Contribution of Enzymes and Other Components in Food in the Formation And

Contribution of enzymes and other components in food in the formation and

destruction of volatile compounds.

Presented in Partial Fulfillment of the Requirements for

The Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Dissertation

Rita A. Mirondo.

Graduate Program in Food Science & Technology

The Ohio State University

2016

Dissertation Committee:

Dr. Sheryl Barringer, Advisor

Dr. Monica Giusti

Dr. John H. Litchfield

Dr. Luis Rodriguez-Saona

Rita Mirondo

2016

Abstract

To assess the effect of peels on the quality of tomatoes and mangoes, these foods were processed into tomato juice and sauce and mango pure from peeled and unpeeled tomatoes or mangoes and the products were analyzed for volatiles, color, viscosity and sensory. Tomato juice with peel made by cold break contained higher levels of some lipoxygenase-derived volatiles, some carotenoid and amino acid derived volatiles, than the juice without peel whereas mango puree made with peel had lower levels of the lipoxygenase-derived volatiles and higher levels of the monoterpenes. Both the tomato juice and mango puree made with peel was less preferred in terms of flavor, aroma and overall liking than those made without peel.

Deodorization of undesirable garlic breath was achieved by consumption of water

(control), raw, juiced or heated apple, raw or heated lettuce, raw or juiced mint leaves or green tea after eating garlic. The levels of the garlic volatiles on the breath were analyzed from 1 to 60min by selected ion flow tube mass spectrometry (SIFT-MS).

Garlic was also blended with water (control), polyphenol oxidase (PPO), rosmarinic acid, quercetin, catechin, peppermint, spearmint or chocolate mint and the volatiles in the headspace analyzed from 3 to 40min by SIFT-MS. Mint leaves and rosmarinic acid produced the highest deodorization compared to other foods and phenolic compounds respectively. Different concentration of phenolic compounds did not produce a significant deodorization and PPO was not the major deodorizing agent.

Other non-phenolic compounds were suggested to take part in the deodorization process in addition to phenolic compounds and enzyme activity.

Dedication

This dissertation is dedicated to my husband, Serge Mawusse Akpalo and my daughter, Xhola Yawa-Atieno Akpalo.

iii

Acknowledgements

I would like to thank:

God, for being the faithful God that I have always known, for his many blessings in my life and for enabling me to successfully complete this work, glory be to HIM.

My Husband, Serge Akpalo and Daughter, Xhola Akpalo, for their support, encouragement and prayers.

My father and mother for the foundation they established in my early life and for encouragment and support. iAGRI for the financial support and for giving me the opportunity to attend this university for graduate school.

My advisor, Dr. Sheryl Barringer, for allowing me to work under her guidance, and for the great support she offered during my entire course of study. May my good Lord bless you.

My mother-in-law, Yoele Mathey, father-in-law, Maurice Akpalo and sister-in-law,

Jenniffer Akpalo for all the support and prayers.

My sister Rachel Atieno, brothers, Reeves Omiende, Rudin Osoro and Fadhili Odete and aunty, Risper Atieno who have always pushed me to do my best in everything and supported me in every way possible.

Committee Members, Dr. Monica Giusti, Dr, Luis Rodriguez-Saona and Dr. John H.

Litchfield for bringing out the best of me.

Hardy Castada and Gregory Sigurdson for their fervent assistance in my research work.

Vita

November 2009………………………………………. B.S. Food Science and

Technology, Sokoine University

of Agriculture. Tanzania.

June 2012……………………………………………... M.S Food Science, Sokoine

University of Agriculture.

Tanzania.

August 2012 to present………………………...... PhD. Food Science and

Technology, Department of Food

Science and Technology, The

Ohio State University

Fields of Study

Major Field: Food Science & Technology

Table of Contents Abstract ...... ii Dedication ...... iii Acknowledgements ...... iv Vita ...... v INTRODUCTION...... 1 Chapter 1: Literature Review ...... 3 1.0 Volatile compounds...... 3 1.1 Mangoes and their products ...... 3 1.2 Tomatoes and their products ...... 7 1.3 Volatile compounds important to garlic breath ...... 12 1.3.1 Diallyl disulfide ...... 13 1.3.2 Allyl Mercaptan ...... 16 1.3.3 Allyl methyl sulfide ...... 19 1.3.4 In vivo and in vitro deodorization of garlic breath ...... 20 1.4 Total phenolic compounds ...... 22 1.4.1 Phenolic compounds in mangoes ...... 22 1.4.2 Phenolic compounds in deodorization of garlic breath or odor ...... 24 1.5 Other factors affecting the quality to foods ...... 25 1.5.1 Rheological nature of mango juice and factors affecting its flow ability 25 1.5.2 Effect of break temperatures on the viscosity of tomato juice...... 27 1.5.3 pH and titratable acidity of tomato and mango juice...... 28 1.5.4 Total soluble solids in mango juice and tomato juice ...... 29 1.5.5 Color ...... 31 1.5.6 Fo-Value ...... 33 1.6 Commercial peeling ...... 34 References ...... 36 Chapter2: Effect of peels on quality attributes of hot and cold break tomato juice and sauce ...... 46 2.1 Introduction ...... 48 2.3 Materials and methods ...... 50

2.3.1 Tomato juice processing ...... 50 2.3.3 Volatiles ...... 52 2.3.4 Viscosity ...... 53 2.3.5 Color ...... 53 2.3.6 Titratable acidity ...... 54 2.3.7 pH 54 2.3.8 Brix ...... 54 2.3.9 Vitamin C determination...... 54 2.3.10 Sensory analysis ...... 55 2.3.11 Statistical analysis ...... 56 2.4 Results and Discussion ...... 56 2.4.1 Effect of peels on volatile levels ...... 56 2.4.2 Effect of thermal treatments on volatile levels ...... 63 2.4.3 Effect of presence of peel and thermal treatment on viscosity ...... 65 2.4.4 Total soluble solids (TSS), pH, titratable acidity (TA) and vitamin C .. 66 2.4.4 Effect of peel and thermal treatments on color ...... 66 2.4.6 Sensory analysis of cold break juice and sauce ...... 67 2.5 Conclusion ...... 70 Reference ...... 72 Chapter 3: Effect of peels on quality attributes of mango puree held at different times ...... 75 3.1 Introduction ...... 76 3.2 Materials and methods ...... 78 3.2.1 Mango puree processing ...... 78 3.2.2 Determination of total phenolics ...... 81 3.2.3 Viscosity ...... 82 3.2.4 Color ...... 82 3.2.5 pH 82 3.2.6 Brix ...... 83 3.2.7 Sensory analysis ...... 83 3.2.8 Statistical analysis ...... 84 3.3 Results and Discussion ...... 84 3.3.1 Effect of peel on lipoxygenase derived volatiles ...... 84 3.3.2 Effect of peel on esters, alcohols and acids ...... 86 3.3.3 Effect of peel on terpene hydrocarbon volatiles ...... 88 3.3.4 Effect of peel on other important volatiles ...... 90 3.3.5 Effect of peel and holding time on total phenolics ...... 91 3.3.6 Effect of peel and holding time on viscosity ...... 93 3.3.7 Effect of peel and holding time on color ...... 93 3.3.8 Sensory results ...... 94 3.4 Conclusion ...... 97

vii

References ...... 99 Chapter 4: Deodorization of garlic breath and odor by the use of food materials with different types and different amounts of phenolic compounds and polyphenol oxidase ...... 103 4.1 Introduction ...... 105 4.2 Materials and Methods ...... 109 4.2.1 Effect of pure Polyphenol oxidase (PPO) and phenolic compounds on garlic volatile levels ...... 109 4.2.2 Headspace volatile levels with different mint varieties ...... 110 4.2.3 Analysis of total phenolic compounds ...... 112 4.2.4 Quantitative analysis of rosmarinic acid by HPLC...... 113 4.2.5 Statistics ...... 114 4.3 Results and Discussion ...... 115 4.3.1 Garlic breath/ odor and deodorization process ...... 115 4.3.2 Contribution of the type of phenolic compound on garlic breath and odor deodorization ...... 116 4.3.3 Concentration of phenolic compounds in deodorization process ...... 121 4.3.4 Contribution of enzymes to the deodorization process of garlic breath 135 4.4 Conclusion ...... 139 Reference ...... 141 General conclusion and recommendation ...... 148 Bibliography ...... 150 Appendix: Tables, figures and questionnaires ...... 165

viii

List of Tables

Table 1: Effect of Processing and Storage on the Formation of Aroma Compounds in

Mango Juice (E1-Nemr and Ismail 1988) ...... 5

Table 2: Volatile flavor components of mango fruit (Macleod and Troconis 1982) . 6

Table 3: The Major Volatiles in Fresh Tomato (Hayes and others 1998) ...... 11

Table 4: The Major Volatiles in Tomato Paste (Hayes and others1998)...... 11

Table 5: Deodorizing rate of different food materials (Negishi and Negishi 1999;

Negishi and others 2002; Negishi and others 2004) ...... 17

Table 6: Total polyphenolic compounds in mango peel (mg/Kg) on dry matter basis

(Lakshminarayana and others 1970) ...... 23

Table 7:Volatile compounds in tomato juice and sauce with peel and no peel after different thermal treatments and break temperatures...... 58

Table 8: Physicochemical properties of cold and hot break juices and sauces with and without peel after different thermal treatments ...... 66

Table 9: Color properties of cold break and hot break juice and sauces with and without peel at different thermal treatment ...... 67

Table 10: Sensory preference for color, aroma, texture, flavor and overall liking of cold break HTST tomato juice and sauce ...... 68

Table 11: Kinetics of mango volatiles measured ...... 80

Table 12: Lipoxygenase derived volatile levels of mango puree made with and without peel at different holding times ...... 85

Table 13: Monoterpenes and other important volatiles level of mango puree made with and without peel at different holding times ...... 89

Table 14: Total phenolics, pH, total soluble solids, viscosity and L*, a*and b* values of mango puree made with and without peel at different holding times ...... 92

Table 15: Sensory results for mango puree made with and without peel at different holding times ...... 95

Table 16: Volatiles measured in the headspace scan ...... 111

Table 17: Difference in volatile concentration (ppb) between water and foods at 5 and

20 min...... 117

Table 18: Total phenolic content found in the various food materials analyzed ...... 122

Table 19: Rosmarinic acid content of different mint varieties ...... 127

Table 20: Garlic volatile compounds detected at 30 min ...... 128

Table 22: The concentration on different volatile compounds present in unpeeled and peeled tomato juice and pulp...... 165

Table 23: Rosmarinic acid content and total phenolic content of different mint varieties ...... 174

List of Figures

Figure 1: Formation of Hexanol, Z-3- Hexanal and E-2-Hexanal through

Lipoxygenase pathway. HPL (Hydro peroxide lyase), ADsH (Alcohol dehydrogenase) and LOX (Lipoxygenase) ...... 9

Figure 2 : Suggested mechanism for diallyl disulfide degradation (Negishi and others

2002) ...... 15

Figure 3: Mechanism of enzymatic deodorization. (Negishi and Negishi 1999) ...... 19

Figure 4: Processing steps for tomato juice and sauce ...... 51

Figure 5: Just About Right responses of panelists on tomato juice color, aroma, freshness and texture. Much too little = -2, slightly too little= -1, Just about right=0, slightly too much=1 and much too much= 2...... 69

Figure 6: Just About Right responses of panelists on tomato sauce color and aroma.

Much too little = -2, slightly too little= -1, Just about right=0, slightly too much=1 and much too much= 2...... 70

Figure 7: Processing treatments for mango puree...... 79

Figure 8: Change in ester concentration with increased holding time and the presence of peel...... 87

Figure 9: Decrease in alcohol and acid concentration with increased holding time and inclusion of peel ...... 88

Figure 10: Just About Right responses of panelists on mango puree made with and without peel held for 5min or 1h 45min on color, aroma, freshness and texture. Much

xi too little = -2, slightly too little= -1, Just about right=0, slightly too much=1 and much too much= 2...... 97

Figure 11: Effect of raw apple, raw lettuce, mint leaves, mint juice and apple juice on diallyl disulfide. Data points at the same time bearing different letters are significantly different (p ≤0.05) ...... 117

Figure 12: Effect of catechin, quercetin, rosmarinic acid, polyphenol oxidase and polyphenol oxidase -catechin on diallyl disulfide, allyl mercaptan and allyl methyl sulfide. Data points at the same time and same volatile bearing different letters are significantly different (p ≤0.05) ...... 120

Figure 13: Deodorization of garlic odor by use of peppermint, spearmint and chocolate mint ...... 124

Figure 14: HPLC Chromatograms of rosmarinic acid quantification of 3 different types of mint...... 127

Figure 15: Effect of pure rosmarinic acid (RA) and a combination of RA and peppermint on the garlic headspace volatiles...... 130

Figure 16: Effect of rosmarinic acid (RA) in peppermint on the garlic headspace volatiles ...... 132

Figure 17: Effect of different quantity of peppermint on garlic odor volatiles ...... 134

Figure 18: Effect of heated apple, heated lettuce and green tea on diallyl disulfide.

Data points at the same time bearing different letters are significantly different (p

≤0.05) ...... 135

Figure 21: Differences in volatile compounds present in peeled and tomato unpeeled juice and pulp ...... 168

xii

Figure 22: The concentration of E-2-hexenal in hot and cold break juices and sauces and at different thermal treatments. Values in the same row bearing different superscript letters are significantly different (p ˂ 0.05) ...... 168

Figure 23: The concentration of (Z)-3-hexen-1-ol in hot and cold break juices and sauces and at different thermal treatments. Values in the same row bearing different superscript letters are significantly different (p ˂ 0.05)...... 169

Figure 24: The concentration of hexanal in hot and cold break juices and sauces and at different thermal treatments. Values in the same row bearing different superscript letters are significantly different (p ˂ 0.05) ...... 169

Figure 25: The concentration of 6-methyl-5-hepten-2-one in hot and cold break juices and sauces and at different thermal treatments. Values in the same row bearing different superscript letters are significantly different (p ˂ 0.05) ...... 170

Figure 26: The concentration of beta-ionone in hot and cold break juices and sauces and at different thermal treatments. Values in the same row bearing different superscript letters are significantly different (p ˂ 0.05) ...... 170

Figure 27: The concentration of methional in tomato and sauces at different heat treatments. Values in the same row bearing different superscript letters are significantly different (p ˂ 0.05) ...... 171

Figure 28: The concentration of dimethyl sulfide in hot and cold break juices and sauces and at different thermal treatments. Values in the same row bearing different superscript letters are significantly different (p ˂ 0.05) ...... 171

Figure 29: The concentration of dimethyl disulfide in hot and cold break juices and sauces and at different thermal treatments ...... 172

Figure 30: The concentration of furfural in hot and cold break juices and sauces and at different thermal treatments ...... 172

xiii

Figure 31: Standard Curve for rosmarinic acid content ...... 173

Figure 32: Standard curve for total phenolics for different mint varieties ...... 173

Figure 33: Effect of raw apple, raw lettuce and mint leave on allyl mercaptan. Data points at the same time bearing different letters are significantly different (p ≤0.05)

...... 174

Figure 34: Effect of mint juice and apple juice on allyl mercaptan. Data points at the same time bearing different letters are significantly different (p ≤0.05) ...... 175

Figure 35: Effect of green tea, heated apple and heated lettuce on allyl mercaptan.

Data points at the same time bearing different letters are significantly different (p

≤0.05) ...... 175

Figure 36: Effect of raw apple, raw lettuce and mint leaves on allyl methyl disulfide

Data points at the same time bearing different letters are significantly different (p

≤0.05) ...... 176

Figure 37: Effect of Mint juice and apple juice on allyl methyl disulfide Data points at the same time bearing different letters are significantly different (p ≤0.05) ...... 176

Figure 38: Effect of heated apple, heated lettuce and green tea on allyl methyl disulfide Data points at the same time bearing different letters are significantly different (p ≤0.05) ...... 177

Figure 39: Effect of different food materials on allyl methyl sulfide. Data points at the same time bearing different letters are significantly different (p ≤0.05) ...... 178

Figure 40: HPLC Chromatograms of rosmarinic acid quantification of 3 different types of mint blended with garlic at 0 and 30min...... 179

xiv

INTRODUCTION

There are several factors that affect the quality and acceptability of food products. One of the major factors being flavor. Flavor is the interaction of taste, odor and textural feeling and it results from the volatile and nonvolatile components (Belitz and others 2009). The volatile components are those that are responsible for odor (aroma) and the non-volatiles are responsible for taste.

Volatile compounds are perceived by the odor receptor sites, the olfactory tissue of the nasal cavity. They reach the receptors when drawn in through the nose

(orthonasal detection) and via the throat after being released by chewing

(retronasal detection) (Belitz and others 2009). On the other hand, the nonvolatile compounds (at room temperature) in food interact with the taste receptors of the taste buds in the tongue. The four important taste perceptions are sweet, sour, bitter and salty (Belitz and others 2009). Sugars and acids are mostly responsible for sweet and sour tastes of food respectively.

Formation of the different volatile compound is as a result of different enzymatic or non-enzymatic pathways which are sometimes enhanced or decreased during food processing. Different foods have different volatile compounds and a combination of different volatile compounds at a specified concentration results in a specific aroma. This explains the differences in aroma and flavor in different foods. Different parts of the plant also contain different amounts of components

1 that act as precursors for the formation of volatile compounds. Therefore, the different parts for example, the peels of a fruit may be used to produce more of the volatiles responsible for the good aroma and flavor or vice versa.

Understanding the contribution of the different parts of a fruit or a vegetable to the volatile composition of a certain food may assist in exploiting natural sources of the volatile compounds in improving the quality of foods in terms of flavor.

Volatile compounds are responsible for either desirable or undesirable flavor or aroma in food. Therefore, it would be beneficial to determine means of enhancing desirable flavor or aroma and suppressing the undesirable aroma. The peels of tomatoes and mangoes have been reported to contain higher levels of compounds that act as precursors for the formation of important volatile compounds (Ties and

Barringer 2012). Therefore, peels can be used to increase the amount of some volatile compounds. On the contrary, the suppression of undesirable odor has been achieved by the use of raw food in deodorizing the garlic breath whose deodorization mechanism is not clear (Negishi and others 2002).

Chapter 1: Literature Review

1.0 Volatile compounds

1.1 Mangoes and their products

Mango is one of the important and popular tropical fruits and has an attractive flavor. Different mango cultivars have different flavor characteristics and their flavor volatile profile greatly depends on the level of maturity, part of the fruit and sample preparation. Volatile compounds are essential in the sensory quality of fruit products since they determine consumer’s acceptability. There are about 452 volatile compounds that have been identified in 20 mango cultivars (Pino and others 2005). The total concentration of volatiles was 18-123 mg/kg of fresh fruit

(Pino and others 2005). Terpene hydrocarbons were the major volatiles of all cultivars, the dominant terpenes being δ-3-carene (cvs. Haden, Manga amarilla,

Macho, Manga blanca, San Diego, Manzano, Smith, Florida, Keitt, and Kent), limonene (cvs. Delicioso, Super Haden, Ordon˜ez, Filipino, and La Paz), terpinolene (cvs. Obispo, Corazo´ n, and Huevo de toro), and R-phellandrene (cv.

Minin) (Pino and others 2005).

Processing affects the volatile profile of mango products, whereby there was a sharp decrease in all volatile fractions after extraction of mango juice and heat treatment at 85oC for 10 min then bottled and stored (E1-Nemr and Ismail 1988).

This decrease is probably due to evaporation of volatiles due to the heat treatment, 3 especially of the ester fractions. The decrease of the volatile compounds was also accompanied by the formation of other compounds, which were not present in fresh juice, such as acetyl furan and 5-methylfurfural due to reactions between the reducing sugars and amino acids during the heat processing of mango juice. Upon storage of the processed juice for two and four months at room temperature, there was formation of other flavor compounds namely, Ethyl laurate, Ethyl myristate,

Ethyl palmitate, and Seline- 11-ene-4-ol that were absent in fresh juice and in heat processed juice. The volatiles that were formed during processing also increased within the two months of storage, after which there was no further increase except for butyl-3-hydro butanoate (table 1).

In guava nectar, there was also a significant decrease on some volatiles with heat pasteurization at 85oC for 42 sec. Hexanal decreases with pasteurization (Correa and others 2010). Studies have focused on the general effect of processing and heat treatment on volatiles in mango juice and guava nectar but were not specific on the effect of different thermal processing on the volatile profile.

Table 1: Effect of Processing and Storage on the Formation of Aroma

Aroma Compounds Concentration (ppm) Fresh mango After Storage time juice processing (months) (85oC/10min) Acetyl furan 0 0.015 0.03 0.03 5-Methyl furfural 0 0.01 0.03 0.03 β-Terpineol 0 0.01 0.035 0.035 Butyl-3-hydroxy butanoate 0 0.025 0.03 0.04 Ethyl laurate 0 0 0.01 0.01 Ethyl myristate 0 0 0.01 0.1 Ethyl palmitate 0 0 0.028 0.35 Seline- 11-ene-4-ol 0 0 0.017 0.2 Compounds in Mango Juice (E1-Nemr and Ismail 1988)

Important volatiles in mango include γ-octalactone, which gives a sweet herbaceous, coconut-like odor and taste and contributes to the overall aroma of the ripe fruit with an odor detection threshold (ODT) of 7ppb (Pino and others

2005).

(E)-β-ionone is another volatile present in mangoes and it is a product of carotenoid oxidation with a violet like aroma and ODT of 0.007 ppb. It contributes to the overall aroma of the mango cultivars (Pino and others 2005).

Other important volatiles include hexanal which contributes to a fatty- green, grassy odor, (E)-2-hexenal, with a green fruity, fresh-green odor, (E, Z)-2,6- nonadienal, (E)-2-nonenal, (Z)-11-pentadecenal, pentadecanal and hexadecanal.

These are all produced via lipid metabolism. The general quality odor of some volatiles present in mango fruit is indicated in Table 2.

