The Effect of Ph, Temperature, and Food Additives on Tomato Product

The Effect of Temperature, pH, and Food additives on Tomato Product Volatile Levels

Thesis

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

Pakanat Patana-anake

Graduate Program in Food Science and Technology

The Ohio State University

2014

Graduate Committee:

Prof. Sheryl Barringer, Advisor

Prof. Luis Rodriguez-Saona

Asst. Prof. Farnaz Maleky

Pakanat Patana-anake

2014

Abstract

In processed tomato products, temperature, NaCl, protein, sucrose, and oil are varied due to consumer consumption conditions and preference while pectin is used as a thickener and pH may be adjusted to keep tomato products out of the low acid food category. The goal of this study was to determine how temperature, pH, and food additives affect the headspace volatile concentration and consumer acceptability of the aroma of flavored tomato products. Temperature (5, 25, 50 °C), pH (2.5, 4.3, 8.5), 1% pectin, 1% whey, collagen or milk protein, NaCl (5, 10%) sucrose (5, 10%), and oil (5,

10%) were varied in tomato juice, as a model for flavored tomato sauces, to determine the effect on volatile levels. The headspace concentrations of different tomato juice samples were measured by selected ion flow tube-mass spectrometry and sensory evaluation was conducted with fifty untrained consumers. Temperature produced the greatest increase, followed by the addition of NaCl. pH and pectin produced no significant difference, while protein, sucrose, and oil decreased volatile levels. 10% Oil changed the order of the odor activity values of important tomato volatiles while the rest did not, which can change the aroma perceived by the consumer. Sensory testing showed that NaCl, control, and sucrose had the highest aroma intensity and consumer preference followed by pectin and milk protein and finally oil. The higher the volatile concentration the stronger the consumer preference.

Vita

2012…………………………………B.S. Food Science, Kasetsart University

2012 to present……………………....M.S. Food Science, Ohio State University

Fields of study

Major Field: Food Science and Technology

iii

Table of Contents Abstract ...... ii

Vita...... iii

List of Tables ...... vii

List of Figures ...... viii

Chapter 1- Literature Review ...... 1

1.2 Flavor chemistry of tomato volatiles ...... 1

1.2.1 Lipoxygenase and alcohol dehydrogenase ...... 1

1.2.2 Other enzymatic, chemical reaction and processing induced volatiles ...... 4

1.2.3 Odor active volatiles ...... 5

1.3 Flavor retention in food matrix ...... 9

1.3.1 Partition coefficient and Henry’s law constant phenomenon ...... 10

1.4.1 Non-volatile compounds ...... 13

1.4.1.1 Salt ...... 13

1.4.1.2 Sugar ...... 16

1.4.1.3 Protein ...... 21

1.4.2 Oil ...... 24

1.5 The involvement of pH with volatiles formation and release ...... 26 iv

1.6 The influence of pectin on flavor intensity and release ...... 28

Chapter 2- The effect of temperature, pH, and food additives on tomato product volatile levels ...... 31

Practical Application ...... 31

Introduction ...... 31

2.1 Materials and Methods ...... 34

2.1.1 Sample preparation ...... 34

2.1.1.1 Temperature and pH ...... 34

2.1.1.2 Sodium chloride, pectin, protein, sucrose, and oil ...... 34

2.1.2 Measurement of volatile compound concentration of different samples ...... 35

2.1.3 Viscosity ...... 36

2.1.4 Sensory evaluation ...... 37

2.1.5 Statistical analysis and odor active value calculation ...... 37

Chapter 2.3- Results and Discussion ...... 38

2.3.1 Temperature ...... 38

2.3.2 NaCl ...... 45

2.3.3 pH ...... 51

2.3.4 Pectin ...... 54

2.3.5 Protein ...... 56

2.3.6 Sucrose ...... 57

2.3.7 Oil ...... 58

2.3.8 Sensory ...... 59

2.4 Conclusions ...... 60

References ...... 62

Appendix: Figures and Tables ...... 74

List of Tables

Table 1 Compounds previous identified in tomatoes ...... 8

Table 2 Important volatiles in tomatoes and their water/air threshold ...... 9

Table 3 The effect of temperature (5, 25, and 50 °C) on tomato juice headspace volatile concentration (ppb) ...... 43

Table 4 The effect of NaCl (5% and 10%), sucrose (5% and 10%), oil (5% and 10%), and 1% protein (whey protein isolate (WPI), milk protein isolate (MPI), and collagen) on tomato juice headspace volatile concentration (ppb) ...... 49

Table 5 The effect of pH (2.5, 4.26, and 8.5) and 1% pectin on tomato juice headspace volatile concentration (ppb) ...... 52

Table 6 Average ranking score for aroma intensity and consumer preference ...... 60

Table 7 Kinetics parameters for SIFT-MS analysis of selected volatile compounds in tomato juice

...... 75

Table 8 The effect of 1% pectin and 1%oil on tomato juice viscosity (Pa-s) ...... 77

Table 9 The effect of 10% NaCl and sucrose on tomato juice volatile (percent increase) ...... 77

vii

List of Figures

Figure 1 Lipoxygnease pathway illustrated by Stone and others (1975) ...... 3

Figure 2 The effect of temperature on odor activity value of volatiles important to tomato aroma

...... 42

Figure 3 The effect of NaCl, pectin, milk protein isolate (MPI), sucrose, and oil on volatile important to tomato aroma ...... 48

Figure 4 The % increase correlation of volatile compounds between 10% NaCl and 10% Sucrose

...... 79

Figure 5 The effect of NaCl on important volatiles in tomato juice ...... 80

Figure 6 The effect of NaCl on alcohols and ketones in tomato juice ...... 81

Figure 7 The effect of NaCl on aldehydes in tomato juice ...... 82

Figure 8 The effect of temperature on the important tomato volatile levels ...... 83

Figure 9 The effect of temperature on the important tomato volatile levels ...... 84

Figure 10 The effect of protein on (E)-2-hexenal, (Z)-3-hexenal, hexanal, and (Z)-3-hexen-1-ol level ...... 85

Figure 11 The effect of protein on (E)-2-pentenal, furfural, 1-penten-3-one, acetaldehyde, benzaldehyde, and 6-methyl-5-hepten-2-one ...... 86

Figure 12 The effect of sucrose (E)-2-hexenal, (Z)-3-hexenal, hexanal, and (Z)-3-hexen-1-ol level

...... 87

viii

Figure 13 The effect of sucrose on 6-methyl-5-hepten-2-one, acetaldehyde, 2-isobutylthiazole, benzaldehyde, phenylacetaldehyde, furfural and (E)-2-pentenal levels ...... 88

Figure 14 The effect of oil on (E)-2-hexenal, (Z)-3-hexenal, hexanal, (Z)-3-hexen-1-ol and levels

...... 89

Figure 15 The effect of oil on 6-methyl-5-hepten-2-one, furfural, acetaldehyde, benzaldehyde, 2- isobutylthiazole, 1-penten-3-one, and (E)-2-pentenal levels ...... 90

Chapter 1- Literature Review

1.2 Flavor chemistry of tomato volatiles

Many volatile compounds have been found in tomato. This includes aldehydes, alcohols, ketones, acids, and terpene-related compounds (Kaseniac and Hall 1970). Most of the volatiles come from enzymatic and chemical reaction in tomato.

1.2.1 Lipoxygenase and alcohol dehydrogenase

Lipoxygenase is oxidative enzyme in tomato which has an important role in the formation of volatile compounds. The substrates of lipoxygenase are linoleic and linolenic acids which are the result of acyl hydrolase enzymatic reaction breaking down the acryl lipids in tomato (Xu and Barringer 2009). Firstly, lipoxygenase oxidized the free fatty acid to hydroperoxides which convert to volatile compounds. (Z)-3-hexenal,

(E)-2-hexenal, and hexenal were reported by Kazeniac and Hall (1970) as the important

C6 aldehydes which mainly associate with fresh green note of tomato. The formation of

C6 aldehyde depends on the fatty acid precursor of lipoxygenase

The formation of aldehyde and alcohol in tomato from linolenic and linoleic has been discussed (Stone and others 1975). The researchers explain that (Z)-3-hexenal is a predominant volatile compound from the degradation of linolenic acid and then it isomerized to (E)-2-hexenal while the high amounts of hexanal were converted from linoleic acid (Figure 1). The isomerized enzyme has lower rate of reaction which explains by the formation of (E)-2-hexenal is much slower than (Z)-3-hexenal (Stone and others 1975). Moreover, (Z)-3-hexenol was formed from cis-3-hexenal by alcohol dehydrogenase, (E)-2-hexenol was formed form (E)-2-hexenal, and hexanol was formed from both (Z)-3-hexenal and (E)-2-hexenal but mainly from hexanal. Although hexanal was mainly degraded from linoleic acid, the result in this study show that small amount of (Z)-3-hexenal is degrade to hexanal; it is possible that the higher content of hexanal from the degradation of linoleic acid inhabit the formation of hexanal from (Z)-3-hexenal

(Stone and others 1975).

Figure 1 Lipoxygnease pathway illustrated by Stone and others (1975)

Furthermore, other than (Z)-3-hexenal, (E)-2-hexenal, and hexanal, lipid related volatiles which produce from an alternative pathway of lipoxygenase has been reported

(Xu and Barringer 2009). The researchers explain that (E)-2-pentenal may produce from the enzymatic cleavage of 12-hydorperoxides of linoleic acid and there is a possibility that (E)-2-octenal decompose from 10-hydroperoxides of linoleic acid. Finally, both 1- penten-3-one and (E)-2-heptenal may produce from the degradation of 13- hydroperoxides of linolenic acid (Xu and Barringer 2009). 3

Additionally, the factors affect the formation of C6 aldehyde were emphasized

(Kazeniac and Hall 1970). The researchers state that oxygen level is very critical for the formation of C6 aldehyde. The formation of (Z)-3-hexenal, (E)-2-hexenal, and hexanal was inhibited in the absence of oxygen. Moreover, there is a good possibility that at high or low temperature, heat or cooling inhibit the enzymatic activity as no formation of C6 aldehyde was occurred (Kazeniac and Hall 1970). The researchers also explain that the rate of cell rapture and the maturity stage also affect the formation of C6 aldehyde. Fully rapture tomato will increase the rate of reaction between air and tomato particles which show the higher amount of (E)-2-hexenal. Finally, firm fully ripe tomato contains the highest concentration of (Z)-3-hexenal, (E)-2-hexenal, and hexenal.

1.2.2 Other enzymatic, chemical reaction and processing induced volatiles

Although the important volatiles associates with tomato flavor are produced in lipoxygenase and alcohol dehydrogenase pathways, many volatiles produce from the chemical or the unknown enzymatic reaction, and the processing has been reported such as many volatiles associates with “cooked” flavor are developed by heat processing

(Kazeniac and Hall 1970). The researchers present that 6-methyl-5-hepten-2-one and acetone are products of the degradation of lycopene oxidation during heating; benzaldehyde may produce from mandelic acid or phenylglyoxylic acid; phenylacetaldehyde produced from phenylalanine; methanol produced from the 4

demethylation of pectin substance; 3-methylmercaptopropanal is a product of the degradation of methionine. Moreover, the volatile compounds which produced by chemical reaction has also been reported. The chemical reaction of valine and luecine may produce 2-methylpropanal and 3-methylpropanal respectively; β-ionone is a product of the oxidative degradation of carotenoid. In addition, linalool, furfural and α-terpineol were found in heated tomato as well as dimethyl sulfide (Buttery and others 1971).

Linalool is the precursor of 2-methyl-3-buten-2-ol (Kazeniac and Hall 1970).

1.2.3 Odor active volatiles

Many volatiles have been identified in tomato but not many volatiles contribute to tomato aroma and flavor. Some example compounds previous identified (Table 1), important volatiles in tomatoes (Table 2), and their water/air threshold in tomato is provided (Buttery and others 1971). Many studies report that lipoxygenase pathway products such as (Z)-3-hexenal, (E) -2-hexenal and hexanal associate with “fresh” and

“green” note of tomato (Kazeniac and Hall 1970, Buttery and others 1971, Xu and

Barringer 2009, Goodman and Barringer 2002, Stone and others 1975). Due to low water and air threshold (Table 2), these compounds are considered to be very important volatiles for tomato aroma. (Z)-3-hexenal give desirable aroma which effective in the range of 0.3-0.5ppm in tomato juice while the concentration of 1 ppm or higher of this compound create strong “green” rancid type flavor which is undesirable. (E)-2-hexenal

is similar to (Z)-3-hexenal but it has been described as “less intense” than (Z)-3-hexenal

(Kazeniac and Hall 1970). The desirable level of (E)-2-hexenal is between 0.5-2 ppm in canned juice and 3-10 ppm in homogenates tomato; higher than these levels it will give rancid note which is objectionable. Hexanal also give desirable “green type flavor” in the range of 0.1-0.5 ppm; higher than these levels, it will give rancid vegetable fat off flavor.

