Analysis of Strawberry Volatiles in Different Hydrocolloids and Different

Analysis of strawberry volatiles in different hydrocolloids and different

conditions using Selected Ion Flow Tube – Mass Spectrometry

THESIS

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

Yachen Zhang

Graduate Program in Food Science and Technology The Ohio State University 2016

Master's Examination Committee:

Dr. Sheryl Barringer, Advisor

Dr. Dennis Heldman

Dr. Christopher Simons

Yachen Zhang

2016

ABSTRACT

Hydrocolloids and additives in gummy candies bind flavors, thus it is important to know how these additives affect flavor release. Selected Ion Flow Tube- Mass Spectrometry

(SIFT-MS) was used to perform static headspace and mouthspace tests. The release of strawberry flavor in different hydrocolloids (gelatin, pectin, and starch) and conditions were analyzed. The factors that were considered were the type of hydrocolloid (gelatin, pectin, and starch), the concentration of the pectin (0, 2, 3, 5 g), sugar content (0, 55, 64,

74g), and acidity (pH 3.86, 3.65, 3.55, 3.47). Volatile release into the headspace of the samples containing no hydrocolloids was significantly higher than samples that contained hydrocolloids. The type of hydrocolloid significantly affected volatile compound concentration released into the headspace. Volatile levels in pectin and starch were lower than when no hydrocolloid was present, but they were not significant different with each other. Gelatin had the lowest volatile concentrations released into the headspace for most compounds. Increasing pectin decreased volatiles release compared to no hydrocolloids present. When the pectin content was further increased from 2g to 5g, most of volatiles had no significant difference. It may be because of the plateau value being reached or the amount pectin added was not sensitive enough to influence further volatile release. Sugar had the greatest effect on volatile release. Increasing sugar content from 0g to 55g caused the amount of volatiles released to drop significantly. This may be due to sugar-water

ii interactions; however, further increasing sugar from 55g to 74g produced no significant difference in the amount of volatiles released. This may be because the amount of volatiles reached a maximum release above certain amount of sugar. pH 3.86 had the greatest volatile release for most volatiles, in part because pectin didn’t form a gel at that pH. A high concentration of citric acid (from pH 3.65 to 3.47) decreased the release of volatiles. A reason for this may be because large amounts of the dissociated forms were present to interact with volatile compounds as pH was decreased. Hardness alone had no significant effect on flavor release. The mouthspace test yielded that the amount of volatiles being released from different types of hydrocolloid gummy gels did not experience a significant difference. Sensitivity of the mouthspace test might not be as sensitive as the headspace test, which lead to the differences being negligible.

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Practical Application

Strawberry gummy candy is very popular. Different recipes can influence the volatile release. The results of this study could be applied to obtain the best flavor release of strawberry flavored candies, or to further research on how hydrocolloids react with strawberry flavors. Pectin, starch, and gelatin have different influence on flavor and texture. Companies can choose different ingredients to make suitable gummy candies based on consumer needs. Gelatin can improve the texture of gummy candies, but it released the lower amount of volatiles than pectin or starch. Pectin has good flavor release and tastes better than starch. Due to this reason, pectin is the most ideal hydrocolloid to use in the confectionary industry to create gummy candy. Sucrose is another important ingredient in the making of gummy candies, but adding more than 55g of sucrose did not have any significance on the amount of volatiles released. Decreasing pH decreased volatiles release. This research demonstrated how physical and chemical properties of hydrocolloids could be related to strawberry volatiles release. The best condition recommended from this study is 5g pectin with 74g sugar in pH 3.65, which achieved the largest strawberry volatile release.

Dedicated to my family and friends

ACKNOWLEDGEMENTS

Firstly, I would like to thank my parents Dan Lu and Desheng Zhang to support me to go abroad to study. I really appreciated what they did for me. I also want to thank my husband, Mark Umpenhour, he always encourages me to try something new. He gave me a family in America. My baby brother, Kevin Lu brought a lot of fun to my family.

My friends, Xiaohu Hu, Hao Wang, Congcong Zhang and Xuequn Ren, they are my best friend in the food science and technology department at OSU. They gave me a lot of guides and suggestions about my study.

Second, I would like to thank my advisor Dr. Sheryl Barringer. She is a super nice person to her students. Because of her, I had an opportunity to come to U.S.A. to study. I learnt a lot from her, it is not just from the course she taught but also how to finish an assignment efficiently and how to solve problems from different sides. Meanwhile, she taught me how to think and how to write a scientific paper.

I want to say thank you to my committee members Dr. Heldman, Dennis, Dr.

Simons, Christopher. They supported and guided me about my research.

I also thank the food science and technology department at OSU, including all professors, staff and students. I met a lot of friends here, and they help me to get through many difficulties.

VITA

JULY 1991 ...... BORN, BEIJING, CHINA SEPTEMBER 2006 ...... BEIJING N0.41 HIGH SCHOOL September 2009 ...... B.S. Food Quality and Safety, Jinan University

FIELDS OF STUDY

Major Field: Food Science and Technology

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Table of Contents

ABSTRACT ...... II PRACTICAL APPLICATION ...... IV ACKNOWLEDGEMENTS ...... VI VITA ...... VII FIELDS OF STUDY ...... VII TABLE OF CONTENTS ...... VIII LIST OF TABLES ...... X LIST OF FIGURES ...... XI CHAPTER 1: INTRODUCTION ...... 1 CHAPTER 2: LITERATURE REVIEW ...... 4 2.1 STRAWBERRY AND STRAWBERRY FLAVOR ...... 4 2.1.1 STRAWBERRY CHEMISTRY ...... 5 2.1.2 BIOSYNTHESIS OF STRAWBERRY VOLATILES ...... 7 2.1.3 FORMATION OF ESTERS FROM ALDEHYDE VIA ALCOHOL ...... 8 2.2. THE INTRODUCTION OF PECTIN, GELATIN AND STARCH ...... 11 2.2.1 PECTIN ...... 11 2.2.2 GELATIN ...... 12 2.2.3 STARCH ...... 12 2.3. THE EFFECTS OF DIFFERENT HYDROCOLLOIDS ON FLAVOR RELEASE ...... 13 2.3.1 CHEMICAL BINDING ...... 13 2.3.2 PHYSICAL ENTRAPMENT ...... 15 2.3.3 THE EFFECT OF TEXTURE ON FLAVOR RELEASE ...... 18 2.4 THE EFFECTS OF CHANGING HYDROCOLLOID CONCENTRATION ON FLAVOR RELEASE 21 2.5 THE EFFECTS OF DIFFERENT AMOUNT OF SUGAR ON FLAVOR RELEASE ...... 23 2.6 THE EFFECTS OF DIFFERENT ACIDITY ON FLAVOR RELEASE ...... 25 2.7 THE EFFECTS OF SALIVA ON FLAVOR RELEASE ...... 26 2.8 SELECTED ION FLOW TUBE – MASS SPECTROMETRY (SIFT-MS) ...... 28 viii

2.8.1 PRINCIPLES OF SIFT-MS ...... 28 2.8.2 MOUTHSPACE ...... 29 CHAPTER 3: MATERIALS AND METHODS ...... 31 3.1 MATERIALS ...... 31 3.2 GEL PREPARATION ...... 32 3.3 HEADSPACE MEASUREMENTS ...... 33 3.4 BREATH MEASUREMENTS ...... 34 3.5 SIFT-MS INFORMATION ...... 34 3.6 TEXTURE ANALYSIS ...... 35 3.7 STATISTICAL ANALYSIS ...... 35 CHAPTER 4: RESULTS AND DISCUSSION ...... 37 4.1 EFFECT OF TYPE OF HYDROCOLLOID ON VOLATILE RELEASE ...... 37 4.2 EFFECT OF PECTIN CONCENTRATION ON VOLATILE RELEASE ...... 41 4.3 EFFECT OF SUGAR CONCENTRATION ON VOLATILE RELEASE ...... 43 4.4 EFFECT OF PH ON VOLATILE RELEASE ...... 46 4.5 MOUTHSPACE ...... 48 4.6 HARDNESS ...... 51 CHAPTER 5: CONCLUSIONS ...... 53 REFERENCES ...... 54 APPENDİX: ADDITIONAL VOLATILE INFORMATION ...... 72

LIST OF TABLES

TABLE 1 STRAWBERRY AROMA COMPOUNDS THRESHOLDS AND ODOUR CHARACTERIZATION FROM LITERATURE REVIEW ...... 10 TABLE 2 COMPOSITION OF THE DIFFERENT GUMMY GELS PER 100G ...... 32 TABLE 3 IMPORTANT STRAWBERRY VOLATILES, REAGENT IONS AND MASSES OF THE COMPOUNDS USED IN THIS STUDY ...... 36 TABLE 4 THE RELATIONSHIP BETWEEN HARDNESS AND VOLATILES CONCENTRATION ...... 52 TABLE 5 THE PARAMETERS OF THE SIFT/MS MEASUREMENTS ...... 72 TABLE 6 THE EFFECT OF THREE TYPES OF HYDROCOLLOIDS (GELATIN, PECTIN, AND STARCH) ON HEADSPACE CONCENTRATION OF VOLATILES ...... 73 TABLE 7 THE EFFECT OF PECTIN CONCENTRATION ON HEADSPACE CONCENTRATION OF VOLATILES ...... 74 TABLE 8 THE EFFECT OF SUGAR CONCENTRATION ON HEADSPACE CONCENTRATION OF VOLATILES ...... 75 TABLE 9 THE EFFECT OF ACIDITY ON HEADSPACE CONCENTRATION OF PECTIN GELS VOLATILES ...... 76 TABLE 10 THE EFFECT OF THREE TYPES OF HYDROCOLLOIDS (GELATIN, PECTIN, AND STARCH) ON HEADSPACE CONCENTRATION OF HYDROCOLLOIDS GELS VOLATILES IN MOUTHSPACE ...... 77

LIST OF FIGURES

FIGURE 1 SHARE OF WORLD EXPORTS AND PRODUCTION OF STRAWBERRY BY COUNTRY 2004 ...... 5 FIGURE 2 REPEATING STRUCTURE IN GELATIN RESPONSIBLE FOR TRIPLE HELIX STRUCTURE ...... 17 FIGURE 3 SIFT-MS CONFIGURATION FOR HEADSPACE (LEFT) AND MOUTHSPACE (RIGHT) MEASUREMENTS ...... 34 FIGURE 4 CONCENTRATION OF IMPORTANT VOLATILES IN NO HYDROCOLLOIDS AND THREE HYDROCOLLOIDS. *CONCENTRATION FOR EACH VOLATILE THAT DOES NOT SHARE A LETTER IS SIGNIFICANTLY DIFFERENT ...... 38 FIGURE 5 CONCENTRATION OF VOLATILES IN DIFFERENT AMOUNT OF PECTIN *CONCENTRATION FOR EACH VOLATILE THAT DOES NOT SHARE A LETTER IS SIGNIFICANTLY DIFFERENT ...... 42 FIGURE 6 CONCENTRATION OF VOLATILES IN DIFFERENT AMOUNT OF SUGAR *CONCENTRATION FOR EACH VOLATILE THAT DOES NOT SHARE A LETTER IS SIGNIFICANTLY DIFFERENT ...... 45 FIGURE 7 CONCENTRATION OF VOLATILES IN DIFFERENT PH *CONCENTRATION FOR EACH VOLATILE THAT DOES NOT SHARE A LETTER IS SIGNIFICANTLY DIFFERENT ...... 47 FIGURE 8 CONCENTRATION OF IMPORTANT VOLATILES IN DIFFERENT HYDROCOLLOIDS IN MOUTHSPACE TEST *CONCENTRATION FOR EACH VOLATILE THAT DOES NOT SHARE A LETTER IS SIGNIFICANTLY DIFFERENT ...... 49 FIGURE 9 CONCENTRATION OF VOLATILES VS. HARDNESS IN DIFFERENT HYDROCOLLOIDS (PECTIN, STARCH AND GELATIN) ...... 78 FIGURE 10 CONCENTRATION OF VOLATILES VS. HARDNESS IN DIFFERENT AMOUNT OF PECTIN (2-5G) ...... 78 FIGURE 11 CONCENTRATION OF VOLATILES VS. HARDNESS IN DIFFERENT AMOUNT OF SUGAR (55G-74G) ...... 79 FIGURE 12 CONCENTRATION OF VOLATILES VS. HARDNESS IN DIFFERENT PH (3. 65-3.47) 79 FIGURE 13 CONCENTRATION OF METHYL BUTANOATE VS. HARDNESS ...... 80 FIGURE 14 CONCENTRATION OF ETHYL HEXANOATE VS. HARDNESS ...... 80