Table 2: Volatile flavor components of mango fruit (Macleod and

Troconis 1982)

Component Amount Odor quality (μg/kg) Dimethyl sulphide Trace Sulphurous, cabbage-like Methyl cyclohexane 0.31 None Octane 0.06 None A dimethyl cyclohexane Trace None Hydrocarbon 0.12 Sweet fruity Trichloroethylene 0.43 Slightly fruity α-Pinene 5.01 Fragrant, scented, floral Toluene Trace Buttery, caramel Car-3-ene 15.88 Green, pungent, mango leaves α-Phellandrene + C,- 0.31 Estery alkylbenzene Limonene 3.67 Lemon-like β-Phellandrene 1.65 Fatty, oily (Z)-Ocimene + C,-alkylbenzene 1.59 Fragrant, herbal γ-Terpinene 4.7 Flat, dull p-Cymene 0.79 Herbal, slightly minty A dimethylstyrene 3.79 Mango Furfural 7.94 Cold meat, gravy Acetylfuran 0.06 Green, resinous, woody Benzaldehyde 0.73 Wood bark. green twigs 5-Methyl-2-furfural 0.12 Caryophyllene 0.31 Floral, fragrant Acetophenone 3.36 Sweet, floral, wallflowers Phenylacetaldehyde α-Humulene 2.75 Floral, fragrant Benzenoid compound 0.12 Unpleasant floral Valencene 0.12 Floral, green p-Selinene 5.31 Floral, nutty, almonds A methyl propenyl benzene 0.98 Sickly Cereal-like, hay Hydrocarbon 0.31 Sweet fruity

1.2 Tomatoes and their products

Tomato peels have been found to have significant higher levels of volatile than the fleshy part (Ties and Barringer 2012). The volatiles that were higher in tomato peels included (Z)-3-hexenal and methanol in all 10 varieties tested. 1- Hexanol,

2- methyl propanal, dimethyl sulfide, hexanal, hexen-1-ol, methional, methyl benzoate, and propanoic acid were significantly greater in 6 of the 10 varieties

(Ties and Barringer 2012). Other volatiles that are also significantly higher in peels in the majority of the varieties are the lipid-derived volatile (C-6 aldehyde volatiles) that is formed through the lipoxyganase pathway. These volatiles included (Z)-3-hexenal, 1-hexanol, hexanal, hexen-1-ol, nonadienal, and (E)-2- hexenal (Ties and Barringer 2012).

The peel contained significantly higher levels of total fatty acids tested in six varieties out of the 10 varieties tested. The fatty acid content of whole tomato is between 0.5 and 1.9g/kg. Linoleic acid was found in highest concentration (39% to51%), followed by Palmitic acid (26% to 30%), then linolenic acid (5% to 19%) and oleic (4% to 19%). Stearic acid (2% to 4%) was always present in the lowest concentration (Ties and Barringer 2012). The peel contained a significant higher concentration of linolenic acid than the flesh part whereas for oleic, the content was higher in the flesh part than the peel. There were minimal differences for palmitic, stearic and linoleic acid. The fatty acid acids are important since they are precursors for lipid-derived volatiles in lipoxyganase pathway (Ties and Barringer

2012). These volatiles produced during the lipoxygenase pathway are responsible

7 of the green fresh aroma of tomatoes. Volatiles such as hexanol, (Z)-3- hexanal and (E)-2-hexanal are formed through lipoxygenase pathway as shown in Figure

1. Lipoxygenase (LOX) activity is another factor that affects the volatile concentration. LOX activity was found to be greater in the peel than in the flesh in all the 10 varieties tested. It was determined that the LOX activity was greatest in the fleshy tissues adjacent to the peel since when all the flesh was removed from the skin, the LOX activity was drastically lowered. Therefore, the high LOX activity of the peel may be attributed by the small amount of the fleshy part that had remained on the skin. The cultivar also affects the concentration of volatiles since different cultivars have varying fatty acid content and LOX activity.

Therefore, tomato juice produced from unpeeled tomatoes may have more volatiles than the peeled tomatoes.

Tomato acyl lipids

Phospholipids Galactolipids triacylglycerols

(Acyl hydrolase enzymes)

Free fatty acid (C16:0, C18:2, C18:3)

Linoleic (C18:2) Linolenic (C18:3)

LOX LOX

9-+ 13-hydroperoxy-C18:2 9-+ 13-hydroperoxy- C18:3

HPL HPL

Hexanal Z-3-Hexanol

ADH ADH Isomerase

Hexanol (Z)-3-Hexanal (E)-2-Hexanal

Figure 1: Formation of Hexanol, Z-3- Hexanal and E-2-Hexanal through Lipoxygenase pathway. HPL (Hydro peroxide lyase), ADsH (Alcohol dehydrogenase) and LOX (Lipoxygenase)

The processing of tomato impacts the volatile profile of the final product. During thermal processing, there is formation of some volatile compounds that are not present in fresh tomato juice. These volatile compounds are thermal products that are formed during the degradation of carbohydrates. They include furfural derivatives such as methyl-5-acetyl-2 furan and furfuryl alcohol and ethyl 9 benzene and trimethyl benzene (Sieso and Crouzet 1977). There are also reports of loss and considerable decrease of some volatiles that are present in fresh juice such as hexanal, pentanol and of (Z)-3-hexen-1-ol in canned tomato juice that was processed at 80oC for 10 min (Sieso and Crouzet 1977; Xu and Barringer 2009).

Some volatiles are associated with the aroma of fresh tomato namely 3-methyl butanol, (E)-2-hexenal, (Z)-3-hexen-l-ol, E-2-hexen-l-ol and 2-isobutylthiazol which decrease during the concentration process in vacuum concentration at 63--

65 cm pressure and 55-60°C to 28o Brix and a canning temperature of 94 °C and the cans immediately cooled with fresh water (Sieso and Crouzet 1977). Other reports on the volatiles associated with fresh tomato juice and tomato paste were reported by Hayes and others (1998) (Table 3 and Table 4). The concentrations of the volatiles were determined for both tomato juice and tomato paste. To obtain the odor activity value (OAV), the concentrations were divided by their odor threshold concentrations in water. The logarithm of odor units for each volatile was calculated and used to rank the volatiles in order of importance. All components in the paste at a concentration lower than their odor threshold were considered unlikely to contribute significantly to fresh tomato or tomato paste odor (Hayes and others 1998).

Table 3: The Major Volatiles in Fresh Tomato (Hayes and others 1998)

Compound Concentration Odor threshold Log odor units (ppb) in water (ppb) (Z)-3-hexenal 12000 0.25 4.7 β-Ionone 4 0.007 2.8 Hexanal 3100 4.5 2.8 β-Damascenone 1 0.002 2.7 1-Penten-3-one 520 1 2.7 3-Methylbutanal 27 0.2 2.1 Furaneol 700 31 (at pH 4.5 1.4 (at pH 4.5) (E)-2-hexenal 270 17 1.2 2-Isobutylthiazole 36 3.5 1.0

Table 4: The Major Volatiles in Tomato Paste (Hayes and others1998)

Compound Concentration Odor threshold Log odor (ppb) in water (ppb) units Dimethyl sulphide 2000(0) 0.3 3.8 β-Damascenone 14 (1) 0.002 3.8 1-Octen-3-one 2 0.005 2.6 β-Ionone 2 (4) 0.007 2.5 Dimethyl trisulphide 2 0.01 2.3 Furaneol 1000 (700) 31 (at pH 4.5) 1.5 (at pH 4.5) 3-Methylbutanal 24 (27) 0.2 2.1 1-Nitro-2-phenylethane 66 (17) 2 1.5 Eugenol 100 6 1.2 Methional 3 (0) 0.2 1.2 6-Methyl-5-hepten-2-one 370 (130) 50 0.87 3-Methylbutyric acid 2000 (200) 250 0.9 Phenylacetaldehyde 18 (15) 4 0.65 Linalool 20 (2) 6 0.5 (Z)-3-hexenal 0.7 (12000) 0.25 0.4 Hexanal 8 (3100) 4.5 0.3 2-Isobutylthiazole 5 (36) 3.5 0.16

It is also essential to note that different processing methods have different effects on the volatile profile (quality) of tomato juice. Vallverdú-Queralt and others

(2013) confirmed the presence of the following volatiles as major markers in 11 tomato that are said to greatly contribute to the tomato aroma: hexanal, (E)-2- hexenal, (E,E)-2,4-decadienal, (Z)-3-hexenol, hexanol, 1-penten-3-one, 6-methyl-

5-hepten-2-one, geranyl acetone, and 2-isobutylthiazole. Research was conducted to compare the effect of High intensity pulsed electric field (HIPEF) and thermal treatment (TH) of tomato juices on volatiles. HIPEF better maintained the individual volatiles just after processing and during storage than thermally treated

(TH) juices at 90oC for 60 s. (Z)-3-hexenol and hexanol are better retained in

HIPEF than in TH. There is an increase of (E, E)-2, 4- decadienal and it was higher in thermally treated tomato juice than in HIPEF. This compound when present in concentration higher than its threshold value can be responsible for fatty and rancid notes in the juice (Vallverdú-Queralt and others 2013).

1.3 Volatile compounds important to garlic breath

Garlic is a bulb that is widely used, as a condiment due to is attractive flavors formed after it had been either cut or crushed. The volatiles responsible for the garlic flavor are associated with the sulfur glucoside containing plants. Garlic consumption has been linked to various health benefits such as lowering of blood cholesterol, enhancing blood circulation and lowering blood sugar (Fleischauer and Arab 2001; Hsing and others 2002; Challier and others 1998; Amagase and others 2001). This has led to the consumption of raw garlic by a number of individuals. Consumption of raw garlic results to a strong undesirable garlic breath that lingers for almost 24h. Therefore, it is important to develop means of deodorizing the volatile compounds responsible for the garlic breath. Four 12 volatiles have been associated with garlic breath; these include diallyl disulfide, allyl methyl disulfide, allyl mercaptan and allyl methyl sulfide (Minami and others 1989; Suarez and others 1999; Tamaki and Sonoki 1999; Tamaki and others 2008).

1.3.1 Diallyl disulfide

Diallyl disulfide is formed from allicin. This volatile has a detection threshold of

0.22ppb. Diallyl disulfide originated from the garlic particles that are trapped in the mouth (Suarez and others 1999). This volatile increases rapidly to its maximum concentration after ingestion (Taucher and others 1996; Suarez and others 1999) and there is a significant decrease an hour after ingestion (Minami and others 1989). It becomes negligible in two hours (Taucher and others 1996;

Suarez and others 1999 Tamaki and Sonoko 1999).

Negishi and Negishi 2002 investigated removal of diallyl disulfide. In their study, a mixture of diallyl disulfide and crushed food were analyzed for concentration of this volatile compound in the headspace by the use of gas chromatography (GC).

There was reduction of this volatile by approximately 70% by the use of lettuce, mushroom, spinach, parsley and basil. The reduction of diallyl disulfide has been achieved by its degradation by vegetables to form thiol gases. This degradation process was caused by action of reductase. The thiol gases formed then bond to phenolic compounds in the presence of polyphenol oxidase (Negishi and Negishi

2002).

Bad odor has also been deodorized by raw apple and three possible mechanisms were suggested. These mechanisms included addition reaction, degradation reaction and physical and chemical interaction (figure 2). In addition, reaction, O- quinones are formed from phenolic compounds that bind with the thiols. This process is referred to as enzymatic deodorization since polyphenol oxidase (PPO) and peroxidase (POD) catalyze the reaction (Negishi and others 2002). The degradation reaction is achieved by the action of reductase on diallyl disulfide to form thiols and the thiols are then eliminated by addition reaction to o-quinone

(Negishi and others 2002). Physical and chemical interaction is another proposed mechanism that involves affinity of the sulfur compounds to food molecules by trapping them in the porous polymers contained in food (Negishi and others

2002). This may also be a possible deodorization mechanism for ally methyl disulfide.

Figure 2 : Suggested mechanism for diallyl disulfide degradation (Negishi and others 2002)

Researchers have also reported higher level of deodorization of the volatiles responsible for garlic breath with raw food materials than the microwaved food

(Munch and Barringer 2014). Raw apple achieved a greater deodorization effect of most volatiles than cooked or microwaved apple. Other raw foods that led to a decrease in volatile levels on an in vivo test included parsley, spinach and mint

(Munch and Barringer 2014). The deodorization achieved by raw food material was suggested to be due to both enzymatic and phenolic compounds (Munch and

Barringer 2014). The microwaved or cooked foods included green tea apple and lemon juice all had a deodorization effect but not as high as the raw foods. This deodorization was suggested to be a polyphenolic deodorization (Munch and

Barringer 2014). Research on the effect of milk on garlic breath volatiles 15 demonstrated higher deodorization level of whole milk than fat-free milk on an in vivo test (Hansanugrum and Barringer 2010). This deodorization effect was suggested to be by physical and chemical interaction between the sulfur compounds and milk component (protein and lipids) (Negishi and others 2002).

1.3.2 Allyl Mercaptan

Allyl mercaptan is another volatile responsible for garlic breath that is produced by reduction of diallyl disulfide. Of the four garlic breath volatiles, it has the lowest odor detection threshold of 0.05ppb. Thus, it has been reported as the main volatile sulfur compound in breath immediately after ingesting raw or cooked garlic (Suarez and others 1999; Tamaki and others 2008). Just like diallyl disulfide, allyl mercaptan comes from the garlic particles that are trapped in the mouth. It also reaches is peak concentration rapidly, decreases significantly an hour after digestion and becomes negligible in two hours, (Suarez and others

1999).

The main proposed deodorization mechanism of allyl mercaptan is in the presence of polyphenol oxidase (PPO) and phenolic compounds. Raw fruits and vegetables have been used for enzymatic deodorization (Table 7) because of being high in phenolic compounds and PPO (Negishi and Negishi 1999;). Different fruits and vegetable have varying deodorizing rate whereas the different cultivar of same fruit and vegetable vary in deodorizing rate (Negishi and Negishi 1999).

Table 5: Deodorizing rate of different food materials (Negishi and Negishi

1999; Negishi and others 2002; Negishi and others 2004)

Foods & % Deodorization Beverages Methyl Allyl Diallyl Diallyl sulfide mercaptan mercaptan disulfide Apple 45-100 35 14 3 Plums 100 - - - Prune 100 100 38 8 Apricot 100 - - - Loquat 100 - - - Lettuce 100 - 70 21 Endive 100 - - - Chicory 100 - - - Celery (leaf) 100 - - - Udo 100 - - - Perilla 100 - - - Peppermint 100 - - - Basil 100 100 73 24 Burdock 100 100 33 27 Eggplant 100 100 27 10 Cherry 95 - - - Peach 93 - - - Grape 73-88 - - - Potato 88 - - - Blueberry 37-76 41 57 21 Okra 76 - - - Avocado 51 - - - Banana 49 - 58 11 Red cabbage 42 - - - Parsley 34 - 70 33 Cucumber 34 - - - Ginger 29 - - - Lemon 27 - - - Horseradish 27 - - - Melon 27 - - - Grapefruit 24 - - - Nectarine 22 - - - Continued

Table 5 continued

Foods & % Deodorization Beverages Methyl Allyl Diallyl Diallyl sulfide mercaptan mercaptan disulfide Wasabi 17 - - - Spinach 15 - 70 6 Tomato 15 - - - Mandarine orange 12 - - - Bell pepper 12 - - - Radish 7 - - - Water melon 7 - - - Strawberry 2 - - - Pineapple 0 - - - Kiwi 0-15 35 85 18 Yam 0 - 41 19 Cooked rice 0 5 70 22 Persimmon - - 12 0 Asparagus - - 38 11 Cow’s milk - 46 95 91 Egg - - 84 88 Carrot 39 - - - Green tea 0 7 - -

By chewing raw fruits and vegetables, the reaction between polyphenols and PPO take place. Phenolic compounds are oxidized by abstracting hydrogen atom from phenolic hydroxyl group to form negatively charged phenoxide ions. An electron from each phenoxide ion is lost to form radicals which are delocalized and the resulting radicals react with CH3S· formed from methyl mercaptan molecules

(Negishi and Negishi 1999). The radicals then bind to the radicals formed from allyl mercaptan to form conjugates that are either odorless or have a different odor. Oxidation of phenolic compounds is still possible in the absence of PPO to form radicals. This reaction is referred to as free addition reaction as illustrated in figure 3. Theoretically, deodorization of allyl mercaptan would still take place

18 with the presence of phenolic compounds and absence of PPO. Nevertheless, researchers have reported PPO and phenolic compound to better deodorize allyl mercaptan than just phenolic compounds. Studies have reported a complete removal of bad breath caused by allyl mercaptan after eating fresh raw apple.

Other food materials such as green tea show a deodorizing effect on methyl mercaptan only after 3 h of ingestion (Lodhia and others 2008).

Figure 3: Mechanism of enzymatic deodorization. (Negishi and Negishi 1999)

1.3.3 Allyl methyl sulfide

Allyl methyl sulfide is associated with the persistent garlic odor in breath after ingestion of both raw and cooked garlic (; Suarez and others 1999; Tamaki and others 2008). It originates from gut and could not be metabolized by liver and gut tissues of rat (Suarez and others 1999). Unlike diallyl disulfide and allyl mercaptan, ally methyl sulfide increased slowly after ingestion and become a 19 predominant compound three hour after ingestion (Suarez and others 1999). Also, it took more than a 24h for allyl methyl sulfide to drop to its baseline level

(Taucher and others 1996).

When garlic was just chewed and not swallowed, allyl methyl sulfide was present in low levels in the breath for 30min only and not detected in the urine (Suarez and others 1999). Therefore, Suarez and others (1999) hypothesized that allyl methyl sulfide was absorbed from gut to the blood circulation and transported other parts of the body such as lungs and urine.

Eating of garlic and ku-ding-ka tea would decrease allyl methyl sulfide for the first hour but the levels would increase again in the next 2 to 4h (Negishi and

Negishi 2004). From these results, Negishi and others (2004) proposed that polyphenols were responsible for the deodorization of allyl methyl sulfide in breath by apple and ku-ding-ka tea. Since ku-ding-ka tea had a higher content of phenolic compounds than apple, it attained a longer deodorizing effect. Also, the deodorization of allyl methyl sulfide by apple during the first hour after ingestion might be caused by enzymatic deodorization of allyl mercaptan, a precursor of allyl methyl sulfide.

1.3.4 In vivo and in vitro deodorization of garlic breath

Foods such as apples, parsley, mint leaves and green tea, were tested in vivo and have been reported to have a deodorizing effect on garlic breath (Munch and

Barringer 2014). Microwaved and raw apples both produced a significant

20 decrease on the levels of garlic breath volatiles (Munch and Barringer 2014).

However, raw apple exhibited a greater reduction than microwaved apple. By microwaving the apple all the enzymes present are deactivated hence the deodorization taking place is attributed to the presence of phenolic compounds.

Furthermore, deodorization by both enzyme and phenolic compounds has been reported to be better than that of phenolic compounds alone (Negishi and Negishi

1999). Fat free milk, 2% fat milk and whole had a significant decrease on the garlic breath volatile (Hansanugrum and Barringer 2010). Consumption of milk mixed with garlic had a greater deodorizing effect than when the milk was consumed after the consumption of garlic. Deodorization of methyl mercaptan was effective by eating raw apple, plum, prun, apricot and cherry whereas lower deodorization was achieved by pear, grapes, kaki and citrus fruits (Negishi and

Negishi 1999). Lettuce, chicory, celery, potato, peppermint, basil and mushroom were some of the vegetables that were very effective in removing methyl mercaptan (Negishi and Negishi 1999). The deodorization of methyl mercaptan by raw fruits and vegetables was attributed to presence of phenolic compounds and polyphenol oxidase. Green tea powder also showed a reduction in concentration of methyl hydrogen sulfide and hydrogen sulfide but this reduction was observed at 1,2 and 3h after ingestion (Lodhia and others 2008).

In an in vitro test, fat-free and whole milk had a significant reduction on the concentrations of the analyzed volatile compounds in the headspace of chopped garlic (Hansanugrum and Barringer 2010). Whole milk was more effective on hydrophobic compounds, diallyl disulfide and allyl methyl disulfide whereas fat-

21 free milk was more effective on the reduction of the hydrophilic compounds allyl mercaptan and allyl methyl sulfide (Hansanugrum and Barringer 2010). Similarly, an in vitro testing on lettuce, apple, pear, mushroom and eggplant also had a deodorizing effect on most of the garlic volatiles (Negishi and others 2002;

Negishi and Negishi 1999). Unripe apple and pear achieved the highest deodorization; these green foods contain high concentration of phenolic compounds and polyphenol oxidase (Negishi and Negishi 1999). Other food compounds that are low in phenolic content such as lettuce has also shown a decrease in the levels of these volatiles (Negishi and others 2002).

1.4 Total phenolic compounds

1.4.1 Phenolic compounds in mangoes

Polyphenols are secondary metabolites of plants and have been linked to health promoting and antioxidative properties. Bioactive compounds found in mangos among other plants have been related to a number of health benefits such as; antioxidative, antimutagenic, anticarcinogenic antiatherosclerotic and angiogenesis (Cao and Cao 1999). There has been an increased interest in the study of mango phenolic compounds from mango fruit, peels, seeds, leaves and kernels due to the health promoting properties that make consumption of mango and its products a healthy habit. The polyphenolic compounds already identified in mango pulp include, mangiferoin, gallic acids, gallotannins, quercetin, isoquercetin, ellagic acid and β-glucogallin. Gallic acid had been identified as one of the major polyphenol present in mango fruits (Scheiber and others 2000) 22 whereas mangiferin has been reported to be the main polyphenolic compound in the leaves and stem bark with medicinal properties. Total phenolics and their associated antioxidative capacity decrease with ripening of the fruit (Kim and others 2010). Total phenols have been found to be higher in the mango peel than mango flesh in all stages of mango development (Lakshminarayana and others

1970) with an estimated total phenolic content of mango peel of 4066mg

(GAE)/Kg (dry matter) (Table 5). Unlike mango flesh, ripe mango peels have also been reported to have higher levels of total phenolic content than raw peels (Ajila and others 2007).