Furthermore, lipid related volatiles which produce from an alternative pathway of lipoxygenase has shown to be contributed to tomato aroma due to low threshold. (E)-2- pentenal which produces from the enzymatic cleavage of 12-hydorperoxides of linoleic acid also shows “fresh green” notes with low water and air odor threshold (Kazeniac and

Hall 1970). However, it contains only small amount in tomato volatile and the intensity is far less than (Z)-3-hexenal and (E)-2-hexenal. (Z)-3-hexenol was formed from (Z)-3- hexenal by alcohol dehydrogenase and it contributes to “green” note. Hexanol forms from (Z)-3-hexenal, (E)-2-hexenal and, hexanal. It gives “green leafy” aroma as well as decrease in “tomato like” note at the concentration of 0.3 ppm.

Moreover, enzymatic, chemical reaction and processing induced volatiles such as

6-methyl-5-hepten-2-one, linalool, dimethyl sulfide, furfural, and acetaldehyde are very important to canned and processed tomato products since they contribute to “cooked” flavor (Kazeniac and Hall 1970). 6-Methyl-5-hepten-2-one is products of the degradation of lycopene during heating which has low water and air threshold. It gives “cooked stewed tomato flavor” and “heated pasted note” at concentration of 0.75 ppm.

Phenylacetaldehyde, furfural, 3-methylmercaptopropanal and acetaldehyde have been reported to give “cooked” flavor to heated tomato juice. Dimethyl sulfide has very low 6

water threshold which is more noticeable than other volatiles (Kazeniac and Hall 1970).

It gives “unique cook flavor” note depending on it concentration which is considered to be off-flavor in canned tomato and the intensity of this compound quite strong which decreases the desirable “green” notes in heated tomato juice. In addition, methyl salicylate gives “canned tomato” note due to very high concentration in tomato; 2- isobutylthaizole give typical green note and 2,4-decadienal produces acidic type note of tomato juice. (E)-2-octenal decompose from 10-hydroperoxides of linoleic acid and it produced cardboard type flavor at the range of 0.1 ppm.

Table 1 Compounds previous identified in tomatoes

ALIPHATIC COMPOUNDS Alkadienals Esters Phenols (E,E)-2,4- Methyl Alkanal decadienal Ethyl acetate salicylate (E,Z)-2,6- Acetaldehyde nonadienal Butyl acetate Phenol Propanal Pentyl acetate Guaiacol Butanal Free acids Hex3-enyl acetate Eugenol 2-Methylbutanal Acetic Methyl hexanoate 3-Methylbutanal Propanoic TERPENOIDS Pentanal Pentanoic AROMATIC AND Glyoxal 2-Methylbutyric HETEROCYCLIC Hydrocarbon Hexanal COMPOUNDS Limonene Heptanal Alkenols Myrcene 1-Penten-3-one Aldehydes Alkenals (Z)-3-Hexen-1-ol Benzaldehyde Alcohols Pent-2-enal 1-Hexanol Cinnamaldehyde Linalool Pent-3-enal Phenylacetaldehyde (E)-2-Hexenal Sulfur compounds Aldehydes (Z)-3-Hexenal Hydrogen sulfide Heterocyclic Citral Hept-2-enal Dimethyl disulfide Furfural Dimethyl sulfide 2-Isobutythiazole Ketones 6-methyl-5- Acetals 2-Ethylfuran hepten-2-one 1-Ethoxy-1- isopentoxyethane Ketones β-ionone 1-Ethoxy-1-pentoxyethane Acetone Alcohols 1,1-Dipropoxyethane 2-Butanone Benzyl alcohol 2-Pentanone 2-Phenylethanol Alkanols 3-Pentanone Methanol 2,3-butanedione Hydrocarbons Ethanol Benzene Propanol Others Toluene Propa-2-nol Butylnitrile Butanol Decane Others Pentanol Undecane Phenylacetonitrile Hexanol 2-Methylpropanol 2-Methylbutanol 3-Methylbutanol (Buttery and others 1971)

Table 2 Important volatiles in tomatoes and their water/air threshold

Air Odor Threshold Water Odor Threshold (mg/m3) (mg/kg)

(E)-2-Hexenal 0.79000 0.11 (Z)-3-Hexenal 0.00120 0.00012 Hexanal 0.23000 0.005 (Z)-3-Hexen-1-ol 0.01300 0.0039 (E)-2-Pentenal 1.40000 0.98 2-Isobutylthiazole N/A 0.029 6-Methyl-5-hepten-2-one 0.30000 0.068 Acetaldehyde 0.00270 0.0251 Eugenol 0.20000 0.006 Methyl salicylate 0.02300 0.04 Benzaldehyde 0.10000 0.75089 2-Pentanone 0.35000 1.38 1-Penten-3-one 0.00100 0.023 (E)-2-Heptenal 2.40000 0.05 (E)-2-Octenal 0.25000 0.003 (E)-2-Pentenal 1.40000 0.98 (E,E)-2,4-Decadienal 0.00230 0.000077 Guaiacol 0.00150 0.00048 Phenylacetaldehyde 0.00170 0.0063 Dimethyl sulfide N/A 0.003 Linalool 0.00240 0.006 Furfural 0.25 0.282 (Kazeniac and Hall 1970, Van Gemert 2011)

1.3 Flavor retention in food matrix

Aroma compounds can be released from food depending on many parameters such as intrinsic characteristics of an aroma compound such as shape, molecular size, and

functional groups, thermodynamic properties such as vapor pressure, solubility, activity coefficient, partition coefficient (Henry’s law constant), and kinetic parameters such as mass transfer coefficient (Voilley and Etiévant 2006). The volatility of aroma compounds is affect not only by these factors but by interaction in food matrix (Voilley and Etiévant 2006). Because of the heterogeneity of food matrix, the resistance to mass transfer, which can be described by diffusion coefficient and mass transfer coefficient), arises by the interaction between food component such as protein, pectin, oil, and etc. thus, aroma compounds need some driving force (boiling point, partial and vapor pressure) to overcome the resistance to mass transfer to release from the food matrix.

Therefore, these are some connection between intrinsic characteristics and thermodynamic parameters of an aroma compound. For example, the affinity of an aroma compounds to solution (or food) is described by activity coefficient (Martínez and others 2012). The activity coefficient decreases or increases depending on the intrinsic characteristic of an aroma compound and this change the solubility thus, affecting the partition coefficient. The following section will discuss briefly the partition coefficient and the interaction between food additives and aroma compounds.

1.3.1 Partition coefficient and Henry’s law constant phenomenon

Partition coefficient is thermodynamic parameter, which explains the partitioning of aroma compounds between air and liquid phase. The definition of the partition coefficient was well-described (Bakierowska and Trzeszczynski 2004). It can be depicted as a ratio of the concentration in the liquid phase to the concentration in the 10

headspace or the inverse. The partition coefficient is described by K = CL/CG; where K is a water/gas partition coefficient, CL and CG are compound concentrations in the liquid and gas phases, respectively. The partition coefficient also can be defined in the inverse dimensionless Henry’s law constant, H = CG/CL (1) (Bakierowska and Trzeszczynski

3 −1 2004). However, Henry’s constant (atm- m -mol ) is often depicted as Hp = RT/K (2); where K is the water/gas partition coefficient value, T is the system temperature (K), R is the gas constant equaling 0.08206 × 10−3 (m3-atm-mol−1-K−1) (Bakierowska and

Trzeszczynski 2004).

The alternative Henry’s law equilibrium is depicted as H= Pi/xi, (3) where Pi is the partial pressure of the volatile compounds in gas phase and xi is the mole fraction of the

∞ sat volatile compounds in aqueous phase or it can also be depicted as H = γi P (4) where

∞ sat γi is acitivity coefficient of the volatile compound, i, at infinite dilution in water and P is the vapor pressure of the pure compound i (Klooster and others 2005). The

∞ sat combination of (3) and (4) results in an equation (5), Pi = xi γi P and finally, partition

coefficient (K) can be related with partial pressure (Pi) by

(6) where Mi is the molecular mass of the volatile, is the

mass fraction of the volatile in solution, P is the total pressure in the system, is the density of the aqueous phase, and Vg is the molar volume of the gas (Klooster and others

2005). 11

The temperature dependent aroma partition coefficient can also explain with

Clausius-Clapeyron equations, which can explain an approximate temperature dependence of the air-solution partition coefficient (Klooster and others 2005);

where Ki,ref is the partition coefficient at Tref (reference temperature (K)), R is the gas constant, and enthalpy of hydration is decribed by Tref and ΔHhydr (Klooster and others

2005). Previous studies of odor and flavor perception in a flavored model system have shown that increasing temperature increased perceived flavor intensity (Ventanas and others 2010a). The odor intensity of cheese soup was stronger at 63 °C than at 33 °C, and the odor and flavor intensity of carrot, meat patty, and mashed potato increased as serving temperature increased from 25 to 65 °C (Kähkönen and others 1995; Ryynanen and others 2001). In addition, the partition coefficient value changes according to the composition of the mineral solutes such as salt and sugar in the water and the temperature of the system (Bakierowska and Trzeszczynski 2004).

1.4 Factor affecting the volatility of aroma compounds from food matrix

1.4.1 Non-volatile compounds

1.4.1.1 Salt

Sodium is an element in many natural substances such as plants, animal, and human which is essential to maintain the human body’s fluid and electrolyte balance, acid-base balance, muscle contractions, and nerve transmission. However, the excesses of sodium content in human body may increase blood pressure and also weaken the bones by promoting calcium excretion. Consequently, many food products have been designed for an additional way to circumvent this problem by formulating the low-salt content products. Although the low-salt products will promote consumer health, there are many evidences showing that the reduction of salt affects flavor of food products which is considered to be one of the most important factors in consumer preference and the food quality. The effect of NaCl, umami compounds, and serving temperature on meat broth system was discussed (Ventanas and others 2010a). In this study, the presence of salt increased volatility but the presence of proteins, the presence of polysaccharides and the presence of lipids degraded the volatility of aroma compounds. The addition of salt

(0.5% NaCl) increased the intensity of overall flavor, broth-like flavor, and saltiness in all samples.

Moreover, the effect of salt reduction on the volatile compounds and sensory evaluation of a vegetable soup have been reported (Mitchell and others 2011). In this study, the commercial regular and low salt vegetable soups, which contained limonene, p-cymene, terpinolene, β-caryophyllene, α-patchoulene, dimethyl sulfide, isopropyl disulfide, 3,3-dimethyl hexane, propanol-1, and hexanal were usedLimonene, p-cymene, terpinolene, β-caryophyllene, α-patchoulene, dimethyl sulfide, isopropyl disulfide, 3,3- dimethyl hexane, propanol-1, and hexanal, were used (Mitchell and others 2011). The salt reduction result in lower concentration of terpene, β-caryophyllen, and p-cymene.

Terpinolene and hexanal showed a slightly higher, but not significant, concentration in regular salt soup while isopropyl disulfide showed significantly higher (Mitchell and others 2011). Only α-patchoulene showed almost double concentrations in low salt soup.

The sensory data shows propanol-1, hexanal, limonene, p-cymene, isopropyl disulfide, and β-caryophyllen have a strong correlation with overall flavor, aftertaste, yellow color, salt flavor, overall flavor complexity, and carrot aroma (Mitchell and others 2011).

These attributes were found in regular salt soup and lower in the low salt soup. α- patchoulene which was found in low salt soup, it shows negative correlations on sensory attributes which were found in regular salt soup (Mitchell and others 2011). It appears that α-patchoulene may associate with off-aroma of the soup. In conclusion, the salt reduction will result in lower concentration of the most volatile compounds being release into headspace of the soup samples thus, lowering the sensory attributes and increasing off-aroma attributes.

Furthermore, the effect of salt addition on gas chromatography (GC) measurement and volatile compounds concentration of apple juice in headspace were explained (Poll and Flink 1984). The addition of various salts to liquid food systems can increase the concentration of components in the headspace. However, the increases in headspace concentrations can be different for different compounds when salt is added.

In this study, alcohols had the highest sensitivity for the addition of salt than aldehydes and esters; aldehydes had higher sensitivity than esters. Moreover, the sensitivity on salt additions appeared to depend on carbon chain length of a compound which, the volatility of compounds increases as chain length increases (Poll and Flink 1984). In addition, this study assumes that salt had a greater effect on headspace at lower temperature (20°C) when compares with higher temperature (40°C). Furthermore, the sensory evaluation results show that the salt addition increases aroma intensity and off-aroma, which increased as the increase of alcohols levels in the soup samples (Poll and Flink 1984).

Ultimately, the salting out effect involves increasing mobility and releasing of flavor compounds induce by salt binds water molecules with strong dipole interaction and the formation of hydration shells during soluble in solute thus, the depleted availability of water molecules for flavor compounds to soluble in water (Ventanas and others 2010a,

Mitchell and others 2011). The affinity of water for sodium and chloride ions are stronger than volatiles ions thus, the decreased in water activity while the initial volatiles concentration increased resulting in an increase in dynamic flavor release. The explanation of the salting out effect was also described (Bakierowska and Trzeszczynski

2004). The researchers discussed the effect of NaCl salt on partition coefficient of

organic volatile compounds and method for the determination of partition coefficient.