Chapter 1: INTRODUCTION

Strawberry is a popular fruit after bananas, apples, oranges, and grapes (Ozcan and Barringer 2011). Strawberry flavor is distinct, unique, and commonly used in yogurt, jam, candy, soft drinks, and so on. Flavor is the mixture of taste and odor, which is important for the consumer perception of a product (Pyssalo and others 1979). There are more than 360 volatile chemicals that are composed of alcohols, esters, aldehydes, ketones, and furanones compounds identified in strawberry flavor (Larsen and Poll

1992).

The variation of ingredients in gummy candies made from hydrocolloid gels is due to the desire to improve the flavor. Consequently, acidity, sugar, and amount of hydrocolloid are also varied. Gelatin, starch, and pectin have been widely used as a forming agent. Altering these additives can lead to different levels of aroma volatiles being released.

Physical entrapment and chemical binding both influence flavor release (Boland and others 2006). Hydrocolloids like gelatin, pectin, starch all decrease the concentration of flavor release in foods (Boland and others 2004; Boland and others 2006; Guinard and

Marty 1995). Entangled polymer networks in hydrocolloids inhibit the transportation of small molecules, so volatiles are entrapped in the system (Baines and Morris 1987; Hau

1 and others 1998). The chemical binding of volatiles by hydrocolloids can also decrease flavor release (Pangborn and Lundgren 1978). The hydrophobic and hydrophilic parts in hydrocolloids can interact with volatiles (Golovnya and others 2001). The triple helical structure in gelatin can have cross-linking and three- dimensional network formation, which can entrap volatiles. Pectin also can entrap flavor chemicals in the food system, react with volatiles, or form hydrophobic micelles to capture hydrophobic compounds

(Chinachoti 1995). The flavor can be kept in the amylose helix in starch by hydrophobic bonding (Boutboul and others 2000). The interaction between hydroxyl groups of starch and flavor through hydrogen bonding also explained why flavor interact with starch.

The greater the concentration of pectin the more binding sites are present to bind with volatile compounds in the gel (Hansson and others 2001b). Increasing amount of pectin provide a barrier for diffusing flavor and interact with flavor (Lubbers and

Guichard 2003). Increasing the concentration of hydrocolloids can increase the concentration of volatiles (Golovnya and others 2001). The addition of pectin from 0,

0.05, 0.1, 0.2 to 0.4% of jam can cause a significant decrease the concentration of most volatile compounds in jam (Guichard and others 1991).

When sugar is added, the pectin precipitates out and forms insoluble fibers

(Hansson and others 2001b). The insoluble fibers produce a mesh-like structure that traps the volatiles enabling a gel to form. Adding sugar allows pectin chains to force together to form a strong network system based on hydrophobic interactions, which decrease flavor release (Hershko and Nussinovitch 1998). The increasing sugar concentration from

0 to 60% w/v in an aqueous sugar solution in the gel leads to a decreased release of the volatiles (ethyl acetate, methyl butanoate, ethyl butanoate, hexanal, and octanal) in short gas chromatography/ flame ionisation detection (GC/FID) (Nahon and others 1998).

When the pH is lowered, more dissociated forms will be in the acid (Van Ruth and Roozen 2000). The dissociated form attracted more to react with volatile compounds than non-dissociated forms (Hansson and others 2001a). Therefore, more volatiles release at higher pH compared to lower pH (Hansson and others 2001a). When organic acids added into fruit nectars, the sensory scores of flavor were increased (Valdes and others

1956) and high concentrations (0g, 0.002, 0.1 and 1g) of citric acids decreased the concentration of esters (Hansson and others 2001a).

The objective of this study was to investigate the volatile change in gummy candies with gelatin, pectin and starch, the concentrations of pectin, sugar content, and acidity in the headspace, to know volatile compounds concentration changes in the mouthspace during chewing. In this study, the most important strawberry volatiles- esters, alcohols, aldehydes, ketones and acids were chosen to be analyze how strawberry flavorchanged in different hydrocolloids and conditions.

Chapter 2: LITERATURE REVIEW

2.1 Strawberry and strawberry flavor Strawberries in the market are hybrids of the large and aromatic Fragaria chiloensis (Whitaker and others 2011). Strawberries are the fourth highest ranked U.S. fruit production value just behind grapes, oranges, and apples (Boriss and others 2006)

For demands, strawberries experienced the increasing consumption of all fruits for over the last two decades. The consumption of strawberries are the fifth highest in fresh fruits in U.S just behind bananas, apples, oranges and grapes (Boriss and others 2006). For production, the production of strawberry in the United States is the world’s largest, which has 28.3 percent of world supply in 2004 followed by Spain, Russia, Korea, Japan and

Poland (Food and Agricultural Organization of the United Nations (FAO) (Figure 1)

(Boriss and others 2006)

Source: Food and Agricultural Organization of the United Nations

Figure 1 Share of World Exports and Production of Strawberry by Country 2004 Strawberry flavor is very popular flavoring added in the food manufacture industry. However, strawberry flavor is very complex. Although there are plenty of volatiles compounds in the strawberry, only certain chemicals contribute to make the strawberry flavor and aroma. Carbohydrates, esters, and furanones are most important classes in strawberry flavor (Bood and others 2002). 50% of strawberry volatiles are in esters, alcohols, aldehydes, carbonyls and acid. Both C10 monoterpenes and C15 sesquiterpenes normally comprise <10% and sulfur compounds <2% contribute to the volatile in wild and cultivated strawberry species (Schreier 1980).

2.1.1 Strawberry Chemistry

There are more than 360 volatile flavors in strawberry flavor (Pino and others

2001; Forney and Jordan 1995). Carbon compounds mostly come from sugars, which are abundant nonvolatile volatiles in strawberry fruits. 99% of total sugars in the strawberry flavor include sucrose, fructose, and glucose (Bood and others 2002). The minor carbohydrates in strawberries are inositol and sorbitol (Makinen and Soderling 1980).

The soluble sugars increase the carbon compounds available to produce aroma compounds.

There are considerable amounts of volatiles that contribute to producing the obvious strawberry aroma, aldehydes and esters (Montero and others 1996). Esters and furanones are important strawberry volatiles. The high amounts of methyl acetate and methyl butanoate were contributed to the strawberry flavor in raw strawberries

(Azodanlou and others 2004). Esters are the most plentiful volatile compounds in strawberry flavors, which comprised from 25% to 90% of strawberry volatiles, 131 different types esters are found in strawberry flavors (Perkins and others 1992). Propyl butanoate, 3-phenyl-1-propanol, butyl butanoate, isobutyl butanoate, 3-methyl butyl butanoate and isopropyl hexanoate were predominant in the strawberry flavor

(Azodanlou and others 2004).

Aldehydes are one of the important volatile flavors in fresh strawberry flavor.

During ripening, the C6 aldehydes can be formed through lipoxygenase (LOX) and hydroperoxidelyase (HPL) enzyme activity in the lipid oxidation pathway (Ozcan and

Barringer 2011). The alcohol can be formed from aldehydes through enzymatic 6 biosynthesis. However alcohol doesn’t have a big influence on strawberry aroma and flavor (Olias and others 2002).

Although the content of 2,5-dimethyl-4-hydroxy-3 (2H)-furanone (DMHF) is tiny, it is important to contribute overall strawberry flavor (Roscher and others 1996).

The low threshold value in water is 4 × 10–5 mg/kg. There are four forms of DMHF,

DMHF-glucoside, mesifuran, DMHF-malonyl-glucoside, and the free aglycone DMHF

(Bood and others 2002). Each of them plays a very important role to provide a distinctive strawberry flavor. The first two compounds are impact volatile compounds, which are demonstrated by other authors (Sanz and others 1995). However, DMHF-glucoside and

DMHF-malonyl-glucoside are not volatiles, but they contributed to the strawberry flavor release (Schwab 1998).

2.1.2 Biosynthesis of Strawberry Volatiles The volatile compound of strawberry is very complicated. The main volatile compounds in strawberry flavor: 8 aldehydes, 40 alcohols and 91 esters (Yamashita and others 1977; Zabetakis and others 1999b). During incubation, most aldehyde and fatty acid convert to ester and alcohols, like acetaldehyde to ethyl acetate, butanal to I-butyl acetate, hexanal to hexanol (Yamashita and others 1977). The important seven chemical compounds within fresh fruit are acetaldehyde, propanal, 2-methylpropanal, butanal, 3- methylbutanal, pentanal, hexanal (Yamashita and others 1977; Perez and others 1992).

The corresponding alcohols produced were changed to their esters (Perez and others

1996a).

2.1.3 Formation of Esters from Aldehyde Via Alcohol The most important groups of volatiles in strawberry are esters. The most plentiful esters quantified by GC-MS are methyl and ethyl butanoates, ethyl hexanoate, hexyl acetate, and trans-2-hexenyl acetate (Yamashita and others 1976). The formations of esters are based on enzyme alcohol acyltransferase (AAT), which accelerated the transformation of an acyl moiety of an acyl CoA on the corresponding alcohol

(Yamashita and others 1996).

The branched chain amino acids synthesize esters (Yamashita and others 1996;

Pyssalo and others 1979). The enzymes used to form ester include a- aminotransferase, a- ketoacid decarboxylase, a-ketodehy- drogenase, alcohol dehydrogenase (ADH), and alcohol acyltransferase (AAT). Amino acid plays the role as precursors, which determine strawberry flavor development of ester. All the alcohols presented as acetate esters in strawberry flavor. The enzyme ATT is an important enzyme to react with alcohols to change to esters (Zabetakis and others 1997). Alcohols and carboxylic acids cause esterification to form esters, which constitute the major group of volatiles in fruit flavor

(Olias and others 2002).

Cis-3-hexenal and trans-2-hexenal were faster than the formation of hexanal in the pureed strawberries (Ozcan and Barringer 2011). Different processing can have a big impact on volatiles formation (Couey and others 1966). In refrigerated storage, there was a significant increase in LOX-derived aldehydes and fruity esters (Ozcan and Barringer

2011). Chewing can help LOX- derived volatiles to form in the mouth. Volatiles

8 compounds can be ingested while eating food (Ke and others 1991). The volatile compounds would be released during the chewing process, so the concentration of those compounds was much lower in the nosespace (Bood and others 2002).

