds a Crippen log P Clog P coefficient (log Pow) (R)-(+)-limonene 3.15 ± 0.07 3.01 4.35 p-cymene 3.50 ± 0.42 3.76 4.07 trans-anethole 2.95 ± 0.16 2.91 3.32 Menthol 2.73 ± 0.16 2.75 3.23 Menthone 2.80 ± 0.12 3.01 2.83 Thymol 2.71 ± 0.09 3.37 3.20 (L)-(-)-carvone 2.49 ± 0.08 1.75 2.01 Pulegone 2.33 ± 0.05 2.02 2.50 Terpinen-4-ol 2.30 ± 0.15 2.10 2.75 Guaiacol 1.39 ± 0.02 1.52 1.32 aExperimentally determined, mean values ± standard deviation, n=3. bData were obtained from CambridgeSoft ChemDraw Ultra 8.0 (Massacussettes, USA).

Figure 3.5 The relationship between flavor retention (%)(bars), hydrophobicity (log Pow ) ( ) and saturation concentration (μL/L) ( ).

47 hydrophobicity would influence the flavor retention. Menthol, menthone and thymol were shown to have significantly higher flavor retention compared to the other compounds. One similarity among these three retained compounds is that they have intermediate values of both concentration and hydrophobicity.

The model used in the current study was 0.7% aqueous starch dispersion. Starch molecules, particularly amylose, have the characteristic of random coil with conceivably, a few helical regions under these conditions (Ring et al., 1985). Upon flavor addition and cooling, formation of starch-flavor single helices can occur under favorable conditions. Based on my interpretation, one favorable condition would be that there is a sufficient amount of flavor compounds in the aqueous system. The second favorable condition would be that the flavor compounds possess sufficient hydrophobicity to overcome the unfavorable entropy of the random coil by inducing the formation of single helixes. Thus, the combination of these two conditions would yield significant flavor retention in our starch aqueous model system.

According to this interpretation, limonene, cymene, and anethole would have enough hydrophobicity to induce single-helix amylose. However, only a relatively small amount of these flavor compounds would be dissolved and in contact with starch molecules to induce formation of helices. As a consequence, flavor retention would be low. On the other hand, for flavor compounds with higher solubility (carvone, pulegone, terpinenol and guaiacol), there would be a sufficient amount with the aqueous starch, but they would not have enough hydrophobicity to interact with the hydrophobic region of starch. Therefore, flavor retention would be low.

Menthol, menthone and thymol, having sufficient concentration and hydrophobicity, would be superior at interacting with starch, resulting in higher retention than the rest.

To further investigate the effect of concentration on flavor retention, the flavor retention was measured as a function of flavor concentration for menthone and thymol (Figure

3.6). As menthone concentration increased from 0 towards 150μL/L, flavor retention measured was less than 5%. However at 200μL/L the flavor retention increased to approximately 20% and started to plateau. The same trend was also observed for the thymol. Figure 3.6 suggested that for each flavor compound, a certain flavor concentration is required to initiate an interaction between the flavor compound and starch molecules. Once the initiation started, the likelihood of more starch-flavor interaction is enhanced.

Figure 3.7 illustrates the binding curves for menthone and thymol in the native starch dispersion. The amounts of bound and free flavor (μL/L) were calculated from Table 3.5. The shapes of the curves (Figure 3.7) are similar with those reported for iodine binding to amylose

(Yamamoto, et al., 1982). Previous studies have also suggested the interaction between amylose with menthone was the same type as the interaction between amylose-iodine, which is the formation of an inclusion complex (Rutschmann et al., 1989). When the data of Figure 3.7 were plotted as Bjerrum plots (see Figure A.4 in Appendix), the values of log ([free flavor]y=90% / log

([free flavor]y=10% ) were 0.08 and 0.06 for menthone and thymol respectively. These values are far smaller than 1.91, suggesting that there is a strong positive cooperatively in menthone and thymol binding to starch (Chang et al., 1985). Based on this interpretation, the binding of the first menthone or thymol molecule to amylose will induce the configuration change of amylose from random to helix which might simplify the successive formation of complexes on the amylose chain.

In summary, starch-flavor interactions appear to be influenced by both concentration and hydrophobicity of the flavor compounds. The flavor retention for thymol and menthone was suggested to be due to an acceptable balance of sufficient hydrophobicity combined with sufficient concentration. For flavor compounds with practically no % retention, the reason was suggested to be either insufficient concentration or insufficient hydrophobicity.

65% Menthone A A 55% 45% 35% 25% 15%

Flavor retention (%) retention Flavor 5% -5% 0.0 1.0 2.0 3.0 4.0 -15% Flavor added (μL)

B B 65% Thymol 55% 45% 35% 25% 15%

Flavor retention retention (%) Flavor 5% -5% 0.0 2.0 4.0 6.0 8.0 -15% Flavor added (μL)

Figure 3.6 Flavor retention (%) as a function of flavor added (μL) for A) menthone and B) thymol. Mean values were plotted, n=2.

Thymol 260 Menthone 220 L/L) μ 180

140

100

Bound flavor ( flavor Bound 60

-20 0 200 400 600 Free flavor(μL/L)

Figure 3.7 Binding isotherms of menthone and thymol in starch. The data points were derived from Table 3.5. The units are expressed as amount of flavor compounds (μL) in starch dispersion (L). 10 mL of native starch dispersion was used and the dispersion contained 0.7% (db) of starch.