Table 6: Total polyphenolic compounds in mango peel (mg/Kg) on dry matter basis (Lakshminarayana and others 1970)

Compound Amount (mg/kg) Mangiferin 1690.4 Mangiferin gallate 321.9 Isomangiferin 134.5 Isomaneferin gallate 82 Quercetin 3-O-galactoside 651.2 Quercetin 3-O-glucoside 557.7 Quercetin 3-O-xyloside 207.3 Quercetin 3-O-arabinopyranoside 101.5 Quercetin 3-O-arabinofuranoside 103.6 Quercetin 3-O-rhamnoside 20.1 Kaempferol3-O-glucoside 36.1 Rhamnetin 3-Ogalactoside/glucoside 94.4 Quercetin 65.3 Total Phenolics 4066.0

1.4.2 Phenolic compounds in deodorization of garlic breath or odor

Phenolic compounds are antioxidants, compounds that delay or inhibit oxidation of substrate when present in low concentration compared to the oxidizable substrate (Apaka and others 2007). Different phenolic compounds have different antioxidant capacities depending on the structure of a particular phenolic compound. In researching on a modified method of Folin-Ciocalteu in comparison to the original method, different phenolic compounds had different antioxidant capacities (Berker and others 2013). Rosmarinic acid (4.08) was reported to have a higher trolox equivalent antioxidant capacity (TEAC) than catechin (3.23) and catechin was higher than quercetin (2.78) (Berker and others

2013) in the modified method. Different methods reported different values of

TEAC of different phenolic compounds (Berker and others 2013). Antioxidant capacity increases with increased concentration and rosmarinic acid reported higher antioxidant capacity than rutin and BHT (Vladimir-Knežević and others

2011). Therefore, the differences in total antioxidant capacities may also affect the level of deodorization by different phenolic compounds. Foods may vary in the types of phenolic compounds and may also have more than one type of phenolic compound. Rosmarinic acid is a major phenolic compound in mint leaves (Tahira and others 2011), quercetin in apples (Lee and others 2003) and catechin in tea (Nagao and others 2007). Different apple cultivars were tested of polyphenol oxidase activity at different developmental stages (Holderbaum and others 2010). Fuji and Elster showed the highest PPO values followed by mellow cultivar at the initial and intermediary stages (Holderbaum and others 2010).

There was no significant difference in the PPO activity of Fuji, Elster and mellow at the final stage. Aori27 showed the lowest PPO activity of all the cultivars in all the developmental stages (Holderbaum and others 2010).

1.5 Other factors affecting the quality to foods

1.5.1 Rheological nature of mango juice and factors affecting its flow ability

Viscous fluids tend to deform continuously under the effect of applied stress.

They can be categorized as Newtonian fluids or non-Newtonian fluids. For

Newtonian fluids, the viscosity is constant and is independent of shear rate. Fluids such as gasses, oils, water and most liquids containing 90% water such as fruit juices, carbonated beverages, milk, coffee and tea exhibit Newtonian behavior

(Sahin and Sumnu 2006). The viscosity of non-Newtonian fluid however changes with changing rate of shear and hence should be characterized by more than one parameter (Dak and others 2007).

The rheological properties of fruit juices appear to be very much dependent of their variety, state of ripeness, and concentration of juice/pulp and temperature variation during storage and processing. Knowledge of rheological properties of fluid foods are important for quality, understanding the texture, process engineering applications, correlation with sensory evaluation, designing of transport systems, equipment design (heat exchanger and evaporator), deciding pump capacity and power requirements for mixing (Dak and others 2007).

The flow nature of mango juice and other fruit juices including papaya, melon and water melon (no thermal treatment) are said to be shear thinning (pseudoplastic) fluids since their flow behavior index (n) is less than 1 (Dak and others 2007;

Sahin and Sumnu 2006). The viscosity of mango juice has been reported to be

936.00 m Pas with total solids of 14.8%, moisture content of 85.2%, and there is a decrease in viscosity with increasing moisture content (Ikegwu and Ekwu 2009).

Thus, high viscosity is attributed to solubility and quantity of high total solids in juice (Ikegwu and Ekwu 2009). The activation energy of mango juice varies from 1.66 to 11.35 kJ/mol K, at various concentration levels estimated using the

Arrhenius model. The lowest concentration, 5.17% total solids, had the highest activation energy and the highest concentration, 17% total solids, had the lowest activation energy. Therefore, the viscosity of mango juice at a low concentration of total solids is more affected by temperature than those with high concentration of total solids (Dak and others 2006; Dak and others 2007; Singh and Eipeson

2000).

Pectin content affects the viscosity/flow characteristic of mango pulp. Fruit juices with high pectin content are higher in viscosity than those with low pectin.

However, depectinization had no effect on the flow characteristics of the mango pulp and suggested that this may be due to the low level of depectinization, which was about 7.5% (Manohar and others 1990). Depectinization of the mango pulp was carried out for 15, 30 and 90 min. One gram of pectinase enzyme was added to l00g of mango pulp heated to 40°C (Manohar and others 1990). After the

26 depectinization period, the pulp was inactivated by heating the pulp to 85°C and holding for 15 min. Conversely, an effect of depectinization by the use of hot and cold break processing of tomato concentrate and a significant effect on the viscosity since depectinization was about 35% (Fito and others 1983). The means of depectinization impacts pectin level which affects the viscosity of the juice.

1.5.2 Effect of break temperatures on the viscosity of tomato juice

Tomato juice and paste, like any other fruit juice, also exhibits pseudoplastic flow. Break temperature during processing greatly influences the viscosity of tomato products regardless of the cultivar. High break temperature produces products of higher viscosity whereby hot-break juice has a significantly higher viscosity at 1986 mPa.s, than cold-break juice at 1547 mPa.s (Hsu 2008). The apparent viscosity of pastes made from tomatoes was highest at a break temperature of 107°C and lowest at 85°C. This observation may be explained by the greater degree of inactivation of the pectic enzymes polygalacturonase (PG) and pectin methylesterase (PME) (Xu and others 1986). Under favorable conditions after homogenization of tomatoes, PME demethylates pectin to methanol and polygalacturonic by removal of the methyl groups from the polygalacturonic acid chain (Anthon and others, 2002). Pectin becomes more vulnerable to further degradation by PG because this enzyme acts on the segments that have been demethylated by PME, i.e. to produce oligomeres and monomers of galacturonic acid (Anthon and others, 2002). The low viscosity in cold break is a result of shortened pectin chains.

Similar results were also reported by Goodman and others (2002) that hot break juice (0 min) had a significantly higher viscosity than cold break juice (held for 2 to 20 min at room temperature after being chopped before thermal processing at

93oC) for all of the cultivars tested. In cold break treatment, the enzymes are active and therefore pectin is degraded and results in a lower viscosity compared to the hot break juice. In hot break treatment, all the enzymes are inactivated and thus there is a presence of pectin hence more viscous.

1.5.3 pH and titratable acidity of tomato and mango juice pH and titratable acidity (TA) have a dual role in fruit juices by acting as a flavor promoter and preservative factor (Akhtar others 2010). TA is a measure of the sum of all the organic acids in juice whose composition varies depending on the nature of the fruit and maturity. Mature unripe fruits are high in organic acid and therefore have a lower pH than ripe fruits. The pH also varies with the type of fruit, whereby there are fruits which are high in organic acid such as the citrus fruits (Oranges) and others such as water melon are low in organic acid and thus have a higher pH. Most fruit juice contains tartaric, malic, citric, succinic, lactic and acetic acids (Tasnim and others 2010). The TA of mango pulp ranges between 0.31 and 0.456% and a pH of between 3.77 and 4.27 depending on the cultivar (Veerenjaneya and Sarathi 2009). The TA and pH of tomato juice is between 0.38 and 0.64 % and 4.15 and 4.46 respectively (Tandon and others

2003).

A decrease in pH of the fruit pulp sample occurs in proportion to an increase in acidity (Akhtar and others 2010; Rivera and Carbornida 2008). An increasing trend in pH and a decreasing trend in titratable acidity was observed in thermal treatment of grape juice at all processing times (30, 45 and 60 min) and temperatures (60, 70, 80 and 90oC) (Cabrera and others 2009). No significant change was reported in pH and titratable acidity of mango pulp that had been heat treated at 95oC for 1 min (Hal and others 2012).

A significant decrease in pH in the juice that had undergone a High Pressure

Carbon Dioxide treatment (HPCD) was noted. The decrease in pH in HPCD may have been caused by CO2 dissolving into juices or solutions, which further dissociated to bicarbonate, carbonate and H+ ions. There has also been a significant decrease in titratable acidity in apple juice that had undergone high temperature short time (HTST) treatment (Aguilar-Rosas and others 2007). This may be due to the evaporative effect of organic acids as a function of temperature increase. Similar results were also observed when pasteurization temperatures of

73, 80 and 83oC were tested at a holding time of 27 s (Charles-Rodriguez 2007).

1.5.4 Total soluble solids in mango juice and tomato juice

Total Soluble Solids (TSS) is one of the most important quality factors and it is one of the parameters that strongly affect flavor of food (Moneruzzaman and others 2011). Sugar in juice is measured using the Brix scale. Brix is reported as

29 degrees Brix and is equivalent to percentage. One-degree Brix is equal to one gram of sugar per 100 grams of juice. The TSS varies depending on the type of fruit used in the juice since fruits differ in their amount of sugar. (Byarugaba-

Biaruzake 2008). Common sugars present in fruits and vegetables are fructose, glucose, and sucrose. The TSS of mango pulp ranges between 11.50-20.5± 0.75% for different mango cultivars and that of mango juice ranges between 14.2% and

20.5% (Veeranjaneya and Sarathi 2009; Bhardwaj and Mukherjee 2010; Hui and

Evranuz and others 2012). The TSS of industrially processed mango juice from

5.1 to 10.3% (Saeed and others 2012). Tomato juice has a TSS ranging between

4.15 and 6.62 % (Kaur and others 2006). TSS also affects other physical properties of fruit juice such as viscosity whereby the higher the TSS the higher the viscosity in mango juice (Singh and Eipeson 2000).

Researchers have reported the effect of temperature and holding time on TSS. An increase of total soluble solids and sugar to acid ratio was observed from 60oC to

80oC but decreased when temperature reached 90oC for 60 min in grape juice

(Cabrera and others 2008). The increase may be attributed to an evaporative effect and the decrease may be due to non-enzymatic browning reactions at the expense of the sugars. Thus, high processing time and temperatures may greatly affect the

TSS of grape juice (Cabrera and others 2008). Contrary to the reports by other researchers, Threlfall and others (2005) reported no change in TSS on heating the must of Black Beauty and Sunbelt grapes.

1.5.5 Color

Color is an important attribute because it is usually the first property the consumer observes. Color pigments called carotenoids are responsible for the characteristic color of mango skin and pulp. Mango carotenoids (Most common being β-

Carotene) and tomato carotenoids (lycopene) are synthesized in the fruit during ripening (Schwarts and others 2007).

Carotenoids are moderately steady within plant cells, but they are more labile during postharvest handling practices or processing (Forget and others 2000). The loss of carotenoid could be a quality indicator from a processing perspective.

Carotenoids are affected by light, oxygen, temperature, peroxides, type of packaging (modified atmosphere packaging), and duration of storage (Moore

2003). Exposing the carotenoids to these elements may lead to undesirable alterations in structure and bioactivity of carotenoids (in terms of isomerization, oxidation, or degradation). The alteration caused by these elements may in turn change the UV-VIS properties of carotenoids but the color appearance to the eye may remain unaffected (Moore 2003). In addition, antioxidant activity may be reduced extensively.

Color in foods may be evaluated with respect to hue angle, lightness, and chroma.

Lightness is a measure of a color's lightness or brightness. Hue angle is a measure of the color angle or actual color. A decrease of hue angle in edible mango flesh represents a change from green to yellow to red and chroma is a measure of a

31 color's intensity (Ahmed and others 2002). Hue angle (h°) is calculated by equation 1.

b∗ h° = tan−1 [1] a∗

Since the major color of mango puree is yellow, Hunter b value is the physical parameter to describe the yellow color degradation of mango puree in thermal processing, and thus the degradation can be calculated by the following equation

ln 푏 =-k1t [2] ⁄푏′

Where, b is the Hunter color b value at time t (dimensionless); b’ is the Hunter color b value at zero time (dimensionless) and k1 is the rate constant for yellow color degradation (min-1) (Ahmed and others 2002). During thermal processing of mango puree at 50, 60, 70, 80 and 90oC for holding times 0, 5, 10, 15 and 20min, it was observed that the Hunter b value and both L and a values decreased with time at the given temperature as the puree turned brown (Ahmed and others

2002). Whereby, L is lightness or darkness, a, is redness or greenness and b yellowness or blueness. Enzymatic browning may have caused the browning of the puree.

Color in the tomato is due to the presence of carotenoids; lycopene is the major carotenoid, comprising about 83% of the total pigment present with beta-carotene accounting for about 3 to 7% of the total. The main cause of color change is oxidation, which depends on: availability of oxygen, low water activity, high temperature, the destabilizing presence of pro-oxidants, metal ions such as copper

32 and iron, the stabilizing presence of antioxidants and lipids (Hayes and others

1998). The color of tomato juice may also change due to the formation of brown compounds caused by maillard browning mainly due to the presence of amino acids and reducing sugars (Hayes and others 1998). No overall significant difference was found between the color of hot break (0 min) and cold break juice

(held for 2 to 24 min after being chopped before thermal processing at 93oC)

(Goodman and others 2002).

1.5.6 Fo-Value

The Fo value is the number of minutes of equivalent sterilization at 121.11°C. If another temperature is used this is normally stated after the symbol F. In practice the F-Value is typically set at 12D values for low acid foods. The F-Value is therefore the duration (in minutes) of the entire sterilization or pasteurization process, at a defined temperature. Fo is calculated by equation 3.

Fo= Δt Σ 10(T-Tb)/Z [3]

Where Δt is the Measurement interval, T is the heating temperature, Tb the reference temperature normally121.1°C (for commercial sterilization) and Z is the temperature unit of logarithmic sterilization capability changes (generally, 10 °C is used).

1.6 Commercial peeling

There is availability of machines, which automatically peel stone fruits and particularly mangoes. An example of such a machine is described in the

Australian Patent No. 602073. This machine has a curved peeling blade that conforms to the concave outer surface of the mango. The mango is first halved and seeded (scraping all the flesh from the pit) and then transported by a conveyer belt towards the peeling blade. However, such arrangement is unsuitable if it is desirable for the fruit to be peeled as single piece rather than slice (Harding 2001).

Three methods are generally desired for the peeling of tomatoes for processing, these are the chemical lye peeling (using Sodium hydroxide, NaOH or Potassium hydroxide, KOH), steam or scalding hot water. These methods remove the cuticle and most of the external cell layers of the tomatoes. Steam is most commonly used method in California. In terms of world production this makes steam the most common method used in the world. Lye peeling with sodium hydroxide on the other hand removes most of the pee but the creation of wastewater and use of large amounts of water, eliminate it in use in some places. Steam and scalding hot water are an expensive method and that’s why lye peeling has gained more popularity in some areas too. Lye peeling is associated with over peeling which impacts on the color. Over peeling caused by improper lye peeling results to a light red color and exposure of some superficial yellow vascular bundles. It is therefore important to monitor operation to avoid the undesirable appearance and also reduce excessive flesh loss. One major disadvantage of lye peeling is the disposal problems that may

34 be destructive to the environment and costly (Schlimme and others 1984; Corey and others 1986).

For lye peeling, the tomatoes are normally treated with a hot lye solution (18%

NaOH w/v) at approximately 95oC in steam-jacketed kettles for periods ranging from 30 to 75 s. The tomatoes are then sprayed with cold water, causing the peel to split. Removal of the peel is much easier following this procedure (Garcia and

Barrett 2006; Knoblich and others 2005).

References

Aguilar-Rosas SF, Ballinas-Casarrubias ML, Nevarez-Moorillon GV,

Martin-Belloso O, Ortega-Rivas E. 2007. Thermal and pulsed electric

fields pasteurization of apple juice: Effects on physicochemical properties

and flavour compounds. J Food Eng. 83(1):41-46.

Ahmed J, Shivhare US, Kaur M. 2002. Thermal colour degradation kinetics of

mango puree. Int J. Food Properties 5(2): 359–366.

Ajila CM, Bhat SG, Prasada Rao UJS. 2007. Valuable components of raw and ripe

peels from two Indian mango varieties. J Food Chem. 102(4): 1006-1011.

Akhtar S, Riaz, M, Ahmad A, Nisar A. 2010. Physico-chemical, microbiological

and sensory stability of chemically preserved mango pulp. Pakistan

Journal. Bot. 42(2): 853-862.

Amagase H, Petesch BL, Matsuura H, Kasuga S, Itakura Y. 2001. Intake of garlic

and its bioactive components. Journal of Nutrition; 131(3s):955S–962S.

Anthon GE, Barrett D M. 2008. Combined enzymatic and colorimetric method for

determining the methanol and uronic acid content of pectins application to

tomato products. J Food Chem110: 239–247.

Apak R, Güçlü K, Demirata B, Özyürek M, Çelik SE, Bektaşoğlu B, Berker K,

Özyurt D. 2007. Comparative Evaluation of Various Total Antioxidant

Capacity Assays Applied to Phenolic Compounds with the CUPRAC Assay.

J Mol 12:1496-1547.

Barrett DM, Weakley C, Diaz JV, Watnik M. 2007. Qualitative and Nutritional

Differences in Processing Tomatoes Grown under Commercial Organic and

Conventional Production Systems. J Food Sci. 72(9):441-451.

Belitz HD, Grosch W, Schieberle P. 2009. Food Chemistry. Spinger. Heidelberg,

Berlin.

Berker KI, Ozdemir Olgun FA, OzyurtD, Demirata B, and Apak R 2013.

Modified Folin−Ciocalteu Antioxidant Capacity Assay for Measuring

Lipophilic Antioxidants. J Agric Food Chem. 61: 4783-4791.

Bhardwaj, R.L. and Mukherjee, S. 2011. Effects of fruit juice blending ratios on

kinnow juice preservation at ambient storage condition. African Journal of

Food Science 5(5): 281 - 286.

Byarugaba-Biaruzake, G. W. 2008. Effect of Enzymatic Processing on Banana

Juice and Wine. Dissertation for award of PhD at Stellenbosch University,

Russia, 110pp.

Cabrera SG, Jang JH, Kim St, Lee YR, Lee HJ, Chung HS, Moon KD. 2009.

Effects of processing time and temperature on thequality components of

campbell grape juice. J Food Processing and Preservation 33(1):347-360.

Cao YH, Cao RH. 1999. Angiogenesis inhibited by drinking tea. Nature, 6726,

381-398.

Challier B, Perarnau JM, Viel JF. 1998. Garlic, onion and cereal fibre as

protective factors for breast cancer: A French case-control study. European

J. Epidemiology; 14(8):737–747.

Charles-Rodrı´guez AV, Neva´rez-Moorillo GV, Zhang QH, Ortega-Rivas E.

2007. Comparison of thermal processing and pulsed electric fields treatment

in pasteurization of apple juice. J Chem Eng 85(2): 93-97.

Correa MIC, Chaves JBP, Jham GN, Ramos AM, Minim VPR, Yokota SRC.

2010. Changes in guava (Psidiumguajava L. var. Paluma) nectar volatile

compounds concentration due to thermal processing and storage. Ciência e

Tecnologia de Alimentos 30(4): 1061-1068.

Corey KA, Schlimme DV, Frey BC. 1986. Peel removal by high pressure steam

from processing tomatoes with yellow shoulder disorder. J. Food Sci. 51:

388–390, 394.

Dak M, Verma RC, Sharma GP., 2006. Flow characteristics of juice of

‘‘Totapuri’’ mangoes. J. Food Eng 76 (1) 557–561.

Dak M, Verma RC, Jaafrey SNA. 2007. Effect of temperature and concentration

on Rheological properties of ‘‘Kesar’’ mango juice. J. Food Eng 80 (2007)

1011–1015.

E1-Nemr SE, Ismail IA. 1988. Aroma changes in mango juice during processing

and storage. J Food Chem 30: 269-275.

Fito PJ, Clemente G, Sanz, FJ. 1983. Rheological behavior of tomato concentrates

(Hot break and Cold break). J Food Eng. 2: 51-62.

Fleischauer AT, Arab L. 2001. Garlic and cancer: A critical review of the

epidemiologic literature. J Nutri; 131(3s):1032S–1040S.

Forget, F. L.; Ggauillard, F.; Rigal, D. Changes in the carotenoid content of

apricot

(Prunus armeniaca, var. Bergeron) during enzymatic browning: β-carotene

inhibition of chlorogenic acid degradation. J. Sci. Food Agric. 2000, 80,763-

768.

Garcia E, Barrett D M. 2006. Peel ability and yield of processing tomatoes by

steam or lye. J Food Process Pres 30 (10): 3–14.

Goodman CL, Fawcett S, Barringer SA. 2002. Flavor, viscosity, and color

analyses of hot and cold break tomato juices. J Food Sci 67(1): 404-408.

Harding GJ. 2001. Peeler for fruits and vegetables, U. S. patent No. 6324969. U. S.

patent and trade mark office, Washington, D.C.

Hal PH, Bosschaart C, Twisk C, Verkerk R, Dekker M. 2012. Kinetics of thermal

degradation of vitamin C in marula fruit (Sclerocarya birrea subsp. caffra) as

compared to other selected tropical fruits. J Food Sci Technol 49(1): 188-19.

Hansanugrum A, Barringer SA. 2010. Effect of milk on the deodorization of

malodorous breath after garlic ingestion. J Food Sci 75(6):549–58.

Hayes WA, Smith PG, Morris AEJ. 1998: The Production and Quality of Tomato

Concentrates, Critical Reviews in Food Science and Nutrition, 38(7): 537-

564.

Holderbaum DF, Kon T, Kudo T and Guerra MP 2010. Enzymatic browning

polyphenol oxidase activity, and polyphenols in four apple cultivars:

Dynamics during fruit development. HortScience 45(8): 1150-1154.

Hsing AW, Chokkalingam AP, Gao YT. 2002. Allium vegetables and risk of

prostate cancer: A population-based study. J of the National Cancer

Institute; 94(21): 1648–1651.

Hsu KC. 2008. Evaluation of processing qualities of tomato juice induced by

thermal and pressure processing. LWT, 41; 450-459.

Hui YH, Evranuz EO. 2012. Hand Book of plant base fermented food and

beverage technology.2nd ed. New York: Taylor and Francis. 802p.

Ikegwu OJ, Ekwu FC. 2009. Thermal and physical properties of some tropical fruits

and their juice in Nigeria. J Food Techn 7(2): 38-42.

Kaur D, Sharma R, Wani A, Gill BS, Sogi DS. 2006. Physicochemical changes in

seven tomatoes (Lycopersicon esculentum) of different cultivars during

rippening in short term storage. Int J Food Protect 9:747-757.

Kim HJY, Moon D, Kim M, Lee H. Cho YS, Choi AK, Cho SK. 2010.

Antioxidant and antiproliferative activities of mango (mangifera indica L.)

flesh and peel. J Food Chem, 121:429-436.

Knoblich M, Anderson B, Latshaw D. 2005. Analyses of tomato peel and seed

byproducts and their use as a source of carotenoids. J Sci Food Agric

85(10):1166–1170.