Furaneol and mesifurane provide sweet caramel-like aroma in the fruit (Sanz and others 1995). Two compounds 2, s-dimethyl-4-hydroxy-3 (2H)-furanone (Furaneol), and dimethyl-4-hydroxy-3(2H)-furanone (Mesifurane) are most important aroma constituent of strawberries so far in wild strawberries (Pyysalo and others 1979). 2,5-dimethyl-4- methoxy-3(2H)-furanone (odor threshold value 0.01 mg/L, Mette and others 1992) is a important volatile compound in wild strawberries (Given and others 1988a). 2,5- dimethyl-4-methoxy-3 (2H)-furanone is a important contributor to the aroma of the berries in the sensory test (Larsen and Poll 1992).

Although there are plenty of volatiles compounds in the strawberry, only certain chemicals contribute to make the strawberry flavor and aroma. Alcohols and carbonyl compounds and esters are important to wild strawberry aroma. The threshold values were presented with odor characteristics and the compounds divided into four groups. (Table

1) (Pyysalo and others 1979).

Table 1 Strawberry aroma compounds thresholds and Odour characterization from literature review

Odour threshold Odor characteristic Reference decade interval (mg/kg) Ester ethyl acetate 0.1-1.0 contact glue a,b,d,e,g,h methyl butanoate 0.001-0.01 fruity,cheese a,c,f 0.000001- ethyl butanoate fruity,sweet,cheese a,c,d 0.00001 methyl hexanoate 0.01-0.1 fruity a,b,d,g ethyl hexanoate 0.00001-0.0001 fruity,fruit gum a,b,d,g butyl acetate 0.01-0.1 apple and glue a,b,c,d,e,f,g isopropyl butanoate 0.01-0.1 pungent a Alcohol linalool 0.0001-0.001 lemon peel,flowers a,b,d,f,g,h (+/-)-nerolidol 0.01-0.1 fir,pine,linoleum a,d,f 1-hexanol 0.01-0.1 green,heavy,nuts a,d,e,f (E)-2-hexen-1-ol 0.1-1.0 green,fruity,burnt a,c,f,h Aldehyde Hexanal 0.01-0.1 green,sour a,d,f (E)-2-hexenal 0.01-0.1 green,grass,almond a Hexanal 0.01-0.1 kerosene,solvents a,c,f (E)-2-hexenal 0.001-0.01 danish blue cheese a,c,d Acid acetic acid 10-100 vinegar a,b,h propanoic acid 0.1-1.0 fruity,silage a hexanoic acid 1.0-10 stuffy,sour,sweet a,d,f,h 2-methylbutanoic acid 0.01-0.1 fruity,sourish a Furaneol 0.001-0.01 burnt,sweet,caramel a,b,d a Mette and others 1992, b Williams and others 2004, c Hakala and others 2000, d Lambert and others 1999, e Yamashita and others 1976, f Egea and others 2014, gBlanch and others 1970, hYun-Tao and others 2009

The highest proportions were measured for acetone and 2,5-dimethyl-4-hydroxy-3

(2H)-furanone (Sanz and others 1995). The most important compounds of cultivated berries were ethyl butanoate, ethyl hexanoate and 2,5-dimethyl-4-hy-droxy-3 (2H)- furanone (Larsen and Poll 1992; Zabetakis and others 1999b). The threshold values of some compounds are low, but they had some influence on the odor, which include methyl butanoate, linalool, 2-heptanone and to a lesser extent 2-methyl butanoic acid (Larsen and

Poll 1992). Methyl and ethyl butanoates and ethyl 2-methyl butanoate are important contributors in strawberry (Ulrich and others 1997).

2.2. The introduction of pectin, gelatin and starch

2.2.1 Pectin Pectin is an important natural gelling polysaccharide, and is most likely obtained from citrus peel, apple (Lubbers and Guichard 2003) or lime peel (according to Hercules

Copenhagen A/S, Lille Skensved, Denmark). The texture of pectin-based gummy candies is soft and less chewy than gelatin-based (Boland and others 2004). Pectin is a common gel polysaccharides used in food product (Barfod and Pedersen 1990). Pectin relies on many factors including pH and sugar content, because its carbohydrate molecules need help to form a bond. Pectin can be divided into two groups depending on the degree of esterification (DE): high methyoxyl (HM) pectin and low methyoxyl (LM) pectin. Pectin is a natural polysaccharide. The individual pectin stick together due to hydrogen-bonds and hydrophobic interactions (Dickinson 1995). Low methoxylated pectin (LMP) is used in relative lower sugar content food. High methoxylated pectin (HMP) can form gels with sugar and acids. They need at least 55grams of sugar, which binds up most of the water 11 molecules, and then pH less than 3.5, which can reverse the electrical charge that ordinarily makes them repel each other. High methoxyl pectin is mainly used to make candies, which form gels in the pH range of 3.0 - 3.6. Normally, the concentration of sugar by weight is greater than 55% (Oakenfull 1987). The hydrophobic part to the free energy of formation of junction zones in typical HM pectin is -18.6 kJ/mol while the hydrogen bonding is -37.5 kJ/ mol. Hydrogen bonds are not enough to offset the entropic barrier which is +41.1kJ/mol (Oakenfull and Scott 1984).

2.2.2 Gelatin Gelatin, which is rich in collagen of animals, likes bovine hides, and cattle bones

(Bakker and others 1998). The application of gelatin includes desserts, yoghurts, and gummy candies among others. Because of its gelatinizing properties, gelatin is good for its unique flavor and special texture (Palazzo and Bolini 2009). Gelatin can be added into almost any solution, as long as the liquid and gelatin are heated and dissolved together

(Bakker and others 1998). Almost half of all gelatin products in the world were used to make gummy candies (Gelatin Manufacturers Institute of America Inc. 1993). The gelatin is very flexible. With the appropriate portion, gelatin can be changed into different texture and shape. Gelatin is a protein that can form a gel with different texture.

2.2.3 Starch Starch includes high amount of branched amylopectin molecules and linear amylose molecules (Chinachoti 1995; Boutboul and others 2000).). Starch is popular to use in the food industry to entrap and support volatile compounds. There are several main types of starches used in confectionary, including thin-boiling (acid-thinned) starches, 12 high amylose cornstarch and starch-hydrolysis products, such as low-DE maltodextrins etc. starch can entrap molecules as carriers and stabilize the system (Boutboul and others

2000). The starch-based jelly had for many years been a very common and popular candy in the United States, well known for its unique and well-known texture (Boutboul and others 2000). Inclusion of starch with different volatiles was studied by amperometric iodine titration (Nuessli and others 1997), X-ray diffraction and differential scanning calorimetry (Nuessli and others 1997).

2.3. The effects of different hydrocolloids on flavor release Interactions between flavor and food components are very important for perception of flavor (Franzen and Kinsella 1974). There are two mechanisms that can explain how hydrocolloids effect on volatiles release. The first mechanism is the chemical binding between volatiles and the gel components, e.g. starch, gelatin and pectin (Bakker and others 1998; Hansson and others 2001b; Boland and others 2004).

The second is the physical entrapment of volatiles compounds within the hydrocolloid system. The entangled three-dimensional network in the hydrocolloids systems prevents the transition of flavor volatiles into air (Boland and others 2004).

2.3.1 Chemical binding Both pectin and starch contain polysaccharides. One of important function of polysaccharides is binding volatiles compounds. Binding of volatiles includes hydrophobic interactions and hydrogen bonding and also influenced by the type of the hydrocolloids. Changing position of the functional group can influence binding ability.

For example, the keto group shifted in octanone from position 2 to 3 leads to a decrease 13 binding with starch. When the keto group changed to position 4, the binding with starch was enhanced (Golovnya and others 2001).

The interactions of the flavor compounds with the food matrix can cause flavor release to decrease (Yven and others 1998). The effect of gelatin concentration on the release of diacetyl in solutions was investigated by using headspace analysis. The release of diacetyl decreased when gelatin increased from 0 to 20% w/v, indicating an interaction between gelatin and diacetyl (Bakker and others 1998).

Pectin contain a linear chain of galacturonic acid units, the molecular weights of pectin is around 110,000–150,000 (Burey and others 2008). For high methyoxyl pectin, the stretching of pectin molecules with water form micelles, which are more hydrophobic

(1995). The more nonpolar compound may be entrapped in the hydrophobic parts of the pectin (Hansson and others 2001b).

The influence of pectin concentration on flavor release and the sensory perception of five volatiles compounds, isopentyl acetate, cis-3-hexenyl acetate, ethyl hexanoate, L- menthone, linalool in fruit pastille model systems was inverstigated. The concentration of isopentyl acetate, cis-3-hexenyl acetate, ethyl hexanoate, L-menthone, linalool decreased in 2.5% pectin in the fruit pastille model systems compared with no pectin because they are more hydrophobic (Hansson and others 2001b).

The release of 11 flavor compounds (diacetyl, 2-butanone, ethyl acetate, 1- butanol, 3-methyl-1-butanol, ethyl butyrate, hexanal, 2-heptanone, heptanal, 2-octanone

14 and 2-decanone, 10 uL kg-1) from starch and pectin was investigated using model mouth/proton transfer reaction-mass spectrometry analysis. There was greater release of hydrophilic flavor compounds (Diacetyl, 2-Butanone, Ethyl acetate) in pectin than starch at the same concentration in the model gel system (Boland and others 2004).

Starch contains linear amylose and highly branched amylopectin (Rutschmann and Solms 1990). There are two types of starch-volatile interaction. The first one involves flavor is captured in the amylose helix by hydrophobic bonding (Boland and others 2004). It is called as an inclusion complex. Amylose binds flavor compounds by formation of inclusion complexes. The second one is interaction between hydroxyl groups of starch and flavor through hydrogen bonding. It explains the mechanism of why flavor compounds have retention in starch gel (Chinachoti 1995). Interaction between hydroxyl groups of starch and flavor through hydrogen bonding constitutes the most probable retention for volatiles in starch gels (Arvisenet and others 2002). More hydrophilic compounds could react with hydrophilic parts into the starch network (Hau and others 1998). Starch and pectin gels were compared in a model mouth test. 2- octanone and 2-decanone in the model mouth analysis, which are hydrophobic volatiles, had lower release in the pectin than starch (Boutboul and others 2000).

2.3.2 Physical entrapment The entanglement of the polygalacturonic chains in pectin entrapped volatiles in the macromolecule network, which decrease volatiles release (Rega and others 2002).

The physical structure of pectin is a three-dimensional network of cross-linked polymer

15 molecules with consists of junction zones (Hwang and Kokini 1992; Lotzkar and others

1946). Most volatiles are hydrophobic, which can bind hydrophobic parts in the pectin.

Hydrogen bonds exist between functional oxygen atoms in the pectin system and bind hydrophilic volatiles (Thakur and others 1997).