Table 3.5 Flavor added (μL), flavor concentration (μL/L), flavor retention (%), bound flavor (μL/L) and free flavor (μL/L) for menthone and thymol in native starch dispersion.

Menthone Bound d a Flavor c Free flavor Flavor concentration b flavor Flavor added (μL) retention (μL/L) (μL/L) (μL/L) 0.6 60 -2% -1 61 1.5 150 -6% -8 158 2.0 200 22% 44 156 3.0 300 47% 140 160 3.5 350 54% 190 160 4.0 400 57% 228 172 Thymol Bound Flavor concentration a Flavor flavor c Free flavor d Flavor added (μL) (μL/L) retention b (μL/L) (μL/L) 1.0 100 -6% -6 106 4.0 400 -10% -39 439 6.0 600 2% 13 587 6.5 650 20% 133 517 8.5 850 30% 251 599 7.5 750 30% 221 529 aThe units are expressed as amount of flavor compounds (μL) in starch dispersion (L). 10 mL of starch dispersion was used and the dispersion contained 0.7% (db) of starch. Calculated as 80% of the saturation concentration. bMean values, n=2. cBound flavor = Flavor retention*Flavor concentration aFree flavor = Flavor concentration – Bound flavor

2 The binary-flavor starch system

2.1 Results

2.1.2 Flavor retention by native starch dispersion

Figure 3.8 illustrates flavor retention by native starch dispersions in both single-flavor and binary-flavor systems. When a flavor compound with a high flavor retention, menthone or thymol, was mixed a compound of low flavor retention, limonene, in the single-flavor system

(Figure 3.8), limonene retention increased from <5% to about 40% and 70%, respectively.

Conversely, menthone and thymol retention decreased by 11-17% when added with limonene.

The effect of cymene in a binary-system was different from that of limonene. Cymene retention in the binary-flavor system did not change significantly in comparison to the single flavored system (Figure 3.8). Flavor retention for menthone and thymol decreased by about 17-

22% in the binary system containing cymene.

When menthone and thymol, both with high flavor retention values, were mixed together, no significant change in flavor retention was observed for either flavor compound compared the values in the single-flavor system (Figure 3.8). Similarly the flavor retention for all flavors in the binary-flavor system consisting of limonene-carvone and menthone-carvone remained unchanged compared to the values of those in the single-flavor system (Figure 3.8).

2.2 Discussion

2.2.1 Possible explanations of flavor retention by native starch dispersion

A significant increase of limonene retention (%) in a binary system in the presence of menthone or thymol indicated that both thymol and menthone enhanced the interaction between limonene and starch (Figure 3.8). This observation suggests the possibility of co- inclusion of both ligands, as shown in Figure 3.9 (Mechanism 1). The enhanced retention of

A A

* * B *

Figure 3.8 Flavor retention (%) by native starch dispersion in A) single-flavor system (data shown earlier in Figure3.3), B) binary-flavor system. Mean values are expressed with standard error, n=3. An asterisk (*) means the values are significantly different from those in the single- flavor system. The number in the bracket is flavor bound (μL/L) calculated by flavor retention (%)*flavor concentration.

Mechanism 1

Mechanism 2

Mechanism 3

Figure 3.9 Schematic of possible explanations to form menthone-starch-limonene complexes.

55 limonene in the presence of either menthone or thymol might be due to the presence of pre- formed single helices that are able to accommodate a limonene molecule when these molecules are otherwise in insufficient concentration to form stable single helices. Once incorporated into the helices, limonene might have a stronger binding affinity to starch than menthone. A limonene-starch complex was reported having lower flavor compound dissociation value (Kd)

(2.74 x 10-4 mol/L) than that of menthone (4.93 x 10-4mol/L) (Rutschmann and Solms, 1990). A similar suggestion of co-inclusion was also made by Rutschmann and Solms (1991) using a precipitation method. In a binary-flavor system containing equal concentrations of limonene and menthone, they found that menthone can stabilize the co-inclusion of limonene, resulting in a synergistic effect in which retention for both compounds increased.

A second possible mechanism may be that limonene might interact with menthone, and the formation of limonene-starch inclusion complexes might enhance the interaction between menthone and starch (Figure 3.9, Mechanism 2). Based on the conclusion from the study of the single-flavor system, limonene, having insufficient concentration in the aqueous starch system, would not be able to induce formation of helical structure to allow subsequent binding of menthone. Therefore, mechanism 2 is unlikely to be a plausible explanation for higher limonene retention by dispersed starch observed in the binary-flavor system.

The observed higher limonene retention in these binary flavor systems suggests that limonene molecules would be more stable in the aqueous menthone than in water alone through the change in the solvent quality. This interpretation is based on the observation that headspace concentration of limonene decreased in the aqueous menthone system as compared to water alone (data not shown). This would mean that limonene concentration in the aqueous system increased. Such phenomenon has been reported by Schober and Peterson (2004) in which L-menthol, a more hydrophilic compound enhances the solubility of a more hydrophobic

56 compound, 1,8-cineole. An increase concentration of limonene in the aqueous phase would enhance the interaction of limonene with menthone and starch, forming inclusion complexes

(Figure 3.9, Mechanism 3).