The researcher states that the partition coefficient increased as salt concentration increased (0-3.5%). The salting effect can be decribed by NaCl salting coefficient (ks), log(KW/KR) = ksμ; where KW is the partition coefficient in redistilled water, KR is the partition coefficient in NaCl solution. The ionic strength, μ, can be calculated from Lewis

and Randall’s equation , and ( ) (Bakierowska

and Trzeszczynski 2004).

1.4.1.2 Sugar

Sugar also very important ingredient and additive. It has been used in many kinds of food products and influences many characteristics such as texture, viscosity, sweetness, color, and microbial activity (Hansson and others 2001b). However, many studies show that the addition of sugar may affect the partition coefficient of aroma volatiles.

Generally, Henry’s law equation (5) and (6) can be used to describe the effect of sugar on the partition coefficient of volatiles. Sugar increases the partition coefficient of volatiles because it increase activity coefficient of the volatiles in solution (Klooster and others 2005, Voilley and others 1977) and it also increases mole fraction of volatile

(Klooster and others 2005). Piccone and others (2012) discuss the effect of sugar on liquid-vapour partition of volatile compounds in coffee beverages. The researchers found

that the addition of sugar led to a change in headspace concentration of important volatiles contain in coffee such as a significantly increase in furan compounds (2-methyl furan, 2-furfuryl furan) and furfuryl methyl sulfide and significant reduction in pyrazine type compounds (Piccone and others 2012). There is a possibility that sugar may affect the partition coefficient of polar compound than less polar since it diminish free water, this refers to salting-out effect. However, this effect may depend on sugar type since each sugar has different in water binding capacity (Piccone and others 2012). Sucrose shows significantly increased while glucose and fructose decreased the volatile levels

(Piccone and others 2012). The researchers conclude that coffee is very complex mixture containing many non-volatile compounds such as carbohydrate, protein which may interact with each other so the addition of sugar may cause only minor change compare with other interaction occurred in coffee beverage (Piccone and others 2012).

Moreover, the experiment on the effect of sugar on flavor release from soft drink shows that the addition of sucrose increased the concentration of isopenthyl acetate, ethyl hexanoate, linalool, L-menthone, and (Z)-3-hexenyl acetate while limonene showed no significant difference because sucrose interacts with free water, this increases the concentration of flavor compounds in remain free water so non-polar compounds not affect by addition of sugar since they are not interact with free water (Hansson and others

2001b). The researchers conclude that the increased of the volatile concentrations is depended on water binding capacity of sugar. Invert sugar has more water binding capacity than sucrose and glucose syrup has less water binding capacity because it contain less water binding site (Hansson and others 2001b).

In contrast, the addition of sugar may involve with salting-in effect. The addition of sucrose, glucose, and fructose decreased volatile levels in some literature (Piccone and others 2012, Hansson and others 2001b). Glucose, with its polar characteristic, can change the structure of water around the non-polar substance, this induces intra- molecular non-polar environment for hydrophobic molecules thus, improving the solubility of hydrophobic molecules (Copolovici and Niinemets 2007). The interactions between the co-solute and water and co-solute and the non-polar molecules affect the overall effect of co-solutes on the solubility of non-polar molecules. The polar co-solutes can reduce the polarity of water. Additionally, the presence of hydrophobic regions in the co-solute molecule affects the interaction with non-polar solute. The concentration of co-solute, polarity, and molecular configuration of co-solute and solute are very important for which co-solutes can increase or decrease the solubility (Copolovici and

Niinemets 2007). The interaction of glucose and water molecules is caused by the hydroxyl groups which result in an increased of hydrogen bonds in solution; the solubility of both substrate and volatiles increase in water which result in lower release of volatiles (Covarrubias-Cervantes and others 2004).

Sucrose can also reduce the volatility of aroma compounds in the headspace. This may due to sucrose-water interaction and sucrose-sucrose interaction (Starzak and others

2000, Richardson and others 1987, Roberts and others 1996). Hydrogen bonding between sucrose and water creates hydrophobic region thus, decreasing water activity and water mobility. The water mobility deceased as the concentration of sucrose increased from 0-40% because an increase in viscosity of solution (Richardson and others 1987).

Also, a linear increase of viscosity (from 0.00067 to 0.0012 Pa-s) has been reported from

0 to 20% sucrose in model solution, thus decreasing mass transfer of volatile compounds in sucrose solution (Nahon and others 2000). Viscosity may play an important role. The volatility of ethyl hexanoate decreased in 57.5% sucrose solution than in water because sucrose solution had higher viscosity compare to water thus, reducing the mass transfer of aroma compound to the headspace (Covarrubias-Cervantes and others 2004). In addition, the addition of invert sugar produced the highest volatile levels followed by sucrose, and glucose syrup in soft drink system (Hansson and others 2001b). The researchers conclude that the increased of the volatile concentrations is depended on water binding capacity of sugar or depicted as the average hydration number, n¯ (Starzak and others 2000). The average hydration number defined as the average number of molecules of hydration water attached to a single molecule of sucrose.

Sucrose and water interaction involves with hydrogen bonding of hydroxyl group of both molecules. Sucrose has eight hydroxyl groups (Starzak and others 2000). “These include three, which have intra-molecular bonding with ring oxygen atom, and five are involved with inter-molecular bonding” (Starzak and others 2000). However, the exact binding sites of these hydroxyl groups on sucrose molecules with oxygen atoms on water molecules are still unclear because sucrose has more than one formations in solution, normal, fold, and spherical, and different hydroxyl groups have different affinity to water molecules (Starzak and others 2000).

The addition of sucrose also decreases amorphous free volume (Roberts and others 1996). For small molecules in a fully amorphous system, the molecules mobility 19

has been related to the amorphous free volume (Roberts and others 1996). The decrease in amorphous free volume affects the movement of aroma compounds thus, decreasing mass transfer and volatility of aroma compounds.

Sucrose-sucrose interaction can be occurred in high concentration and the probability of sucrose-sucrose collisions can be increased thus, creating a higher degree of structural order of the solution (Starzak and others 2000). Pair interactions of sucrose could form a hydrophobic region because sucrose has a α-glycosidic linkage, which moves to form a hydrophobic region with the non-polar aroma compounds molecules

(Roberts and others 1996). Aroma compounds are also hampered to move in the free water phase due to the decreased distances between hydrated sucrose molecules and sucrose oligomers (Rabe and others 2003b).

Finally, the polarity of aroma compounds also affects sucrose/flavor interaction.

The higher polar aroma compounds showed an increasing volatility but non-polar aroma compounds showed a decrease (Roberts and others 1996). In model solution, the volatility of the polar compounds, acetone, ethyl acetate, 2-propanol, ethanol and polar acetates increased as the sucrose or glucose concentration was increased (Roberts and others 1996). Ketones decreased in volatility in a sucrose solution as the carbon number increased. In contrast, the headspace of non-polar compounds, 2-heptanone, 2-heptanal,

β-ionone, and nonpolar limonene decreased in volatility with added sucrose (Roberts and others 1996).

1.4.1.3 Protein

Tomatoes contain a good source of vitamin. However, the total protein amount in tomato is only 1g per 90g of fresh, chopped or sliced tomatoes, thus diet food made by tomatoes includes tomato sauce will lacking of protein (Thakur and Singh 1996).

Moreover, proteins are used as a fat replacer for low-fat food due to their properties and are commonly added to tomato sauce. Since this study focuses on the effect of protein on tomato volatiles, the understanding of protein physicochemical which contributes to flavor binding is essential.

Generally, primary and secondary binding sites of protein can interact with aroma compounds by many interactions and the protein-flavor binding depending on aroma compounds and type of proteins. Because of protein has many primary and secondary binding site, the interaction between exact specific site and aroma compound still remain unclear; only possible interaction has been found in the studied of many proteins and aroma compounds. The interactions between soy protein and aroma compounds were described (Macleod and others 1988). The authors explain that aroma compounds were bound to soy protein isolate or denature soy protein due to hydrophobic interaction on their specific hydrophobic region. The affinity of aroma compounds to protein was depended on hydrophobic nature of aroma compounds and structure of protein. Soy protein isolate bound to aroma compounds less than soy mostly concentrate because the binding ability of carbohydrate, lipid components and protein fractions. The reaction of volatiles and protein may involve free amino acid groups on the protein and carbonyl

group in aldehyde or hydroxyl group in alcohol (Macleod and others 1988). For an example, the Schiff bases are induced by carbonyl group of aldehyde react with free amino acid, the carbonyl group of aldehydes react with the primary amine, usually lysine by Michael addition, the double bond of alkenal react with the imidazole ring of histidine, and the reaction of alkenal with lysine-containing peptides produced pyridinium derivatives (Macleod and others 1988 and Meynier and other 2004). The reversible or irreversible bindings were depended on the polarity, carbon chain length, the ligand of volatiles, ionic strength, pH, and temperature of the system (Macleod and others 1988).

Additionally, the longer carbon chain length increased van der Waals interactions, thus more blinding of volatiles to the protein. The authors also present an protein-flavor binding equation;

ῡ = KC,

where K is a measure of the binding affinity of the ligand for the protein, C is the molar concentration of free ligand at equilibrium, and ῡ is the average number of moles of ligand bound per mole of protein (Macleod and others 1988). From this the equation which applied to many studies on the interaction between soy protein and aroma compounds, it can be conclude that the strongest affinity to soy protein is aldehydes, followed by ketones, alcohols, but acid shows no affinity for binding (Macleod and others 1988). It is possible that the competitive interaction between alcohols and water was higher than alcohol and proteins. However, in low water activity system, the affinity of volatile compounds to protein is different. Alcohols were strongly bound to protein than aldehydes and ketones due to competitive interaction between alcohol and water was 22

not found. In higher temperature system, heat changed protein structure (denature) because heat unfolded the soy protein molecules, which increased nonpolar regions and decreased in the polar regions (Macleod and others 1988). The author conclude that in low water activity system, alcohols were strongly bound to protein than aldehydes because aldehydes, ketones, and methyl esters interacted with protein by van der Waals forces and only one hydrogen bond while alcohol interacted by two hydrogen bonds and van der Waals forces.

Moreover, the interactions between milk proteins such as β-lactoglobulin, α- lactalbumin, bovine serum albumin, whey, and caseins with volatile compounds have been studies (Khun and others 2006, Thanh and others 1992, Hansen 1997, Landy and others 1995, Mottram and others 1996, and Meynier and other 2004). Comparing to other proteins, β-lactoglobulin has used to described the interaction between protein and aroma compounds because of its known structure and properties (Khun and others 2006).

Many research show that β-lactoglobulin has the hydrophobic interaction with alkanes, ketones, aldehyde, lactones, and esters (Khun and others 2006). The primary binding sites of β-lactoglobulin were suspected to be central hydrophobic cavity and a groove near outer surface of the protein. Hydrophobic interaction played an important role but the binding affinity may decrease with the introduction of polar group or increase with addition of non-polar group. Consequently, bovine serum albumin also interacts with volatile compounds by many binding sites with both hydrophobic and electrostatic force

(Khun and others 2006).

Although many critical reviews and articles discuss the protein flavor binding, most of the previous studies usually develop by using model system which may not explains in all food systems especially, tomato products. The knowledge of flavor binding in tomatoes system is still limited. Only the sensory and the quality test on the effect of added soy protein to tomato sauce was presented (Thakur and Singh 1996). The results show that the concentration of soy protein between 0.25-1 percent is acceptable while the acceptability is gradually decreased when protein concentration increased.

Moreover, the quality such as viscosity and serum separation is increased. However, the studies by Thakur and Singh (1996) did not present the volatile analysis which shows the effect of protein on tomato volatiles. Ultimately, the experiment which illustrates the association between volatile compound levels and protein is needed.

1.4.2 Oil

Oil is liquid fat at room temperature and semi-solid at low temperature depending on fatty acid composition and the position of fatty acid on glycerol backbone. More importantly, the non-polar characteristic of oil is induced by long chain unsaturated fatty acid. Since some food products contain water in the system such as soup, ice cream, sausages, sauces, and juices; adding oil will affect the water activity of the system, viscosity, and induce the chemical interaction between compounds. The effect of fat content on the temporal changes of flavour and texture in cooked bologna type sausages

was well-reported (Ventanas and others 2010b). The researchers state that the decreased in flavor intensity of 1-octen-3-ol, which is hydrophobic, with higher fat content due to solubility in fat content (oil droplet), thus diminishing their release. Moreover, the oil/air partition coefficient has also been reported (Sato and Nakajima 1979, and Kaneko and others 1994). The researcher state that oil/air partition coefficient of alcohol and ketone are lower than water/air partition coefficient while acetate esters show the inverse. This implies that more hydrophilic compounds were retained in oil more than in water. This also supported by a study, which discusses the temporal release of flavor compounds from low-fat and high-fat ice cream during eating (Chung and others 2003). The researchers state that fat also reduce the flavor intensity of lipophilic compounds such as hexanal, benzaldehyde. Moreover, the decrease of fat content diminished garlic and pepper flavor intensity in salad dressing (Guinard and others 2002). It is possible that that the resistance to mass transfer of volatile compounds in fat and oil is higher than in water. The hydrophobic aroma compounds are trapped in oil droplet thus, the release of aroma compounds is more complex because aroma compounds need to be released from many phases, lipid and aqueous, to the headspace (Jo and Ahn 1999, Guinard and others

2002). This results in less volatility of hydrophobic aroma compounds in fat containing system than in water system (Jo and Ahn 1999, Guinard and others 2002). For hydrophilic aroma compounds, the volatility might decrease due to an increase in viscosity caused by the addition of oil.