Lakshminarayana S, Subhadra NV, Subramanyan H. 1970.Some aspects of

developmental physiology of mango fruit. J Hort Sci 45:133-42.

Lodhia P, Yaegaki K, Khakbaznejad A, Imai T, Sato T, Tanaka T, Murata T,

Kamoda T. 2008. Effect of green Tea on volatiles sulfur compounds in

mouth air. J. Nutrition Sci Vitaminol.

Lee K, Kim Y, Kim D, Lee H, Lee C 2003. Major phenolics in apple and their

contribution to the total antioxidant capacity. J Agric Food Chem, 51:6516–

6520.

Macleod AJ, Troconis NG. 1982. Volatile flavour components of mango fruit. J

Phytochemistry, 21(10): 2523-2526.

Manohar B, Ramakrishna P, Ramteke RS. 1990. Effect of pectin content on flow

properties of mango pulp concentrates J of Texture Studies 21: 179-190.

Medlicott PA, Thompson KA. 2006. Analysis of sugar and organic acids in

ripening mango fruit (Mangifera indica L.var Keitt). J Sci Food Agric.

36(7):561-566.

Minami T, Boku T, Inada K, Morita M, Okazaki Y. 1989. Odor components of

human breath after the ingestion of grated raw garlic. J Food Sci 54 (3):

763-765.

Munch R, Barringer S.A. 2014.Deodorization of garlic breath volatiles by food ad

food components. J Food Sci. 79(4):526-533.

Moneruzzaman, K.M., Al-Saif, A.M., Alebidi, A.I., Hossain, A.B., Normaniza, O.

and Nasrulhaq, B.A. 2011. An evaluation of the nutritional quality

evaluation of three cultivars of Syzygium samarangense under Malaysian

conditions. African Journal of Agricultural Research 6(3): 545-552.

Moore J P. 2003. Carotenoid synthesis and retention in mango (mangifera indica)

fruit and puree as influenced by postharvest and processing treatments.

Thesis presentation, University of Florida.

Nagao T, Hase T, Tokimitsu I, 2007. A green tea extract high in catechins reduces

body fat and cardiovascular risks in humans. Obesity 15(6):1473–1483.

Negishi O. and Negishi Y. 1999. Enzymatic deodorization with raw fruits,

vegetables and mushrooms. Food Sci. Technol. Res 5(2):176-80.

Negishi O, Negishi Y, Ozwa T 2002. Effects of food materials on removal of

Allium- specific sulfur compounds. J. Agric. Food Chem.50:3856-3861.

Negishi O, Negishi Y, Yamaguchi F, Sugahara T. 2004. Deodorization with ku-

ding-cha containing a large amount of caffeoyl quicin and acid dervatives. J.

Agric. Food Chem 52:5513-8.

Pino JA, Mesa J. 2006. Contribution of volatile compounds to mango (Mangifera

indica L.) aroma. J. Flav. Frag., 21(2): 207–213.

Pino JA, Mesa J, Munoz Y, Martia MP, Marbot R. 2005. Volatile components

from mango (Mangiferaindica L.) cultivars. J Agric Food Chem. 53(6):

2213-2223.

Rivera, A.P. and Cabornida, I.M.B. 2008, Development of ready-to-drink green

mango juice. USM R & D J 16(1): 71-77.

Saeed A, Shahid M, Haider S J, Ijaz A, Shaukat U. 2012. Quality evaluation of

different brands of Tetra Pak mango juices available in market. PAK. J.

Food Sci., 22(2), 2012:96-100.

Sahin S, Sumnu GS. 2006. Physical properties of foods. New York: Springer. 257p.

Schieber A, Ullrich W, Carle R. 2000. Characterization of polyphenols in mango

puree concentrate by HPLC with diode array and mass spectrometric

detection. Innovative Food Science and Emerging Technologies 1:161-166.

Schlimme DV, Corey KA, Frey BC. 1984. Evaluation of lye and steam peeling

using four processing tomato cultivars. J. Food Sci. 49: 1415–1418.

Schwartz SJ, Von Elbe JH, Giusti MM. 2007. Colorants. Fennema OR,

Damodaran S, Parkin KL. 2007. Food Chemistry. New York: Taylor and

Francis Group. 597p.

Sieso V., Crouzet J. 1977. Tomato volatile components: effect of processing. J Food

Chem 2: 241-252.

Singh' NI, Eipeson WE. 2000. Rheological behaviour of clarified mango juice

concentrates. J. of Texture Studies 31: 287-295.

Sharma SK, Le Maguer M. 1996. Lycopene in tomatoes and tomato pulp

fractions. Italian J Food Sci 8(2): 107- 113.

Suarez F, Springfield J, Furne J, Levitt M. 1999. Differentiation of mouth versus

gut as site of origin of odoriferous breath gases after garlic ingestion. Am J

Physiol 276: G425–30.

Tahira R, Naeemullah M, Akbar F, Masood MS. 2011. Major phenolic acids of

local and exotic mint germplasm grown in Islamabad. Pak. J. Bot., 43: 151-

154.

Tamaki T, Sonoki S. 1999. Volatile sulfur compounds in human expiration after

eating raw or heat-treated garlic. J Nutr Sci Vitaminol 45: 213-222.

Tamaki K, Sonoki S, Tamaki T, Ehara K. 2008. Measuremet of odour after in

vitro or in vivo ingestion of raw or heated garlic, using electronic nose,

gas chromatography and sensory analysis. Intl J Food Sci Technol

43:130–9.

Tandon KS, Baldwin EA, Scott JW, Shewfelt RL. 2003. Linking sensory

description to volatile and non volatile componets of fresh tomato flavor. J

Food Sci 68(7):2366-71.

Tasnim, F, Anwar HM , Nusrath S , Kamal HM, Lopa D, Formuzul HKM. 2010.

Quality assessment of industrially processed fruit juices available in Dhaka

City, Bangladesh. J Mal Nutr 16 (3): 431-438.

Taucher J, Hansel A, Jordan A, Lindinger W. 1996. Analysis of Compounds in

Human Breath after Ingestion of Garlic Using Proton-Transfer-Reaction

Mass Spectrometry. J. Agric. Food Chem. 44: 3778-3782.

Ties P, Barringer S. 2012. Influence of lipid content and lipoxygenase on flavor

volatiles in the tomato peel and flesh. J Food Sci 77(7): 830-837pp.

Threlfall RT, Morris JR, Howard LR, Brownmille RCR, Walker TL. 2005. Pressing

effects on the yield, quality and nutraceutical content of juice, seeds and skins

from Black Beauty and Sunbelt grapes. J. Food Sci. 70: 167–171.

Veeranjaneya RL, Sarath VRO. 2009. Production, optimization and

characterization of wine from mango (Mangifera indica Linn.). Natural

Product Radiance, Research paper 8(4): 426-435.

Vallverdú-Queralt A, Bendini A, Barbieri S, Di Lecce G, Martin-Belloso O,

Gallina Toschi T. 2013. Volatile profile and sensory evaluation of tomato

juices treated with pulsed electric fields. J Agric Food Chem 61:

1977−1984.

Vladimir-Knežević S, Blažeković B, Štefan MB, Alegro A, Kőszegi T, Petrik J.

2011. Antioxidant Activities and Polyphenolic Contents of Three Selected

Micromeria Species from Croatia. J Mol 16: 1454-1470.

Xu S, Shoemaker CF, Luh BS. 1986. Effect of break temperature on rheological

properties and microstructure of tomato juices and pastes. J. Food Sci. 51(2)-

399.

Xu Y, Barringer S. 2009. Effect of temperature on lipid-related volatile production

in tomato puree. J Agric Food Chem. 57(19): 9108–9113.

Chapter2: Effect of peels on quality attributes of hot and cold break tomato juice and sauce

ABSTRACT

Tomato peels have been shown to produce more lipoxygenase-derived volatiles that are associated with the fresh flavor of tomatoes, than the tomato flesh.

Tomatoes were processed into tomato juice from peeled and unpeeled tomatoes using hot break or cold break. The juices were thermally treated by high temperature short time (HTST), low temperature long time (LTLT) or retort.

Fresh juice was the control with 10% calcium chloride to stop enzymatic activity in the tomato juice. Sauces were then made from the respective juice and the tomato products were analyzed for volatiles, color, viscosity and sensory. Juice with peel made by cold break contained higher levels of some lipoxygenase- derived volatiles such as (E)-2-hexenal, (Z)-3-hexen-1-ol and hexanal and some carotenoid and amino acid derived volatiles, than the juice without peel. Because of the lack of enzyme activity, hot break juices had lower levels of these volatiles and there was no significant difference between hot break juices with and without peel. Fresh and HTST juice had higher levels of most of the volatiles including the lipoxygenase- derived volatiles than LTLT. The presence of peel produced a significant decrease in the viscosity of the cold break juice and sauce. There was no significant difference in the hue angle, total soluble solids, pH, titratable 46 acidity and vitamin C in most of the treatments. The texture, flavor and overall liking of cold break juice with no peel was preferred over that with peel whereas the color was less preferred. No significant differences in the preference were obtained between the sauces.

Practical application

There is an increased level of lipoxygenase-derived volatiles in the peel than the fleshy part, which is linked with the fresh flavor of tomatoes. Peeling the tomatoes before juicing decreases the concentration of some of these important volatiles but increases viscosity and overall acceptability when cold break is used.

No difference is seen in the hot break juices or sauces.

2.1 Introduction

Tomatoes are the second-most consumed vegetable around the world (Min and others 2003). Most tomatoes are consumed as processed products such as tomato juice, paste, puree, ketchup, sauce, and canned tomatoes. Processed tomato products are important sources of minerals and vitamins in the diet. Flavor, color, taste, and nutritional value are major quality attributes of foods and influence the consumer’s choice.

There have been over 400 flavor volatiles identified in tomato and a combination of 10 of these volatiles at varying concentrations produces the aroma of a ripe tomato (Petro- Turza 1987). A majority of these volatiles in tomatoes are lipid derived and originate from the lipoxygenase pathway. The important volatiles in tomato peel and flesh can be broken down into three categories: lipid derived, amino acid derived and carotenoid derived. During processing of tomatoes into juice, some parts such as skin and seeds are removed as product waste but their presence may impact the volatile profile of tomato products.

Tomato peels produce higher levels of lipid-derived volatiles than the fleshy part through lipoxygenase activity (Ties and Barringer 2012). Thus unpeeled cold break tomato products are likely to have fresher flavor than peeled tomato products. Cold break juice is usually high in lipoxygenase derived volatiles mainly because at these temperatures enzymatic activity is allowed which results in the increased production of these associated volatiles. On the other hand, hot break juice will have decreased levels of these volatiles because the enzymes are inactivated not allowing their production to take place. The lipoxygenase derived

48 volatiles include hexanal, (Z)-3-hexenal, (E)-2-hexenal, hexanol, (Z)-3-hexenol and (E)-2-hexenol which are typically associated with a fresh or green note in tomato aroma (Ties and Barringer 2012).

Thermal treatment of tomato juice helps to preserve the product and at the same time makes the food safe for consumption. Tomato juice can be retorted; whereby it is filled in cans and placed in a retort in either a batch system or a continuous retort process. Other forms of heat treatment that can be used are high temperature short time (HTST) treatment at 72oC for 15 sec and low temperature long time

(LTLT) treatment at 63oC for 30 min. HTST treated juice is expected to be of better quality than LTLT due to its shorter heating time.

Viscosity of juice is dictated by a number of factors, the presence of pectin being one of them. High pectin levels usually results in high viscosity. In fruits the level of pectin can be reduced by the action of enzymes including pectin methyl esterase (PME) and polygalacturonic acid (PG). These enzymes break down the structure of pectin resulting in loss of its functionality. Break temperatures have an effect on PME and PG enzymes, whereby hot break treatments leads to their deactivation whereas cold break increases activity of these enzymes. One of the ways to manipulate the viscosity of tomato juice is by changing the break temperature.

Hedonic sensory testing is a method that attempts to quantify the degree of liking or disliking of a given product by consumers. The 9 point hedonic scale is commonly used and consist of the following options; like extremely, like very much, like moderately, like slightly, neither like nor dislike, dislike slightly

49 dislike moderately, dislike very much and dislike extremely (Lawless and

Haymann 2010). The objective of this study was to determine the effect of presence of peel, different thermal treatment, concentration of juice to sauce and hot and cold break temperatures on volatile compounds, viscosity, color, titratable acidity, total soluble solids and pH of tomato juice and tomato sauce.

2.3 Materials and methods

2.3.1 Tomato juice processing

Fresh and fully ripe tomatoes (Guard PS696) were harvested in Wooster, Ohio

USA, in September 2013. The tomatoes were washed and for peeled tomato juice samples were treated with a hot lye solution (18% NaOH w/v) at approximately

95oC in the lye peeler for 60 s (Figure 1). They were then sprayed with cold water on a rubber disc peeler, which helped in removing the peels. Juice was extracted from peeled and unpeeled tomatoes separately using a juicer (Omega juicer model

8000, Harrisburg, PA, USA). The extracted juice then underwent hot break processing at 93oC or cold break at 65oC in a tube and shell heat exchanger

(Groen, model D10, San Francisco, CA, USA). Fresh juice, 1500ml, was treated with 10% calcium chloride for both with peel and no peel juice to stop the enzymatic reaction.

Fresh

Sauce Cold Break

HTST Peeled Tomatoes

Retort Sauce Hot Break

Tomatoes

Fresh

HTST Sauce

Cold Break LTLT

Retort Unpeeled Tomatoes Sauce

Hot Break Retort

Figure 4: Processing steps for tomato juice and sauce

2.3.1.1 Thermal processing

Three thermal treatments were performed: high temperature short time (HTST), low temperature long time (LTLT) and retort. The juice that was to be HTST treated (38 L) was held for 5- 10 min after being juiced. It was then thermally treated through a tube and shell heat exchanger for a high temperature short time

(HTST) at 72oC for 15s. The HTST equipment used was micro-Thermics Inc.

(5024-F; model 25HV hybrid UHT/HTST Raleigh, NC, USA) operating at a

51 production rate of 2000ml/min. One holding tube was used to achieve a holding time of 15s. The juice was cooled to 25 °C by the heat exchanger following the holding tube. For LTLT, a steam jacketed kettle (Dover, model TDC/2-10,

Chicago, IL, USA) was used. The juice, 3000 ml, was held for 17min before the

LTLT; it was then heated for 20 min to get to 63oC and held for 30 min. About

2L of juice was held for approximately 30 min before retorting and 280 ml per can was retorted at 220oC for 30 min. The juices were refrigerated for 15-19 h and the analysis for volatiles and vitamin C was done. Juice used for viscosity, titratable acidity, pH and total soluble solids was stored in the refrigerator for 2 d and that for tomato sauce was also stored in the refrigerator for 4 d.

2.3.2.2 Tomato Sauce

Cold or hot break unpeeled tomato juice, 1.5 L, were placed in a rotary vacuum evaporator (Model WU 23012-12, Cole-Parmer Chicago, IL). The water in the tomato juice was evaporated for 2 h 15 min, at a temperature of 50oC and pressure of 6.6 mbar until total solids of 11% was reached. Dilution was not done to the sauce before carrying out the analytical measurements.

2.3.3 Volatiles

The tomato juice or sauce sample (60ml) was put in a 500ml Pyrex culture bottle covered with tops enclosed with septum then placed in a water bath at 50oC for 1h in a water bath (precision circulating bath model 260, Winchester, VA, USA).

Two needles were used; one was connected to the SIFT-MS machine and was

52 pierced through the septum into the bottle to allow the flow of volatiles through the machine. The second one was pierced through the septum into the bottle and was left in open air for ventilation. The volatiles were measured using a Selected

Ion Flow Tube Mass Spectrometer (SIFT-MS) (SYFT Voice 200, Christchurch,

New Zealand) using the method described in Ties and Barringer (2012).

2.3.4 Viscosity

The viscosity of tomato juice or sauce was measured using a viscometer (LV

DVII+, Brookfield Engineering Laboratories, Inc., Stoughton, MA) with a UL adapter. Viscosity was determined at 22 °C, 4 rpm and spindle number 1 with 500 ml of juice placed in the UL adapter.

2.3.5 Color

Tomato juice or sauce samples (200ml) were filled into an optical cuvette and the color was measured by the use of light reflectance specular component included,

D65 illuminant, 10 degree observer angle with a colorimeter (Color Quest XE,

Hunter Associates Laboratory, Inc., Reston, VA, USA). All samples were measured in triplicate and the hue angle and lightness (L, a, b) was then reported

(Tongnuanchan and others 2010).

2.3.6 Titratable acidity

The titratable acidity was carried out as per method 942.15 (AOAC 1995). The tomato juice or sauce (25ml) was diluted to ca 250 ml with distilled water and 0.3 ml phenolphthalein indicator was added for each 100ml solution then titrated against 0.1 N sodium hydroxide to a pink end point.

2.3.7 pH

The pH of the tomato juice or sauce was directly determined using a pH meter

(Accumet, Fisher scientific 300025, Denver, Co, USA).

2.3.8 Brix

The total soluble solids of the tomato juice or sauce were determined using a refractometer (Reichert Abbe Mark II 10480, Buffalo, NY, USA) according to

Method 932.12 (AOAC 1995). The tomato juice was filtered through a kimwipe to remove the insoluble solids, a drop was put on the glass and the value was read.

2.3.9 Vitamin C determination

Tomato juice or sauce (20ml) was pipetted into a 250 ml conical flask and 150 ml of distilled water, 5 ml of 0.6 mol L−1 potassium iodide, 5 ml of 1 mol L−1 hydrochloric acid and 1 ml of starch indicator solution were added. The sample was titrated with 0.002 mol L−1 potassium iodate solution. The endpoint of the

54 titration was the first permanent trace of a dark blue-black color due to the starch- iodine complex. Titration was repeated 6 times with further aliquots of sample solution until concordant results were obtained.

2.3.10 Sensory analysis

A sensory test was conducted with an untrained group of 51 people that consisted of both males and females from the ages of 18 to 66 and over. This group was made up of people from the department of Food Science and Technology at the

Ohio State University. They were requested to rate the liking of color, texture, flavor, and overall acceptability by looking, tasting and smelling the tomato juice and only looking and smelling the tomato sauce. A hedonic scale of 1 to 9 was used for each attribute. The higher number represents a higher preference for the attributes. Just about right (JAR) testing on the color, aroma, freshness and texture was done which included responses on much too little, slightly too little, just about right, slightly too much and much too much for aroma and freshness. A sample is considered to be too high or too low in an attribute when less than 60% of the panelists marked it as being just about right. For color it ranged from much too light, slightly too light, just about right, slightly too light and much too light and for texture, much too thin, slightly too thin, just about right, slightly too thin and much too thin. Thermally processed HTST, peeled and unpeeled cold break processed juice and sauce, 30ml, were served in randomly numbered plastic cups on a tray with a bottle of water and random three digit numbers were assigned to the samples.

2.3.11 Statistical analysis

The data was coded, entered and analyzed using JMP version 10.0.2 64-bit edition

(Statistical discovery, Cary, NC, USA). Data were analyzed by using one-way analysis of variance (ANOVA) and comparison of all pairs using Tukey-Kramer

HSD. Significance was defined as p ≤ 0.05.

2.4 Results and Discussion

2.4.1 Effect of peels on volatile levels

When peel was present in fresh and cold break juices, there was a significantly higher level of half of the volatiles than in samples with no peel (Table 7). With peel cold break juice showed higher levels of most lipoxygenase-derived volatiles including (E)-2- heptane, (E)-2- pentenal, (E)-2-octanal, (Z) -3-hexen-1-ol, (Z)-3- hexenal and hexanal than no peel cold break juice. These are most of the major lipoxygenase-derived volatiles that are associated with green fresh flavor in tomato juice and are known to contribute to the tomato flavor (Sieso and Crouzet

1977; Hayes and others 1998; Vallverdú-Queralt and others 2013). Studies have reported more lipoxygenase-derived volatiles in peels than in the fleshy part of tomatoes (Ties and Barringer 2012). Therefore, we expected higher levels of these volatiles in juice when peels are present.

The increased amount of volatile compounds when peel is present in cold break juice may be due to either the presence of higher levels of total fatty acid in the peels than in the fleshy part or higher lipoxygenase (LOX) activity in the fleshy

56 part adjacent to the peel than the rest of the flesh. Fatty acids in tomatoes are important because they are the precursors that are converted to the lipid-derived volatiles. Linolenic acid levels have been reported to be higher in concentration in the peels of tomatoes than in the flesh (Ties and Barringer 2012). It is therefore possible that the differences observed in the products with peel are caused by the higher concentrations of linolenic acid present in the peel than in the fleshy part.

The LOX activity is another factor affecting the concentration of lipid-derived volatiles. LOX activity is higher in the fleshy part adjacent to the peel than the rest of the flesh (Ties and Barringer 2012). During peeling this part is removed resulting in decreased LOX activity. Therefore, products with peel may have had a higher LOX activity since the skin adjacent to the peel was retained. Thus there is a higher level of lipid-derived volatiles in with peel than in no peel products.