The menthone and thymol retention in the binary-flavor system remained similar as the single-flavor system, showing an additive effect of menthone and thymol retention in the binary-flavor system.

3 Conclusion

For single-flavor starch systems, a combination of sufficient hydrophobicity and sufficient solubility, leading to sufficient concentration, would generate high flavor retention observed among menthol, menthone and thymol. Flavor compounds with low retention would be due either to insufficient hydrophobicity or insufficient concentration.

In the binary-flavor starch system, the enhanced retention of limonene in the presence of either menthone or thymol would be due to the presence of pre-formed single helices that are able to accommodate a limonene molecule when these molecules are otherwise in insufficient concentration to generate stable single helices.

4 References

Arvisenet, G., Le Bail, P., Voilley, A., Cayot, N. 2002. Influence of physiochemical interactions between amylose and aroma compounds on the retention of aroma in food-like matrices. J. Agric. Food Chem. 50:7088-7093.

Chang, C.H., Chen, J.G., Govindjee, R., and Ebrey, T. 1985. Cation binding by bacteriorhodopsin. Proc. Natl. Academia Sci. 82:396-400.

Gunning, Y.M., Parker, R., Ring, S.G., Rigby, N.M., Wegg, B., Blake, A. 2000. Phase behavior and component partitioning in low water content amorphous carbohydrates and their potential impact on encapsulation of favors. J. Agric. Food Chem. 48:395-399.

Jouenne, E., Crouzet, J. 2000. Determination of apparent binding constants for aroma compounds with α- lactoglobulin by dynamic coupled column liquid chromatography. J. Agric. Food. Chem. 48:5396 5400.

Leo, A., Hansch, C., and Elkins, D. 1971. Partition Coefficients and their uses. Chem. reviews. 71(6):525- 616.

Lun, R., Varhanickova, D., Shiu, W.Y, Mackay, D. 1997. Aqueous solubilities and octanol-water partition coefficients of cymenes and chlorocymenes. J. Chem. Eng. Data. 42:951-953.

Mannhold, R., Waterbeemd, H. 2001. Substructure and whole molecule approaches for calculating log P Journal of Computer-Aided Molecular Design. 15:337–354.

Piraprez, G., Herent, M-F., Collin, S. 1998. Determination of the lipophilicity of aroma compounds by RP- HPLC. Flavor and Fragr. J. 13:400-408.

Ring, S.G., l’Anson, K.J., Morris, V.J. 1985. Static and dynamic light scattering studies of amylose solutions. Macromol. 18:182-188.

Ruelle, P., Kesselring, U. W. 1998. The hydrophobic effect 3: A key ingredient in predicting n-octanol- water partition coefficients. J. Pharma. Sci. 87:1015-1024.

Rutschmann, M.A., Solms, J. 1990. Formation of inclusion complexes of starch with different organic compounds. II studying of ligand binding in binary model systems with decanal, 1-napthol, monostrearate, monopalmitate. Lebensm-Wiss Technol. 23:70-79.

Rutschmann, M.A., Solms, J. 1991. Inclusion complexes of potato starch- a binding model with synergism and antagonism. Lebensm-Wiss Technol. 23:70-79.

Sangster, J. 1997. In Octanol-water partition coefficients: Fundamentals and physical chemistry. England: John Wiley & sons Ltd. England.113-124.

Schober, A.L., Peterson, D.G. 2004. Flavor release and perception in hard candy: Influence of flavor compound-compound interactions. J. Agric. Food Chem. 52:2623-2627.

Shiu, P W. Y., Mackay, D. 1986. A critical review of aqueous solubilities, vapor pressures, Henry's Law constants, and octanol-water partition coefficients of polychlorinated biphenyls. J. Phys. Chem. Ref. Data. 15:911-929.

Tanemura, I., Sarro, Y., Ueda, H., Sato, T. 1998. Solubility method using static head-space gas chromatography for determination of the stability constants of fragrance materials with 2- hydroxypropyl-β-cyclodextrin. Chem. Pharm. Bull. 46(3):540-542.

Yamamoto, M., Sano, T., Yasunaga, T. 1982. Interaction of amylose with iodine. 1. Characterization of cooperative binding isotherms for amyloses. Bull. of Chem. Society of Japan. 55:1886. 58

Chapter 4

Influence of Native Lipids on Flavor Retention and Precipitated Starch Yield

1 Effect of presence of native lipid on flavor retention

1.1 Results

As described earlier (pg 40), by statistical analysis using three-way ANOVA, the type of starch dispersion (native or lipid-free) had no significant overall effect on flavor retention (Figure

4.1, Table 3.2, Table A.2, Appendix Table A.3), whereas the type of flavor compound and time did have a significant effect on flavor retention.

1.2 Discussion

No significant difference in flavor retention for menthol, menthone and thymol between native starch and lipid-free starch dispersion at day one (Figure 4.1). These data differ from the report by Tapanapunnitikul et al. (2008) which suggested that the presence of lipid enhanced the retention of limonene and cymene, but decreased the retention of thymol molecules.