However, some researchers report that fat reduced both hydrophilic and hydrophobic volatiles levels. The concentration of alcohols, aldehydes, and ketones were

decreased by increased the lipid content (Jo and Ahn 1999). There is a possibility that fat increase viscosity, thus inhibit flavor release. Undoubtedly, fat content affects the volatiles concentration of food which depends on the chemical of volatiles and the nature of the food system but the understanding of fat content to tomato product is also limited; with only the sensory was done on the effect of fat, protein, and mineral tomato soup quality but the researchers did not mention the correlation between the volatiles profile and those treatments (Rosett and others 1997).

1.5 The involvement of pH with volatiles formation and release

Many studies have been reported on the effect of pH on volatiles formation, and flavor perception. The formation of volatile compounds in heated garlic including allyl alcohol, diallyl trisulfide(DATS), dially tetrasulfide (DATTS), diallyl disulfide (DADS), diallyl pentasulfide (DAPS), and other heterocyclic sulfur compounds was affected by pH (Kim and others 2009). The heating garlic at 120°C, allyl alcohol is formed by thermal degradation of alliin and alliin continue decomposing to diallyl polysulfides.

Then, diallyl polysulfides transform to heterocyclic sulfur compounds. pH has an important role in volatile compounds formation, when lowering the pH (pH 2), allyl alcohol was formed right from the start of heating with very high concentration because an increased in the thermal degradation of alliin (Kim and others 2009). However, it was unstable and started decreasing after 45 minutes, diallyl polysulfides were formed earlier

by decomposition of alliin which later transformed to heterocyclic sulfur compounds but at higher pH condition (pH 6).

The similar purpose on the effect of pH on flavor formation has been studied on shiitake mushroom (Chen and others 1984). The formation of eight-carbon compounds and sulfurous compounds in shiitake were affected by different pH conditions. The concentration of the eight –carbon compounds, 3-octanone, 1-octen-3-ol, 1-octanol, and

2-octen-1-ol reached the highest concentration at around pH 5-5.5 and the sulfurous compounds reached at pH 7. In addition, 1-octen-3-ol had highest concentration of all the eight-carbon compounds. The formation of eight-carbon compounds may be involved with the enzymatic reaction of linoleic acid and the optimum pH for this reaction was

5.0-5.5, dimethyl disulfide and dimethyl trisulfide had high concentration among the other sulfurous compounds (Chen and others 1984). Lenthionine was reported as the characteristic compound of shiitake, it was not found due to this compound unstable when pH was more than 5.0 and it may degradation to dimethyl disulfide and dimethyl trisulfide.

The effect of changes in pH on the release of flavor compounds form a soft drink related model system has been studied (Hansson and others 2001a). The variation in pH significantly affected volatile concentration in headspace. The addition of 0.002 g citric acid (pH 4) increased ethyl hexanoate, isopentyl acetate, limonene, and (Z)-3-hexenyl acetate headspace concentration compared to sample without citric acid (pH 5) (Hansson and others 2001a). The addition of 1 g citric acid (pH 2), however, significantly

decreased (Z)-3-hexenyl acetate and limonene because a large amount of citric acid was added also contains a lagre amount of it dissociated form (RCOO-), which might be able to form complex with these compounds (Hansson and others 2001a). The addition of sodium hydroxide (pH 7 and9) increased the release of (Z)-3-hexenyl acetate due to both concentration of hydroxyl group and salting-out effect of sodium hydroxide. The researchers stress that the effect of basic pH on flavor release was not clarify in their study (Hansson and others 2001a).

1.6 The influence of pectin on flavor intensity and release

Polysaccharides or hydrocolloids are used as thickeners, stabilizers, and gelling agents (Hansson and others 2001b). Pectin is one of the most important gelling polysaccharide in food. Pectin is block copolymer, which “smooth block” consisted of

D-galacturonic acid unit linked together by L-1,4- glycosidic linkage (galacturonan chain) and “hairy block” consisted of galctorunan chain and branched rhamnose sugar units (Thakur and others 1997). Generally, high methoxylated pectins (HMP), which have 50 to 80 % degree of methylation, are used to form gels in acidic and high sugar content media while low methoxylated pectins (LMP), which have 25 to 50% degree of methylation, are used in products with wide range of pH and suitable for moisture products but presence of Ca2+ is needed (Guichard 1996, Thakur and others 1997). LMP gel formed mainly by ionic bond via Ca2+ bridge between two different carboxyl group

but HMP gel formed by hydrophobic interaction between methyl groups and hydrogen bond between oxygen atoms of pectin. The strength of gels obtained of LMP depends on not only concentration of calcium ions in the medium but also the molecular characteristics of the polysaccharide. The molecular weight, degree of methylation, number of changes groups, pH, co-solute content, and temperature also influence both

HMP and LMP gel strength characteristics (Guichard 1996; Thakur and others 1997).

Many studies have illustrated that hydrocolloids not only modified viscosity, but also reduced the intensities of odor, taste, and flavor (Guichard 1996). Pectin decreased the release of hydrophobic compounds in flavor model (Boland and others 2004). The overall intensity and typical flavor note of jam decreased with high amount of HM pectin

(Gichard 1996). Headspace analysis shows that the addition of 0.05% pectin drastically decreased the amount of ethyl hexanoate, butyl acetate, hexyl acetate, nonanal, (E)-2- hexenal decreased from 0 to 0.2%. Another study of strawberry flavor release from pectin gel presents that the partition coefficient of ten compounds (ethyl butyrate, ethyl hexanoate, (Z)-3-hexen-1-ol, ethyl iso-pentanone, (Z)-3-hexenyl acetate, benzyl acetate, hexanal, methyl anthranilate, styrallyl acrtate, and β-ionone) was also significantly decreased with higher concentration of pectin (Boland and others 2006). Moreover, the addition of 2.5% decreased the release of limonene in soft-drink related model system as compare to the sample containing smaller amount of pectin (Hansson and others 2001b).

Finally, the pectin addition caused a decrease in the partition coefficients of the aroma compounds studied as compared with the sugar syrup samples (Lubbers and Guichard

2003).

The influence of pectin on flavor intensity and release has been shown in literature above (Guichard 1996; Hansson and others 2001b; Lubbers and Guichard 2003;

Boland and others 2004; Boland and others 2006) and can be conclude that the release of hydrophobic compounds are affected more than the release of hydrophilic compounds.

There are two possible mechanisms, which may affect flavor intensity (Boland and others 2004); the physical entrapment of flavor molecules within the food matrix, and the interactions between the aroma compounds and the gel components. More non-polar environment is induced by the increase in pectin concentration and hydrophobic compounds may trap in hydrophobic region of pectin polymers. When pectins dissolve in water, theirs molecules aligning together. This event forms micelles, which are more hydrophobic because intermolecular hydrogen bonds replace bond water between pectin molecules (Chinachoti, 1995), thus hydrophobic compounds such as limonene may be trapped in the hydrophobic micelles of the pectin solution while other compounds were not affected in soft drink system (Hansson and others 2001b) and a large retention of more hydrophobic compound, such as methyl anthranilate, than less hydrophobic compounds, such as hexanal, in strawberry study (Boland and others 2006).

The macromolecule cross-linked network seems to play a vital role in the flavor release. Cross- linking of polygalacturonic chains may be responsible for the decrease of flavor mobility in pectin solution because pectin cross-linked polymers provide a barrier to diffusion, thus reducing the release rate, mass transfer, of volatiles into the air

(Lubbers and Guichard 2003).

Chapter 2- The effect of temperature, pH, and food additives on tomato product volatile levels

Practical Application

An increase of NaCl concentration and an increase in temperature increased headspace concentration of tomato juice while the addition of pectin, protein, sucrose, and oil decreased headspace concentration. Both sensory and headspace information in this study can be used by the food industry and consumers to improve aroma quantity and quality of processed tomato products.

Introduction

Tomatoes are an important agricultural commodity, which possess a characteristic taste and aroma. The production of tomato products rose from 6 to 14 million metric tons from 1980 to 2012 (USDA 2012a). Processed tomato products account for 80% of total tomato consumption. Americans consume three-fourths of their tomatoes in processed form. In the late 1980s, the increasing popularity of pizza, pasta, and salsa led to an increase in U.S. consumption of processed tomatoes (USDA 2012b).

The largest use of processed tomatoes is for sauces which account for 35%, followed by tomato paste, 18%; canned whole tomato products, 17%; and catsup and juice, 15% (USDA 2012b).

Many ingredient variations in tomato products are due to a desire to improve the flavor. Consequently, salt, sugar, and oil contents are varied among different brands, and proteins and pectin have been widely used as a fat replacer, thickener, and forming agent.

Altering these additives, however, results in different levels of aroma volatiles being released. Salt increases flavor release due to the salting-out phenomenon (Bakierowska and Trzeszczynski 2004; Ventanas and others 2010a; Martínez and others 2012). NaCl decreases the availability of water in food, thus increasing volatility of aroma compounds

(Rabe and others 2003b). Sucrose, however, decreases aroma compounds due to sucrose- sucrose interaction and sucrose-water interaction (Richardson and others 1987; Roberts and others 1996; Starzak and others 2000).

Oil and protein are frequently added to tomato products and also have been reported to have a large effect on flavor volatile levels. Higher contents of either oil

(Rosett and others 1997; Chung and others 2003; Ventanas and others 2010b) or protein

(Macleod and others. 1988; Thanh and others 1992; Kuhn and others 2006) reduce the volatility of some aroma compounds due to their hydrophobic characteristics. The resistance to mass transfer of volatile compounds in fat and oil is higher than in water, thus the release of fat-soluble flavor compounds is delayed because flavor compounds must be released from the lipid phase to the aqueous phase and then released from the aqueous phase to the headspace; thus the volatility of fat-soluble volatile compounds in 32

high-fat-content foods is lower than in low-fat foods (Jo and Ahn 1999; Guinard and others 2002). Protein has hydrophobic interactions with volatile compounds depending on the polarity of the protein structure, temperature, and degree of denaturation (Macleod and others 1988).

Pectin and pH may be adjusted intentionally to change flavor or texture. pH can either increase or decrease flavor perception and volatility (Chen and others 1984;

Hansson and others 2001a; Kim and others 2009); pectin can physically entrap flavor molecules within the food matrix, interact with aroma compounds, or form hydrophobic micelles, which capture hydrophobic compounds. These interactions reduce the volatility of some aroma compounds (Chinachoti 1995; Guichard 1996; Boland and others 2006).

Tomato products are consumed at different temperatures and the temperature of food is an important factor for food volatile levels (Cardello and Maller 1982; Zellner and others 1988; Ventanas and others 2010b). Flavor release, which is a function of temperature according to Henry’s law, is increased by temperature (Bakierowska and

Trzeszczynski 2004, Klooster and others 2005). Although many studies were done in many types of food and model systems, the knowledge of the effect of food additives, pH, and temperature on tomato product is still limit thus, the goal of this research was to determine how temperature, pH, and food additives affect the headspace volatile concentration and consumer acceptability of the aroma of flavored tomato products, using tomato juice as a model system.

2.1 Materials and Methods

2.1.1 Sample preparation

2.1.1.1 Temperature and pH

To adjust temperature to simulate different serving temperatures, samples were placed into a 25 °C or 50 °C water bath or refrigerated at 5 °C for 1 h to fully equilibrate.

Tomato juices (pH 4.26) were adjusted to pH 2.5 and 8.5 with 2 M citric acid solution

(Archer Daniels Midland, Decatur, Illinois, U.S.A.) and 1 M sodium hydroxide (Sigma,

St.Louis, MO, U.S.A.) using a Model 10 pH Meter (Fisher Scientific, Waltham, Mass.,

U.S.A.). For 350 mL juice, 40 mL citric acid solution was used to adjust to pH 2.5 and

30mL NaOH was used to adjust to pH 8.5.

2.1.1.2 Sodium chloride, pectin, protein, sucrose, and oil

To 350 mL tomato juice was added one of the following additives: 1% (w/v) whey protein isolate (Industrial Food Ingedients, Minneapolis, MN, USA), milk protein

(Protient Inc., Norfolk, NE, U.S.A.), or collagen (Arnhem Group, Cranford, NJ, USA);

5% or 10% olive oil (Kroger CO., Cincinnati, OH, USA) plus 1% mono and diglycerides

(Continental Custom Ingredient, Inc., West Chicago, IL, USA); 5% or 10% (w/v) NaCl 34

(Sigma-Aldrich, Co., St. Louis, MO, USA); 5% or 10% (w/v) sucrose (Domino Food,

Inc., Yonkers, NY, USA) or 1% (w/v) low-methoxyl pectin (TIC Gums, Inc., Belcamp,

MD, USA). To test volatile levels in the pectin and proteins themselves, 1% pectin, 1% milk protein isolate, 1% whey protein isolate or 1% collagen was added to deionized water.