Table 7:Volatile compounds in tomato juice and sauce with peel and no peel after different thermal treatments and break temperatures

Hot break Cold break Hot break Analyte Cold break juice juice sauce sauce Fresh HTST Retort LTLT Retort HTST Retort Concentration with with with with with with no with (ppb) no peel peel no peel peel peel peel no peel peel no peel peel peel peel (E)-2-heptenal 2.00c 3.02b 1.59cd 3.05b 3.16b 1.55cd 1.64cd 1.24d 6.56a 4.25b 1.32cd 1.20d (E)-2-hexenal 28.98def 57.79cd 56.66cde 112.47c 133.8b 28.44def 13.14f 14.11f 174.12a 172.56a 19.88f 22.86ef (E)-2-nonenal 0.44a 0.63a 0.23a 0.54a 0.64a 0.44a 0.38a 0.43a 1.72a 0.85a 0.35a 0.35a (E)-2-octenal 0.61d 0.92bcd 0.61d 1.1b 1.05bc 0.69d 0.70d 0.66d 4.74a 2.25a 0.63d 0.73cd c b c b a def c de a a ef ef 58 (E)-2-pentenal 2.89 3.88 2.81 3.580 4.88 1.71 2.13 2.03 11.41 5.07 1.53 1.54

(E,E)-2,4-decadienal 0.64cde 0.95c 0.55cde 0.80cd 0.94c 0.60cde 0.49de 0.47de 4.72a 2.03b 0.33e 0.42de (E,Z)-2,6-nonadienal 1.16abcd 1.64a 0.59d 0.84bcd 1.31abc 1.00bcd 1.04bcd 0.76cd 3.60a 1.36ab 0.88bcd 0.92bcd (Z)-3-hexen-1-ol 5.82b 6.68a 4.21de 5.20bc 5.66bc 3.12fg 3.54ef 2.83fg 7.29a 4.84cd 2.92fg 2.46g (Z)-3-hexenal 78.58b 170.72a 18.20de 25.13cd 29.34c 11.66ef 12.04ef 9.94ef 57.18c 27.22cd 12.11ef 9.67ef 1-butanol 7.75b 8.59a 4.77d 5.38cd 6.03c 3.14ef 3.84e 2.99f 3.05f 2.16gh 2.07h 1.84h 1-hexanol 3.12a 2.89a 2.45a 3.68a 3.15a 1.77a 1.79a 1.49a 2.47a 2.79a 1.64a 1.53a 1-octen-3-ol 4.11ab 4.93a 2.57cde 3.46bcde 3.59bc 3.33bcde 3.31bcde 2.48cde 4.36ab 3.96ab 2.33e 2.36de 1-penten-3-one 9.00b 14.47a 4.65d 6.91c 4.31df 2.61f 2.59f 2.26f 6.00cd 3.24ef 2.45f 2.21f 1-propanol 13.09a 13.62a 8.47a 9.413a 9.94a 3.88a 5.61a 2.88a 2.37a 1.25a 1.19a 0.67a 2,3-butanediol 3.83a 3.61a 2.89a 3.22a 3.38a 3.17a 3.20a 2.54a 6.62a 10.94a 4.98a 4.76a 2,3-butanedione 10.09b 8.22cd 6.88ef 9.38bc 11.90a 7.93de 6.53fg 5.54gh 202.32a 7.59def 5.35gh 4.61h 2-isobutylthiazole 1.25a 1.55a 1.37a 1.65a 1.67a 0.34b 0.53b 0.40b 0.511b 0.42b 0.25b 0.25b 2-methylpropanal 4.69b 5.63b 4.63b 5.62b 6.55ab 4.60b 6.11ab 5.32b 10.12a 10.20a 6.78ab 5.00b Continued

Table 7 continued.

Hot break Cold break Hot break Analyte Cold break juice juice sauce sauce Fresh HTST Retort LTLT Retort HTST Retort Concentration with with with with with with no with (ppb) no peel peel no peel peel peel peel no peel peel no peel peel peel peel 2-pentanol 4.90ab 5.24ab 3.68bc 4.20b 4.46b 2.32cd 2.25cd 1.78d 2.03d 6.03a 2.28cd 2.55cd 2-pentanone 2.40ab 2.68ab 2.10b 2.53ab 2.48ab 1.95b 2.05b 1.91b 4.02ab 6.86a 2.68ab 3.78ab 2-pentylfuran 1.72bc 2.06ab 1.33c 1.74bc 2.39a 1.55bc 1.51c 1.33c 3.72a 2.50a 1.48c 1.61bc 3-methylbutanal 10.15e 13.64d 14.01d 21.30b 25.44a 6.89fg 7.76f 8.10f 16.13c 17.55c 6.93fg 5.28gh 6-methyl-5-

59 hepten-2-one 8.40de 10.89bc 6.89e 9.37cd 11.92ab 4.21f 8.02de 7.87de 37.99a 14.15a 2.21f 1.83f

Acetaldehyde 27.32e 38.31d 55.57c 89.06a 86.26a 23.72e 30.00e 28.61e 35.07de 69.89b 24.48e 18.59f Acetone 70.68bcd 71.35bcd 67.93bcd 69.73bcd 78.34bc 62.59cd 55.64d 33.91e 171.51a 162.20a 86.38b 34.53e Benzaldehyde 6.58b 7.41a 4.84c 5.41c 6.36b 2.51d 2.24de 1.54ef 2.68d 2.53d 1.70ef 1.66ef benzene ethanol 1.13bc 1.07bc 0.68bc 0.89bc 1.51ab 1.39abc 1.35abc 1.20bc 5.17a 1.93b 0.95bc 1.34abc benzyl alcohol 1.60bc 1.59bc 1.19c 1.35c 1.34c 1.28c 1.19c 1.03c 3.52b 7.24a 1.78bc 2.75bc beta-ionone 0.41bc 0.56bc 0.31b 0.34b 0.58bc 0.41bc 0.37c 0.38c 2.38a 0.72b 0.47bc 0.53bc Citral 2.45cd 3.63b 2.09cd 3.10cd 3.59c 2.29cd 1.89cd 1.82cd 18.12a 7.78b 1.28d 1.61cd cyclic terpenes 0.80bc 0.99b 0.63cd 0.79bc 1.02b 0.65cd 0.54cde 0.62cd 3.12a 1.12b 0.44de 0.57cd Decanal 0.89bcd 1.11ab 0.52e 0.54e 0.72cde 0.68bcd 0.58de 0.48e 1.26a 0.91abc 0.57de 0.43e dimethyl disulfide 0.83f 1.37cde 0.87f 1.12def 1.84bcd 1.05ef 1.55cde 2.06bc 2.88ab 2.88a 0.82f 1.16ef 329.09 dimethyl sulfide 49.12ef 67.05e 50.54fg 58.27ef a 41.45g 204.89c 229.96b 95.80d 14.62h 6.65h 7.54h dodecanal 0.51abc 0.56ab 0.27bc 0.27bc 0.50ab 0.39abc 0.29bc 0.23c 0.63a 0.42abc 0.28bc 0.48abc Ethanol 60.18a 28.03d 46.79b 18.21e 34.19c 11.83fg 48.22b 30.09cd 32.25cd 16.76ef 10.72gh 6.05h Continued

Table 7 continued.

Hot break Cold break Hot break Analyte Cold break juice juice sauce sauce Fresh HTST Retort LTLT Retort HTST Retort Concentration with with with with with with no with (ppb) no peel peel no peel peel peel peel no peel peel no peel peel peel peel ethyl acetate 5.91cd 6.83bc 5.23de 5.57cd 5.53cd 3.99ef 5.27de 3.81f 9.18a 8.16ab 7.75b 6.04cd Eugenol 0.68abc 0.79ab 0.25cd 0.29cd 0.34cd 0.38bcd 0.59abcd 0.30cd 0.88a 0.64abcd 0.37bc 0.60abcd Furfural 1.58fg 2.59bc 1.45g 2.76b 5.19a 1.91ef 2.25cde 2.49bcd 2.04de 1.58fg 0.96h 0.93h Guaiacol 8.06b 9.20b 3.54g 4.27efg 5.41cde 4.96def 5.95cd 4.37efg 11.13a 6.40c 5.09cdef 3.93fg 32.96e 73.68d 74.96d 206.25a 175.34b 23.19ef 14.49f 22.91ef 35.20e 146.75c 10.06f 15.81f

60 Hexanal cde bc f def ef def cde ef a a bc b hexanoic acid 3.34 3.65 2.03 2.70 2.65 2.80 3.13 2.46 5.48 5.54 3.98 4.36 hexyl acetate 2.86bc 3.05b 1.36e 1.71de 1.68de 2.27bcde 2.58bcd 1.97cde 4.98a 4.27a 2.99b 3.03b Isobutanal 4.69f 5.63de 4.63f 5.62de 7.80b 4.60f 6.11cd 5.32def 10.12a 10.20a 6.78c 5.00ef isobutyl alcohol 7.10b 7.88a 4.37d 4.94cd 5.52c 2.88ef 3.52e 2.74f 2.79f 1.98gh 1.90h 1.69h Methanol 4756.5c 6191.5a 4731.9c 4888.1c 5334.9b 3173.9e 1537.7f 1162.2f 4035.4d 108.6g 60.0g 99.9g Methional 1.53cde 1.94bc 1.11ef 1.73cd 1.96bc 1.08ef 1.05ef 0.80f 2.52a 2.32ab 1.16ef 1.27cde methyl benzoate 13.66b 15.38a 10.04c 11.21c 13.18b 5.20d 4.64de 3.20e 5.29d 5.25d 3.53e 3.45e methyl hexanoate 2.96cdef 3.10bcd 1.98f 2.06ef 2.15ef 2.68cdef 2.96bcde 2.40def 3.81ab 4.62a 3.40bc 3.55bc methyl salicylate 1.09bc 1.38b 0.71cde 0.95bcd 1.35b 0.73cde 0.77cde 0.59de 4.42a 1.40b 0.38e 0.40e methylbutanoic acid 6.23bc 7.08ab 4.72de 5.38cde 6.01bcd 4.57e 4.96cde 4.28e 9.23a 8.27a 6.04bc 7.13ab Nonanal 5.72a 6.67a 1.25cd 1.51cd 2.20c 2.15c 1.48cd 1.22cd 3.28b 2.23bc 1.39cd 1.05d Octanal 3.94c 5.06b 1.81fg 2.52ef 3.09cde 2.37def 3.59cd 2.71def 6.65a 3.58cd 2.04fg 1.55g Phenylacetaldehyde 8.40b 8.50b 7.96b 8.41b 8.51b 7.99b 8.30b 7.10b 27.36a 11.05b 9.48b 10.28b Propanal 3.61e 4.31de 4.41d 7.30c 8.42ab 3.79de 4.15de 4.00de 9.10a 7.95bc 4.44d 3.66e propanoic acid 4.38a 4.33a 3.55a 3.26a 3.30a 2.78a 3.87a 2.99a 5.62a 6.85a 3.32a 4.09a Concentrations in the same row bearing different superscript letters are significantly different (p ≤ 0.05) 60

Hot break juice usually contains lower levels of lipoxygenase derived volatiles in

comparison to cold break juice. Hot break juice had significantly lower levels of (Z)-3-

hexen-1-ol, (E)-2-hexenal, (Z)-3-hexanal, (E)-2-octenal, (E)-2 pentenal, (E, E)-2, 4-

decadienal, 1-hexanol and hexanal than cold break juice (Table 7). The lower level of

lipid derived volatiles in hot break than cold break juice is due to the inactivation of

the lipoxygenase and other enzymes responsible for the formation of these volatiles.

Immediately after grinding, the puree was heated to 90oC to make sure all the enzymes

present were inactivated thus not allowing formation of these volatiles. Because of the

inactivated enzymes during the hot break process, there was no difference between

with peel and no peel hot break juice and sauce in concentration of lipid-derived

volatiles: (Z)-3-hexen-1-ol, (E)-2-hexenal, (Z)-3-hexanal, (E)-2-octenal, (E)-2

pentenal, (E,E)-2,4-decadienal and hexanal in these samples.

Carotenoid derived volatiles include 6-methyl-5-heptene-2-one and beta-ionone. Both of these volatile compounds are major compounds in tomatoes that contribute to tomato aroma (Sieso and Crouzet, 1977; Hayes and others 1998; Vallverdú-Queralt and others

2013). 6-Methyl-5-heptene-2-one was higher in with peel cold break HTST and fresh juice, and cold break sauce than those with no peel (Table 7). Lycopene is degraded into 6-methyl-5-heptene-2-one (Lewinsohn and others 2005). The significant difference between with peel and no peel juice in the level of 6-methyl-5-heptene-2-one may be because there is significantly more lycopene in the peel than in the flesh

(Markovic and others 2010). There was no significant difference between hot break with peel and no peel in the level of 6-methyl-5-heptene-2-one. There is a high association between color and aroma compounds in fruits and vegetables because of the

61 degradation of carotenoids facilitated by lipoxygenase enzyme activity (Schwartz and others 2007). In hot break processing, the enzymes are inactivated hence no significant difference between hot break with peel and no peel. Beta-ionone on the other hand showed no significant difference in any samples. It is derived from an oxidative product of β-carotene and none of the samples had any significant differences likely because the levels were very low.

The peel also increased the level of amino acid derived volatiles. Methional and dimethyl sulfide are both produced from methionine and are associated with boiled like and tomato juice aroma respectively (Table 7) (Lindsay 2007). This amino acid reacts with dicarbonyl compounds that are formed from strecker degradation to form methional. Methionine also undergoes biosynthesis to produce S-methyl methionine, which thermally degrades to dimethyl sulfide (Lindsay 2007). There was a significantly higher level of methional in with peel than in no peel cold break juice. The cold break

HTST and fresh juice with peel also showed higher levels of dimethyl sulfide than the juice with no peel (Table 7). It is likely that the levels of amino acids are higher in the skin than the fleshy part of tomatoes. These results were in agreement with other researcher who reported higher levels of amino acid derived volatiles in the skin than the flesh (Sieso and Crouzet 1977).

The cold and hot break juices for both with and without peel were concentrated to sauces. The sauce with and without peel made by cold break, had higher levels of most of the volatiles including the C-6 compounds; (E)-2-hexenal, (E)-2-heptenal, (E)-2- octenal, (E)-2 pentenal and (E,E)-2,4-decadienal, carotenoid derived volatiles, 6- methyl-5-heptene-2-one and beta- ionone and amino acid derived volatile methional 62 than their respective juices due to the concentration process (Table 7). A few volatiles, such as the amino acid derived volatile dimethyl sulfide was lower in sauce than the juice. This may be because dimethyl sulfide is volatile with a boiling point of 37oC and readily escaped during the concentration process. Concentration of hot break juice to sauces on the other hand had no significant effect on the levels of volatiles. This may be due to the low starting levels of most of the volatiles in hot break juices.

2.4.2 Effect of thermal treatments on volatile levels

Thermal treatment caused the destruction of some volatiles (Table 7). Fresh juice generally had the highest levels of volatile compounds since no thermal treatment was applied. HTST and retorted juice generally had lower volatile levels since these juices were heated. LTLT had the lowest level of most volatiles and the longest heating time of all the thermal treatments. Other researchers also reported a decrease in these volatile compounds with thermal treatment (Sieso and Crouzet 1977; Correa and others 2010).

The time between juicing and thermal treatment is essential in controlling the level of the volatiles formed in cold break juice. Prolonging this time results in increased volatile levels due to the prolonged enzymatic activity (Goodman and others 2002). The higher volatiles in retorted juice may have been due to the longer time (approximately

30min) allowed for the juice to stand before retorting which permitted production of more volatile compounds compared to HTST which was held for approximately 10 min and LTLT which was held for approximately 17 min. Goodman and others (2002) reported an increased amount of volatile compounds with longer holding time before any heat treatment, with the volatiles being highest at 20 min. than at 0 min. Fresh juice was expected to have higher levels of volatiles than the thermally treated juice as 63 reported by other researchers (Goodman and others 2002) but this was not the case for many of the lipoxygenase derived volatiles because the additional of calcium chloride at less than 2 min after the cold break juice was collected from the juicer, which did not permit production of volatiles. Thus it is difficult to distinguish the effect of holding time to create the volatiles from thermal treatment destroying the volatiles.

Retorted juice had higher levels of furfural, whereas there was no significant difference for HTST and fresh juice while LTLT juice showed the lowest level of the compound.

This was in contrast to Sieso and Crouzet (1977) who reported that furfural derivatives are thermal products formed during degradation of carbohydrates and increase with thermal processing. However, this volatile compound was present in fresh juice, which was not thermally treated, and there was a significant decrease with LTLT.

Of the four heat treatments, cold break with peel LTLT had the least level of hexanal probably due to the longer heating time, followed by cold break with peel fresh, the reason being due to the stopped enzymatic reaction which is responsible for the lipoxyganase pathway that helps in the formation of this volatile compound. Thermal treatment had no effect of on methional except for LTLT that had a significant loss and concentration to sauce lead to the increase of this volatile compound (Table 1). Another important volatile that is thermally induced is dimethyl disulfide. There was no significant difference in all the treatments of juices and sauce except for cold break sauces that had higher levels for dimethyl disulfide.

2.4.3 Effect of presence of peel and thermal treatment on viscosity

The viscosity of hot break juice was greater than the viscosity of cold break juice

(Table 8). Similarly, the viscosity hot break sauce was greater than cold break sauce.

Hot break juice had a significant higher viscosity than cold break juice because of the

inactivation of enzymes during hot break processing. Pectin is crucial in controlling

the viscosity of tomato juice. There are many enzymes involved in pectin hydrolysis

but the major ones are pectin methyl esterase (PME) and polygalaturonase (PG). These

enzymes decrease pectin polymer size that in turn results in loss of fluid viscosity

(Goodman and other 2002; Anthon and Barrett 2008; Molwane and Gunjal 1985).

During hot break, these enzymes are inactivated and hence the pectin remains intact.

In cold break, the enzymes are still active leading to the fragmentation of pectin hence

the loss of viscosity (Fito and others 1983; Anthon and Barrett 2008).

With peel had significantly lower viscosity than no peel for fresh cold break juice and cold break sauce (Table 8). The lower viscosity of the juice with peel may be due to an increased amount of enzymes in the peels than the flesh, which would have caused the breakdown of pectin in the tomato juice. Thermal treatment did not have a significant effect on the viscosity of the juices (Table 8).

Table 8: Physicochemical properties of cold and hot break juices and sauces with and without peel after different thermal treatments

Treatment Viscosity TSS Titratable pH Vitamin C (mPa.s) (oBrix) acidity (g/100ml) (g/L) Cold Break no peel fresh 189.41d 19a 0.325f 2.50f 0.018a Cold Break with peel fresh 58.98e 19a 0.380de 2.55f 0.022a Cold Break no peel HTST 82.94e 6.5g 0.397de 4.24de 0.021a Cold Break with peel HTST 68.38e 6.5g 0.421cd 4.36cd 0.022a Cold break with peel retort 54.64e 6.8f 0.424cd 4.52ab 0.021a Cold Break with peel LTLT 60.77e 6.8f 0.461c 4.22e 0.019a Hot Break no peel retort 168.07d 6.8f 0.37e 4.52ab - Hot Break with peel retort 185.50d 6.8f 0.37e 4.58a - Cold Break no peel HTST sauce 392.99b 11.4b 0.685a 4.18e - Cold Break with peel HTST sauce 315.22c 11.2c 0.665a 4.28de - Hot break no peel retort sauce 868.05a 11.1d 0.645a 4.43abc - Hot break with peel retort sauce 855.30a 11.0e 0.585b 4.48ab - Values in the same column bearing different superscript letters are significantly different (p ≤0.05)

2.4.4 Total soluble solids (TSS), pH, titratable acidity (TA) and vitamin C

The presence of peel did not have any significant effect on the TSS, pH, TA or vitamin

C (Table 8). Thermal treatment also did not have any significant effect on these physic- chemical parameters. Concentration of juice to sauce increased titratable acidity of all the sauces but there was no significant change in their pH. The increase of titratable acidity is due to the concentration of the organic acid present in tomato juice.

2.4.4 Effect of peel and thermal treatments on color

Hue angle is a measure of the color angle or actual color. Hue is an angular measure from 0 to 360, which represents basic color and a decrease of hue angle in tomato represents a change from green to yellow to red (Darrigues and others 2008). The presence of peel and thermal treatment did not have any significant effect on the hue angle, L*and a* of the juices or sauces (Table 9). These results were contrary to the

66 expectation that juice with peel should have a redder color than that without peel since there is additional lycopene content, in peels than in tomato pulp which is responsible for the red color in tomato, (Sharma and Le Maguer, 1996; Reboul and others 2005;

Markovic and others 2010).

Table 9: Color properties of cold break and hot break juice and sauces with and without peel at different thermal treatment

Treatments L* a* b* h* ab Cold Break No peel fresh 38.09e 23.23de 12.77h 32.00 Cold Break with peel fresh 37.96e 22.89e 11.95i 31.42b Cold Break no peel HTST 39.39cd 23.57cd 13.23g 32.55ab Cold Break with peel HTST 39.34cd 23.88c 12.6h 30.91ab Cold break with peel Retort 39.52c 23.00e 13.83f 34.47ab Cold Break with peel LTLT 39.24d 23.77c 13.1g 32.07ab Hot Break no Peel 39.82b 23.06e 14.91c 36.54a Hot Break with peel 39.89ab 23.71c 14.61d 35.16a Cold Break no peel sauce 39.47cd 25.64b 14.1e 31.99ab Cold Break with peel sauce 39.41cd 26.57cd 13.83f 30.56ab Hot break no peel sauce 40.15a 26.29a 16.31a 35.35a Hot break with peel sauce 40.8ab 26.41a 15.74b 34.21ab Values in the same column bearing different superscript letters are significantly different (p ≤0.05)

2.4.6 Sensory analysis of cold break juice and sauce

The variation in the chemical properties of the juices and sauce with peel and without peel are only important if they can be detected by consumers hence the need for sensory analysis. The color, aroma, flavor and overall liking were liked slightly to liked moderately on most samples (Table 10). With peel juice texture and overall liking was between neither like nor dislike and like slightly.

The color of juice with peel was more preferred than that of no peel (Table 10) even though there was no significant difference in the hue angle, L* and a* of the two juices

(Table 9). The with peel juice was just about right; while the no peel juice was too light

(Figure 3). With peel juice had higher levels of lipoxygenase –derived volatiles (Table

7) and therefore was expected to have a fresher, more desirable aroma and flavor but there was no difference in the aroma presence (Table 10). However, the texture, flavor and the overall liking of with peel juice were less preferred than the juice with no peel.

The flavor of the no peel juice was preferred (Table 10). There was no difference in soluble solids, titratable acidity or pH but it did have lower volatile levels (Table 7, 8).

The with peel juice had lower viscosity (in a t-test between the two samples) and the texture was less preferred than no peel. Once the juices were concentrated into sauce, the differences in preference disappeared and there was no significant difference between the peel and no peel sauce in color, aroma and overall liking.

Table 10: Sensory preference for color, aroma, texture, flavor and overall liking of cold break HTST tomato juice and sauce

Treatment Color Aroma Texture Flavor Overall liking No peel Juice 6.4b 6.5a 6.7a 7.0a 6.7a With peel juice 7.0a 6.2a 5.5b 6.0b 5.7b No peel sauce 7.0a 6.0a - - 6.0a With peel sauce 7.0a 6.0a - - 6.0a Values in the same column bearing different superscript letters are significantly different (p ≤0.05)

The freshness of the juice with peel was perceived to be little and the texture thin by some panelist and thus based of these results, the viscosity and the freshness of the juice should be increased (figure 5). On the other hand the juice with no peel had aroma, freshness and texture that had more than 60% of people who thought these attributes are just about right and only 28% and 31% of the panelist thought aroma and freshness was little respectively and 21% thought the texture was thin. Juice with no 68 peel was generally more preferred because many thought that the aroma, freshness and texture were just about right. After concentration to sauce both the with peel and no peel scored lower than 60% in just about right for color and above 60% for the aroma

(Figure 6).