Analysis of a system containing varying proportion of menthone, monostearate (17:0) and potato starch showed that the binding of monostearate inhibited the inclusion of menthone

(Rutschmann and Solms, 1990). They suggested that in the presence of monostearate,

-5 formation of starch-menthone inclusion complex is less because Kd (4.27x10 ) for lipid complex

-4 is lower than menthone complexes (4.93x10 ). The Kd value for starch-glycerol monostearate complex would be similar to that of starch-lipid complexes in dispersed HAMS. The native lipids in HAMS consist that of starch-lipid complexes in dispersed HAMS. The native lipids in HAMS consist exclusively of free fatty acids (FFAs) and lysophospholipids, which both are the monoacyl lipids (Morrison, 1988). The major components of these monoacyl lipids are linoleic (18:2) and palmitic acid (16:0).

85% 75% 65% 55% 45% 35% 25% 15% Flavor retention (%) retention Flavor 5% -5% -15%

Figure 4.1 Flavor retention (%) for native ( )and lipid –free ( ) starch dispersions at day 1. Mean values are expressed with standard errors, n=3.

The discrepancy between the data from current study and data in the literature could be due to the different conditions in dispersing the starches. Tapanapunnitikul et al. (2008) dispersed HAMS in a pressure vessel in 200oC for 75 min in a convection oven and Rutschmann and Solms (1990) dispersed potato starch using sodium hydroxide. In the current study, HAMS was heated to 160oC for 30 min in a pressure vessel wrapped with an electric band heater.

Different techniques in dispersing starch might affect the molecular structure of starch or native lipids. In order to form inclusion complexes, a minimum length of the linear starch chain would be required to induce the helical structure. Perhaps some of the unsaturated fatty acids in the starch might be degraded thermally or oxidized and therefore account for different levels of ability for flavor compounds to interact with starch.

The other possible explanation for the discrepancy between the data could be the methods used to measure flavor retention values. Studies by Tapanapunnitikul et al. (2008) and

Rutschmann and Solms (1990) used a precipitation method, which required a large amount of flavor addition to induce formation of precipitate. In the current study, a headspace method using lower flavor concentration was used to measure flavor retention in both aqueous and precipitated starch.

2 Effect of presence of native lipids on precipitated starch yield over time without flavor addition

2.1 Results

The proportion of the initially dispersed starch that was recovered as precipitate is referred to as starch yield in the following figures and tables. By three-way ANOVA, there was a significant effect of type of starch dispersion, flavor compound, and time on starch yield

(Appendix Table A.4). Even without addition of flavor compound, some starch precipitated at one day (Figure 4.2, Table 4.1). Starch yield for native and lipid-free starch dispersion at one day

61 was less than 10%. At one week, lipid-free starch had a greater increase in starch yield (79%) than the lipid-free starch (57%). At one month, the starch yields for native and lipid-free starch were 86% and 97% respectively.

2.2 Discussion

The starch yield for dispersed native starch without flavor addition might result from either the precipitation of associated single-helical lipid-starch complexes or associated starch double helices (retrogradation) or both; while the starch yield for lipid-free starch would only result from precipitation of associated double helices (retrogradation). Without flavor compound addition, a higher starch yield was found in the lipid-free starch than native starch after one week or one month (Figure 4.2, Table 4.1). This suggests that, in the presence of native lipid, single helices of starch-monoacyl lipid complexes may form and therefore partly inhibit the formation of starch-starch double helices (retrogradation). In literature, it is well-known that monoacyl lipids (0.5%) can retard firming in bread (Boyle, 1997; Zeleznak and Hoseney, 1986).

However, the exact mechanism is unclear. The addition of monoacyl lipid would form an inclusion complex with amylose and therefore the amylose would not take part in the crystallization of starch double helices (Gudmundsson and Eliasson, 1990). It was also suggested that the formation of these amylose-lipid complexes would restrict the mobility of those starch chains that would otherwise be involved in forming double helices during retrogradation

(Hoover, 1995).

3 Effect of flavor addition to native starch dispersion on precipitated starch yield over time

3.1 Results

For native starch dispersions to which flavor compounds were added, at one day terpinen-4-ol had the highest starch yield (66%), followed by menthol (50%), thymol (43%) and

Figure 4.2 Starch yield (%)for naitve starch an lipid-free starch dispersion without flavor addition a one day ( ), one week ( ), one month ( ). Mean values are expressed with standard error, n=3.