2.1.2 Measurement of volatile compound concentration of different samples

Treatments used 50 mL tomato juice filled in 500-mL Pyrex bottles and closed with a silicon septum cap. Hot break tomato juice (5.72 ± 0.05 %TS and 4.76 ± 0.11

%SS) was produced from fresh Roma tomatoes, which were washed, chopped, and pumped through a 88 °C hot break system and extracted into juice. Juice has been filled in canned and stored at 25c since September 2011. The samples were equilibrated in a

45 °C water bath for 1 h before measurement unless specified otherwise.

The concentrations of volatile compounds of samples were analyzed by selected ion-flow tube mass spectrometry (SIFT-MS) (SYFT Voice 200, Syft LTD, Christchurch,

New Zealand). During the measurement, 2 needles were used. The short passivated needle (5.5 cm), which is the inlet to the apparatus, was placed in the middle of the septum. The long needle (27 cm), which allowed air inside to maintain atmospheric pressure in the bottle, was placed next to the short needle. Water (45 °C) was used as a blank between each sample and all samples were measured for 3 min. The flow tube 35

pressure during the machine run was 0.060±0.002 torr. The temperature of the capillary and arm was automatically maintained at 120 °C. Each sample was measured using 5 replicates.

+ + + The method used a selected ion mode with H3O , O2 , and NO as precursor ions, developed based on volatile compounds which contribute to tomato flavor and derived from Xu and Barringer (2010) and Azcarate and Barringer (2010). Each volatile compound concentration was calculated by using known kinetic parameters. The concentration [M] of selected volatiles was calculated using the product count rate [Ip], reaction rate constant [k], precursor ions count rate [I], and reaction time [t] as shown in the equation: [M]=Ip/Ikt (Spanel and Smith 1999). Some compounds produce the same mass for a given precursor ion, in which case a mixture of the interfering compounds were reported. The following pairs are mixtures: isobutanal and 2-methylpropanal, 1- butanol and isobutyl alcohol, benzaldehyde and methyl benzoate, and, (E,E)-2,4- decadienal and citral. Cyclic terpenes are a mixture of alpha-terpinene, terpinolene, β- pinene R-limonene, α-pinene, S-limonene, (+)-aromadendrene, and (E)-cayophyllene.

Compounds with irresolvable conflicts or low concentrations are not reported.

2.1.3 Viscosity

Viscosity was measured from 300 mL of sample in a 600 mL beaker using a viscometer (Brookfield model DV-II+, Stoughton, MA). Low viscosity (LV) spindle no.

2 (Brookfield Engineering Labs, Inc., Stoughton, MA) was used at 10 rpm; at 25°C.

Each sample was measured every 30 sec for 3 min then the result reported as an average.

2.1.4 Sensory evaluation

The experiment was divided into 2 parts; aroma intensity and preference evaluation. Fifty untrained participants were asked to sniff 20 mL tomato samples with

10% NaCl, 10% sucrose, 1% pectin, 10% oil, 1% milk protein or the control at room temperature and rank the aroma intensities and their preference for each sample from 1 to

6. To avoid bias, three-digit random numbers were used to represent each treatment and the positions of the samples on the tray were randomized. The samples were filled in an opaque container and closed with a small 6 cm x 6 cm aluminum cap for sniffing.

2.1.5 Statistical analysis and odor active value calculation

Odor active values (OAV) of individual compound were calculated as the ratio of the headspace concentrations obtained and their odor thresholds in water. One-way analysis of variance (ANOVA) was performed on volatile and OAV data by Minitab

(Minitab Inc., State college, PA, USA), and Tukey’s test was carried out to determine significant differences among mean values of volatile concentrations, OAV, and sensory data. The independent, two-sample T-test was performed on the pectin result. The

Friedman test was carried out for statistical difference among groups for sensory data. A significance level of 0.05 was applied throughout the study.

Chapter 2.3- Results and Discussion

The various treatments had different effects on the volatile levels that appear in the headspace, and thus the samples may be perceived differently by the consumer during consumption. Increasing temperature and NaCl increased volatile levels, pH and pectin had no significant effect, and protein, sucrose and oil decreased volatile levels.

2.3.1 Temperature

Different temperatures were used to simulate different consumer consumption conditions for different tomato products. Comparing the effect of temperature to the other additives, it is apparent that temperature had a much larger effect on the volatile levels. When the temperature increased, the volatile levels all increased, but by different amounts (Table 3). From 5 to 25 °C, the increase varied from 45 to 530% (average

188%), depending on the volatile. There was a 65-518% increase (average 209%) when the temperature increased from 25 to 50 °C. The air/water partition coefficient of volatile compounds in the headspace increases as temperature increases, thus the concentration of volatile compounds in the headspace increases and the flavor intensity is stronger

(Jouquand and others 2004).

The volatiles considered important to create tomato aroma are (Z)-3-hexanal, (Z)-

3-hexanol, hexanal, 1-penten-3-one, 3-methylbutanal, (E)-2-hexenal, 6-methyl-5-hepten-

2-one, methyl salicylate, 2-isobutythiazole, methional, eugenol, and β-ionone (Buttery

1993). The odor activity value (OAV) of methional, which was described as “boiled potato like” (Guen and others 2000), was less than 1 for all treatments, and so should not be detectible to the consumer. All of the other compounds are at detectible levels (Figure

2 and 3). (Z)-3-hexanal, (Z)-3-hexanol, (E)-2-hexenal, and hexanal are associated with

“fresh green” aroma in tomato juice (Kazeniac and Hall 1970; Goodman and others 2002;

Xu and Barringer 2009). 3-methylbutanal was described as “malty” (Fickert and others

1998) and 2-isobutythiazole has a spoiled wine-like, slightly horseradish- type flavor, becoming rancid, medicine like and metallic if it is used at levels above 50 ppm in tomato juice (Kazeniac and Hall 1970). The use of 2-isobutythiazole has been patented for use in tomato products as a means of enhancing tomato flavor (Christiansen and others 2011).

“Fruit-like” aroma and “cooked-stewed tomato”, at concentrations around 0.75 ppm, was used to describe 6-methyl-5-hepten-2-one (Kazeniac and Hall 1970). Eugenol was described as “clove” aroma (Laing and others 1984; De Wijk and Cain 1994) and β- ionone was described as floral and raspberry (Perez-Cacho and Rouseff 2008). Methyl salicylate was described as “maraschino-cherry” or “almond” aroma (De Wijk and Cain

1994) and gave a “canned tomato” note (Kazeniac and Hall 1970). 1-penten-3-one was described as “fresh/sweet” (Tandon and others 2000) or “green” (Krumbein and

Auerswald 1998).

(Z)-3-hexanal, 3-methylbutanal, hexanal and (Z)-3-hexen-1-ol had the highest

OAV in all treatments, and so should have the greatest aroma impact (Figure 2 and 3).

The other volatiles varied slightly in order depending on the temperature or additive used.

This might change aroma perceived by consumer; however, the ratios between the volatiles did not change greatly.

The overall effect of temperature on volatile levels is explained by the partition coefficient and Henry’s law equations (Bakierowska and Trzeszczynski 2004; Klooster and others 2005). The partition coefficient K = CG/CL (Bakierowska and Trzeszczynski

3 −1 2004) and the Henry’s law constant (atm- m -mol ) is often described as Hp = RT/K; where K is the water/gas partition coefficient value, T is the system temperature (K), and

R is the gas constant equaling 0.08206 × 10−3 (m3-atm-mol−1-K−1). An increase in temperature increases the Henry’s law constant of volatile compounds. The temperature dependent aroma partition coefficient can be described with Clausius-Clapeyron equations (Covarrubias-Cervantes and others and others 2004; Klooster and others 2005).

According to these equations, the logarithm of the partition coefficient is proportional to the inverse of temperature.

Because of the change in concentration of volatiles and ratio of volatiles in the headspace, temperature plays an important role in the flavor perception of tomato products. Previous studies of odor and flavor perception in a flavored model system have shown that increasing temperature increased perceived flavor intensity (Ventanas and others 2010a). The odor intensity of cheese soup was stronger at 63 °C than at 33

°C, and the odor and flavor intensity of carrot, meat patty, and mashed potato increased 40

as serving temperature increased from 25 to 65 °C (Kähkönen and others 1995;

Ryynanen and others 2001).

180,000 a 160,000 5°C

140,000 25°C

120,000 50°C

100,000

80,000 a b

60,000 c Odor Active OdorActive Vvalue 40,000 b c 20,000 a a c b c b 0 (Z)-3-hexen-1-ol hexanal 3-methylbutanal (Z)-3-hexenal

4,000 a 3,500 5°C

25°C 3,000

2,500 50°C

2,000

1,500

Odor Active OdorActive Vvalue b 1,000 a a a a a 500 b c b c c b a c b c b a c b 0

*Odor activity value for each volatile that does not share a letter is significantly different

Figure 2 The effect of temperature on odor activity value of volatiles important to tomato aroma

Table 3 The effect of temperature (5, 25, and 50 °C) on tomato juice headspace volatile concentration (ppb)

Volatile compounds (ppb)/ 5°C 25°C 50°C treatments Aliphatic alcohols

(Z)-3-hexen-1-ol 6.02c 16.36b 44.19a 1-hexanol 3.85c 7.76b 22.66a 1-octen-3-ol 4.68c 7.71b 15.66a 1-propanol 3.33c 16.70b 51.19a 2,3-butanediol 4.09c 12.55b 47.68a 2-pentanol 3.59c 10.36b 33.93a ethanol 136.07c 628.35b 2071.74a methanol 1,947.90c 6,388.76b 19,514.43a Heterocyclic and aromatic alcohols benzene ethanol 0.66c 1.60b 5.41a benzyl alcohol 0.60c 3.13b 18.22a

Aliphatic aldehydes

(E)-2-heptenal 1.71c 3.64b 7.41a (E)-2-hexenal 0.87c 1.87b 7.32a (E)-2-nonenal 0.53c 1.22b 3.46a (E)-2-octenal 0.91c 1.84b 4.94a (E)-2-pentenal 1.20c 3.48b 12.67a (E,Z)-2,6-nonadienal 1.51b 2.35b 5.59a (Z)-3-hexenal 2.80c 6.71b 18.38a 3-methylbutanal 7.88c 25.23b 76.87a acetaldehyde 27.73c 57.83b 117.67a decanal 0.94c 2.62b 7.04a dodecanal 0.46c 0.73b 1.75a hexanal 6.81c 19.62b 55.86a (Continue)

(Table 3 continue) isobutanal 11.89c 30.71b 65.47a methional 1.51c 3.80b 13.01a nonanal 2.97c 5.28b 11.54a octanal 5.85c 13.94b 29.00a propanal 9.87c 21.73b 49.52a Hetrocyclic and aromatic aldehydes benzaldehyde 2.51c 9.92b 37.69a furfural 2.01c 4.71b 13.64a phenylacetaldehyde 4.15c 6.96b 17.00a

Aliphatic ketones

1-penten-3-one 3.65c 5.48b 9.09a 2,3-butanedione 5.87c 11.45b 30.71a 2-pentanone 4.08c 7.47b 16.62a acetone 91.87c 357.85b 1,099.10a Terpene ketones

6-methyl-5-hepten-2-one 11.48c 62.80b 220.16a beta-ionone 0.40c 0.72b 3.83a

Acids hexanoic acid 29.11c 113.08b 298.20a methylbutanoic acid 26.16c 77.56b 174.76a propanoic acid 8.91c 16.72b 33.44a

Heterocyclic and aromatic phenols eugenol 0.84b 3.16a 2.88a guaiacol 10.18c 14.84b 30.89a methyl salicylate 0.85c 3.40b 19.43a (Continue)

(Table 3 continue) Esters ethyl acetate 18.2c 54.97b 119.68a hexyl acetate 19.05c 73.65b 297.57a methyl hexanoate 20.67c 80.26b 235.26a

Sulfur containing compounds 2-isobutylthiazole 0.51c 0.89b 2.04a dimethyl disulfide 1.61c 3.70b 12.32a dimethyl sulfide 33.60c 118.16b 541.33a

Others heterocyclics cyclic terpenes 7.56 b 28.91 b 134.34 a 2-pentylfuran 2.25c 4.38b 11.50a *Means in the same row that do not share a letter are significantly different.

2.3.2 NaCl

NaCl is frequently added to foods to improve the flavor. NaCl and temperature were the only 2 treatments in this study that increased volatile levels (Tables 2 and 3). As

NaCl increased from 0 to 10%, half of the volatile levels significantly increased while the other half of the volatiles showed no significant difference. The increase was 0-60%

(average 10%) depending on the volatile. The addition of 10% NaCl produced the highest % increase for alcohols (average 29%) followed by ketones (average 22%), aldehydes (average 19%), and esters (average 16%) (Table 4). In apple juice aroma analysis, an increase from 0-37.5% salt increased the off-aroma because of higher alcohol 45

levels in the headspace (Poll and Flink 1984). Alcohol showed the highest increase with the addition of salt followed by aldehydes and esters producing an increase in both aroma intensity and off-aroma (Poll and Flink 1984).