Color Aroma 90 90 80 80 70 70 60 60 50 50 40 40

30 30

Percentage (%) Percentage Percentage Percentage (%) 20 20 10 10 0 0 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 No peel juice With peel juice No peel juice With peel juice

Freshness Texture 90 90 80 80 70 70 60 60 50 50 40 40

30 30

Percentage(%) Percentage (%) Percentage 20 20 10 10 0 0 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 No peel juice With peel juice No peel juice With peel juice

Color Aroma 70 70 60 60 50 50 40 40

30 30 Percentage Percentage (%) Percentage Percentage (%) 20 20 10 10 0 0 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 No peel sauce With peel sauce No peel sauce With peel sauce

Figure 6: Just About Right responses of panelists on tomato sauce color and aroma. Much too little = -2, slightly too little= -1, Just about right=0, slightly too much=1 and much too much= 2.

2.5 Conclusion

The with peel cold break juice showed higher levels of most of the volatiles, particularly the lipoxygenase derived volatiles which are associated with the fresh green note tomato flavor. This was probably because of high lipoxygenase activity in the fleshy part adjacent to the peel and higher fatty acid in the peel than in the fleshy part.

Cold break juice and sauces had higher level of volatiles than the hot break juices and sauces because of enzymatic activity. There was no significant difference between the peel and no peel hot break juice because the enzymes were inactivated. The viscosity was lower in the with peel cold break juice than with no peel probably because of higher pectinolytic enzymatic activity in the peels than the fleshy part. From the

70 sensory results, with peel cold break juice was less preferred in the flavor, texture and overall liking than the no peel juice probably due to the fact that most of people were not tomato juice drinkers or the higher levels of volatiles were less desirable. Therefore, different results may be obtained if sensory was done by regular tomato juice drinkers.

The viscosity of with peel juice was higher than that of no peel. There was no significant difference between with peel and no peel sauce, which may indicate concentration of the juice, decreased the differences.

References

Anthon GE, Barrett D M. 2008. Combined enzymatic and colorimetric method for

determining the methanol and uronic acid content of pectins application to tomato

products. J Food Chem110: 239–247.

AOAC 1995. Official Methods of Analysis, 16th edition, Association of Official Analytical

Chemists, Washington D.C, 138pp.

Correa MIC, Chaves JBP, Jham GN, Ramos AM, Minim VPR, Yokota SRC. 2010.

Changes in guava (Psidiumguajava L. var. Paluma) nectar volatile compounds

concentration due to thermal processing and storage. Ciência e Tecnologia de

Alimentos 30(4): 1061-1068.

Darrigues A, Hall J, Knaap EV, Francis MD. 2008. Tomato Analyzer-color Test: A New

Tool for Efficient Digital Phenotyping. J. Amer. Soc. Hort. Sci. 133(4):579–586.

Del Valle M, Cámara M, Torija ME. 2007. The nutritional and functional potential of

tomato by-products. Acta Hort. (ISHS) 758:165-172.

Fito PJ, Clemente G, Sanz, FJ. 1983. Rheological behavior of tomato concentrates (Hot

break and Cold break). J Food Eng. 2: 51-62.

Goodman CL, Fawcett S, Barringer SA. 2002. Flavor, viscosity, and color analyses of hot

and cold break tomato juices. J. Food Sci 67(1):404-408.

Hayes WA, Smith PG, Morris AEJ. 1998: The Production and Quality of Tomato

Concentrates, Critical Reviews in Food Science and Nutrition, 38(7): 537-564.

Lawless HT and Heymann N. 2010. Sensory evaluation of food. 2nd ed. New York:

Springer. 88p.

Lewinsohn E, Sitrit S, Bar E, Azulay Y, Ibdah M, Meir A,Yosef E, Zamir D and Tadmor Y

2005. ‘Not just colors-carotenoid degradation as a link between pigmentation and aroma in

tomato and watermelon fruit. Trends Food Sci. Technol 16:407-415.

Lindsay RC. 2007. Flavors. Fennema OR, Damodaran S, Parkin KL. 2007. Food

Chemistry. New York: Taylor and Francis Group. 597p.

Markovic K, Panjkota KI, Krpan M, Bicanic D, Vahcic N. 2010. The lycopene content in

pulp and peel of five fresh tomato cultivars. Acta Alimentaria 39(1):90-98.

Min S, Jin Z T, Zhang QH. 2003. Commercial scale pulsed electric field processing of

tomato juice. J Agric Food Chem 51 (11): 3338–3344.

Molwane, S. D. and Gunjal, B. B. 1985. Viscometric Characteristics of Cold-break and

Hot-break Extracted Tomato Juice Concentrates. J Food Sci. Tech., 22: 353-357.

Petro-Turza M. 1987. Flavor of tomato and tomato products. Food Rev Int 2(3):309–51.

Reboul E , Borel P , Mikail C , Abou L , Charbonnier M *, Caris-Veyrat C , Goupy P ,

Portugal H , Lairon D , Amiot M. (1996) Enrichment of Tomato Paste with 6%

Tomato Peel Increases Lycopene and β-Carotene Bioavailability in Men. J Nutr.

vol. 135(4): 790-794

Schwartz SJ, Von Elbe JH, Giusti MM. 2007. Colorants. Fennema OR, Damodaran S,

Parkin KL. 2007. Food Chemistry. New York: Taylor and Francis Group. 597p.

Sieso V, Crouzet J. 1977. Tomato volatile components: effect of processing. J Food Chem

2: 241-252.

Sharma SK, Le Maguer M. 1996. Lycopene in tomatoes and tomato pulp

fractions. Italian J Food Sci 8(2): 107- 113.

Ties P, Barringer S. 2012. Influence of lipid content and lipoxygenase on flavor volatiles in

the tomato peel and flesh. J Food Sci 77(7): 830-837.

Tongnuanchan P. Benjakul S. Prodpran T. 2010. Roles of lipid oxidation and pH on

properties and yellow discolouration of red tilapia muscle based film. Food

Innovation Asia Conference: Indigenous Food Research and Development to Global

Market, June 17-18, 2010, BITEC, Bangkok, THAILAND.

Vallverdú-Queralt A, Bendini A, Barbieri S, Di Lecce G, Martin-Belloso O, Gallina Toschi

T. 2013. Volatile profile and sensory evaluation of tomato juices treated with pulsed

electric fields. J Agric Food Chem 61: 1977−1984.

Xu Y, Barringer S. 2009. Effect of temperature on lipid-related volatile production in

tomato puree. J Agric Food Chem. 57(19): 9108–9113.

Chapter 3: Effect of peels on quality attributes of mango puree held at different times

Abstract

Mangos are usually peeled during processing, but this is a labor-intensive step and may not always improve the quality of the final product. Mangoes were processed into puree from peeled and unpeeled mangoes, and then held for 5min or 1h 45min before pasteurization at

104oC for 30min. The mango puree was analyzed for volatiles, viscosity, total phenolics, color, pH, total soluble solids and sensory. Puree without peel showed higher levels of most lipoxygenase-derived volatiles. Terpene hydrocarbons, which have been associated with mango flavor, especially with green mango flavor, were lower in the puree made without peel. The two hydrocarbons carveol and carvone, which are responsible for off flavor in mangoes, were also lower in the puree made without peel. Puree made without peel had lower viscosity and total phenolics, higher pH and lower Total Soluble solids (TSS) while color showed no significant difference. Generally, the puree made without peel was more preferred in flavor, aroma, texture, color and overall liking and it was considered to be fresher with less off flavor.

Practical Application

Peeling of mangoes during processing is a common practice but skipping this step is cost

effective and increases the level of total phenolics and viscosity. Peels increase the level of

terpene hydrocarbons that are important in mango flavor but also some of the hydrocarbons

responsible for off flavor. Peel removal increases the level of lipoxygenase-derived

volatiles, which is linked with the fresh flavor, and also lowers viscosity. Puree made

without peel is more preferred in terms of texture, color, flavor and aroma.

3.1 Introduction

Mango (Mangifera indica L.) is one of the most important and popular of tropical fruits, mainly due to its attractive flavor, succulence and delicious taste. Mango fruits are perishable and therefore postharvest losses are reduced through processing which ensures a sustainable supply of a wide range of high quality fruit products. Some of the mango- based products include: juice-based products such as nectar, juice, jam, jelly powder, fruit bars and flakes.

About 270 volatiles have been identified in mango fruits. Out of the 270 only a few have

been classified as important volatiles that contribute to mango flavor: (E)-2-hexenal, (Z)-

3-hexen-1-ol, (E)-2-heptenal, (E)-2-pentenal, methyl benzoate, 3-carene, alpha-pinene,

limonene, myrcene, ocimene, beta-ionone and gamma-octalactone (Pino and Mesa 2006;

Pino and others 2005; El hadi and others 2013). The lipoxygenase derived volatiles

include (Z)-3-hexenal, (E)-2-hexenal, (Z)-3-hexen-1-ol, (E)-2-heptenal and (E)-2-

pentenal, that are a result of linolenic and linoleic acid breakdown via the lipoxygenase 76 pathway and produce the characteristic green fresh aroma in most fruits and vegetables

(Galliard and others 1977).

Mangoes have a characteristic leathery peel with a large seed right at the center of the fruit. The polyphenolic content of the peels range from 55 to 110 mg/g dry peel (Ajila and others 2007). Higher levels of these bioactive compounds are beneficial to human health, acting as antioxidants and free radical scavengers. There are some mango peels, which are edible; these edible peels are a good source of polysaccharides as fiber sources and pectin (Iagher and others 2002). Therefore, unpeeled mango juice may have increased nutritional value by increasing the bioactives and reducing the cost of production compared to the peeled mango juice.

Most fruits contain enzymes that speed up a number of important reactions responsible for the formation and destruction of some flavor compounds, loss of viscosity and many other major factors. Industrially when making mango puree, enzymes are added to the mango puree and it is left to stand for 2h to break down the pectin and make it less viscous. During this holding time several enzymatic reactions take place that may or may not affect the physicochemical quality of the fruit puree, in terms of color, viscosity and flavor. Therefore, the objective of this study is to assess the effect of peels and different holding times on the quality of mango puree.

3.2 Materials and methods

3.2.1 Mango puree processing

Fresh and fully ripe mangoes, Alphonso variety grown in India, were procured from

Mango ZZ Inc. (Chicago, IL, USA) during the month of May 2014. The mangoes were divided into two batches (Fig 7). One batch was washed, peeled and passed through a lye peeler (18%NaOH w/v), at approximately 95oC for 60 s. Any remaining part of the peel was removed by a knife and the fleshy part was scrapped off the seed. The second batch was washed and the flesh plus the peel were scraped from the seed. The mango flesh and flesh plus peel were ground using a juice extractor (Omega juicer model 8000 Harrisburg,

PA, USA). The puree was packed in size 303 X 406 cans and was held for 5min or 1hr

45min before pasteurization at 104oC for 30min. The puree was refrigerated for 15-19 h and the analysis for volatiles was done. Puree used for viscosity, titratable acidity, pH and total soluble solids was stored in the refrigerator for 2 d.

Held for 5min

Peeled mangoes

Held for 1h 45min

Mangoes Pasteurized

Held for 5min

Unpeeled mangoes

Held for 1h 45min

Figure 7: Processing treatments for mango puree

Mango puree (60ml) was put in a 500ml Pyrex culture bottle covered with tops enclosed with septum then placed in a water bath (precision circulating bath model 260,

Winchester, VA, USA) at 50oC for 1h. Two needles were used; one was connected to the

SIFT-MS machine and was pierced through the septum into the bottle to allow the flow of volatiles through the machine. The second one was pierced through the septum into the bottle and was left in open air for ventilation. The volatiles were measured using a

Selected Ion Flow Tube Mass Spectrometer (SIFT-MS) (SYFT Voice 200, Christchurch,

New Zealand) using the method described in Ties and Barringer (2012). The kinetics of all the volatiles measured is shown in Table 11.

Table 11: Kinetics of mango volatiles measured

Analyte Reaction Mass Precursor Product ion rate x10-9 (m/z) ion + + (E)-2-heptenal 3.9 111 NO C7H13O .H2O + + (E)-2-Hexenal 3.8 97 NO C6H9O + + (E)-2-Nonenal 3.8 139 NO C9H15O + + (E)-2-Octenal 4.1 125 NO C8H13O + + (E)-2-Pentenal 4.0 83 NO C5H7O + + (Z)-3-Hexen-1-ol 2.9 82 NO C6H11O + + (Z)-3-Hexenal 3.1 70 NO C4H6O + + Alpha-pinene, myrcene 2.2 92 O2 C7H8 + + Alpha-terpinene, terpinolene 2.0 136 O2 C10H16 + + Beta-ionone 2.5 192 NO C13H20O + + + Carvacrol and ethyl benzoate 3.1 151,169 H3O C10H14O.H , + + Carveol 2.5 152 NO C10H16O + + Carvone 2.8 180 NO C10H14O.NO + + Cuminal 2.8 147 NO C10H11O + + Cyclohexanone 3.3 128 NO C6H10O.NO + + 3-Carene 2.2 81 NO C10H16 + + Decanal, menthol, undecane 3.3 155 NO C10H19O + + Delta or gamma-decalactone 2.5 200 NO C10H18O2.NO + + Diallyl disulfide 3.0 147 H3O (C3H5)252.H + Dihydrocarvone 2.5 182 NO+ C10H16O.NO + + Dodecane 2.8 189 H3O C12H26. H3O + + Ethanol 2.3 46 O2 C2H6O + + Ethyl 2-methylbutanoate 2.5 130 O2 C7H14O2 + + Ethyl decanoate 3.0 201 H3O C12H24O2.H + + Ethyl heptanoate 2.5 188 NO C9H18O2.NO + + Ethyl hexanoate 2.5 174 NO C8H16O2.NO + + Ethyl maltol 2.5 140 NO C7H8O3 + + Ethyl nonanoate 2.5 216 NO C11H22O2.NO + + Ethyl octanoate, octanal 3.0 127 NO C8H15O + + Eucalyptol, lemonol, 2.8 154 NO C10H18O menthone + + Eugenol 2.4 164 NO C10H12O2 + Farnesol 2.0 71 O2+ C5H8 + + Gamma-octalactone 2.5 172 NO C8H14O2.NO Continued

Table 11 continued.

Analyte Reaction Mass Precursor Product ion rate x10-9 (m/z) ion + + Gamma-terpinene 2.1 135 NO C10H15 + + Guaiacol 2.5 124 NO C7H8O2 + + Heptanal 3.3 113 NO C7H13O + + Hexanal, cyclohexanol 2.5 99 NO C6H11O + + Hexanoic acid 2.5 146 NO C6H12O2.NO + + Limonene 2.2 68 O2 C5H8 + + Methanol 2.7 33 H3O CH5O + + Methyl 2-methylbutanoate 3.0 117 H3O C6H12O2.H + + Methyl benzoate 3.5 105 O2 C6H5CO + + Methyl cinnamate 2.5 162,163 NO C10H10O2 + + Methyl hexanoate 3.0 131 H3O C7H14O2.H + + Nonanal 2.7 141 NO C9H17O + Ocimene 2.1 92 NO C7H8 + + Octane 1.9 114 O2 C8H18 + + p-isopropenyl toluene 1.8 132 O2 C10H12 + + pentanal,1-penten-3-ol, 3.2 85 NO C5H9O + + Phenylacetaldehyde 2.5 120 NO C8H8O + + Styrene 1.8 104 O2 C8H8 + + Toluene 2.2 93 H3O C7H8.H

3.2.2 Determination of total phenolics

Determination of total phenolics was performed as described by Barrett and others (2007) by the use of Folin-Ciocalteau’s reagent. Five milliliters of acetone, 0.5 ml mango puree and 1.0 ml Folin–Ciocalteau reagent were added to a 25 ml volumetric flask. The contents were mixed and permitted to stand for 8 min at room temperature. Ten milliliters of 7% sodium carbonate solution was added, followed by the adding distilled water filled to volume. Solutions were mixed thoroughly and permitted to stand at room temperature for 2 h. Sample aliquots were filtered through a Whatman 0.45-μm polytetrafluoroethylene filter before determination of total phenols concentration using a

81 spectrophotometer (50 BIO UV-Visible Varian, LA,USA) monitoring Abs 760 nm. Total phenolics content was standardized against gallic acid and expressed as gallic acid equivalents. The linearity range for this assay was determined as 0.05 to 1.4 mg/100ml gallic acid equivalent (R2 = 0.9811).

3.2.3 Viscosity

The viscosity of mango puree was measured using a viscometer (LV DVII+, Brookfield

Engineering Laboratories, Inc., Stoughton, MA, USA) with a UL adapter. Viscosity was determined at 22 °C, 4 rpm and spindle number 1 with 500 ml of juice placed in the UL adapter; the values were read after every 30s for 3min.

3.2.4 Color

Mango puree samples (200ml) were filled into an optical cuvette and the color was measured by the use of light reflectance specular component included, D65 illuminant,

10-degree observer angle with a colorimeter (Color Quest XE, Hunter Associates

Laboratory, Inc., Reston, VA, USA). The L*, a* and b* were reported.

3.2.5 pH

The pH of the mango puree was determined directly using a pH meter (Accumet, Fisher scientific 300025, Denver, CO, USA).

3.2.6 Brix

The total soluble solids of the mango puree were determined using a refractometer

(Reichert Abbe Mark II 10480, Buffalo, NY, USA) according to Method 932.12 (AOAC

1995). The juice was filtered through a kimwipe to remove the insoluble solids, a drop was put on the glass and the value was read.

3.2.7 Sensory analysis

A sensory test was conducted with an untrained group of 98 people which consisted of both males and females from the ages of 18 to 66 and over at the department of Food

Science and Technology in The Ohio State University. They were requested to rate the liking of color, texture, flavor, aroma, and overall acceptability by looking, tasting and smelling the mango puree. Pasteurized purees, 30ml, made with and without peel held at different times were served in randomly numbered plastic cups on a tray with a bottle of water and random three digit numbers were assigned to the samples. A hedonic scale of

1 to 9 was used for each attribute. The higher number represents a higher preference for the attributes. Just about right (JAR) testing on the color, aroma, freshness and texture was done which included responses on much too little, slightly too little, just about right, slightly too much and much too much for aroma and freshness. A sample is considered to be too high or too low in an attribute when less than 60% of the panelists marked it as being just about right. For color it ranged from much too light, slightly too light, just about right, slightly too dark and much too dark and for texture, much too thin, slightly too thin, just about right, slightly too viscous and much too viscous.

3.2.8 Statistical analysis

The data was coded, entered and analyzed using JMP version 10.0.2 64-bit edition

(Statistical Discovery, Cary, NC, USA). Data were analyzed by using one-way analysis of variance (ANOVA) and comparison of all pairs using Tukey-kramer honestly significant difference (HSD). Significance was defined as p ≤ 0.05. There were six replicates for volatiles analysis and three replicates for total phenolics, total antioxidant, viscosity, color, TSS and pH.

3.3 Results and Discussion

3.3.1 Effect of peel on lipoxygenase derived volatiles

Most lipoxygenase derived volatiles, which are known to contribute to the fresh flavor of fruits and vegetables, were higher in the puree made without peel than the puree made with peel (Table 12). This class of volatiles includes (E)-2-hexenal, (Z)-3-hexen-1-ol,

(E)-2-heptenal, hexanal and (E)-2-pentenal, which are important to mango flavor (Pino and Mesa 2006; Pino and others 2005; El hadi and others 2013). Quercetin, a compound found in mangoes, has been reported to inhibit lipoxyganase enzymes (De Pascual 2004).

Like most polyphenols in mangoes, quercetin compound has been detected in higher concentration in the peel than the fleshy part (Lakshminarayana and others 1970). Hence this may account for the higher level of most individual lipoxygenase-produced volatiles, as well as the sum total of all the lipoxygenase-derived volatiles in the puree made without peel.

(Z)-3-Hexenal is the first volatile formed through the lipoxygenase pathway from linolenic acid (Croft and others 1993; Hatanaka and others 1987; Buttery and Ling 1993). 84

However, there are lower levels of (Z)-3-hexenal in the puree made without peel which may be because the alcohol dehydrogenase is higher in the peel.

Table 12: Lipoxygenase derived volatile levels of mango puree made with and without peel at different holding times

Without Without peel With peel With peel Analyte peel (5min) (1h 45min) (5min) (1h 45min) (E)-2-heptenal, 1-octen-3-ol 7.60b 8.77a 5.42c 5.67c (E)-2-hexenal 13.7a 13.4a 8.27b 7.18b (E)-2-nonenal 4.88a 6.06a 3.04b 3.51b (E)-2-octenal 3.49bc 4.45a 3.16c 3.79b (E)-2-pentenal 91.6b 112a 38.8c 45.3c (Z)-3-hexen-1-ol 395a 351b 317c 213d (Z)-3-hexenal 34.0c 32.8c 59.3a 43.5b Heptanal 8.35a 8.69a 9.13a 12.2a Hexanal, cyclohexanol 21.5a 22.6a 20.8a 20.8a Concentrations in the same row bearing different superscript letters are significantly different (p ≤ 0.05)

Longer holding time decreased the concentration of (Z)-3-hexanal (Table 12). (Z)-3-

Hexanal is rapidly converted to (E)-2-hexenal and (Z)-3-hexen-1-ol, thus the decreasing trend with time.

Holding time had a significant effect on (Z)-3-hexanal, (E)-2- heptenal, (E)-2-octenal,

(E)-2-pentenal and (Z)-3-hexen-1-ol (Table 2). For (Z)-3-hexenal and (Z)-3-hexen-1-ol as time increased these volatiles decreased. The loss with time is because these volatiles continue along the LOX pathway with conversion of (Z)-3-hexenal to (E)-2-hexenal and

(Z)-3-hexen-1-ol and (Z)-3-hexen-1-ol, reacting with acids to form esters (Croft and others 1993). (E)-2- Heptenal, (E)-2-octenal and (E)-2-pentenal form from linoleic acid via minor side branches of the LOX pathway and increased with holding time. The

85 formation of most of these volatiles was time dependent hence the higher levels in longer holding time.

3.3.2 Effect of peel on esters, alcohols and acids

The presence of peel produced no clear pattern on the ethyl ester content (Figure 8).