Table 4.1 Starch yield (%) for native and lipid-free starch dispersions with and without flavor addition at one day, one week or one month. Flavor 1 day 1 week 1 month compounds Starch yielda for native starch dispersion(%) None 8.6 ± 5.9 DEc 57.4 ± 3.1 Cb 85.5 ± 0.5 Ba Limonene 5.1 ± 4.6 DFc 57.7 ± 2.2 Cb 86.3 ± 0.5 Ba Cymene 4.6 ± 2.0 EFc 57.0 ± 5.1 Cb 86.0 ± 0.5 Ba Anethole 2.3 ± 4.1 EFc 56.5 ± 11.3 BCb 86.6 ± 1.4 ABa Menthol 50.4 ± 3.2 Ba 54.3 ± 1.8 Ca 54.5 ± 1.1Da Menthone 35.1 ± 1.1 Cc 45.6 ± 1.4 Cb 64.8 ± 1.2 CDa Thymol 42.9 ± 1.3 BCa 51.8 ± 1.4 Ca 56.9 ± 3.0 Da Carvone 3.4 ± 2.1 EFc 34.6 ± 1.2 Db 81.6 ± 2.9 Ba Pulegone 13.4 ± 1.5 Dc 23.5 ± 0.6 Eb 54.1 ± 0.9 Da Terpinen-4-ol 66.1 ± 2.1 Aa 67.4 ± 1.9 Ba 67.7 ± 0.5 Ca Guaiacol -1.5 ± 4.3 Fa 4.7 ± 2.9 Fa 8.1 ± 2.2 Ea Starch yielda for lipid-free starch dispersion(%) None 5.8 ± 4.6 DFc 78.6 ± 3.5 Ab 96.5 ± 0.5 Aa Limonene -2.9 ± 3.3 EFc 79.4 ± 4.0 Ab 93.5 ± 1.4 Aa Cymene 0.7 ± 2.5 Fc 80.0 ± 4.4 Ab 93.7 ± 1.7 Aa Anethole -2.8 ± 3.9 EFc 80.8 ± 0.9 Ab 92.7 ± 1.0 Aa Menthol 47.0 ± 0.9 Bb 52.3 ± 1.1 Cab 60.9 ± 1.4 CDa Menthone 36.5 ± 1.1 Cb 49.7 ± 2.5 Cb 65.6 ± 1.8 CDa Thymol 49.3 ± 3.4 Bb 54.4 ± 0.8 Cb 61.4 ± 1.2 Ca arvone -3.5 ± 5.8 EFc 53.6± 1.1 Cb 91.9 ± 1.9 Aa Pulegone 12.8 ± 0.6 Dc 34.6 ± 3.5 Db 70.1 ± 2.4 Ca Terpinen-4-ol 66.7 ± 1.8 Aa 66.3 ± 1.4 Ba 67.7 ± 1.0 Ca Guaiacol 3.5 ± 3.6 DFa 1.6 ± 1.8 Fa 0.4 ± 1.0 Ea aMean values with standard deviations, n=3. Statistical analysis by three-way ANOVA (flavor compounds, starch type, time) was performed. Significance was determined using Tukey’s pair wise comparison. Means in the same column (across flavor compounds) with the same capital letter are not significantly different at P>0.05. Means in the same row (across time) with the same small letter are not significantly different at P>0.05. “None” refers to starch dispersion without flavor addition.

95.0% 85.0% 75.0% 65.0% 55.0% 45.0% 35.0%

Starch yield yield (%) Starch 25.0% 15.0% 5.0% -5.0% -15.0%

Figure 4.3 Starch yield (%) for native starch dispersion with and without flavor addition at one day ( ), one week ( ), or one month ( ). “None” refers to starch dispersion without flavor addition. Mean values are expressed with standard deviation, n=3.

65 menthone (35%) (Figure 4.3, Table 4.1). For the remaining flavor compounds, at one day, the starch yields were <14% and they were not significantly different from each other.

Figure 4.3 and Table 4.1 show that, over time, the addition of limonene, cymene or anethole had no effect on starch yield relative to the control (without flavor compound addition). For these three flavor compounds, the starch yield increased from less than 5% for one day to about 57% for one week, and to about 86% for one month. For menthol, menthone and thymol, the starch yield for one day was higher (35% to 50%). It increased either relatively slowly or not at all over one month. For carvone, pulegone, terpinen-4-ol and guaiacol, the changes in starch yield for each flavor compound varied tremendously (Figure 4.3, Table 4.1).

X-ray diffractograms of precipitates formed from mixture of a flavor compound and a dispersion native starch at one week are shown in Table 4.2. Samples at one week, instead of those at one day, were chosen for X-ray analysis because insufficient precipitate was formed at one day for most of mixtures of flavor compound and native starch dispersion. Lipid-free starch precipitate was not analyzed because no significant difference was observed in flavor retention between lipid-free and native starch dispersion. The precipitates formed with limonene, cymene, anethole and carvone showed the same XRD pattern as those formed without flavor compound addition. The XRD pattern shows reflection at 5, 17, 22 and 23 2θ, which are characteristics of a

B-pattern (Zobel, 1988). The precipitates formed with menthol, menthone, thymol, pulegone and terpinen-4-ol showed V type XRD patterns. The reflections at 7.1, 12.9, 17.8 2θ are the characteristics of V pattern (Tozuka et al., 2006).

Table 4.2 The flavor bound (μL/L), flavor retention (%) and starch yield (%) determined from the mixture of a flavor compound and native starch dispersion at one week are shown alongside with the XRD diffractogram. Flavor Flavor Flavor Starch XRD Retentiona Boundb Compounds Yielda (%) (%) (μL/L)

None - 57.4 ± 3.1 - Limonene 6 ± 2 57.7 ± 2.2 2 Cymene 2 ± 6 57.0 ± 5.1 1 Anethole 0 ± 4 56.5 ± 11.3 0 Menthol 65 ± 4 54.3 ± 1.8 234 Menthone 67 ± 0 45.6 ± 1.4 269

Thymol 32 ± 0 51.8 ± 1.4 275 Carvone 9 ± 8 34.6 ± 1.2 89 Pulegone 4 ± 3 23.5 ± 0.6 42

11 ± 2 67.4 ± 1.9 284 Terpinenol

aMean values with standard deviations, n=3. bThe units are expressed as amount of flavor compounds (μL) in starch dispersion (L). 10 mL of starch dispersion was used and the dispersion contained 0.7% (db) of starch. Bound flavor = Flavor retention(%)*Flavor concentration (μL/L). Flavor concentration is 80% of the saturated concentration for each flavor compound. “None” refers to starch dispersion without flavor addition. The starch-guaiacol complexes were not analyzed due to insufficient amount of precipitate formed.