Among the volatiles considered to be most important, only the headspace concentration of (Z)-3-hexanal, (Z)-3-hexen-1-ol, hexanal, and 3-methylbutanal significantly increased at 10% NaCl. These volatiles also had the highest OAV values among important tomato volatiles.

Others have also found an increase in flavor perception and intensity by NaCl addition. In meat broth, the addition of 0.5% NaCl increases the intensity of overall flavor and broth-like flavor (Ventanas and others 2010a). The mushroom flavor intensity of cooked bologna sausage was shown to be increased by an increase from 1.4 to 2.0% salt (Ventanas and others 2010b). A reduction from 0.37 to 0.18% salt has previously been shown to decrease total volatile compound levels by 50% in vegetable soup

(Mitchell and others 2011). The partition coefficient of volatiles in a model system increases as salt concentration increases, therefore producing a higher concentration in the headspace (Bakierowska and Trzeszczynski 2004).

Na+ and Cl- bind water molecules with a strong dipole interaction and form hydration shells thus depleting the availability of water molecules for the solubilization of flavor compounds (Rabe and others 2003a; Ventanas and others 2010a; Mitchell and others 2011). NaCl decreases the free water of solubilization available for polar compounds as well as creating the iceberg effect around hydrophobic molecules (Shinoda

1976; Blokzijl and Engberts 1993; Rabe and others 2003a). This results in an increased concentration of the volatile compounds in the remaining free water. The iceberg effect occurs when numerous layers of water molecules orient around the organic solute, inducing solubility of a nonpolar organic molecule in water. However, the iceberg effect depends on both solvent properties and intrinsic thermodynamic properties of a compound. Therefore, this effect does not occur for all compounds.

700 a 10% NaCl 600 Control ab

500 1% Pectin bcd bc 1% Milk d 400 protein isolate 10% Sucrose 300 a a a 10% Oil a ab ab abc ab d Odor Active OdorActive Vvalue ab 200 abcd ab dcd bcd a ab cd bcd bc 100 a a a abb ab b bab a abc bc b bc b b 0

60,000 10% NaCl a

50,000 Control b

a 1% Pectin 40,000 c c c c 1% Milk protein b b bc 30,000 isolate bc c 10% Sucrose

20,000 Odor Active OdorActive Vvalue

10,000 ab a bc cdbcd a b bcbcd bcd cd de 0 (Z)-3-hexen-1-ol hexanal 3-methylbutanal (Z)-3-hexenal

* Odor active value for each volatile that does not share a letter is significantly different

Figure 3 The effect of NaCl, pectin, milk protein isolate (MPI), sucrose, and oil on volatile important to tomato aroma

Volatiles/Treatments Control 5%NaCl 10%NaCl 5% Sucrose 10% Sucrose 5%Oil 10%Oil WPI MPI Collagen Aliphatic alcohols

(Z)-3-hexen-1-ol 13.71b 13.71b 17.41a 10.89cd 10.52d 8.67e 8.18e 12.29bc 11.65cd 12.28bc 1-hexanol 4.85b 4.85bc 6.00a 3.97d 3.76de 3.17e 3.22e 3.89d 3.96d 4.22cd 1-octen-3-ol 6.76a 5.62abc 6.63ab 4.91cd 4.75cd 3.83d 4.5cd 4.95cd 4.94cd 5.52bc 1-propanol 15.49c 18.57b 24.88a 14.8cd 14.13d 14.81cd 14.56cd 13.92d 14.53cd 14.35cd 2,3-butanediol 14.35b 14.12bc 18.02a 12.65cde 11.55e 12.38de 12.85bcde 13.71bcd 13.3bcd 13.34bcd 2-pentanol 8.02c 9.63b 12.66a 7.89cd 7.21de 7.07de 6.62e 7.56cd 7.36cde 7.82cd ethanol 579.41c 544.51b 638.80a 481.19d 464.09d 367.28e 344.33e 474.81d 478.88d 481.89d methanol 10,556.66bcd 10,945.79bc 13,086.63a 10,223.26cde 9,699.44e 10,504.15bcd 11,147.12b 10,149.26de 10,364.96cde 10,107.51de Heterocyclic

and aromatic alcohols benzene ethanol 2.29ab 2.07abc 2.69a 1.83bc 1.77bc 1.35bc 1.67c 1.82bc 1.71bc 1.80bc benzyl alcohol 4.42ab 3.73bc 4.90a 3.73bc 3.16c 3.27c 4.05abc 4.15abc 3.75bc 3.92abc

Aliphatic aldehydes

(E)-2-heptenal 2.66a 2.34abc 2.59ab 2.33abc 2.07c 2.08c 2.10c 2.16bc 2.15c 2.29abc (E)-2-hexenal 3.32ab 3.02bc 3.69a 2.87cd 2.48def 2.27f 2.33ef 2.73cdef 2.76cde 2.86cd 49 (E)-2-nonenal 1.02a 0.80abc 0.88ab 0.73bc 0.71bc 0.57bc 0.61c 0.75abc 0.80abc 0.85abc a b a c c d d c c bc

(E)-2-octenal 5.35 4.35 5.55 3.50 3.31 1.90 1.65 3.50 3.59 3.81 (E)-2-pentenal 8.97b 8.11c 9.84a 7.36c 7.00cd 5.71e 5.43e 7.05d 7.12d 7.19d (E,Z)-2,6-nonadienal 2.21a 1.52bc 1.91ab 1.18c 1.36bc 1.38bc 1.58bc 1.52bc 1.51bc 1.84ab (Z)-3-hexenal 4.98b 4.71bc 5.97a 4.28cd 4.13cd 3.71d 4.32cd 4.12cd 4.20cd 3.96d 3-methylbutanal 29.66de 34.72bc 41.67a 37.68b 29.62de 32.93cd 29.68de 32.84cd 29.94de 26.91e acetaldehyde 58.59bcd 60.72bc 70.55a 59.45bc 49.45e 59.2bcd 55.56cd 61.41b 58.71bcd 53.49de decanal 1.21c 0.80d 0.93cd 0.70d 0.64d 2.34b 3.11a 0.77d 0.88cd 0.93cd dodecanal 0.63a 0.40b 0.53a 0.38b 0.34b 0.40b 0.47ab 0.41b 0.49ab 0.50ab hexanal 22.34cd 20.72de 25.16bc 18.89de 16.90e 21.83cd 20.88d 27.11b 31.19a 19.76de isobutanal 23.62cd 21.90de 25.77ab 19.30fg 17.79g 18.91fg 17.68g 27.21a 24.65bc 20.74ef methional 3.50ab 3.19abc 3.86a 2.58cde 2.48cde 2.18e 2.43de 3.00bcd 3.03bcd 2.92bcd nonanal 3.71a 2.15bcd 2.68abcd 1.78cd 1.58d 2.36abcd 2.88abcd 3.61ab 2.47abcd 3.16abc octanal 9.43a 6.25bcd 7.59ab 5.27cde 4.89de 3.84e 4.00e 6.19bcd 5.79bcde 7.10bc propanal 15.54d 15.09d 17.95c 12.89fg 11.56g 19.75b 21.38a 14.37de 13.56ef 13.62ef Heterocyclic

and aromatic aldehydes phenylacetaldehyde 19.08a 16.64bcde 18.48ab 15.13cdef 14.71ef 14.01f 14.94def 16.37cde 16.81bcd 17.07bc benzaldehyde 11.52bc 12.50b 17.64a 10.19cd 9.19de 8.54e 8.16e 11.53bc 12.40b 10.98bc furfural 19.53a 16.36b 18.66a 14.89bc 14.46c 10.22d 9.53d 14.72bc 14.38c 15.44bc Aliphatic ketones

1-penten-3-one 5.47a 4.57cde 5.35ab 4.07def 3.82f 4.82abc 4.78bcd 4.00ef 3.98ef 4.40cdef 2,3-butanedione 13.99bc 14.47b 17.78a 14.86b 12.1de 14.27b 13.59bcd 14.37b 12.63cde 11.89e 2-pentanone 5.66a 4.79b 5.97a 4.61b 4.24b 4.25b 4.56b 4.47b 4.51b 4.82b acetone 537.63bc 564.51b 713.19a 501.8de 467.91e 486.73de 493.41de 512.76cd 511.73cd 500.06de (continue)

(Table 4 continue) Terpene ketones

6-methyl-5-hepten-2- 33.69ab 33.51ab 39.20a 29.60bc 27.87bc 10.74d 8.35d 27.91bc 26.12c 31.16bc one beta-ionone 1.82a 1.56ab 1.92a 1.39ab 1.24bc 0.54d 0.65cd 1.32ab 1.15bcd 1.64ab

Acids

hexanoic acid 6.99a 4.92bcd 5.90b 4.38cd 3.97d 4.19cd 4.52cd 4.17cd 4.55cd 5.03bc methylbutanoic acid 12.74a 11.02b 13.44a 9.80bcd 9.05d 9.09d 9.24d 9.58cd 9.69cd 10.74bc propanoic acid 10.15a 8.73b 10.40a 7.64b 7.45b 7.70b 8.31b 7.80b 8.04b 8.33b

Heterocyclic and aromatic phenols eugenol 1.15 a 0.83 abc 1.03 ab 0.66 bc 0.57 c 0.45 c 0.50 c 0.69 bc 0.68 bc 1.10 ab guaiacol 16.91a 12.72b 15.77a 9.79cd 9.2cd 7.89d 8.5d 12.08bc 11.96bc 12.55b methyl salicylate 2.69a 2.23abc 2.70a 1.99bc 1.82bc 0.69d 0.71d 1.85bc 1.77c 2.33ab

Esters ethyl acetate 14.95 a 13.18 b 16.26 a 11.16 c 10.68 c 10.63 c 10.66 c 11.22 c 11.79 bc 12.85 b hexyl acetate 6.57a 4.65bc 5.38b 3.63e 3.64e 3.52e 3.75de 3.98cde 4.34cde 4.83bc methyl hexanoate 6.52a 4.38bcd 5.27b 3.75d 3.64d 3.61d 3.92cd 3.78d 4.00cd 4.89bc Sulfur containing compounds

50 2-isobutylthiazole 2.34 a 1.98 ab 2.37 a 1.56 b 1.56 b 0.67 c 0.71 c 1.62 b 1.58 b 1.85 ab dimethyl disulfide 6.89b 7.16b 9.64a 5.97b 5.69bc 3.84c 3.88c 9.56a 7.21b 5.41bc dimethyl sulfide 5,191.22a 4,758.39b 5,339.91a 4,252.64c 4,214.55c 2,960.12d 2,651.15e 4,300.67c 4,323.73c 4,242.86c

Others heterocyclics cyclic terpenes 3.61 a 3.05 ab 3.65 a 2.94 ab 2.58 b 0.91 c 0.92 c 2.79 b 2.51 b 3.14 ab 2-pentylfuran 4.68a 3.85bc 4.35ab 3.57c 3.36c 2.16d 2.26d 3.61bc 3.32c 3.79bc *Means in the same row that do not share a letter are significantly different.

2.3.3 pH

The pH of tomato products may be adjusted to keep it out of the low acid food category, but is not typically adjust for flavor. Changing the pH from 2.5 to 8.5 produced anywhere from 155% increase to 55% decrease, depending on the volatile (Table 5).

However, the majority of volatile showed no significant effect of pH. There was also no correlation between pH (2-9) and volatile concentrations in the headspace of a soft drink model system (Hansson and others 2001a). They found that volatile levels in the headspace can either increase or decrease due to the interaction between the dissociated form of citric acid and volatiles but not due to the change in pH, which may explain why the headspace concentration of some volatiles were significantly higher with the addition of citric acid to fruit pulp (Marsh and others 2006).