However, the methyl esters are formed in higher concentrations and are much higher without peel (Figure 8). Esters play a major role in contributing to flavor and aroma of fruits and they are produced from alcohol reacting with carboxylic acids. With increased holding time, this reaction leads to a decrease in concentration of the alcohols and acids as the esters concentration increases simultaneously (Figure 8, 9). The alcohols and acids that contributed to ester formation included ethanol and methanol, hexanoic and butanoic acid. Some of the esters that increased with holding time were ethyl-2-methylbutanoate that has a characteristic fruity sweet green aroma, ethyl heptanoate with sweet floral caramel furan aroma and ethyl hexanoate and ethyl octanoate which gives a sweet fresh floral aroma (Mahattanatawee and others 2005). Ethyl esters were formed in low concentrations and did not change much with peel.

A major ester in mango aroma is methyl benzoate (Pino and others 2005). Methyl benzoate concentration was five times higher in the puree made without peel than the puree made with peel (Figure 1). This ester has a low odor detection threshold of 0.52 ppb (Pino and others 2005) with a characteristic deep floral odor with fruity undertones

(Arctander 1969). Methyl benzoate has also been reported to be a major volatile in many of the mango varieties (Pino and others 2005). It might therefore contribute significantly to the overall aroma of most mangoes cultivars. The formation of methyl benzoate from

86 benzoic acid is an enzyme catalyzed reaction where S-adenosyl-l-methionine is used as a methyl donor (Pino and others 2005). The holding time had no significant effect on this volatile.

25 a Without peel(5min) a ab a Without peel (1h 45min) 20 b bc a With peel(5min) c 15 ab a c ab b With peel (1h 45min) c b 10 c

5 Concentration (ppb) Concentration

Volatiles

300 Without peel(5min) Without peel (1h 45min) 250 a a a With peel(5min) 200 b With peel (1h 45min) 150

100 b Concentration (ppb) Concentration b 50 c c

0 Methyl benzoate Methyl-2-methylbutanoate Volatiles

Concentrations bearing different superscript letters are significantly different (p ≤ 0.05)

Figure 8: Change in ester concentration with increased holding time and the presence of peel. 87

7000 180 a Without peel(5min) a a 160 6000 Without peel (1h 45min) b 140 With peel(5min) 5000 120 b With peel (1h 45min) 4000 b 100 a a a 80 a 3000 b 60 b 2000 c 40 b

Concentration (ppb) Concentration c c Concentration (ppb) Concentration 1000 20 0 0 Hexanoic Butanoic Methanol Ethanol Volatiles Volatiles

Concentrations bearing different superscript letters are significantly different (p ≤ 0.05)

Figure 9: Decrease in alcohol and acid concentration with increased holding time and inclusion of peel.

3.3.3 Effect of peel on terpene hydrocarbon volatiles

The puree made without peel exhibited lower concentrations of most terpene hydrocarbons than puree made with peel (Table 13). The biosynthesis of monoterpene compounds (C10) is catalyzed by specialized monoterpene synthases, which utilize geranyl pyrophosphate (GPP) to form terpenoid compounds. Therefore, it may be speculated that the higher level of terpene hydrocarbons in the puree made with peel is due to the higher terpene synthase and other enzymes plus the precursors that take part in the formation of these volatiles in mango peel than the flesh.

Some of the important monoterpenes are 3-carene, alpha-pinene, limonene, myrcene and terpinolene that contribute to the overall mango aroma (Pino and Mesa 2006). All of

88 these volatiles were lower in the puree made without peel than the puree made with peel

(Table 13). 3-Carene was found to be the major component in 10 mango cultivars (Pino and others 2005). It has a sweet and limonene-reminiscent odor and has been reported to be higher in green than ripe mangoes (Sandoval and others 2007). Alpha-pinene is produced via cyclisation of linaloyl pyrophosphate followed by loss of a proton from the carbocation equivalent. It gives a woody-green pine-like smell and has also been reported to be more important in green mangoes than the ripe mangoes (Sandoval and others

2007). Limonene is associated with citrus fruits such as lemon and oranges (Qiao and others 2008; Buettner and others 2003) and myrcene gives a green, metallic, earthly aroma (Sawamura and others 2004). Ocimene is also another important hydrocarbon, which gives a warm, herbaceous, and floral odor. This volatile was almost five times higher in the puree made with peel than the peel made without peel. In general, the green aroma note typical of mangoes has been correlated to monoterpene hydrocarbons (Lopes and others 1999).

Table 13: Monoterpenes and other important volatiles level of mango puree made with and without peel at different holding times

Without With Without peel (1h With peel peel (1h Analyte peel (5min) 45min) (5min) 45min) 3-carene 489b 395b 1798a 2085a Alpha-pinene, myrcene 415b 439b 520a 582a Carveol 18.0c 22.2c 27.5b 41.5a Carvone 1.89b 1.81b 2.99a 3.00a Limonene 316b 295b 495a 553a Ocimene 1869b 1635b 5008a 5731a Alpha-terpinene, terpinolene 86.5b 75.6b 246a 266a Gamma-octalactone 17.4ab 17.2ab 14.8b 17.6a Beta-ionone 2.86a 2.81a 2.16a 2.61a Concentrations in the same row bearing different superscript letters are significantly different (p ≤ 0.05) 89

Auto-oxidation of limonene results in the formation of carveol and carvone (Bouwmeester and others 1998). Carveol and carvone give a woody turpeny off flavor which is associated with off flavor (Marsili 2007). These two volatiles were lower in the puree made without peel than the puree made with peel therefore the puree made without peel should have less off flavor (Table 3).

3.3.4 Effect of peel on other important volatiles

Beta-ionone is produced via carotenoid oxidation and has been described as having a violet like aroma with an odor detection threshold of 0.007 ppb (Arctander 1969; Fisher and Scott 1997). It is identified as one of the volatiles that play an important role to the overall aroma of most mango cultivars (Pino and others 2005). Absence of peel exhibited no significant effect on the levels of beta-ionone (Table 13).

The compound gamma-octalactone, which gives a sweet herbaceous, coconut-like odor and taste (Arctander 1969), was found to be a major volatile compound and to occur above its odor detection threshold of 7 ppb (Engle and others 1988) in some mango cultivars, so it might contribute to the overall aroma of the ripe fruit. The presence of peel led inhibited the volatile concentration at the shorter holding time but did not affect it at the longer holding time (Table 13).

3.3.5 Effect of peel and holding time on total phenolics

The puree made without peel had lower levels of total phenolics than the puree made with peel (Table 14). Phenols are secondary plant metabolites and one of the major groups of nonessential dietary components appearing in fruits and vegetables. However, the increased interest in these compounds is associated with the inhibition of cancer and atherosclerosis. They act as antioxidants due to their ability to chelate metals, inhibit lipoxygenase and scavenge free radicals. The lower level of total phenolics in the puree made without peel may be due to fewer phenolic compounds in the peel than the flesh the of mango fruit. Other researchers have reported higher level of polyphenols in the peel than the flesh (Kim and others 2010). The major polyphenols in mango fruit in terms of antioxidative capacity and/or quantity are: mangiferin, catechins, quercetin, kaempferol, rhamnetin, anthocyanins, gallic and ellagic acids, propyl and methyl gallate, benzoic acid, and protocatechuic acid (Masibo and He 2008). Holding time on the other hand had no significant effect on total phenolics because these compounds are produced in the course of growth and maturation in the field and not during processing.

Table 14: Total phenolics, pH, total soluble solids, viscosity and L*, a*and b* values of mango puree made with and without peel at different holding times

Total pH TSS Phenolics Viscosity Treatments (mg/100g) (mPa.s) L* a* b* Puree without peel 4.49b 17.1b 21.54 47.98 5min 64.0b c 2956c 55.23b b a Puree without peel 4.54a 16.9c 22.31 47.62 1h 45min 62.7b 1621d 54.95b a a Puree with peel 4.36c 18.3a 55.37a 20.52 46.10 5min 78.9a 3539b b c a Puree with peel 1h 4.37c 17.3b 20.81 48.46 45min 80.3a 4071a 56.26a c a Values in the same column bearing different superscript letters are significantly different

(p ≤0.05)

Puree made without peel had a slight but significantly higher pH than the puree made with peel (Table 14). The major organic acids responsible for the pH in mangoes include citric and malic acid but other acid such at tartaric, ascorbic, oxalic and alpha-ketoglutaric are also present in low concentration (Medlicott and Thompson 2006). Absence of peel significantly lowered the total soluble solids (TSS) (Table 14). TSS is a measure of soluble sugars, salts and acids. Therefore, the lower TSS in the puree made without peel may be explained by the lower soluble solids such as sugars and organic acids in the mango flesh than the peel. Soluble solids have been reported to range from 17 to 23% in the peel and

10.6-11.2% in mango flesh (Medlicott and Thompson 2006; Ajila and Rao 2013). The puree made without peel also had higher pH hence lower organic acid, also affecting the lower TSS in the puree made without peel.

3.3.6 Effect of peel and holding time on viscosity

The puree made without peel had almost half the viscosity of puree made with peel

(Table 14). This may be due to higher levels of pectin in the peel than the fleshy part.

More pectin, 24%, is extracted from mango peel than from the flesh, 5.8% (Kratchanova and others 1991). Holding time also had a significant effect on the viscosity of the puree. For the puree without peel, a longer holding time resulted in a decrease of viscosity. Among the enzymes responsible for pectin breakdown are polygalacturonase and polymethylesterase. These enzymes decrease the length of the pectin chains with time hence reducing the viscosity and therefore the viscosity of the puree made without peel decreased with increasing holding time. However, when the peel was present, the enzymes responsible for pectin breakdown were still active and probably solubilisation of the pectin out of the peel continued simultaneously hence resulting in an increase in viscosity.

3.3.7 Effect of peel and holding time on color

The puree made without peel was the same yellowness, was the same or slightly darker, and was slightly redder, than the puree made with peel (Table 14). Mango peel has a variety of pigmentation, with spots ranging from yellow to green to red to brown while the mango flesh is bright yellow. These spots may have caused the differences obtained between the puree made with and without peel. Color did not change with holding time, even though there are some reactions that consume beta carotene such as its oxidation in the formation of beta-ionone but its depletion during this reaction seem to be insignificant to affect the color of the puree or the concentration of the beta ionone. 93

3.3.8 Sensory results

The puree without peel was more preferred in all the attributes than the puree made with peel (Table 15). The texture of the puree without peel scored higher in liking than the puree made with peel. It appeared to the researchers that the texture of the puree made without peel was smoother and more consistent whereas the puree made with peel was thick and somewhat irregular. Also, the puree made without peel had lower viscosity than the puree made with peel (Table 14). Similarly, the color of puree made without peel was more preferred. There was very little difference in the L*, a* and b* values (Table 14) but the puree made without peel, in the researchers’ opinion, appeared visually brighter with a more attractive yellow color. Similarly, the puree made without peel was perceived to be fresher in flavor and aroma (Table 15). From the quantification of the volatile compounds, most of the lipoxygenase-derived volatiles were higher in the puree made without peel than the puree made with peel (Table 12). These volatiles are associated with the green fresh flavor of most fruits and vegetables. On the other hand, terpene hydrocarbons were lower in the puree made without peel. The puree made without peel was evaluated to have less off flavor than the puree made with peel. Carveol and carvone are two terpene hydrocarbons associated with off flavor that were significantly higher in the puree made with peel thus they may have contributed to the off flavor. Therefore, with the results obtain from all the sensory attributes that is texture, color, aroma and flavor it is sensible that the puree made without peel scored higher in overall liking.

The holding time only had a significant effect on the texture, the color and the freshness

(Table 15). The longer the holding time, the higher the viscosity in the puree made with peel (Table 14) and lower viscosity was more preferred (Table 15). There was no significant difference in the color values of the puree held at different times but the panelists were able to detect the difference between the two treatments. Freshness of the purees increased with holding time (Table 15). This may be explained by more time required for the formation of some of the lipoxygenase-derived volatiles responsible for freshness (Table 12).

Table 15: Sensory results for mango puree made with and without peel at different holding times

Off Overall Most Treatments Texture Color Freshness flavor Aroma Flavor liking Preferred

Without peel 5min 6.63a 7.07a 2.83b 2.32bc 6.94a 7.07a 7.28a 3.32a Without peel 1h 45min 6.84a 7.26a 3.21a 2.01c 6.81a 6.98a 6.94a 3.24b With peel 5 min 6.12b 5.28c 1.78d 2.94a 5.46b 5.09b 5.58b 1.65c With peel 1h 45min 5.57c 6.23b 2.18c 2.72ab 5.99b 5.34b 5.51b 1.95c Values in the same column bearing different superscript letters are significantly different

(p ≤0.05)

The panellists judged the color, aroma, freshness and texture of the puree made without peel that was held for 1h 45min to be just about right whereas the color, aroma, texture and freshness of the puree made with peel was observed to be darker, too little, too viscous and too little respectively (figure 10). From the flavor volatile analysis the puree made without peel was determined to contain more of the lipoxygenase-derived volatiles

95 that is associated with fresh flavor. On the contrary, the puree made without peel that was held for 5 min were marked just about right for color, freshness and texture whereas the aroma was too little (figure 10).

Color Intensity Color Intensity 80 80 70 70 60 60 50 50 40 40 30 30

20 20 Percentage (%) Percentage Percentage(%) 10 10 0 0 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 With peel 1h without peel 1h With peel 5min Without peel 45min 45min 5min

Aroma Aroma 80 60 70 50 60 40 50 40 30 30 20 20

Percentage(%) 10 Percentage (%) Percentage 10 0 0 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 With peel 1h without peel 1h With peel 5min Without peel 45min 45min 5min

Figure 10: Just About Right responses of panelists on mango puree made with and without peel held for 5min or 1h 45min on color, aroma, freshness and texture. Much too little = -2, slightly too little= -1, Just about right=0, slightly too much=1 and much too much= 2.

Viscosity Viscosity 70 70 60 60 50 50 40 40 30 30

20 20 Percentage(%) Percentage (%) Percentage 10 10 0 0 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 With peel 1h without peel 1h With peel Without peel 45min 45min 5min 5min

Freshness Freshness 80 70 70 60 60 50 50 40 40 30 30

20 20 Percentage(%) Percentage (%) Percentage 10 10 0 0 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 -2 -1 0 1 2 With peel 1h without peel 1h With peel Without peel 45min 45min 5min 5min

Figure 11: Just About Right responses of panelists on mango puree made with and without peel held for 5min or 1h 45min on color, aroma, freshness and texture. Much too little = -2, slightly too little= -1, Just about right=0, slightly too much=1 and much too much= 2.

3.4 Conclusion

Peeling of mangoes before processing is better in terms of the associated flavor volatiles than skipping the peeling step. This is because there is production of more lipoxygenase derived volatiles associated with fresh flavor and less off flavor-associated volatiles in the

97 puree made without peel. Puree made without peel was more preferred in all the sensory attributes. The texture, color, aroma and flavor of puree made without peel scored higher in liking and this may also explain its overall liking. Peeling of mango during processing generally improves the quality of the end product. Holding time had an effect on some of the volatiles and these may be due to the enzymatic process in the formation of these volatiles. Peeling on the other hand decreased the total phenolics content and the viscosity of the puree. Total phenolics and viscosity were lower for the puree made without peel. The lower phenolic compounds and pectin levels in the mango flesh than peel explains the results obtained in this study. Hence if the mango processing industry desires to have more phenolics and a more viscous puree, leaving the peel on would be one of the recommendations.

References

Arctander S.1969. Perfume and flavor chemicals; Arctander: Montclair, NJ, 1969

Ajila CM, Bhat SG, Prasada Rao UJS. 2007. Valuable components of raw and ripe peels

from two Indian mango varieties. J Food Chem 102(4): 1006-1011.

Ajila CM, Rao UJS. 2013. Mango peel dietary fiber: Composition and associated bound

phenolics. 5(1):444-450

AOAC 1995. Official Methods of Analysis, 16th edition, Association of Official Analytical

Chemists, Washington D.C, 138pp.

Barrett DM, Weakley C, Diaz JV, Watnik M. 2007. Qualitative and Nutritional Differences

in Processing Tomatoes Grown under Commercial Organic and Conventional

Production Systems. J Food Scie 72(9):441-451

Bouwmeester HJ, Gershenzon J, Konings MC, Croteau R. 1998. Biosynthesis of the

monoterpenes limonene and carvone in the fruit of caraway. I. Demonstration Of

enzyme activities and their changes with development. J Plant physiol 117(3): 901-

912.

Buettner A, Montse M, Fischer A, Guasch J, Schieberle P. 2003. Evaluation of most odor-

active compounds in the peel of clements (citrus recticulate blanco cv. Celmentine)

Eur. Food Res. Technol, 216(1),11-14.

Buttery RG, Ling LC. 1993. Enzymatic production of volatiles in tomatoes. In: Schreir P,

Winterhalter P, editors. Flavor precursors. Wheaton IL: Allured Publications. p 137-

146. 99

Croft KPC, Juttner F, Slusarenko AJ. 2003. Volatile products of the lipoxygenase pathway

evolved from Phaseolus vulgaris (L.) leaves inoculated with Pseudomonas syringae

pv phaseolicola. Plant Physiol; 101:13–24.

De Pascual TS, Johnston KC, Dupont MS. 2004. Quecertin metabolic downregulates

cyclooxygenase 2 transcrition in human lymphocytes ex vivo but not invivo. J.

Nutri.3:552-557

El Hadi MAM, Zhang F, Wu F, Zhou C, Tao J. 2013. Advances in fruit aroma volatile

research. J. molecules 18: 8200-8229

Engel, K.-H.; Flath, R. A.; Buttery, R. G.; Mon, T. R.; Ramming, D. W.; Teranishi,

R.1988. Investigation of volatile constituents of aroma components in some nectarine

cultivars. J. Agric. Food Chem. 36, 549-553.

Fisher, C.; Scott, T. T.1997. Food Flavours: Biology and Chemistry; The Royal Society of

Chemistry: Hunt Valley, MD,

Galliard T, Matthew JA, Wright AJ, Fishwick MJ. 1977. The enzymic breakdown of lipids

to volatile and non-volatile carbonyl fragments in disrupted tomato fruits. J Sci

Food Agric 28:863-868.

Hatanaka A, Kajiwara T, Sekiya J. 1987. Biosynthesis pathway for C6 aldehyde formation

from linolenic acid in green leaves. Chem. phys. Lipids, 44:341-361.

Iagher F, Reicher F, Ganter JL. 2002. Structural and rheological properties of

polysaccharides from mango (Mangifera indica L.) pulp". Int. J. Biol Macromol.

31(1-3): 9-17.

100

Kim HJY, Moon D, Kim M, Lee H. Cho YS, Choi AK, Cho SK. 2010. Antioxidant and

antiproliferative activities of mango( (mangifera indica L.) flesh and peel. Food

Chem, 121:429-436

Kratchanova M, Benemou C, Kratchanov C. 1991. On the pectic substances of mango

fruits. J Carbohydrate Polymers 15(3):271-282

Lakshminarayana S, Subhadra NV, Subramanyan H. 1970.Some aspects of developmental

physiology of mango fruit. J Hort Sci 45:133-42

Lopes, D. C.; Fraga, S. R.; Rezende, C. M. 1999. Aroma impact substances on commercial

Brazilian mangoes by HRGC-OAEDA- MS. Quim 22, 31-36.

Mahattanatawee K, Rouseff R, Valim MF, Naim M. 2005. Identification and aroma impact

of norisoprenoids in orange juice. J Agric Food Chem 53(2):393–7.

Masibo M, He Q. 2008. Major Mango Polyphenols and Their Potential Significance to

Human Health. J Food Science and Food Safety 7(4):309-319.

Marsili R. 2007 (ed). Sensory directed flavor analysis. Florida: Taylor and Francis group

256p.

Medlicott PA, Thompson KA. 2006. Analysis of sugar and organic acids in ripening mango

fruit (Mangifera indica L.var Keitt). J Scie Food Agric. 36(7):561-566

Pino JA, Mesa J. 2006. Contribution of volatile compounds to mango (Mangifera indica L.)

aroma. J. Flav. Frag., 21(2): 207–213.

Pino JA, Mesa J, Munoz Y, Martia MP, Marbot R. 2005. Volatile components from mango

(Mangiferaindica L.) cultivars. J Agric Food Chem. 53(6): 2213-2223.

101

Qiao Y, Xie BJ, Zhang Y, Zhang Y, Fan G, Yao XL, Pan SY. 2008. Characterization of

aroma active compounds in fruit juice and peel oil of jinchen sweet orange fruit

(Citrus sinensis(L) Osbeck) by GC-MC and GC-O. Molecules. 13:1334-1334

Sandoval IS, Hernández AS, Valdez GV, Cruz-López L.2007. Volatiles of Mango var.

Ataulfo Characterized by SPME and Capillary GC/MS Spectroscopy. J. Mex.

Chem. Soc. 51(3): 145-147

Sawamura M, Tu NTM, Onishi Y, Ogawa E, Choi HS. 2004. Characteristic odor

components of citrus reticulate blanco (ponkan) cold-pressed oil. Bioscie,

Biotechnol and Biochem. 68(8):1690-1697

Ties P., Barringer S. 2012. Influence of lipid content and lipoxygenase on flavor volatiles

in the tomato peel and flesh. J Food Sci 77(7): 830-837pp.

102

Chapter 4: Deodorization of garlic breath and odor by the use of food materials with different types and different amounts of phenolic compounds and polyphenol oxidase

Abstract

Garlic causes a strong undesirable garlic breath when eaten raw that may persist for almost a day. Therefore, it becomes important to study deodorization techniques of garlic breath.

The volatiles responsible for garlic breath include diallyl disulfide, allyl mercaptan, allyl methyl disulfide and allyl methyl sulfide. After eating garlic, water (control), raw, juiced or heated apple, raw or heated lettuce, raw or juiced mint leaves or green tea were consumed immediately. The levels of the garlic volatiles on the breath were analyzed from 1 to 60min by selected ion flow tube mass spectrometry (SIFT-MS). Garlic was also blended with water (control), polyphenol oxidase (PPO), rosmarinic acid, quercetin or catechin or three mint varieties, peppermint, spearmint or chocolatemint or a combination of peppermint and rosmarinic acid at different concentration and the volatiles in the headspace analyzed by

SIFT-MS. Different types of phenolic compounds produced different deodorization. Mint leaves exhibited the higher deodorization of all the food tested. Rosmarinic acid which is a major phenolic compound in mint leaves also produced the highest deodorization compared to other phenolics; namely quercetin and catechin. On the contrary, the amount of phenolic compound was not a major factor in the deodorization process. Foods with high or low total phenolic compounds, that is, raw apple and raw lettuce respectively had almost the same deodorizing capacity. Similarly, green tea with a relatively high total phenolic 103 content exhibited no deodorization for most garlic volatile compounds. The insignificant effect of the amount of phenolic compound was also observed in the different mint varieties and different concentrations of rosmarinic acid. Enzyme activity may not be a requirement in the deodorization process. Pure PPO produced a minimal deodorization effect and heated food product still achieved significant deodorization level. Non phenolics may be important in the deodorization process and this may explain why heated lettuce with low to phenolic and no enzyme activity was able to achieve significant deodorization.