3.2 Discussion

Precipitated starch could be due to one or more of three different mechanisms of precipitation: 1) formation of double helices which may associate and precipitate

(retrogradation) 2) single- helical starch-monoacyl lipid complexes which may form and then associate, leading to precipitation 3) single helical starch-flavor complexes which may form and associate, leading to precipitation.

Except for limonene, cymene and anethole, starch yields for other 7 compounds appear to be different from the starch dispersion without flavor addition (Figure 4.3, Table 4.2). Since

<5% of flavor retention (and very small amount of flavor bound) was observed for limonene, cymene or anethole, no or very little starch-flavor single helices were formed, and hence the changes in starch yield were similar to that of the starch dispersion without flavor compound addition. To explore whether the precipitate resulted from association of double helices or single helices, XRD analysis was conducted on the precipitate held at one week (Table 4.2).

Precipitates that formed without flavor compound addition, or with the addition limonene, cymene, anethole and carvone, showed a weak B type XRD pattern. This provides evidence that these precipitates were formed primary by association of double helices.

In a similar study by Tapanapunnitikul et al. (2008), precipitates formed with native starch without flavor compound or with limonene and cymene at one day showed a V type XRD pattern due to the formation of starch-lipid single helices. In the current study, precipitates held at one week were analyzed and showed only a weak B type pattern (Table 4.2). The difference in

XRD pattern is probably mainly due to the storage time. After one week, a large increase in starch yield from <10% to 57% was observed for native starch dispersions formed without flavor compound addition, or formed with the addition of limonene, cymene, and anethole (Figure 4.3,

Table 4.2). This suggests that retrogradation has taken place to a large extent, and might therefore have quantitatively overshadowed any V pattern that might be present.

The high flavor retention (about 29-74%) for menthol, menthone and thymol corresponds to high starch yields, even at one day (Figure 3.4, Figure 4.3). The mutually high values probably resulted primarily from the precipitation of associated starch-flavor complexes, as shown by a V type X-ray XRD pattern (Table 4.2). The formation and association of these starch-flavor single helices reduces the association of double helices, shown by the 20-30% lower starch yields for menthol, menthone and thymol at one month, compared to the control.

Precipitate formed with terpinen-4-ol also showed a V pattern, despite its low flavor retention (11%) (Table 4.2). Since terpinen-4-ol was added into starch dispersion at higher concentration than the other flavor compounds, the amount of bound terpinen-4-ol was relatively high (284 μL/L), similar to the amount of bound menthol, menthone and thymol (234,

269, 275 μL/L respectively). The V pattern suggests that a considerable amount of single helical starch-terpinen-4-ol complexes was formed.

A V pattern was also observed for pulegone, but not for carvone, even though the amount of bound pulegone (42 μL/L) was less than that of bound carvone (89 μL/L). This observation suggests that carvone molecules might be retained by the dispersed starch as soluble single helical inclusion complexes that do not associate and lead to precipitation. A second possible explanation would be that the carvone molecules were retained between the double helices of starch since the precipitate formed was B type. A third possible explanation would be that these single helical starch-carvone complexes were not packed tightly enough to give a X-ray pattern.

Conversely, the amount of bound pulegone (42 μL/L), though less than that of bound carvone (Table 4.2), would exist in single helices which associate readily, form precipitate and pack tightly to give a V pattern.

4 Effect of flavor addition to lipid-free starch dispersion on precipitated starch yield over time

4.1 Results

The starch yields for limonene, cymene and anethole at one day, one week and a month were <5%, 80% and 90% respectively (Table 4.1, Figure 4.4). These values were similar to the starch yield for the lipid-free starch dispersion without flavor addition. For menthol, menthone, thymol and terpinen-4-ol, the starch yield at one day was higher (35% to 65%) than the other flavor compounds. It increased slowly or not at all over one month. The changes in starch yield for carvone, pulegone, and guaiacol varied (Figure 4.4).

4.2 Discussion

Little or no flavor retention was observed for lipid-free starch dispersion with added limonene, cymene or anethole (Figure A.2, Table A.1 in Appendix). Therefore, the changes in the starch yield for these three compounds were similar to that of the lipid-free starch dispersion without flavor compound addition (Table 4.1, Figure 4.4). The high starch yield at one week and one month probably resulted primarily from the precipitation of associated starch double helices.

The mutually high values in flavor retention and starch yield for menthol, menthone and thymol suggest that the starch yield probably resulted primarily from the precipitation of associated starch-flavor complexes (Table 4.1, Figure 4.4).

95.0%

85.0%

75.0%

65.0%

55.0%

45.0%

35.0%

25.0% Starch yield yield (%) Starch 15.0%

5.0%

-5.0%

-15.0%

Figure 4.4 Starch yield (%) for lipid-free starch dispersion with and without flavor addition at one day ( ), one week ( ), or one month ( ). “None” refers to starch dispersion without flavor addition. Mean values are expressed with standard deviation, n=3.