Temperature, pH, and pectin concentration affects the firmness of the gel and its structure development, and the sugar used (Demarty and others 1984). Comparing 0.7,

0.25, 0.10 and 0.05% of pectin, the 2.5% of pectin into the soft drink-related model system decrease the release of limonene (Hansson and others 2001b). The compounds 2- acetyl pyridine, 2,3-diethyl pyrazine, 2-acetyl thiophene and 2-ketones in solutions of high-esterified pectinates at concentrations (10–3 % v/v) were analyzed using direct gas chromatographic analysis. The concentration of all volatiles decreased in the high- esterified pectinates through physical and chemical interaction (Braudo and others 2000).

Gelatin gels are proteins, which are more like polysaccharide gels than those formed by heat-setting globular proteins (Clark and others 1990). The formation of gelatin is the ordered quasi-crystalline triple-helical junction zones by flexible regions form at low temperature. The volatiles are entrapped within the network system (Franzen and Kinsella 1974). Gelatin had repeating glycine-X-Y triplets, X and Y are likely proline and hydroxyproline amino acids (Figure 2). The repeating sequences form triple helical structure in gelatin. The triple helical segments form a three- dimensional network system, which can entrap volatiles (Clark and others 1990).

Figure 2 Repeating structure in gelatin responsible for triple helix structure The release of 11 flavor compounds (diacetyl, 2-butanone, ethyl acetate, 1- butanol, 3-methyl-1-butanol, ethyl butyrate, hexanal, 2-heptanone, heptanal, 2-octanone and 2-decanone, 10 uL kg-1) from three hydrocolloids (pectin, starch and gelatin) was investigated using model mouth/proton transfer reaction-mass spectrometry analysis.

Overall, the release of 11 flavor compounds was significantly decreased in the gelatin gel compared to starch and pectin, which had no significant difference (Boland and others

2004).

Starch form complexes with different functional groups, molecular sizes, and polar and nonpolar molecules. The inclusion complexes can entrap small molecules like volatile compounds in the center by physical capture (Hau and others 1998). The volatiles ketone, aldehyde, ester, alkane, alcohol and fatty acid (total 10ml) in wheat starch gel

(6g) were investigated by using inverse gas chromatography. The result showed a similar

17 binding trend for most volatiles, with 1-hexanol had lower release in starch than ethyl acetate (Hau and others 1998).

The effect of polymer concentration (high, medium and low mol. wt) on volatile release in gels has been investigated using solutions incorporating different concentration of guar gum with fixed sugar and acid, with intrinsic viscosity values of 16.7, 8.4 and

5.04 dl/g, respectively. At lower concentrations guar gum has no significant effect on perception of sweetness or flavor but at higher concentrations, where the chains are forced together to form an entangled system, the perceived intensity of attributes decreases significantly with increasing guar gum concentration (Baines and Morris and others 1987).

2.3.3 The effect of texture on flavor release The firmness of the gel system must be considered as it will have an influence on the rate and degree of flavor release in model gel systems (Boland and others 2004). The hardest gel, gelatin gel, had the lowest flavor release (E (N m-2)=400.25) compared with starch (E (N m-2)=53.15) and pectin (E (N m-2)=48.61) at same concentration (2g)

(Boland and others 2004). The rigidity of the starch and pectin gels was not significantly different from each other. The concentration of diacetyl, 2-butanone, ethyl acetate, 1- butanol, 3-methyl-1-butanol, ethyl butyrate, hexanal, 2-heptanone, heptanal, 2-octanone and 2-decanone significantly decreased in gelatin compared to starch and pectin in the headspace (Boland and others 2004).

The texture is a factor to influence the flavor release. The texture of high- methoxyl pectin gel is quantified by instrumental and sensory techniques. HM pectin (0.0,

0.5, 1.0, or 1.5%) confectionery gels with 33.4% sucrose and 29.8% 42 DE corn syrup solids into oval-shaped samples. The texture of the gel was tested by light and transmission electron microscopy. Adding pectin from 0.5 to 1.0 % increased pectin gels hardness. Further increasing pectin significantly increased to the texture of pectin significantly. Adding pectin from 0.5 to 1.0% can decrease the flavor release. The sensory result showed that 0.5% pectin were more brittle, less chewy, and smoother than

1.0% pectin. When the concentration is 1.0% or 1.5%, the gel were harder and tougher

(Demars and Ziegler 2001).

The release of strawberry flavor compounds from low, medium and high hardness pectin with different Young’s modulus of elasticity 181, 300 and 493 N m-2 was evaluated by instrumental and sensory analysis. The 0.5 g kg-1 strawberry flavor mix include (1 mg/g), hexanal (1 mg/g), benzyl acetate vanillin (5 mg/g), methyl dihydrojasmonate furaneol (5 mg/g), cis-3-hexenyl acetate (5 mg/g), ethyl iso-pentanoate

(10 mg/g), cis-3-hexenol c-decalactone (20 mg/g), ethyl hexanoate, methyl cinnamate (24 mg/g), and ethyl (90 mg/g) in triacetin bionone (1 mg/g), styrallyl acetate (1 mg/g), methyl anthranilate. The concentration of most of volatiles decreased when pectin firmness were increased by changing pectin concentration from 0.75g to 1.0g. The perception of odor, strawberry flavor and sweetness decreased in the sensory test (Boland and other 2006).

The release of 11 flavor compounds (diacetyl, 2-butanone, ethyl acetate, 1- butanol, 3-methyl-1-butanol, ethyl butyrate, hexanal, 2-heptanone, heptanal, 2-octanone and 2-decanone, 10 uL kg-1) in gelatin, starch and pectin gels (2g) was investigated in model gel systems by mouth/proton transfer reaction-mass spectrometry analysis. The hardest gel had the lowest flavor release for all compounds. The texture of the starch and pectin had no significant difference, however the release of hydrophilic volatiles in pectin was higher than starch. There was greater release of hydrophobic volatiles from starch gels than pectin gels. These results showed that chemical binding happened in the hydrocolloid gels (Boland and others 2004).

The effect of pectin on flavor release has been investigated by sensory analysis.

The perceived amounts of aroma, flavor, sweetness and sourness from an orange- flavored pectin decreased by increasing the concentration of pectin from 1.26%, 1.73% to

2.35%, while firmness increased (soft< medium

The assessment of interactions between texture and volatile compounds in gelatin desserts were analzed by free choice profiling. All combinations of 2 concentrations of gelatins (5%; 7%) were used in raspberry flavor. The panelist was trained to analyze flavor and texture. The results indicate that increasing gelatin concentration resulted in a lowering of perceived flavor of all attributes. Gelatin content influenced instrumental evaluation of texture. The Young’s modulus of pectin significantly decreased when the percentage gelatin increased. The Young’s modulus was a parameter used to measure

20 texture of hydrocolloids. 7% of gelatin was harder than 5% gelatin. Panelists didn’t find the texture change in the sensory analysis (Jaime and others 1993).

Lower concentrations of pectin (2%) and high concentration of pectin (10%) were used to analyze flavor change in sensory test. 2% of pectin had high score of aroma and flavor. The soft pectin did not inhibit flavor release into air; 10% of pectin had harder texture to decrease aroma release (Lundgren and others 1986).

2.4 The effects of changing hydrocolloid concentration on flavor release When the concentration of cornstarch increased, the amount of volatiles decreased.

The reduction in volatiles released was probably related to the presence of more binding sites to bind with small molecules like volatile compounds in the gel. While, the decrease of all volatiles were also observed with increasing cornstarch concentration (2, 3, 4, or

6%), and these changes – insignificant for n-hexyl acetate and n-octanol – are more noticeable for n-octyl acetate and, especially for n-hexanol (Golovnya and others 2001).

The pectin types can influence flavor release (Guichard and others 1991). When the pectin was changed from G gel, DE40 gel, DE60 gel, the viscosity order is G gel <

DE40 gel < DE60 gel. Increasing pectin can decrease the release of the flavor compounds. The highest to lowest for volatiles release was: G gel > DE40 gel > DE60 gel. It might due to the thicker gel provide a barrier for diffusing flavor and interact with flavor (Lubbers and Guichard 2003).

Composition of headspace and flavor release was determined in different amount of pectin in jams. 55 volatiles were identified in jam. Compared to adding pectin from 0,

0.05, 0.1 to 0.2, adding 0.4% pectin caused a significant decrease for 1-pentanol, 2- methyl propyl butanoate, hexanoic acid, octanal, octanoic acid. Adding 0.05% of pectin decreased significantly of ethyl hexanoate, and other compounds had no significant difference. Moreover, butyl acetate, ethyl hexanoate and hexyl acetate decreased with the volatile release of the jam’s aroma, from 0 to 0.2% of pectin. The decrease in flavor intensity in the study was probably due to the small molecules being captured in the gel

(Guichard and others 1991).

The release of six different flavour compounds, viz. isopenthyl acetate, ethyl hexanoate, cis-3-hexenyl acetate, linalool, L-menthone and limonene was analyzed in a soft drink-related model system. Comparing smaller amounts of pectin from 0.05%, 0.25,

0.10, 0.25, 0.7%, the addition of pectin (2.5%) decreased the release of limonene in the soft drink-related model system (Hansson and others 2001b). 2.5% of pectin had a significantly decrease in release of limonene. Changing concentration from 0.05%, 0.25,

0.10, 0.7% were not significantly different in the other compounds. The release of the other compounds was not significantly different when more pectin was added (Hansson and others 2001b).

The effect of gelatin concentration on the dynamic release of diacetyl was investigated in gelatin gels using headspace analysis. An increasing gelatin concentration

22 from 2-5 g decreased the concentration of diacetyl, which indicates binding between diacetyl and gelatin (Bakker and others 1998).

The relationship between aroma perception and the volatile concentration were tested in-nose in a model food by using sensory ranking and time–intensity analysis (TI) and atmospheric pressure ionization mass spectrometry. The gelatin concentrations from

2 to 8% w/w were analyzed and furfuryl acetate (300 mg/kg) was added as the flavor in the model food. Sensory result showed decreased flavor intensities as the gelatin concentration increased. Volatiles had no significant differences in nose when the concentration of gelatin was added (Baek and others 1999).

Changing different concentration of corn starch (1.2, 2.5, 5.0, and 12mmol/L) cryotextures was analyzed by capillary gas chromatography. The mixture of alcohols, ketones, alkyl acetates were the flavoring added in aqueous solutions as the concentration of cornstarch increases, the concentration of all volatiles decreases (Golovnya and others

2001).

2.5 The effects of different amount of sugar on flavor release As a sweetener, sucrose is the most important ingredient in confectionery and food industry. It has a lot of effects including, change the flavor, texture and sweetness.

The most common sugars used in foods are sucrose, fructose, and corn syrups (Godshall

1997). Sugars form a HE pectin gel network by hydrophobic interaction that pectin chains are forced together to forming a network (Hershko and Nussinovitch 1998).

Volatile concentration is significantly increased in the air when sucrose is added from 5% 23 to 10% in aqueous sucrose solutions as ‘salting out’ effect (Nahon and others 2000). The sugar interacts with the free water and increases the hydrophilic characteristics of the system. Therefore this salting-out effect increases the aroma compound release in the vapor phase (Hansson and others 2001b; Chen and Joslyn 1967). The relatively high sucrose (60% w/w) levels significantly increased flavor release (Evageliou and others

2000).

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;

Nahon and others 2000). An increase or a decrease of volatiles in the headspace is dependent on hydrophobicity and other physical properties of the volatiles. The addition of sugar can change the solubility of the volatile compound that they force together

(salting-in) (Chiou and others 1986).