Table 5 The effect of pH (2.5, 4.26, and 8.5) and 1% pectin on tomato juice headspace volatile concentration (ppb)

Volatile 1% Pectin compounds (ppb)/ pH 2.5 pH 4.26 pH 8.5 Control 1% Pectin in Water treatments Aliphatic alcohols

(Z)-3-hexen-1-ol 13.16a 12.23a 12.78a 12.77a 13.25a 5.54 1-hexanol 4.44a 4.22a 4.51a 5.23a 5.07a 2.97 1-octen-3-ol 6.63a 6.06b 5.97b 7.54a 6.50b 4.9 1-propanol 47.29a 38.52b 18.38c 14.68b 554.92a 466.37 2,3-butanediol 11.61a 10.40c 11.07b 13.26a 13.27a 3.41 2-pentanol 7.20a 6.47b 7.32a 7.32b 8.19a 2.39 Ethanol 376.53a 310.95b 369.96a 490.39a 383.87a 22.38 8,149.25 Methanol 7,993.39a 6,853.69b 11,647.21a 12,650.70a 80.06 a Heterocyclic and aromatic alcohols benzene ethanol 2.84a 1.63b 1.56b 2.11a 2.12a 0.74 benzyl alcohol 2.11ab 1.71b 2.35a 4.32a 3.98a 2.36

Aliphatic aldehydes (E)-2-heptenal 2.95a 2.79a 2.96a 3.15a 2.65a 2.15 (E)-2-hexenal 2.31b 2.36b 6.03a 2.81a 2.71a 0.81 (E)-2-nonenal 1.16a 1.10a 0.99a 0.96a 1.15a 1.04 (E)-2-octenal 3.20a 2.64b 3.02ab 3.98a 2.38a 1.06 (E)-2-pentenal 6.73a 5.66b 6.72a 7.59a 5.78a 0.64 (E,Z)-2,6- 2.14a 1.81a 1.96a 2.18a 1.69a 1.9 nonadienal (Z)-3-hexenal 3.98a 4.11a 4.00a 4.83a 4.17a 2 3-methylbutanal 22.43a 23.91a 21.09b 26.61a 33.06a 3.97 acetaldehyde 49.99ab 51.84a 46.22b 56.35a 100.33a 53.94 Decanal 1.87a 1.56b 1.52b 1.10a 1.15a 1.32 dodecanal 0.67a 0.59a 0.57a 0.70a 0.50a 0.64 (Continue)

(Table 5 Continue) Hexanal 27.69a 30.14a 24.08a 18.10a 23.95a 11.6 Isobutanal 18.52a 17.65a 15.92b 21.20a 19.83b 6.47 Methional 3.04a 2.62b 3.03a 3.95a 3.75a 2.05 Nonanal 6.58a 5.11a 4.96a 3.76a 3.16a 3.82 Octanal 11.66a 10.49ab 9.42b 9.75a 7.27b 5.94 Propanal 14.13a 14.23a 12.38b 13.97a 15.69a 9.53 Hetrocyclic and aromatic aldehydes phenylacetaldehyd 7.13a 7.65a 8.30a 18.04a 15.96a 15.89 e benzaldehyde 9.87a 8.37b 9.62a 10.94a 12.41a 1.89 Furfural 12.29a 9.26b 11.04b 16.88a 11.95b 1.56

Aliphatic ketones

1-penten-3-one 4.92a 4.74a 4.84a 5.84a 5.19a 4.08 2,3-butanedione 11.04a 11.44a 11.73a 14.66a 15.85a 7.08 2-pentanone 4.86b 5.42a 5.61a 5.56a 5.29b 3.89 Acetone 424.11a 363.11b 403.20a 465.49a 488.48a 11.5 Terpene ketones 6-methyl-5-hepten- 59.20 a 62.11 a 63.40 a 26.48a 23.08a 1.96 2-one beta-ionone 1.40a 1.10a 1.23a 2.07a 1.74a 0.81

Acids hexanoic acid 4.77b 5.81a 4.51b 6.63a 5.96a 6.81 methylbutanoic 11.94a 12.01a 10.39b 14.25a 13.62a 13.61 acid propanoic acid 7.68b 7.69b 10.69a 10.56a 9.70b 6.39

Heterocyclic and aromatic phenols Eugenol 1.70 a 0.90 b 0.84 b 1.26a 0.88b 1.02 Guaiacol 15.71a 13.53a 13.86a 18.05a 14.78b 12 (Continue)

(Table 5 Continue) methyl salicylate 3.49a 3.23ab 2.75b 2.52a 2.18a 0.81

Esters ethyl acetate 12.01 a 13.78 b 12.31 a 14.16a 14.90a 12.02 hexyl acetate 6.21a 6.42a 5.84a 8.23a 6.28a 7.2 methyl hexanoate 4.57a 4.94a 3.97b 6.56a 5.63b 6.75

Sulfer containing compounds 2-isobutylthiazole 0.74 b 1.63 a 1.66 a 1.78a 1.50a 0.57 dimethyl disulfide 5.99a 4.50b 5.80a 8.28a 5.99a 0.75 3,178.46 dimethyl sulfide 3,184.00a 2,413.29b 4485.41a 2883.59a 1.24 a

Others heterocyclics 2-pentylfuran 4.71 a 4.30 a 3.71 b 4.44a 3.89a 2.33 cyclic terpenes 3.31b 3.78b 5.44a 3.38a 5.27a 5.41 *Means in the same row for pH and pectin that do not share a letter are significantly different. Statistical calculation was done separately for pH and pectin treatments.

2.3.4 Pectin

Pectin and other thickeners may be added to increase the viscosity of processed tomato products. The addition of pectin produced a significant decrease in only 18% of the volatile compounds with the rest showing no difference (Table 5). Previous studies have found a decrease in volatile compound levels with the addition of 1-2.5% pectin

(Boland and others 2006, Hansson and others 2001b; Lubbers and Guichard 2003).

Pectin was expected to decrease volatile levels because the overall intensity and typical flavor note in jam and some volatile levels decreased when 0.5% pectin was added

(Guichard 1996). The partition coefficient of compounds in a gel system and a fruit pastille was decreased by 1-2.5% pectin because of flavor entrapment (Lubbers and

Guichard 2003; Boland and others 2006). In this study, the addition of 1% pectin also produced a sharp increase for 1-propanol due to the initial 1-propanol levels in the pectin itself.

Hydrocolloids may delay flavor release due to 2 mechanisms (Boland and others

2004). For flavor intensity, there are two possible mechanisms, which may affect flavor intensity (Boland and others 2004); the physical entrapment of flavor molecules within the food matrix, and the interactions between the aroma compounds and the gel components. More non-polar environment is induced by the increase in pectin concentration and hydrophobic compounds may trap in hydrophobic region of pectin polymers. When pectins dissolve in water, theirs molecules aligning together by hydrogen or hydrophobic bond. This event forms micelles, which are more hydrophobic because intermolecular hydrogen bonds replace bond water between pectin molecules

(Chinachoti, 1995), thus hydrophobic compounds such as limonene may be trapped in the hydrophobic micelles of the pectin solution while other compounds were not affected in soft drink system (Hansson and others 2001b) and a large retention of more hydrophobic compound than less hydrophobic compounds, such as hexanal, in strawberry flavor model system (Boland and others 2006). Pectin also significant increased viscosity of tomato juice from 0.0707 to 0.2991 Pa-s. Cross- linking of polygalacturonic chains of

pectin may be responsible for the decrease of flavor mobility in pectin solution because pectin cross-linked polymers provide a barrier to diffusion, thus reducing the release rate, mass transfer, of volatiles into the air (Lubbers and Guichard 2003).

2.3.5 Protein

Meat, milk, and cheese are commonly added to tomato sauce therefore the addition of collagen, milk protein isolate, and whey protein isolate were tested. The volatile concentrations in tomato juice significantly decreased with the addition of 1% protein (Table 4). Milk protein produced 4-41% decrease (average 24%). Whey protein isolate produced a 2-42% decrease (average 9%). Collagen produced a 2-29% decrease

(average 15%). Others have also found protein-flavor binding interaction by proteins such as whey protein isolate and milk protein isolate (Macleod and others 1988; Mottram and others 1996; Kuhn and others 2006). Different proteins have different binding sites, but for most volatiles in this study there were no significant differences between the 3 types of proteins tested. The strongest affinity to soy protein is aldehydes, followed by ketones and alcohols, with no binding affinity to acid (Macleod and others 1988). In this study, aldehydes and ketones were also decreased by the addition of protein more than alcohols were (Table 4). Therefore, the decrease in volatile levels by proteins is likely due to the hydrophobicity of individual compounds (Macleod and others 1988; Landy and others 1995; Kuhn and others 2006). In sulfide volatiles, sulfhydryl groups form disulfide bridges with cysteine and cysteine amino acid units in the protein (Mottram and 56

others 1996). In this current study, sulfides were affected by collagen more than the other proteins. This may be due to the fact that collagen contains more -SH containing amino groups than milk proteins (Gordon and Ziegler 1955; Steven and Jackson 1967;

Husdan and others 1977)

Although most of the volatiles decreased with the addition of protein, hexanal and acetaldehyde increased in concentration. The increase was probably caused by the initial volatile levels in the proteins themselves. Most volatile levels were low in 1% whey protein isolate, milk protein isolate, and collagen in deionized water but hexanal was 10-

29 ppm and acetaldehyde was 27-31 ppm.

2.3.6 Sucrose

The volatile levels of most aroma compounds significantly decreased as the amount of sucrose increased from 5% to 10% (Table 4). Sucrose at 10% produced a 14-

47% decrease (average 32%). For the important tomato compounds, only 3- methylbutanal and 6-methyl-5-hepten-2-one showed no significant decrease from control.

Interestingly, there was a strong negative correlation (R= 0.92) between the effect of

NaCl and sucrose. The volatiles that had the greatest percent increase with NaCl showed the least change with sucrose, and those that had the lowest increase with NaCl had the greatest decrease with sucrose. Both may be due to the effect of the solute on water mobility. In previous studies, contradictory results have been found, where sucrose has

been shown to either increase volatiles due to the salting-out effect or to decrease volatile levels due to sucrose-water interactions (Roberts and others 1996; Covarrubias-Cervantes and others 2004; Piccone and others 2012). However, the effect of addition of sucrose may be because sucrose’s interaction with water molecules, which effect the interaction between pectin molecules, in tomato juice. The addition of sucrose as a co-solute in pectin solution decreased water activity thus decreasing interaction between water and pectin, and increasing the interaction between pectin molecules (Sato and others 2004;

Sato and Miyawaki 2008). The addition of 5% and 10% sucrose in the current tomato juice study significant reduced the water activity of tomato juice from 1.003 to 0.995.

The reduction in water activity creates a more hydrophobic environment thus increasing the interaction between pectin molecules. This event leads to an increase in cross-link network polymers resulting in more hydrophobic matrices, which may trap more hydrophobic volatiles in the system (Boland and others 2004; Boland and others 2006).

Therefore, a decrease in water activity and an increase in pectin-pectin interaction may affect the volatility of aroma compound in tomato juice.

2.3.7 Oil

Oil is normally added to tomato sauce. Oil at 10% produced a 10-83% decrease

(average 33%). Most of the volatile levels significantly decreased as the amount of oil increased from 0% to 10% and terpenes showed the greatest decrease followed by esters, aldehydes, alcohols, and ketones (Table 4). Oil at 10% also changed the order of the 58

OAV values for the important volatiles compared to the rest of treatments. The order of

β-ionone, eugenol, 1-penten-3-ol, and 6-methyl-5-hepten-2-one was different from the other treatments. Thus, there might be a slight change in aroma perceived by the consumer. Previous studies have also shown a decrease in volatiles with an increase in oil content (Kaneko and others 1994; Chung and others 2003; Ventanas and others

2010b). The higher fat content in cooked bologna decreased the volatility of some volatiles because fat acts as a solvent for hydrophobic compounds, thus diminishing their release (Ventanas and others 2010b). Fat also reduced the release of hydrophobic compounds in ice cream (Chung and others 2003). An increase of fat in salad dressing reduces garlic and pepper flavor intensity due to its viscosity (Guinard and others 2002).

The resistance to mass transfer of volatile compounds in fat and oil is higher than in water. Hydrophobic compounds may trap in oil droplet while hydrophilic compounds were affected by an increase in viscosity (0.0707 to 0.1313 Pa-s), which decreased mass transfer. Hydrophobic flavor compounds are also delayed by fat and oil because compounds must be released from three different phases, the lipid phase to the aqueous phase and the aqueous phase to the air phase. Therefore, the release rate of hydrophobic compounds in high-fat-content foods is slower than in low-fat content foods (Jo and Ahn

1999, Guinard and others 2002).

2.3.8 Sensory

Sensory evaluation was conducted to determine the most preferable tomato juice with the addition of different additives in this study. NaCl (10%), control, and sucrose

(10%) produced the highest aroma intensity followed by pectin (1%) and milk protein

(1%) while oil (10%) had the lowest aroma intensity (Table 6). The aroma intensity was similar to the volatile results from SIFT-MS, with NaCl producing the highest volatile levels (Table 4) and perceived aroma intensity (4.68), and oil producing the lowest

(2.14). Consumer preference was very similar to aroma intensity, with the highest aroma intensity (10% NaCl) being the most preferred (4.68). Samples that were significantly different from the control were pectin, milk protein, and oil, which were significantly less preferred.

Table 6 Average ranking score for aroma intensity and consumer preference

10% 1% 10% NaCl Control 1%MPI 10% Oil Average/treatments Sucrose Pectin Aroma intensity 4.68a 4.08a 3.82ab 3.18b 3.10b 2.14c

Consumer a a a b b c 4.68 4.16 4.30 3.18 2.78 1.82 preference *Means in the same row that do not share a letter are significantly different.

2.4 Conclusions

Temperature and food additives affect tomato juice volatile levels. Temperature produced the greatest increase in volatiles among all treatments. The addition of NaCl also increased volatile levels, due to the salting-out effect. This suggests that NaCl should increase the aroma intensity of tomato products, though sensory aroma results were not significantly different from the control. Pectin and pH produced little significant difference in volatile levels. Proteins decreased volatile levels and aroma

intensity due to protein-flavor binding. Thus, the addition of protein or the applications of protein-based fat replacers decreased the volatility of flavor compounds, changing the flavor profile of low-fat tomato products. Sucrose also decreased volatile levels. Oil produced the greatest decrease in volatile levels and perceived aroma among treatments due to the emulsion system affecting mass transfer of volatile compounds.