In general, the changes in starch yield for flavor compounds with high flavor retention

(%) (menthol, menthone, thymol) were smaller than the changes in starch yield for the other flavor compounds (Table 4.1). Based on this interpretation, the presence of native lipid would affect the formation of double helices more than the formation of starch-flavor single helices.

5 Conclusion

Based on the current study, compared to lipid-free starches, the presence of native lipid did not affect flavor retention for any of the tested flavor compounds in an aqueous starch dispersion system.

The high starch yield observed among flavor compounds with high flavor retention could result from the precipitation of the associated starch-flavor single helices that gave a V pattern. The high starch yield observed among flavor compounds with low flavor retention (%) and low amount of flavor bound could result from the precipitation of associated double helices that gave a B pattern. However, since the flavor compounds were added into the starch dispersion in varied concentrations, a V pattern precipitate could also be obtained if the amount of flavor bound to starch was high, despite low calculated flavor retention (%). The generation of V pattern would depend on whether the single helices can associate to form a precipitate and the packing of these helices.

The formation of single helices, either from starch-flavor or starch-lipid complexes, would reduce the formation of double helices over time.

6 Reference

Boyle, E. 1997. Monoglycerides in food systems: Current and future uses. Food Technol. 51(8)52-59.

Godet, M.C., Bizot, H., Buleon, A. 1995. Crystallization of amylose-fatty acid complexes prepared with different amylose chain lengths. Carbohydrate Polymers. 27:47-52.

Gudmundsson, M., Eliasson, A.C. 1990. Retrogradation of amylopectin and the effects of amylose and added surfactants/emulsifiers. Carbohydrate Polymers. 13:295-315.

Hoover, R. 1995. Starch retrogradation. Food Reviews International. 11(2):331-346.

Morrison, W.R. 1988. Lipids in cereal starches: a review. J. of Cereal Sci. 8:1–15.

Rutschmann, M.A., Solms, J. 1990. The formation of ternary inclusion complexes of starch with menthone and monostearate – a possible food model system. Lebensm-Wiss Technol. 23:451-456.

Tozuka,Y., Takeshita, A., Nagae, A., Wongmekiat, A., Moribe, K., Oguchi, T., Yamamoto, K. 2006. Specific Inclusion Mode of Guest Compounds in the Amylose Complex Analyzed by Solid State NMR Spectroscopy. Chem. Pharm. Bull. 54(8):1097-1101.

Zeleznak, K.J., Hoseney, R.C. 1986. The role of water in the retrogradation of wheat starch gels and bread crumb. Cereal Chem. 63(5):407-411

Zobel, H.F. 1988. Starch crystals transformations and their industrial importance. Starch/Staerke .40:1–7.

Chapter 5

Suggested Future Works

One limitation for this current study is that the low flavor retention values (<15%) could not be distinguished by the static headspace method used. Other methods or adjustments should be explored to overcome this limitation.

Flavor retention in the current study is calculated based on the assumption that the partition coefficient of a flavor compound in a system containing 0.7% starch is the same as that in a system containing water. Therefore, only the flavor concentration in headspace was measured to calculate relative flavor retention. In future work, the gas-aqueous partition coefficients for both the starch and water system should be measured for more accurate calculation. This could be done by measuring the flavor concentration in both gas and aqueous phases.

The conclusion that hydrophobicity and solubility affect flavor retention is based on the study of the ten selected individual cyclic flavor compounds. It would be useful to know if the same conclusion can be applied to not just cyclic, but linear cyclic compounds.

The mechanism of how limonene retention was enhanced by other flavor compounds in a binary-flavor system should be further explored. The study of binary-flavor systems might also be expanded to a multiple-flavor system to understand the mechanisms involved in the selective retention of flavor compounds by starch.

Flavor retention by starch dispersions can have both positive and negative implications.

During food preparation and processing, as well as in the area of flavor encapsulation technology, maximal flavor retention and optimal flavor stability are highly desirable. On the other hand, in order for a flavor to be perceived, flavor that is retained in the food has to be released during food consumption. Only an appropriate combination of retention and release at 74 the point of consumption can provide desired sensory properties. The focus of the current research was to study the retention of flavor compounds in a starch system. An additional study of flavor release might provide a more complete understanding in starch-flavor interaction that is more closely related to flavor perception. For example, to simulate conditions found in the month, enzymatic break down of starch-flavor complexes by salivary α-amylase using the dynamic headspace method might be used to study flavor release from the starch system.

Appendix

Figure A.1 Graphs to determine saturation concentration for flavor compounds.

80%

70%

60%

50%

40%

30%

20%

10% Flavor retention (%) retention Flavor 0%

-10%

-20%

-30%

Figure A.2 Flavor retention (%) by lipid-free starch dispersion at one day ( ), one week, ( ) or one month ( ). Mean values are expressed with standard errors, n=3.