The effect of sugars (sucrose, lactose, glucose, 10%w/v) at similar weight concentration from 0- 10% w/v on the liquid–vapor partition of diacetyl, 2, 3- pentanedione, ethylpyrazine, hexanal 0.005%v/v of coffee beverages investigated by headspace-gas chromatography (HS-GC) and solid phase micro-extraction-HS-GC-mass spectrometry. The concentration of four selected aroma compounds significantly decreased when the sucrose and lactose changed from 0 to10% w/v in the model systems than in the glucose ones (Piccone and others 2012).

The influence of corn syrups (G syrup, DE 40, and DE60, 75%) on flavor release and 3-methyl-1-butanol, 2-phenyl ethanol, and ethyl phenyl glycidate, 0.035g in pectin gel from fruit pastille model systems were studied by sensory test. The result showed significant differences between products with different sugar types in the sensory test.

The presence of corn syrup DE40 in pectin gel had lower flavor concentration than G and

DE60 syrup (Lubbers and Guichard 2003).

An empirical model was used to analyze the 40 volatile releases in gas–liquid partition behavior in aqueous sugar solutions [0–65% (w/v)]. When the sucrose concentration increased from 0 to 65% (w/v), the concentration of isoamyl acetate, ethyl hexanoate, eugenol increased in the headspace test. Others volatiles had no significant difference (Friel and Taylor 2000).

Sucrose can also increase volatiles concentration in the gel system. The significant increase in the release of ethyl hexanoate and methane was shown when sucrose was increased from 20% to 60% w/w in the soft drink model system (Hansson and others 2001b).

2.6 The effects of different acidity on flavor release Citric acid has a low pH and is a commonly used acid in the food industry. It could provide an environment for microbiological stability and improve the taste. Three carboxylic groups and a hydroxylic group form these highly polar bulky molecules

(Valde and others 1956). Citric acid is a strong organic acid and highly water-soluble. It gives people a “burst” of tartness and can be used as flavor modification (Hansson and

25 others 2001a). When pH changed from 4.5 to 4 into a soft drink, there was a big decrease of most volatiles in the beverage (Giese 1992). The change in flavor release may be due to the interaction between volatiles and dissociated form in the acid (Olafsson 1995).

Citric acid is a triprotic acid. Therefore, the dissociated form is the major form in this acid system (Olafsson 1995). Adding more citric acid in the soft drink model system can form more dissociated form. More citric acid in a sample also contains more dissociated form to bind these flavor compounds. The dissociated form has a bigger attraction to react with volatile compounds than non-dissociated forms (Hansson and others 2001a). Therefore, more flavor molecules release in the headspace when pH is higher because less dissociated form presented to interact with volatiles.

The release of six flavour compounds (0.40 w/w % Isopentyl acetate, 0.1 w/w %

Cis-3-hexenyl acetate, 0.2 w/w % Ethyl hexanoate, 1.2 w/w % l-Menthone, 0.1 w/w %Linalool, 0.1 w/w % Limonene) were analyzed by adding different amount of

Citric acid (1.0, 0.1, 0.002, and 0 g to get pH values of 2, 3, 4, and 5) to a soft drink model system. pH= 2 decreased the concentration of esters, because more dissociated form interact with volatiles in the citric acid. The other flavor compounds were not affected (Hansson and others 2001a).

2.7 The effects of saliva on flavor release Three important glands can secrete saliva. The function of saliva is for digestion, dentition protection, mucosal protection and pH adjustment (Van Ruth and Roozen 2000;

Matsuo and others 1997). Saliva is a hypotonic fluid, sodium, calcium, potassium, 26 chloride, phosphate, and bicarbon are the principal ions used as electrolytes in the body fluids (Van Ruth and Roozen 2000). The pH range of saliva is 6.2 to 7.4, the salivary pH rise with increasing rates of secretion. The type of salivary gland, the nature of stimulation and flow rate has a big effect on the bicarbonate concentration of saliva. The human saliva contains salts, enzymes and over 200 different proteins and peptides. The glycoprotein mucin is the major component for the viscosity of saliva. Amylase is an important enzyme in the saliva and it can break starch down in the mouth (Nawar 1971).

Moreover, lingual lipase is also present in human saliva (Van Ruth and Roozen 2000).

The flavor perceived in food is determined by the degree of flavor release in the mouth (Matsuo and others 1997). The concentration of proteins can influence aroma release and enzyme also contributes to degrade of starch compounds in the mouth. The amount of saliva also contributes to hydration and dilution of foods (Taylor and others

2000). Summarizing, chewiness, saliva composition and volume are important contributor to influence aroma release in the mouth. Especially, mastication can break up the food; expand the surface area, which accelerate the diffusion of volatile compounds

(Wilson and Brown 1997; Van Ruth and Roozen 1994). This effect is obvious in the gelatin gel because it is the hardest gel compared with starch and pectin (Boland and others 2004).

The gelatin gel was significantly harder (Young’s modulus of elasticity (E) is=400.25 than the starch (E=53.15) and pectin gels (E=48.61), so it had lower flavor release in model gel systems (Boland and others 2004). Adding saliva in gelatin and

27 pectin decreased hydrophilic compounds and increased the hydrophobic compounds concentration. The presence of saliva may increase hydrophilic nature of the system

(Wilson and Brown 1997). Saliva had an effect to the functional group of volatiles in starch of the compounds. The esters and alcohols increased flavor release and the ketones and alcohols had decreased flavor release with saliva (Wilson and Brown 1997).

Meanwhile, amylase can also break starch down and enhance the flavor release in model gel systems (Boland and others 2004).

2.8 Selected Ion Flow Tube – Mass Spectrometry (SIFT-MS)

2.8.1 Principles of SIFT-MS Selected Ion Flow Tube- Mass Spectrometry (SIFT-MS) is an analytical method that is a quantitative, accurate, fast, real time method to measure complex compounds without degrading chemicals (Spanel and Smith 1998). This machine allows measuring

+ different volatile organic compounds simultaneously. The selected precursor ions H2O ,

+ + NO and O2 do not have reaction with main components in the air, but they do react with most volatile organic compounds at a rapid speed. It is a flow tube based technique that can identify and quantify VOCs in whole air without requiring sample pretreatment or preconcentration. Ion-molecule reactions have very little temperature dependence.

Thermalized reactants give reproducible reactions. The products are always the same, as are the reaction rate coefficients. The collision limited reaction rate (the maximum rate at which the reaction can proceed) is relatively easily calculated. VOCs are detected as the ionic products of a reaction between the VOCs (analyst) and the chemical ionization agent (reagent). This chemical ionization is soft i.e. it only transfers a small amount of 28 energy to the analyst and thus produces a limited number of fragment ions. This can be a double-edged sword for chemical ionization techniques. It allows low detection thresholds but increases the risk of isomers giving identical products (Smith and Spanel

1999). In other MS systems the need for regular calibration is caused by changes in the system. The area, which is critical for this change, is the detection region. The detection region in SIFT-MS is physically isolated from the source region (the main source of contamination) and thus stays clean/stable for a long period. The ion counts are detected as a ratio, minimizing effects of detector instability and drift. SIFT-MS is a fast, real-time analytical method to analyze different complicated chemicals, which do not need to record or pre-process of the volatile of chemicals. It is a quantitate and accurate method that allow the scientist to monitor several volatiles of chemicals at the same time (Syft

Technologies Ltd. 2007).

2.8.2 Mouthspace Physical breakdown strength, air flow, swallowing and volatile complex binding contribute to the nosespace test (Ingham and others 1995a). When the fresh strawberry was eaten, mastication can affect the volatile release at the olfactory epithelium. Hexanal was lower in the nosespace than the headspace. This chemical is formed by homogenization, either by chemical degradation, or enzymatic lipids oxidation (Latrasse

1991).

Chewing can help LOX- derived volatiles to form in the mouth. The persistence of LOX-derived compounds was longer than esters in the mouths (Friel and Taylor

2001). Since the volatile compounds will be released during the chewing process, the concentration of those compounds was lower in the nosespace. Meanwhile, swallowing can also help to decrease overall amount of volatiles compounds (Taylor 1996).

Chapter 3: MATERIALS AND METHODS

3.1 Materials Different hydrocolloid gels (gelatin, pectin and starch) were prepared. The composition of the gels is shown in Table 2. Distilled water was added to produce a final weight of 100g. The unflavored gelatin was supplied by the Kroger Co. (Cincinnati, OH,

U.S.A.), Grindsted Pectin CF 130B was obtained from Danisco USA Inc, (Thomson, IL,

U.S.A.) and Food Grade Unmodified Tapioca Starch was supplied by Avebe America

Inc. (Princeton, NJ, U.S.A.). Granulated sugar was purchased from a local market

(Kroger Co., Columbus, OH, U.S.A.). Citric acid was obtained from Archer Daniels

Midland Co. (Decatur, IL, U.S.A.). Givaudan Flavors Corp (Cincinnati, OH, U.S.A.) supplied strawberry Flavor WONF IP-059-541-5.

Table 2 Composition of the different gummy gels per 100g

Sucrose Citric acid Type Hydrocolloid (g) Water (g) pH (g) (g)

No 0 74 4.5 21 2.61 hydrocolloid Different hydrocolloids Pectin 5 74 4.5 16 3.47 Starch 5 74 4.5 16 3.59 Gelatin 5 74 4.5 16 3.53

No 0 74 4.5 21 2.61 hydrocolloid Different amount of 2 74 4.5 19 3.42 pectin Pectin 3 74 4.5 18 3.45 5 74 4.5 16 3.47 5 0 4.5 90 3.71 Different 5 55 4.5 30 3.53 amount of Pectin sugar 5 65 4.5 25 3.52 5 74 4.5 16 3.47 5 74 0 20.5 3.86 Different pH 5 74 2.5 18 3.65 Pectin value 5 74 3.5 17 3.55 5 74 4.5 16 3.47

3.2 Gel preparation Different hydrocolloid based gels using gelatin, starch or pectin were prepared by

mixing the hydrocolloid with sugar and citric acid, and then combined with boiling water.

The mixture was heated to 80 °C for about 5 min with constant stirring until all of the

solids dissolved. The mixture was cooled to 50 °C and 0.5 g strawberry flavor added by

tuberculin syringe (BD Integra™, NJ, U.S.A.) into the beaker. The mixtures were stirred

for one minute to ensure the volatile compounds were distributed evenly and immediately

placed in 500-mL Pyrex bottles, Pyrex 1395 (Fisher Scientific, Corning, NY, U.S.A.) and 32 sealed with silicon septum. The samples were refrigerated at 4 °C for 24 h prior to analysis. Three replicates were used for every sample.

3.3 Headspace measurements The prepared pyrex bottles with the samples were held for 60 min at 37 °C (body temperature) in a water bath to allow for equilibration of the volatiles released from the samples. Once equilibrium was achieved, the headspace was measured. The samples were analyzed with a Selected Ion Flow Tube – Mass Spectrometer (SIFT-MS) after 1 hour. Selected compounds were measured with Syft VOICE-200 software (v.1.4.9.17754,

Syft Technologies Ltd., Christchurch, New Zealand). A standard gas mixture used to calibrate the instrument during automated validation procedures. The scan time in headspace analysis was 60 sec. A short needle (5.5 cm) was used, which was connected to the SIFT-MS, placed in the middle of the septum with the tip of the short needle 3 cm below the septum to sample the headspace of volatile compounds. The holes were sealed using labeling tape (Fisher Scientific, United Kingdom) and the lid was not removed during the measurement.