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Appendix: Figures and Tables

Table 7 Kinetics parameters for SIFT-MS analysis of selected volatile compounds in tomato juice

k (10- precursor Compound product ion 9 m/z ref ion cm3/s) + + (E)-2-heptenal NO C7H11O 3.9 85 1 + + (E)-2-hexenal NO C6H9O 3.8 97 2 + + (E)-2-nonenal NO C9H15O 3.8 139 1 + + (E)-2-octenal NO C8H13O 4.1 125 1 + + (E)-2-pentenal NO C5H7O 4 83 1 (E,Z)-2,6- NO+ C H O+ 4.2 151 1 nonadienal 10 15 + + (Z)-3-hexenal NO C4H6O 3.1 70 2 + + (Z)-3-hexen-1-ol NO C4H8O 2.5 72 4 + + 1-hexanol NO C6H13O 2.4 101 5 + + 1-octen-3-ol H3O C8H15 3.1 111 4 + + 1-penten-3-one NO C5H8O.NO 2.5 114 6 + + 1-propanol NO C3H7O 2.3 59 5 + 2-isobutylthiazole H3O C7H11NS.H+ 3 142 6 + + 2-pentanol NO C5H11O 2.5 87 6 + + 2-pentanone NO NO .C5H10O 3.1 116 2 + + 2-pentylfuran H3O C9H14O.H 3 139 3 + + 2,3-butanedione NO C4H6O2 1.3 86 2 + + 2,3-butanediol NO C4H9O2 2.3 89 6 NO+ C H O +.H O 2.3 107 6 4 9 2 2 NO+ C H O +.H O 2.3 107 6 4 9 2 2 + + 3-methylbutanal NO C5H9O 3 85 1 6-methyl-5- NO+ C H + 2.5 108 6 hepten-2-one 8 12 + + acetaldehyde O2 C2H4O 2.3 44 2 + + acetone NO NO .C3H6O 1.2 88 2 + + benzaldehyde NO C7H5O 2.8 105 2 + + benzene ethanol NO C8H10O 2.3 122 7 + + benzyl alcohol NO C7H7O 2.3 107 7 (Continue) 75

(Table 7 Continue)

+ + beta-ionone NO C13H20O 2.5 192 6 + + cyclic terpenes O2 C9H13 2 121 8 7 (Terpinolene and (+)- aromadendren e) 6 ((E)- cayophyllene) + + decanal NO C 10H19O 3.3 155 1 + + dimethyl disulfide NO (CH3)2 S2 2.4 94 10 + + dimethyl sulfide NO (CH3)2 S 2.2 62 10 + + dodecanal NO C10H19O 3.3 155 1 NO+.CH COOC ethyl acetate NO+ 3 2 2.1 118 11 H5 + + ethanol NO C2H5O 1.2 45 5 + + NO C2H5O .H2O 1.2 63 5 + + NO C2H5O .2H2O 1.2 81 5 + + eugenol NO C10H12O2 2.5 164 6 + + furfural NO C5H4O2 3.2 96 12 + + guaiacol H3O C7H8O2.H 3 125 6 + + H3O C8H12N2O 3 143 6 + + hexanal NO C6H11O 2.5 99 2 + + hexanoic acid NO C6H12O2.NO 2.5 146 6 + + hexyl acetate NO C8H16O2.NO 2.5 174 6 + + isobutanal NO C4H7O 3.1 71 1 + + methanol H3O CH3OH.H .(H2O)2 2.7 69 5 + + H3O CH5O 2.7 33 5 + + H3O CH3OH.H .H2O 2.7 51 5 + + methional O2 C4H8OS 2.5 104 6 methyl butanoic NO+ C H O .NO+ 2.5 132 6 acid 5 11 2 + + methyl hexanoate NO C7H14O2.NO 2.5 160 6 + + methyl salicylate H3O C8H8O3.H 4.5 153 6 (continue)

(Table 7 continue)

+ + nonanal NO C9H17O 2.7 141 6 + + octanal NO C8H15O 3 127 1 phenylacetaldehyd NO+ C H O+ 2.5 120 6 e 8 8 + + propanal NO C3H5O 2.5 57 2 + + propanoic acid O2 C2H5COOH 2.2 74 12 [1] Spanel and others (2002), [2] Spanel and Smith (1997a), [3] Syft (2005), [4] Schoon and others (2007), [5] Spanel and Smith (1997b), [6] Syft (2011), [7] Wang and others (2004a), [8] Wang and others (2003), [10] Spanel and Smith (1998a), [11] Spanel and Smith (1998b), [12] Wang and others (2004b),

Table 8 The effect of 1% pectin and 1%oil on tomato juice viscosity (Pa-s)

1% Oil + 1% Control 1% Pectin Di,Mono glycerides Vicosity (Pa-s) 0.0707 ± 0.0009 0.2991 ± 0.0155 0.1313 ± 0.0022

Table 9 The effect of 10% NaCl and sucrose on tomato juice volatile (percent increase)

Volatile compounds Sucrose Volatile compounds (ppb) (%increase) (ppb) NaCl (% increase) nonanal -57.41 Nonanal -27.76 eugenol -50.43 Decanal -23.14 octanal -48.14 Octanal -19.51 decanal -47.11 methyl hexanoate -19.17 dodecanal -46.03 hexyl acetate -18.11 guaiacol -45.59 dodecanal {SB} -15.87 hexyl acetate -44.60 hexanoic acid -15.59 methyl hexanoate -44.17 (E)-2-nonenal -13.73 (E,Z)-2,6- hexanoic acid -43.20 nonadienal -13.57 (Continue)

(Table 9 continue) (E,Z)-2,6- nonadienal -38.46 eugenol -10.43 (E)-2-octenal -38.13 2-pentylfuran -7.05 2-isobutylthiazole -33.33 guaiacol -6.74 methyl salicylate -32.34 furfural -4.45 phenylacetaldehyd beta-ionone -31.87 e -3.14 (E)-2-nonenal -30.39 (E)-2-heptenal -2.63 1-penten-3-one -30.16 1-penten-3-one -2.19 1-octen-3-ol -29.73 1-octen-3-ol -1.92 methional -29.14 methyl salicylate 0.37 methylbutanoic acid -28.96 cyclic terpenes 1.11 ethyl acetate -28.56 2-isobutylthiazole 1.28 cyclic terpenes -28.53 propanoic acid 2.46 benzyl alcohol -28.51 dimethyl sulfide 2.86 2-pentylfuran -28.21 (E)-2-octenal 3.74 propanoic acid -26.60 2-pentanone 5.48 furfural -25.96 beta-ionone 5.49 propanal -25.61 methylbutanoic acid 5.49 (E)-2-hexenal -25.30 ethyl acetate 8.76 2-pentanone -25.09 isobutanal 9.10 isobutanal -24.68 (E)-2-pentenal 9.70 hexanal -24.35 ethanol 10.25 (Z)-3-hexen-1-ol -23.27 methional 10.29 phenylacetaldehyde -22.90 benzyl alcohol 10.86 benzene ethanol -22.71 (E)-2-hexenal 11.14 1-hexanol -22.47 hexanal 12.62 (E)-2-heptenal -22.18 propanal 15.51 6-methyl-5-hepten- (E)-2-pentenal -21.96 2-one 16.36 benzaldehyde -20.23 benzene ethanol 17.47 ethanol -19.90 (Z)-3-hexenal 19.88 2,3-butanediol -19.51 acetaldehyde 20.41 dimethyl sulfide -18.81 1-hexanol 23.71 dimethyl disulfide -17.42 methanol 23.97 6-methyl-5-hepten- 2-one -17.28 2,3-butanediol 25.57 (Z)-3-hexenal -17.07 (Z)-3-hexen-1-ol 26.99 acetaldehyde -15.60 2,3-butanedione 27.09 2,3-butanedione -13.51 acetone 32.65 acetone -12.97 dimethyl disulfide 39.91 (continue)

(Table 9 continue) 2-pentanol -10.10 3-methylbutanal 40.49 1-propanol -8.78 benzaldehyde 53.13 methanol -8.12 2-pentanol 57.86 3-methylbutanal -0.13 1-propanol 60.62

80 R² = 0.9227 60

-70 -60 -50 -40 -30 -20 -10 0 NaCl (%increase) NaCl -20

-40

-60 Sucrose (%increase)

Figure 4 The % increase correlation of volatile compounds between 10% NaCl and 10% Sucrose

18 a Control 16 5% NaCl b b 14

10% NaCl

8 a a 6 Concentration (ppb) Concentration b a 4 ab b a a a a a 2 a

0 (E)-2-hexenal (Z)-3-hexen-1-ol 2-pentanone beta-ionone methyl salicylate

* NaCl levels for each volatile that do not share a letter are significantly different

Figure 5 The effect of NaCl on important volatiles in tomato juice

a 25 Control

10% 20 a a NaCl a b b 15 b b a

10 b a a a a a a a a a b Concentration (ppb) Concentration 5 a a

* NaCl levels for each volatile that do not share a letter are significantly different

Figure 6 The effect of NaCl on alcohols and ketones in tomato juice

a 70 0% NaCl

b 10% 60 NaCl

50 a 40

b 30 a a b Concentration (ppb) Concentration a a 20 a a a b b b a a 10 a a a a b a a a a a a a a a a a a a a a a 0

* NaCl levels for each volatile that do not share a letter are significantly different

Figure 7 The effect of NaCl on aldehydes in tomato juice

60 a 5°c 50 25°c a

50°c 40

a b

20 b Concentration (ppb) Concentration

10 a c b c c c b 0 (E)-2-hexenal (Z)-3-hexen-1-ol (Z)-3-hexenal hexanal *Temperature levels for each volatile that do not share a letter are significantly different

Figure 8 The effect of temperature on the important tomato volatile levels

600 a 5°c

500 25°

400

300 a 200 Concentration (ppb) Concentration a b 100 b b c c c 0 acetaldehyde dimethyl sulfide 6-methyl-5-hepten-2-one

*Temperature levels for each volatile that do not share a letter are significantly different

Figure 9 The effect of temperature on the important tomato volatile levels

35 a 0% protein 30 b Whey protein isolate

25 Milk protein isolate b Collagen c 20

15 a

ab b ab Concentration (ppb) Concentration 10

a 5 b b b a b b b

0 (E)-2-hexenal (Z)-3-hexenal hexanal (Z)-3-hexen-1-ol *Protein levels for each volatile that do not share a letter are significantly different

Figure 10 The effect of protein on (E)-2-hexenal, (Z)-3-hexenal, hexanal, and (Z)-3- hexen-1-ol level

70 0% protein a a a 60 Whey protein isolate b

50 Milk protein isolate Collagen 40 a ab 30 ab b a Concentration (ppb) Concentration 20 bc c bc a a a a a 10 b b b a b b b

* Protein levels for each volatile that do not share a letter are significantly different

Figure 11 The effect of protein on (E)-2-pentenal, furfural, 1-penten-3-one, acetaldehyde, benzaldehyde, and 6-methyl-5-hepten-2-one

25 a 0% glucose 5% 20 ab glucose 10%

b glucose

15 a

b b

10 Concemtration (ppb) Concemtration a 5 b b a b b

0 (E)-2-hexenal (Z)-3-hexenal hexanal (Z)-3-hexen-1-ol * Sucrose levels for each volatile that do not share a letter are significantly different

Figure 12 The effect of sucrose (E)-2-hexenal, (Z)-3-hexenal, hexanal, and (Z)-3- hexen-1-ol level

70 0% glucose a a 60 5% glucose

b 50 10% glucose 40 a a 30 a a a

Concentration(ppb) 20 b b b b a a ab b 10 b b a b b 0

* Sucrose levels for each volatile that do not share a letter are significantly different

Figure 13 The effect of sucrose on 6-methyl-5-hepten-2-one, acetaldehyde, 2- isobutylthiazole, benzaldehyde, phenylacetaldehyde, furfural and (E)-2-pentenal levels

25 Control a a a 20 5% Oil+ 1%T,DMG 10% Oil+ 1%T,DMG 15 a

b b Concentration (ppb) Concentration a b 5 a b b b

0 (E)-2-hexenal (Z)-3-hexenal Hexanal (Z)-3-hexen-1-ol

*Oil levels for each volatile that do not share a letter are significantly different

Figure 14 The effect of oil on (E)-2-hexenal, (Z)-3-hexenal, hexanal, (Z)-3-hexen-1-ol and levels

70 0% Oil a a 60 a 5% Oil+ 50 1%T,DMG 10% Oil+ 40 a 1%T,DMG 30 a 20 b a b b b b b a Concentration (ppb) Concentration 10 b b a ab b a b b 0

* Oil levels for each volatile that do not share a letter are significantly different

Figure 15 The effect of oil on 6-methyl-5-hepten-2-one, furfural, acetaldehyde, benzaldehyde, 2-isobutylthiazole, 1-penten-3-one, and (E)-2-pentenal levels