Table A.1 Flavor retention (%) by lipid-free starch dispersion at one day, one week and one month. Flavor 1 day 1 week 1 month Compounds Flavor Retention a (%) Limonene 1.0 ± 8 Ca -12 ± 14 Ca -10 ± 15 Ca Cymene -5 ± 5 Ca -4 ± 9 Ca -2 ± 15 Ca Anethole 1.0 ± 1 Ca 0 ± 6 Ca 9 ± 3 Ca Menthol 66 ± 2 Aa 66 ± 2 Aa 79 ± 2 Aa Menthone 63 ± 3 Ab 72 ± 1 Ab 76 ± 3 Aa Thymol 31 ± 2 Ba 34 ± 2 Ba 40 ± 0 Ba Carvone 4 ± 3 Ca 7 ± 4 Ca 6 ± 2 Ca Pulegone 0 ± 1 Ca 14 ± 2 Ca 11 ± 5 Ca Terpinen-4-ol 2 ± 1 Ca 12 ± 5 Ca 4 ± 3 Ca Guaiacol 1 ± 1 Ca 2 ± 5 Ca -5 ± 6 Ca aMean values with standard deviations, n=3. Statistical analysis by three-way ANOVA (flavor compounds, starch type, time) was performed. Preliminary F test showed significant effect of flavor compounds and time on flavor retention, but not starch type. Only flavor compounds and time for lipid-free starch dispersion are shown in this table. Significance was determined using Tukey’s pair wise comparison. Means in the same column (across flavor compounds) with the same capital letter are not significantly different at P>0.05. Means in the same row (across time) with the same small letter are not significantly different at P>0.05.

Table A.2 Peak area of flavor compound in headspace measured by GC in a water system and an aqueous dispersed starch system, and flavor retention (%). Flavor Water Starch Dispersion Flavor a Compounds GC Peak Area a Retention (%) Limonene 1.15 x 108 ± 4.22 x 106 1.12 x 108 ± 8.89 x 106 3 ± 7 Cymene 1.20 x 108 ± 2.55 x 107 1.15 x 108 ± 1.38 x 107 3 ± 11 Anethole 3.43 x 106 ± 2.92 x 105 3.30 x 106 ± 2.04 x 105 4 ± 3 Menthol 3.33 x 106 ± 1.79 x 105 1.39 x 106 ± 8.26 x 104 58 ± 4 Menthone 1.35 x 107 ± 3.22 x 105 5.82 x 106 ± 2.26 x 105 57 ± 2 Thymol 1.85 x 106 ± 1.04 x 105 1.31 x 106 ± 1.05 x 105 29 ± 2 Carvone 4.08 x 106 ± 4.37 x 105 3.96 x 106 ± 4.14 x 105 3 ± 3 Pulegone 7.05 x 106 ± 1.04 x 105 6.87 x 106 ± 1.62 x 105 3 ± 1 Terpinen-4-ol 9.63 x 106 ± 4.95 x 105 9.81 x 106 ± 5.95 x 105 -2 ± 4 Guaiacol 7.51 x 106 ± 6.30 x 104 7.15 x 106 ± 4.77 x 105 1 ± 6 aMean values with standard deviations, n=3.

Sample calculation of flavor retention in dispersed native starch, R (%):

R (%) = (HS starch- HS water )* 100

HS water

Limonene retention (%):

(1.11 x 108 - 1.10 x 108)* 100 = 0.01 x 108 *100 = 1 %

8 8 (one of the three 1.11 x 10 1.11 x 10 replicate analysis)

Thymol retention (%):

(1.96 x 106 - 1.44 x 106) * 100 = 0.52 x 106 *100 = 26 % (one of the three 1.96 x 106 1.96 x 106 replicate analysis)

Figure A.3 Sample calculation of flavor retention (%) by dispersed native starch relative to water.

100

80 Thymol 60 Menthone

20 Relative bound flavor (%)Relative flavor bound

0 1.70 1.90 2.10 2.30 2.50 2.70 2.90 -20 log [free flavor]

Figure A.4 Bjerrum plots for the binding of menthone and thymol.

Table A.3 ANOVA output for showing the effects of three different factors: starch type1, flavor compound2, time3) on the response (flavor retention).

Source DF F p Flavor compound 9 403.51 0.000 Time 2 14.1 0.000 Starch type 1 2.13 0.147 Flavor compound*time 18 3.31 0.000 Flavor compound*starch type 9 3.41 0.001 Time*starch type 2 1.22 0.299 Flavor compound*time*starch type 18 1.05 0.414 Error 114 Total 173 The output was generated using the General Linear Model (full model) from Minitab. 1native and defatted starch 2limonene, cymene, anethole, menthol, menthone, thymol, carvone, pulegone, terpinenol, guaiacol 3one day, one week and one month

Table A.4 ANOVA output for showing the effects of three different factors: starch type1, flavor compound2, time3) on the response (starch yield).

Source DF F p Flavor compound 10 635.52 0.000 Time 2 6153.74 0.000 Starch type 1 201.37 0.000 Flavor compound*time 20 350.76 0.000 Flavor compound*starch type 10 159.98 0.000 Time*starch type 2 105.54 0.000 Flavor compound*time*starch type 20 47.26 0.000 Error 125 Total 190 The output was generated using the General Linear Model (full model) from Minitab. 1native and defatted starch 2no flavor, limonene, cymene, anethole, menthol, menthone, thymol, carvone, pulegone, terpinenol, guaiacol 3one day, one week and one month