3.4 Breath measurements Breath measurements for gummy candies were obtained using a Bacterial/Viral

Respiratory Filter (Sure Guard Hyper Gard ”TEC200GM”Filteration, Birdhealthcare Co.,

Victoria, Australia). The filter was positioned around the SIFT passivated needle encompassing the entire needle and tight to the end to prevent air escape. Total breath time was 60 sec for the breath measurement. One individual did the breath test. Breath scan patterns of 5 sec exhales and 5 sec inhales were used each time for the span of the breath measurement. All together, each 60 second period had six inhales and exhales.

Three replicates were run for every breath test (Figure 3). The settings for SIFT-MS scans can be found in Appendix A.

Figure 3 SIFT-MS configuration for headspace (left) and mouthspace (right) measurements

3.5 SIFT-MS information The concentration of volatile compounds in this study was calculated using known kinetic parameters (Table 3). The concentration (M) of selected volatiles was calculated using the product count rate (Ip), reaction rate constant (k), precursor ions 34 count rate (I), and reaction time (t) as follows: (M)=Ip/Ikt (Spanel and Smith 1999).

Conflicts were removed by selecting different masses or different precursor ions for each compound in the method. Compounds with irresolvable conflicts or low concentrations are not reported.

3.6 Texture analysis Texture profile analysis (TPA) were determined using a TA-TX2 Texture

Analyzer (Stable Micro Systems Ltd., Surrey, UK), fit- using 2 mm diameter flat-tipped cylinder probe on an Instron 5542 Universal Testing Machine (Instron Corp., Canton,

Mass., U.S.A.) with Bluehill 2 Materials Testing Software (Instron Corp., Norwood,

Mass., U.S.A.). To simulate mastication, TPA with 30% deformation at a rate of 3 mm/s with 25 Kg cell load was used (Daubert and Foegeding 2003). 10 g samples were prepared in a small container and the hardness (N) was tested. Ten replicates of all samples were analyzed because of large variation between samples.

3.7 Statistical Analysis The mean and standard deviation of three replicates of samples were calculated by

2010 Microsoft Excel (Microsoft, Redmond, WA, U.S.A.). All statistical analyses were performed by one-way analysis of variance (ANOVA) using SPSS software (SPSS Inc.,

Chicago, Ill., U.S.A) to determine significant difference among gummy gels with different hydrocolloids. The differences were considered significant when p ≤ 0.05. A significance level of 0.05 was used throughout the study.

Table 3 Important strawberry volatiles, reagent ions and masses of the compounds used in this study

Reaction Precursor Mass rate(k) Analyte Product ion ion (m/z) (10-9 cm3/s) Esters + + ethyl acetate NO 118 NO CH3COOC2H5 2.7 + +. methyl butanoate NO 132 NO C3H7COOC3 2.4 + + ethyl butanoate NO 71 C4H7O 2.5 + + methyl hexanoate NO 99 C6H11O 2.1 + +. ethyl hexanoate NO 174 NO C8H16O2 2.5 + + butyl acetate H3O 61 C2H3O2H2 2.9 + + isopropyl butanoate NO 59 C3H7O 2.2 Alcohols + + linalool NO 136 C10H16 2.6 + + (+/-)-nerolidol NO 204 C15H24 3.0 + + 1-hexanol O2 42 C3H6 2.6 + + (E)-2-hexen-1-ol O2 57 C3H5O 2.9 Aldehydes + + hexanal O2 44 C2H4O 2.0 + + (E)-2-hexenal NO 97 C6H9O 3.8 Ketones + +. 2-pentanone NO 116 NO C5H10O 3.1 + +. 2-heptanone NO 144 NO C7H14O 3.4 + +. acetic acid NO 90 NO CH3COOH 9.0 + +. propanoic acid NO 104 NO C2H5COOH 1.5 + +. hexanoic acid NO 146 NO C6H1202 2.5 + + 2-methylbutanoic acid NO 85 C5H9O 1.8 + + furaneol O2 128 C6H8O3 3.0 + + furfural NO 96 C5H4O2 3.2

Chapter 4: RESULTS AND DISCUSSION

4.1 Effect of type of hydrocolloid on volatile release Gummy gels were made with hydrocolloid, sugar, acid, water, and strawberry flavor. Three types of hydrocolloids were used in this study, gelatin, pectin and starch.

The level of some volatiles was higher in the hydrocolloid itself than in the strawberry flavor (Figure 4). The concentration of butyl acetate, (+/-) nerolidol, 1-hexanol, hexanal and acetic acid was higher in all of the hydrocolloids than in the strawberry flavor. Isopropyl butanoate and 2-pentanone were higher in pectin than in the strawberry flavor. These volatiles were not considered in the following discussion because their naturally high levels in the hydrocolloids made it difficult to determine how different variables affect their release into the headspace.

500 No hydrocolloid No hydrocolloid a 250 Gelan 450 b a Gelan c a a Pecn 400 200 b 350 Pecn a Starch a b 300 Starch 150 b a 250 b c 200 c 100 b a a Concentraon(ppb) a d b c a 150 b b b b b b d 50 c 100 c Concentraon (ppb) 50 0 0 ethyl methyl ethyl methyl ethyl propanoic acid hexanoic acid 2-methylbutanoic acetate butanoate butanoate hexanoate hexanoate acid

300 500 a No hydrocolloid No hydrocolloid a 250 450 Gelan Gelan a 400 Pecn Pecn 200 b b 350 Starch 300 Starch b 150 250 c 200 100 b c b 150 Concentraon (ppb) 50 Concentraon (ppb) 100 a a a c c b b b c bc 50 b c b d 0 0 (E)-2-hexen-1- (E)-2-hexenal linalool 2-heptanone furaneol furfural ol

Figure 4 Concentration of important volatiles in no hydrocolloids and three hydrocolloids. *Concentration for each volatile that does not share a letter is significantly different

In almost all cases, volatile release into the headspace of the samples containing no hydrocolloids was significantly higher than samples that contained hydrocolloids, because the system didn’t form a gel without hydrocolloids (Figure 4).

Others found that when the gelatin increased from 0, 2.5, 5, 10, 15, and 20% w/v, the concentration of diacetyl gradually decreased (Bakker and others 1998). Limonene and most esters and aldehydes significantly decreased in the presence of xanthan due to interaction with the xanthan matrix (Bylaite and others 2005). Physical entrapment and chemical binding both influence flavor release (Boland and others 2006). Entangled polymer networks in hydrocolloids inhibit the transportation of small molecules, so volatiles are entrapped in the system (Baines and Morris 1987; Hau and others 1998;

Pangborn and Szczesniak 1974). The chemical binding of volatiles by hydrocolloids can also decrease flavor release (Pangborn and others 1978). Binding of volatiles is in part due to hydrophobic and hydrophilic interactions (Golovnya and others 2001). In starch, hydrophobic flavor is entrapped in the amylose helix by hydrophobic bonding

(Boland and others 2004). Interaction between hydroxyl groups of starch and flavor compounds through hydrogen bonding can also decrease flavor release (Chinachoti

1995). The stretching of pectin molecules with water form micelles, which are more hydrophobic and the more nonpolar compound may be captured in the hydrophobic parts of the pectin solution (Hansson and others 2001b). The gelatin is protein, which has NH2, OH group to bind hydrophilic volatiles and also has carbon-oxygen bond to bind hydrophobic volatiles (Burey and others 2008).

Comparing the concentration of volatiles in the gummies made from these three hydrocolloids, it is apparent that the type of hydrocolloid had a significant effect on the volatile release (Figure 4). The release of these important strawberry flavor compounds into the headspace was significantly higher for pectin and starch than for the gelatin gel. However, the binding by starch and pectin was similar, 57% of volatiles had no significant difference in volatile release between starch and pectin.

The concentration of most volatiles in pectin and starch were not significantly different in a model gel systems (Boland and others 2004). Pectin has good flavor release and the tastes better than starch. However, 2-octanone and 2-decanone, which are hydrophobic compounds, in a model mouth analysis, had higher release values in the starch gel than pectin, which disagreed with my results (Boutboul and others 2000).

Volatile release in gelatin gel was lowest of the hydrocolloids tested; 64% of volatiles were lower in gelatin than pectin and starch (Figure 4). Similarly, the concentration of most volatiles was significantly lower in gelatin than in pectin or starch in the headspace in a model gel system (Boland and others 2004). Gelatin binds and physically entraps in gelatin more strongly than pectin and starch (Boland and others 2004). The structure of gelatin explains why it is able to effective bind volatiles.

Gelatin is made up of protein, which has the ability to tightly bind molecules. The triple helical segments form the basis for cross-linking and three- dimensional network formation in gelatin, which can tightly entrap molecules (Clark and others 1990).

4.2 Effect of pectin concentration on volatile release High methoxylated pectin (HMP) is a common gelling agent used in the food industry, especially in jelly and fruit based products (Boland and others 2004).

Different amounts of pectin are used in the food industry to meet different consumer expectations for different gummy gels (Lin and others 1978). When pectin was added to the solution to form a gel (from 0 to 2g), 71% of volatiles significantly decreased

(Figure 5). When the pectin content was further increased from 2 to 5g, only 21% of volatiles significantly decreased further. The greater the concentration of pectin the more binding sites is present to bind with volatiles compounds in the gel (Hansson and others 2001b). Increasing pectin from 0.7 to 2.5% decreased the release of limonene in a soft drink-related model system (Hansson and others 2001b). An increase of pectin from 0.1 to 0.4% in jam significantly decreased many but not all of the volatiles release of most esters out of 55 volatiles. Other volatiles aldehydes such as benzaldehyde, nonanal, and 2-hexenal, decreased from 0 to 0.2% of pectin (Guichard and others 1991). Adding pectin from 0.0 to 1.5% can decrease the flavor release in a pectin gummy gel (Demars and Ziegler 2001). Increasing amount of pectin provide a barrier for diffusing flavor and interact with flavor (Lubbers and Guichard 2003).

500 0g Pecn a 250 a a 0g Pecn 450 ab b b b b 2g Pecn 400 2g Pecn 200 3g Pecn 350 3g Pecn a b a c b 5g Pecn 300 5g Pecn 150 a b 250 b b b 200 b 100 b a c b a a 150 a bc a b c c 100 d 50 Concentraon (ppb) 50 0 0 ethyl methyl ethyl methyl ethyl propanoic acid hexanoic acid 2-methylbutanoic acetate butanoate butanoate hexanoate hexanoate acid

0g Pecn 300 350 a 2g Pecn a 300 250 a b a 0g Pecn 3g Pecn 2g Pecn 250 200 c 5g Pecn 3g Pecn 200 150 5g Pecn 150

100 100 Concentraon (ppb) b b

Concentraon (ppb) a 50 a 50 b b b b b b b b b a b bc c b 0 0 furaneol furfural

Figure 5 Concentration of volatiles in different amount of pectin *Concentration for each volatile that does not share a letter is significantly different

4.3 Effect of sugar concentration on volatile release For every 100-gram gummy candy, the commercial level of sugar is between

55 and 69g (Tschumak and others 1976). Sugar was changed from 0 to 74g to investigate the volatile change in gummy gels. When sucrose is added to pectin, the pectin precipitates out and forms insoluble fibers (Friel and others 2000). Sugar had a large effect on flavor release. 100% of the volatiles significantly decreased in concentration when sugar was added to the solution to form a gel (Figure 6). Therefore, adding sugar decreased volatile release. Adding sugar allows the pectin gel network to form by changing the solvent structure such that pectin chains are forced together, thereby forming a network structure based on hydrophobic interactions, which decrease flavor release (Hershko and Nussinovitch 1998). As sugar was further increased from 55 to 74g, 57 % of volatiles had no significant difference in concentration. It may be because the amount of volatiles reached a maximum release above certain amount of sugar.

Sucrose can increase or decrease the concentration of volatiles within a solution (Hansson and others 2001b; Chen and Joslyn 1967). In this study, 29% of volatiles increased while 7% of volatiles decreased with addition of sugar from 55 to

74g (Figure 6). Adding more sugar increases the binding of water by sugar (Nahon and others 2000). The system becomes more hydrophilic because sugar is hydrophilic. The volatile compounds within a hydrophilic system are more easily released to the gas phase compared to a hydrophobic system. This effect is called “salting-out”. The

“salting-out” effect increases the aroma compound release in the vapor phase (Friel 43 and others 2000; Hansson and others 2001b). There was a significant increase in the release of ethyl hexanoate and menthone when sucrose was increased from 20 to 60% w/w in a soft drink model system (Hansson and others 2001b). Depending on the volatile compound, the polarity and other physic-chemical properties of the volatiles can reduce flavor release (Chiou and others 1986). The addition of sugar to the gel can change the solubility of the volatile by attracting it (salting-in) (Chiou and others

1986). The concentration of four selected aroma compounds significantly decreased when the sugar changed from 0 to10% w/v in a model systems (Piccone and others

2012).

3500 a 2000 0g Sugar a 0g Sugar 1800 55g Sugar 3000 65g Sugar 55g Sugar 1600 2500 1400 74g Sugar 65g Sugar 1200 2000 74g Sugar a 1000 a 1500 800 a a 600 a 1000

Concentraon (ppb) b 400 b c c b 500 a b b b b c c 200 b b b b b b c c b c bc c c 0 0 ethyl methyl ethyl methyl ethyl propanoic acid hexanoic acid 2-methylbutanoic acetate butanoate butanoate hexanoate hexanoate acid

800 a 0g Sugar 400 700 0g Sugar a 55g Sugar 350 600 55g Sugar 65g Sugar 300 65g Sugar 500 74g Sugar 250 74g Sugar 400 200 a 300 150

Concentraon (ppb) b 200 c c Concentraon (ppb) 100 a a 100 a b b b b b c d b b b 50 b b b c bc 0 0 (E)-2-hexen-1-ol (E)-2-hexenal 2-heptanone linalool furaneol furfural

Figure 6 Concentration of volatiles in different amount of sugar *Concentration for each volatile that does not share a letter is significantly different

4.4 Effect of pH on volatile release Changing pH had a significant effect on the release of most compounds (Figure

7). When the pH is lowered, more of the acid is in the dissociated form. As volatiles bind to the dissociated form, the amount of volatiles released is decreased (Van Ruth and Roozen 2000). pH 3.86 had the greatest volatile release for most volatiles, in part because pectin didn’t form a gel at that pH. The release of volatiles continued to as more citric acid was added and pH decreased from 3.65 to 3.47. 71.4% of volatiles released decreased. Adding citric acid in small amounts decreases the headspace concentration of the volatiles. When pH decreased from 4.5 to 4 in a soft drink, there was a significant decrease in most of the beverage’s volatiles (Giese 1992). Adding one gram of citric acid per 10 ml of flavored water mixture decreased the release of esters and limonene (Hansson and others 2001a).

The more citric acid is added to the pectin gels; the greater amount of dissociated form (RCOO-) in the citric acid will be present to interact with volatiles.

Citric acid is a triprotic acid, and increasing the amount of citric acid will create a more dominating dissociated form presence. This dissociated form interacted with volatiles more so than the non-dissociated citric acid. Therefore, the pH = 3.47 of the gel was low, a large amount of the citric acid would exist in the dissociated form, which may explain why the headspace concentration of some volatiles were significantly lower than pH=3.65 (Figure 7). However, some of the remaining dissociated form of the citric acid may not have interacted with the other esters like ethyl hexanoate in this study. Some esters exhibit the same volatile release regardless 46 of the pH level because the dissociated form does not react with the esters. Others found that no differences in the release of esters in relation to the pH values from pH 5 to pH 3 (Hansson and others 2001).

1200 450 a pH a pH 3.86 400 a 1000 3.86 pH 3.65 350 a a pH 3.55 pH 800 300 3.65 b b pH 3.47 a b 250 a c b 600 c 200 d b c 400 150 d b c b b b c d Concentraon(ppb) d b 100 c d 200 c a b c 50 0 0 propanoic acid hexanoic acid 2-methylbutanoic ethyl methyl ethyl methyl ethyl acid acetate butanoate butanoate hexanoate hexanoate

700 a 120 a pH 3.86 600 pH 3.86 pH 3.65 100 pH 3.65 500 pH 3.55 pH 3.55 80 400 pH 3.47 b pH 3.47 60 300 c 40 b 200 d Concentraon (ppb) a c Concentraon (ppb) a 20 d 100 b b c c b d a b b b a b c d 0 0 furaneol furfural

Figure 7 Concentration of volatiles in different pH *Concentration for each volatile that does not share a letter is significantly different

4.5 Mouthspace The flavor of foods perceived during eating is determined by the rate and extent of flavor release in the mouth (Friel and others 2000). Mastication and saliva in the mouth influence both the thermodynamic and kinetic components of volatiles release in pectin, gelatin and starch (Boland and others 2004). Comparing the concentration of volatiles in no hydrocolloid, pectin, starch, and gelatin in the mouthspace test (Figure 8), only 36% of the concentration of volatiles were significantly different. However, 100% volatiles were significantly different in the headspace test.

70.00 No hydrocolloid No hydrocolloid a a a 60.00 Starch 60.00 a Starch a Pecn a a Pecn 50.00 50.00 Gelan Gelan 40.00 40.00 a a ab 30.00 ab a a a a a a a b a 30.00 a a a 20.00 a a b a a 20.00 a a a a 10.00 10.00 0.00 0.00 propanoic acid hexanoic acid 2-methylbutanoic ethyl methyl ethyl methyl ethyl acid acetate butanoate butanoate hexanoate hexanoate

No hydrocolloid 25.00 150.00 a a Starch 140.00 a a No hydrocolloid 130.00 Starch 20.00 Pecn a a 120.00 a 110.00 Pecn Gelan 100.00 15.00 90.00 Gelan 80.00 70.00 60.00 10.00 50.00 40.00 b a 30.00 a b b 5.00 b ab b b 20.00 a a a a a 10.00 c b b 0.00 0.00 furaneol furfural

Figure 8 Concentration of important volatiles in different hydrocolloids in mouthspace test *Concentration for each volatile that does not share a letter is significantly different

Salivary composition and chewiness are influenced by the degree of hydration, body position, light, olfaction, stimulation and surroundings (Dawes 1987; Wisniewski and others 1992). All these factors can influence large variations within the panelist on different day(Pangborn and Lundgren 1978). These variables may influence measured concentration changed and make error bar large in the mouthspace.

71% of volatiles released experienced a trend in which the gelatin was lower than the starch or pectin. However, some of them were not significantly different

(Figure 8). Meanwhile 64% of volatiles in gelatin were lowest in headspace test. This result is same with the headspace result demonstrating the binding ability and physical properties of gelatin with volatiles was higher than starch or pectin. This explains the lower release of volatiles from the gelatin gel compared to the starch and pectin gels.

Within starch 14% of volatiles were higher than no hydrocolloid, pectin or gelatin (Figure 8). Physico-chemical changes occurring during mastication may have affected the release of volatiles. Amylase is a major protein in saliva that breakdown starch (Dawes 1987). The protein amylase can promote the release of volatiles within the mouth. Meanwhile, chewing will generally increase amount of surface area exposed to the air increasing the release of volatiles (Van Ruth and Roozen 1994).

Only (E)-2-hexenal and furfural released least in starch. This may indicate starch would like to bind (E)-2-hexenal and furfural in the mouthspace (Figure 8).

4.6 Hardness The hardness of the gel system had an influence on the release of volatiles in model gel systems (Boland and others 2004). Hardness is the force required to compress a food between molars, and to attain a given deformation (Guinard and

Marty 1995). The gelatin gel was significantly harder than pectin and starch at same concentration (Table 4). The concentration of methyl butanoate and ethyl hexanoate had a larger decrease in gelatin than starch and pectin. The concentration of methyl butanoate decreased in starch compared to pectin. However, pectin and starch did not exhibit a significant change for ethyl hexanoate release. When the concentration of pectin was increased from 2-5g, the hardness of pectin increased. The concentration of methyl butanoate had a decrease when the pectin content was further increased from 2 to 5g. However, increasing hardness had no significant effect on ethyl hexanoate release. Adding sugar from 55-74g resulted in increasing hardness. The hardness increased yielded no significant effect on ethyl hexanoate release but increased methyl butanoate release. When pH decreased from 3.65 to 3.47, hardness decreased resulting in higher volatiles release of methyl butanoate. For ethyl hexanoate, decreasing hardness had no significant effect. In this study, the hardness was not considered in the discussion because there is no linear regression for the volatiles decrease when hardness increased. Therefore, texture was not a factor to influence the release of volatiles. Others found that the gelatin gel system (E (N m-2)=400.25) used in model gel systems was significantly more rigid than pectin (E (N m-2)=48.61) and starch (E

(N m-2)=53.15) at same concentration, which was not significantly different from each

51 other. The concentration of most volatiles had a significant decrease in gelatin than starch and pectin in the headspace (Boland and others 2004). Adding pectin from 0.0 to 1.5% can decrease the flavor release in the pectin gummy gel. The sensory evaluation also showed that gels with less pectin were more brittle, less chewy, more smooth, and easier to break up into smaller particles (Demars and Ziegler 2001).

Table 4 The relationship between hardness and volatiles concentration

methyl butanoate- ethyl hexanoate- concentration(ppb) hardness(N) concentration(ppb) hardness(N) pectin 206d 12.57b 68.8a 12.57b starch 173e 13.68b 70.0a 13.68b gelatin 86.8g 35.90a 36.1c 35.90a 2g-pectin 231c 11.50a 69.4a 11.50a 3g-pectin 199d 12.29a 57.0b 12.29a 5g-pectin 206d 12.57a 68.8a 12.57a 55g sugar 125f 10.30a 75.8a 10.30a 65g sugar 122f 10.96ab 50.0b 10.96ab 74g sugar 206d 12.57b 68.8a 12.57b pH 3.65 314a 14.88a 70.3a 14.88a pH 3.55 259b 13.93a 76.5a 13.93a pH 3.47 207d 12.57a 68.8a 12.57a

Concentration for each volatile that does not share a letter is significantly different. Hardness that does not share a letter is significantly different.

Chapter 5: CONCLUSIONS

The type of hydrocolloid, concentration of pectin, sugar content, and pH affected the volatile release in a strawberry gummy gel. The adding of hydrocolloids lowers volatile release; Volatile release in gelatin was the least among all treatments.

Pectin and starch exhibited lower volatile release compared with no hydrocolloids; however, they were not significant different with each other. When the pectin was added, the volatile levels decreased. This suggests that pectin created a thicker system to entrap the volatiles in the gel. The addition of pectin from 2-5g had no significant difference in most volatile levels. Adding sugar significantly decreased volatile levels.

Further increasing sugar from 55 to 74g had no significant effect on volatile release. It may be because the amount of volatiles released reached a plateau. pH produced a significant difference in volatile levels. Lowering the pH from 3.65 to 3.47 shift the equilibrium, and more dissociated form were formed causing decreased release of volatiles. In the mouthspace test, most volatiles had no significant difference in gelatin, starch, and pectin. Human error, instrument error, and other factors may cause this insensitivity. In this study, sugar had the greatest effect on volatile release.

Hardness alone had no significant effect on flavor release.

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APPENDIX: Additional volatile information

Table 5 The parameters of the SIFT/MS Measurements

Carrier Flow (torr L/sec) 5.1611

Sample Flow (torr L/sec) 0.3126

Tube Pressure (torr) 0.6934

Downstream Intensity (pA) 136.8164

Upstream Intensity (nA) 3.7598

Reaction Time (ms) 2.659

Dilution Factor 1

Sample Needle Size (cm) 5.4

Table 6 The effect of three types of hydrocolloids (gelatin, pectin, and starch) on headspace concentration of volatiles

Analyte compounds (ppb) No hydrocolloid Pectin Starch Gelatin Esters ethyl acetate 161 a 65.2b 72.4b 27.8c methyl butanoate 200 a 206a 173 b 86.8c ethyl butanoate 214 a 122 b 135 b 54.4c methyl hexanoate 105 a 41.1b 47.5b 46.0b ethyl hexanoate 65.6a 68.8a 70.0a 36.1b butyl acetate+ 123b 554 a 120 b 104 b isopropyl butanoate* 1012c 2845a 1144b 275 d Alcohols

linalool 15.2a 9.51b 9.64b 4.69c (+/-)-nerolidol+ 14.9c 13.9d 15.7a 15.6b 1-hexanol+ 126d 247a 192b 147c (E)-2-hexen-1-ol 240a 167b 177b 105c Aldehydes Hexanal+ 409b 603a 393b 241c (E)-2-hexenal 205a 4.39c 61.1b 81.1b Ketones

2-pentanone* 9.61b 8.75b 13.4a 9.55b 2-heptanone 24.4a 9.57c 11.8bc 12.9b Acids acetic acid+ 63.9a 54.3ab 52.2b 46.3b propanoic acid 120 a 69.2b 75.4b 51.7c hexanoic acid 295a 153 c 194 b 78.3d 2-methylbutanoic acid 439 a 394 b 362 c 128d Furaneol 22.9a 2.85c 7.65b 10.0b Furfural 274 b 10.5d 139 c 419 a

73 a, b,c,d Values with different superscripts within a row are significantly different. + the concentration of volatile was high in pectin, starch and gelatin naturally. * the concentration of volatile was high in pectin naturally.

Table 7 The effect of pectin concentration on headspace concentration of volatiles

Analyte compounds (ppb) 0g Pectin 2g Pectin 3g Pectin 5g Pectin Esters ethyl acetate 162 a 87.1b 80.5b 65.3c methyl butanoate 201 b 231 a 199 b 207 b ethyl butanoate 214a 140 b 123 b 123 b methyl hexanoate 105a 63.6b 51.9bc 41.2c ethyl hexanoate 65.6a 69.4a 57.0a 68.8a butyl acetate* 124 c 913 a 551 b 554 b isopropyl butanoate* 1012c 3918a 2816b 2845b Alcohols linalool 15.2a 12.8b 11.1bc 9.50c (+/-)-nerolidol* 14.9a 15.4a 15.6a 13.9a 1-hexanol* 126 c 199b 184 b 246 a (E)-2-hexen-1-ol 240 a 227 a 186b 167c Aldehydes Hexanal* 409 c 701 a 547 b 603 b (E)-2-hexenal 205 a 16.3b 15.5b 4.39b Ketones 2-pentanone* 9.61a 8.95ab 8.26b 8.75ab 2-heptanone 24.4a 10.7b 9.68b 9.57b Acids acetic acid* 63.9b 94.3a 57.6b 54.4b propanoic acid 121 a 94.4b 79.5c 69.2d hexanoic acid 295 a 188 b 174 b 153 b 2-methylbutanoic acid 439 a 414 ab 289 c 394 b furaneol 22.9a 4.53b 4.36b 2.85b furfural 274 a 30.3b 32.9b 10.5b a, b,c,d Values with different superscripts within a row are significantly different. * the concentration of

the volatile was high in pectin naturally.

Table 8 The effect of sugar concentration on headspace concentration of volatiles

Analyte 0 g Sugar 55g Sugar 65g Sugar 74g Sugar Esters ethyl acetate 581 a 41.8b 53.9b 65.3b methyl butanoate 731 a 125 c 122 c 207 b ethyl butanoate 1725a 67.4b 70.5b 123b methyl hexanoate 475 a 65.0b 44.2c 41.2c ethyl hexanoate 615 a 75.8b 50.0c 68.8bc butyl acetate* 4429a 2743b 1708c 554d isopropyl butanoate* 9821b 11294a 7479c 2846d Alcohols linalool 76.0a 10.1b 6.71b 9.51b (+/-)-nerolidol* 65.9a 30.3b 22.2bc 13.9c 1-hexanol* 207 cb 214ab 176 c 247 a (E)-2-hexen-1-ol 720 a 132 c 109c 167 b Aldehydes Hexanal* 1180a 1220a 816 b 603 c (E)-2-hexenal 260 a 5.17b 2.74b 4.39b Ketones 2-pentanone* 23.2a 13.6b 10.4b 8.75a 2-heptanone 52.0a 15.9b 12.9c 9.57d Acids acetic acid* 151 a 51.9b 47.4b 54.3b propanoic acid 128a 34.8c 40.9c 69.2b hexanoic acid 3110a 119 b 120 b 153 b 2-methylbutanoic acid 1630a 179 c 158 c 394 b furaneol 49.4a 3.93b 3.51b 2.85b furfural 342a 17.7b 4.67c 10.5bc a, b,c,d Values with different superscripts within a row are significantly different. * the concentration of

the volatile was high in pectin naturally.

Table 9 The effect of acidity on headspace concentration of pectin gels volatiles

Analyte pH 3.86 pH 3.65 pH 3.55 pH 3.47 Esters ethyl acetate 200 a 131 b 81.8c 65.3d methyl butanoate 274b 314a 259b 207c ethyl butanoate 355 a 191b 138 c 123 d methyl hexanoate 377a 74.7b 59.2c 41.2d ethyl hexanoate 309 a 70.3b 76.5b 68.8b butyl acetate* 3224a 1717b 722 c 554 c isopropyl butanoate* 9240a 6529b 3543c 2846d Alcohols linalool 24.9a 17.4b 10.9c 9.51d (+/-)-nerolidol* 51.6a 17.0b 13.1b 13.9b 1-hexanol* 180 c 201 b 240 a 247 a (E)-2-hexen-1-ol 595 a 279 b 219c 167 d Aldehydes Hexanal* 1183a 904 b 650 c 603 c (E)-2-hexenal 68.8a 10.9c 12.7b 4.39d Ketones 2-pentanone* 17.4a 9.18b 8.55b 8.75b 2-heptanone 22.3a 10.2b 9.05b 9.57b Acids acetic acid* 131 a 93.9b 68.3bc 54.3c propanoic acid 74.7c 105 a 88.2b 69.2c hexanoic acid 645 249 b 181 c 153 d 2-methylbutanoic acid 983 a 543 b 462c 394 d furaneol 18.3a 5.86b 4.94b 2.85c furfural 110 a 20.3c 27.4b 10.5d a, b,c,d Values with different superscripts within a row are significantly different. * the concentration of

the volatile was high in pectin naturally.

Table 10 The effect of three types of hydrocolloids (gelatin, pectin, and starch) on headspace concentration of hydrocolloids gels volatiles in mouthspace

Analyte No hydrocolloids Starch Pectin Gelatin Esters ethyl acetate 15.5a 14.4a 14.5a 15.0a methyl butanoate 56.1a 49.0a 53.3a 47.9a ethyl butanoate 21.0a 20.8a 19.9a 15.7b methyl hexanoate 15.1a 14.8a 14.1a 11.9a ethyl hexanoate 26.7a 27.0a 25.1a 19.2a butyl acetate* 75.2b 135a 60.6b 71.1b isopropyl butanoate* 40.5b 328a 41.0b 46.4b Alcohols linalool 3.69a 3.76a 3.63a 3.29a (+/-)-nerolidol* 21.9a 18.1b 18.7ab 17.7b 1-hexanol* 80.0a 76.8a 75.9a 75.6a (E)-2-hexen-1-ol 125a 126a 123a 118a Aldehydes hexanal* 92.8b 109a 88.1b 87.6b (E)-2-hexenal 2.32a 0.92c 1.72b 1.59b Ketones 2-pentanone* 8.13a 6.45b 6.20b 6.62ab 2-heptanone 13.7a 12.2b 12.4b 11.4b Acids acetic acid* 53.3a 47.0a 46.7a 47.8a propanoic acid 28.7a 24.7ab 23.4b 27.3ab hexanoic acid 36.6a 37.8a 34.2a 25.6a 2-methylbutanoic acid 24.5a 24.1a 23.3a 18.7a furaneol 3.27a 2.69b 2.92ab 2.63b furfural 16.3a 1.95b 13.7a 14.5a a, b,c,d Values with different superscripts within a row are significantly different. * the concentration of

the volatile was high in hydrocolloids naturally.

250

200 methyl butanoate

150 ethyl hexanoate

100

Volale concentraon (ppb) 50

0 0 5 10 15 20 25 30 35 40 Hardness(N)

Figure 9 Concentration of volatiles vs. hardness in different hydrocolloids (pectin, starch and gelatin)

250

200

150 methyl butanoate ethyl hexanoate 100

50 Volale concentraon(ppb)

0 11.4 11.6 11.8 12 12.2 12.4 12.6 12.8 Hardness(N)

Figure 10 Concentration of volatiles vs. hardness in different amount of pectin (2- 5g)

250

200

150 methyl butanoate 100 ethyl hexanoate

50 Volale concentraon (ppb)

0 8 9 10 11 12 13 Hardness(N)

Figure 11 Concentration of volatiles vs. hardness in different amount of sugar (55g- 74g)

350

300

250 methyl butanoate 200 ethyl hexanoate 150

100

Volale concentraon (ppb) 50

0 12 12.5 13 13.5 14 14.5 15 Hardness(N)

Figure 12 Concentration of volatiles vs. hardness in different pH (3. 65-3.47)

350 methyl butanoate 300 methyl butanoate 250

200 Linear (methyl butanoate ) 150 R² = 0.16214 100 50 Volale concentraon (ppb) 0 0 10 20 30 40 Hardness (N)

Figure 13 Concentration of methyl butanoate vs. hardness

ethyl hexanoate 90 ethyl hexanoate 80 70 Linear (ethyl 60 hexanoate ) 50

40 R² = 0.52009 30

Volale concentraon 20 10 0 0 10 20 30 40 Hardness(N) Figure 14 Concentration of ethyl hexanoate vs. hardness