104

Practical Application

Garlic has been reported to have a number of health benefits therefore some individuals tend to eat garlic in its raw form. Raw garlic has a strong smell responsible for the undesirable garlic breath that may linger for about 24 hours. Therefore, it is helpful to come up with means of deodorizing the volatiles responsible for garlic breath as the associated health benefits are enjoyed. Chewing mint leaves, and eating apple and lettuce (raw and cooked) will help reduce the garlic breath volatiles. This study also helps to understand part of the deodorization process of garlic odor. This may lead to innovations or improvements of existing deodorization techniques for bad odor or breath. Rosmarinic acid deodorizes the garlic odor, but not as good as a mixture of rosmarinic acid and mint leaves.

4.1 Introduction

Garlic (Allium sativum L. Fam. Liliceae) is a bulb that is widely used as a condiment by most people around the world. Garlic has also been found to have antimicrobial properties in some in vitro tests. Recent studies have shown garlic to be medicinal as it aids in reducing the risk of cancer, particularly stomach cancer, esophagus cancer, colon cancer, breast cancer and prostate (Fleischauer and Arab 2001; Hsing and others 2002; Challier and others 1998; Amagase and others 2001). Researchers have also reported some cases where garlic has led to lower blood cholesterol and blood sugar and it also acts as an anti-aging agent by producing new body cells and assisting in memory enhancement (Thomson and others 2006; Kim and others 2013; Sarkaki and others 2013). Due to these benefits associated with garlic more people consume at least a clove a day for health purposes.

105

Garlic has an imperceptible smell before it has been crushed, but once it has been cut or crushed it gives a strong smell that is distinctive of sulfur glucoside containing plants.

Consumption of garlic may therefore result in undesirable garlic breath that persists for almost 24 hours. Therefore, it becomes important to find ways and mechanisms of deodorizing garlic breath.

There are four volatile compounds associated with garlic breath. These include diallyl disulfide, allyl mercaptan, allyl methyl disulfide and allyl methyl sulfide. Diallyl disulfide with an odor detection threshold (ODT) of 0.22ppb and allyl methyl disulfide whose ODT is still unknown are formed from allicin (Henry and Gehr 1980; Nagata and Takeuchi

1990). Diallyl disulfide is then reduced to form allyl mercaptan which has an ODT of

0.05ppb (Henry and Gehr 1980; Nagata and Takeuchi 1990), allyl mercaptan is then methylated to allyl methyl sulfide with an ODT of 0.14ppb (Henry and Gehr 1980; Nagata and Takeuchi 1990).

Various fruits and vegetables have been shown to successfully lower the levels of the volatiles both in vitro and on the breath. The means of deodorization has been theorized to be due to the presence of phenolic compounds and enzyme activity particularly polyphenol oxidase (PPO) and reductase (Negishi and others 2002). Phenols are oxidized to form negatively charged phenoxide ions. An electron from each phenoxide ion is lost to form radicals that are delocalized and the resulting radicals react with radicals formed from allyl mercaptan or with thiol gasses formed from reduction of disulfides (Negishi and Negishi

1999, Negishi and others 2002) to form conjugates that are either odorless or have a different odor.

106

Different methods have been used to test the deodorization of garlic breath, both in vivo and in vitro. On in vivo testing, some raw fruits and vegetables have been reported to have a deodorizing effect of garlic breath. Raw fruits such as apples, plum, prune, apricot and cherry showed higher deodorizing capacity than citrus fruits, grapes, pear and kaki (Munch and Barringer 2014; Negishi and Negishi 1999). Lettuce, chicory, celery, potato, parsley, mint leaves, peppermint, basil and mushroom were some of the vegetables that were very effective in removing methyl mercaptan and allyl mercaptan (Negishi and Negishi 1999,

Munch and Barringer 2014). Microwaved and raw apples both produced a significant decrease on the levels of garlic breath volatiles (Munch and Barringer 2014). However, raw apple exhibited a greater reduction than microwaved apple. By microwaving the apple all the enzymes present are deactivated hence the deodorization taking place may have been attributed to the presence of phenolic compounds and other components in the food but not enzymes. Furthermore, deodorization capacity by both enzyme and phenolic compounds has been reported to be better than that of phenolic compounds alone (Negishi and Negishi

1999). Fat free milk, 2%fat and whole milk also produced a deodorizing effect on the garlic breath volatiles (Hansanugrum and Barringer 2010). Green tea powder also showed a reduction in concentration of methyl hydrogen sulfide and hydrogen sulfide but this reduction was observed at 1,2 and 3h after ingestion (Lodhia and others 2008).

In an in vitro test, fat-free and whole milk had a significant reduction on the concentrations of the analyzed volatile compounds in the headspace of chopped garlic (Hansanugrum and

Barringer 2010). Whole milk was more effective on hydrophobic compounds, diallyl disulfide and allyl methyl disulfide whereas fat-free milk was more effective on the reduction of the hydrophilic compounds allyl mercaptan and allyl methyl sulfide

107

(Hansanugrum and Barringer 2010). An in vitro testing on lettuce, apple, pear, mushroom and eggplant also produced a deodorizing effect on most of the garlic volatiles (Negishi and others 2002; Negishi and Negishi 1999). Other food compounds that are low in phenolic content such as lettuce has also shown a decrease in the levels of these volatiles

(Negishi and others 2002).

Mint leaves have been reported to produce the highest decrease in the volatile levels compared to other food materials in vivo (Munch and Barringer 2014, Mirondo and

Barringer 2016). The deodorization of garlic breath and odor has been linked to the presence of phenolic compounds and polyphenol oxidase. Rosmarinic acid is the dominant phenolic compound in mint and this compound has been reported to have high antioxidant properties, better than trolox (Cao and others 2005). Therefore, rosmarinic acid may be responsible for the high deodorization capacity exhibited by mint leaves. Mint leaves have a concentration of rosmarinic acid ranging between 26.45 and 362.2mg/100g (Tahira and others 2011). Therefore, the objective of this study was to deodorize garlic breath by the use of different food materials and assess the effect of pure PPO and different pure phenolic compounds on the levels of the volatiles responsible for garlic breath. Also to assess the in vitro contribution of rosmarinic acid at different concentrations, rosmarinic acid plus peppermint leaves and different mint concentrations in the deodorization process of garlic volatile compounds.

108

4.2 Materials and Methods

Garlic, apple (Fuji), mint leaves, lettuce and green tea (Lipton tea bags, Unilever,

Englewood Cliffs, NJ) were purchased from Kroger (Columbus, Ohio. U.S.A). Three grams of garlic clove were chewed for 25s, 100ml of water was drunk immediately and the breath volatile levels were analyzed using the SIFT-MS 200 (SYFT Voice 200,

Christchurch, New Zealand). This was achieved by blowing through a 5cm straw attached to the SIFT-MS 200 starting from 1 to 60min where each scan was 2min. The same procedure was followed with different treatments except no water was used. The treatments included 100g of raw apple, 100g of heated apple (microwaved for 4min), 100 ml apple juice (100g of apple blended with 50ml water for 2min and filtered using a metal mesh strainer) 10g mint leaves, 100ml of mint juice (100g of mint blended with 50ml water for

2min and filtered using a strainer), 100g of raw lettuce, 100g of heated lettuce (microwaved for 4min) or 100 ml green tea (100ml of water was microwaved for 4min, the tea bag was placed in the hot water for 2min) was consumed immediately and the breath was analyzed by SIFT-MS.

4.2.1 Effect of pure Polyphenol oxidase (PPO) and phenolic compounds on garlic volatile levels

Three replicates of 16g of garlic was blended with 100ml water (30oC) for 25s and placed in a 500ml Pyrex bottle. Treatments contained 0.05g of PPO, quercetin, catechin or rosmarinic acid (Fischer Scientific, Kansas, MO. USA) and placed in a Pyrex bottle. They were then analyzed for volatile levels by the use of SIFT-MS after 1min for 1 to 60min at room temperature. A needle was connected to the SIFT-MS machine and was pierced

109 through the septum into the bottle to allow the flow of volatiles through the machine. The kinetics of the volatiles measured is shown in Table 16.

4.2.2 Headspace volatile levels with different mint varieties

Mint of three different varieties, spearmint (Mentha spicata), peppermint (Mentha piperita) and chocolate mint (Mentha piperita, ‘chocolate’) were purchased from Meijer and Kroger

(Columbus Ohio U.S.A.). Pure rosmarinic acid was purchased from Fisher Scientific

(Kansas, MO. USA). Ten grams of the mint leaves was blended using a magic bullet blender (MB 1001B) with 5g of garlic and 50ml of water for 30sec and the mixture was transferred into a 500ml Pyrex bottle for volatile analyses. Selected ion flow tube mass spectrometry (SIFT-MS) was used for volatile analyses from 0 to 30min with 1min scans.

There was 30s between end of blending and the start of the SIFT-MS measurements. The same procedure was followed with different amounts of peppermint, different concentrations of rosmarinic acid and a combination of rosmarinic acid and peppermint.

One, five and ten grams of peppermint were used for the different weights of mint. Two or forty milligrams was used for the different concentration of pure rosmarinic acid and 2 or

40mg of rosmarinic acid was added to 1g of peppermint. The volatiles were analyzed at room temperature, 30s after blending was stopped and the scan took 1min.

110

Table 16: Volatiles measured in the headspace scan

Volatile compound Ion Product Precursor m/z Reaction rate Ion (k) (10-9 cm3/s)

+ + Methyl Mercaptan CH4S.H H3O 49 1.8

+ + Allyl methyl sulfide C4H8S.H H3O 89 2.6

+ + Diallyl sulfide (C3H5)2S.H H3O 115 2.9

+ + Allicin C6H10OS2H H3O 163 2.4

+ + Allyl methyl tetrasulfide C4H8S4.H H3O 185 2.6

+ + Diallyl Tetrasulfide C6H19S4.H H3O 211 2.6

+ + Dimethyl Thioether (CH3)2S NO 62 2.2

+ Allyl mercaptan C3H6S NO 74 2.4

+ + Acetone NO .C3H6O NO 88 1.2

+ + Dimethyl disulfide (CH3)2S2 NO 94 2.4

+ + Dimethyl Thiosulfate C2H6OS2 NO 110 2.4

Allyl methyl disulfide C4H8S2+ NO+ 120 2.4

+ + Dimethyl trisulfide C2H6S3 NO 126 1.9

+ Allyl methyl thiosulfinate C4H8S2O NO 136 2.4

+ + 2-or3-vinyl-4H-1 2or3-dithiin C6H8S2 NO 144 2.4

+ + Diallyl Disulfide (C3H5)2S2 NO 146 2.4

+ + Allyl Methyl Trisulfide C4H8S3 NO 152 2.4

+ Diallyl Trisulfide (C6H10)S2 NO 178 2.4

111

The headspace concentration of volatile compounds present in garlic were measured using selected ion flow tube mass spectrometry (SIFT-MS). This is a method of direct mass spectrometry that analyses volatile organic compounds (VOC s) in air with typical detection limits at parts-per-trillion level (by volume; pptv). Real-time, quantitative analysis is achieved by applying precisely controlled soft chemical ionization and eliminating sample preparation, pre-concentration and chromatography. Chemical ionization of the sample compounds occurs after reaction with precursor ions formed from

+ + microwave discharge (Spanel and Smith 1998). The three precursor ions are H3O , NO or

+ O2 (Spanel and Smith 1998). The concentrations of volatile compounds are obtained via the reaction of the precursor ions with the compound of interest through a predetermined reaction rate constant for the volatiles with the selected precursor ions and accounting for the dilution of the carrier gas in the flow tube (Spanel and Smith 1998).

4.2.3 Analysis of total phenolic compounds

Raw apple, heated apple, raw lettuce, heated lettuce, mint leaves or green tea (0.5g) were measured separately in 15 mL glass tubes. Five milliliters of 80 % acetone-water (0.01%

HCl) was added and then vortexed for 30 seconds. It was then centrifuged at 4500 rpm for

10 min. The supernatant was transferred to a 25 ml glass tube and 5 ml 80 % acetone-water

(0.01% HCl) was added. It was again vortexted for 30 sec, stirred and centrifuged again for10 min. The supernatant was taken out and placed in 25 ml glass tubes. Then chloroform

(10 ml) was added to the 25 ml glass tube. This was mixed and then centrifuged at

4500rpm for 10 min. The top layer was collected into a 5 ml volumetric flask and 2.5 mL of 0.01 % HCl-Water was added into 25 mL glass tubes containing the sample. The

112 mixture was then centrifuged at 4500RPM for 10min. The top layer was collected and transferred to a 5 mL volumetric flask. Chloroform was evaporated using a rotovap (Model

WU 23012-12 Cole-Parrmer, Chicago, Ill., U.S.A.) for 10 min. The volumetric flask was filled to 5 mL with 0.01 % HCl-Water and 5 ml final solution was transferred to 15 mL tube and put in a refrigerator at 5oC until the analysis.

Total phenolics were determined as per Barrett and others (2007) using Folin-Ciocalteau’s reagent. Five milliliters of acetone, 0.5 ml sample and 1.0 ml Folin–Ciocalteau reagent were added to a 25 ml volumetric flask. The contents were mixed and allowed to stand for

5 to 8 min at room temperature. Ten milliliters of 7% sodium carbonate solution was added, followed by the addition of Nanopure water filled to volume. Solutions were mixed and allowed to stand at room temperature for 2 h. Sample aliquots were filtered through a

Whatman 0.45-μm polytetrafluoroethylene filter prior to the determination of total phenols concentration using a spectrophotometer (50 BIO UV-Visible Shimadzu, LA, USA) monitoring Abs 750 nm. Total phenolics content was standardized against gallic acid and expressed as gallic acid equivalents (GAE). The linearity range for this assay was determined as 0.3 to 2.5 mg/100ml GAE (R2 = 0.9853) giving an absorbance range of

0.0321 to 2.5351 AU.

4.2.4 Quantitative analysis of rosmarinic acid by HPLC

4.2.4.1 Sample preparation

Mint leaves of the three varieties was weighed (0.5 g), 10 mL of methanol-H2O (7:3) was added to the mint and the mixture was placed in an ultrasonicator at room temperature for

30 min. The extract was centrifuged at 4000 rpm for 10min. About 10ml of the aliquot was 113 drawn and methanol was evaporated using a rotovap (Model WU 23012-12 Cole-Parrmer,

Chicago, Ill., U.S.A.) The extraction was done twice and the solutions were combined and transferred into a 25-mL volumetric flask and made up to volume with acidified water

(0.01% HCl) and filtered through a syringe filter (0.2 μm, Alltech, Beerfield, IL, USA). An aliquot of 10 μL of the filtrate was injected into HPLC for analysis. The analysis was repeated for a mixture of garlic and spearmint, peppermint and chocolate mint at 0 and

30min.

4.2.4.2 HPLC conditions

The analysis of rosmarinic acid was carried out by HPLC (Shimadzu, model RID-10A, Ill,

U.S.A). 330 nm was selected as the wavelength for UV detection. Elution was carried out at a flow rate of 1.0 mL/min at 25°C. Two mobile phases, A and B were used. Mobile phase A was 0.1% (v/v) formic acid solution in water, while mobile phase B was acetonitrile. A ratio of 88% A and 12% B was applied in the first 30 min. After 30 min, a ratio of 80% A and 20% B was used for the next 15 min. Finally, 70% A and 30% B were used after 45 min for an additional 15 min and standard was developed by the use of rosmarinic acid and the concentration calculated from the standard curve.

4.2.5 Statistics

Statistical Analysis System (SAS, v9.3) (Cary, NC: SAS® Institute Inc) and JMP version

10.0.2 64-bit edition (Statistical Discovery, Cary, NC, USA) was used for data fitting and analysis. Five replicates of in vivo data and three replicates of in vitro data were obtained.

Data were analyzed by using one way and two-way analysis of variance and significance

114 was defined as p ≤ 0.05. Comparison was done using Tukey-kramer honestly significant difference (HSD).

4.3 Results and Discussion

4.3.1 Garlic breath/ odor and deodorization process

Allicin is a major thiosulfinate that is responsible for both the odor and antibacterial properties of freshly cut garlic. It is also a precursor to many of the important garlic volatile compounds responsible for the garlic breath namely diallyl disulfide, allyl methyl disulfide, allyl mercaptan and allyl methyl sulfide. After cutting or crushing of garlic, two of the first volatiles formed from allicin in the pathway is diallyl disulfide with an odor detection threshold (ODT) of 0.22 ppb (Henry and Gehr 1980; Nagata and Takeuchi 1990) and allyl methyl disulfide whose ODT is still unknown. Allyl mercaptan is formed from diallyl disulfide and has a detection threshold of 0.05ppb (Henry and Gehr 1980; Nagata and

Takeuchi 1990) whereas allyl methyl sulfide is formed in the body through methylation of allyl mercaptan by S-adenosylmethionine and gut microflora (Lawson 1998). Allyl methyl disulfide is also formed outside the body from diallyl disulfide through an enzymatic reduction process (Iciek and others 2009). The deodorization process of garlic breath has been proposed to be caused by the reaction of phenolic compounds with garlic volatile compounds to form odorless compounds or compounds with a different odor. The phenolic compounds are oxidized by abstracting a hydrogen atom from the phenolic hydroxyl group to form negatively charged phenoxide ions. The disulfides are degraded to thiol gas by reductase, an electron from each phenoxide ion is lost to form radicals which react with thiol gas (Negishi and Ozawa 1997; Negishi and Negishi 1999; Negishi and others 2002). 115

This deodorization reaction is catalyzed by polyphenol oxidase (PPO) and peroxidase

(POD) (Negishi and others 2002; Lu and others 2006). Therefore, Negishi and others suggest that the presence of both phenolics and enzymatic activity may be essential for the deodorization of diallyl disulfide. Other researchers reported a significant decrease in diallyl disulfide by the consumption of raw foods such as apple, lettuce and mint leaves

(Munch and Barringer 2014; Negishi and others 2002). Deodorization of allyl mercaptan occurs by the same mechanism as for the disulfides, except the initial reduction of allyl mercaptan is not needed (Negishi and Ozawa 1997; Negishi and Negishi 1999).

4.3.2 Contribution of the type of phenolic compound on garlic breath and odor deodorization

4.3.2.1 Effect of different food material on garlic breath

Most of the treatments tested led to a decreased level of diallyl disulfide, allyl mercaptan and allyl methyl disulfide, compared to the control (Fig 11 table 17). Raw apple, raw lettuce and mint leaves decreased the concentration of these volatiles in breath by 50% or more compared to the control for the first 30min (Figure 11). Different foods have different types and profiles of phenolic compounds and this may contribute significantly to the difference in the deodorizing capacity observed in the different foods. Usually, mint leaves showed higher deodorization than the other foods tested. The higher deodorization capacity by mint leaves compared to other food has also been reported by other researchers (Munch and Barringer 2014).

116

a 8000 8000 a Water Water 7000 a 7000 a b a Raw apple 6000 6000 ab a Mint juice Raw lettuce 5000 5000 b Apple juice a a Mint leaves c 4000 4000 c dd c cd b 3000 a 3000 a d d c

d Concentration (ppb) 2000 2000 e e cd b a Concentration Concentration (ppb) c a cd a d c a 1000 c ab a a 1000 d c a a c c a a b a a 0 c 0 0 20 40 60 0 20 40 60 Time (min) Time (min)

Figure 12: Effect of raw apple, raw lettuce, mint leaves, mint juice and apple juice on diallyl disulfide. Data points at the same time bearing different letters are significantly different (p ≤0.05)

Table 17: Difference in volatile concentration (ppb) between water and foods at 5 and 20 min. Diallyl Allyl Analyte disulphide mercaptan Allyl methyl disulfide Allyl methyl sulfide Concentration 5min 20min 5min 20min 5min 20min 5min 20min b c c d a a b e Raw apple 4340 2144 4220 1464 829 124 19.8 * 8.98 * Raw Lettuce 4617b 2772ab 5890bc 3064b 808a 139a 37.7a* 116a Mint Leaves 5860a 3011a 9134a 4844a -141e -809d -300d -402f Mint Juice 1398d 1632d 2380d 2209c 200c* 80.3b* 31.2a* 122.6a c b b a b a d c Apple juice 3778 2414 6988 4044 666 125 -14.0 * 76.2 Green tea 349e* 632d -740e 548e 81.7d* 22.0c* -2.14c* 35.2d* b b d c b ab c ab Heated apple 4224 2537 3270 2216 654 98.8 7.96 * 104 Heated Lettuce 5566a 3002a 8714a 3554b -11604f -1357e -2646g -9060g

*Values are not significantly different from water

117

4.3.2.2 Effect of different phenolic compounds on garlic breath/odor

Rosmarinic acid is a major phenolic in mint leaves (Tahira and others 2011), quercetin in apples (Lee and others 2003; Holderbaum and others 2010) and catechin in green tea

(Nagao and others 2007). Thus, the major phenolic compound in a food is likely to contribute sinificantly to the deodorization process. To investigate the effect of different phenolic compounds in the deodorization processes of garlic odor, pure phenolic compounds, catechin, quercetin and rosmarinic acid were blended with garlic and the headspace levels were analyzed. All the phenolic compounds showed a significant deodorization where rosmarinic acid had the highest deodorization and quercetin and catechin had a significantly smaller deodorization (Fig 12).

Diallyl disulfide being one of the first volatiles formed was significantly reduced by the presence of rosmarinic acid (Fig 12). The significantly lower levels of diallyl disulfide may have resulted in the lower levels of allyl mercaptan and allyl methyl sulfide, which are formed from diallyl disulfide and allyl mercaptan respectively (Fig 12). Similarly, higher deodorization of allyl methyl disulfide was achieved by addition of rosmarinic acid compared to quercetin and catechin. The headspace results from pure phenolic compounds supported some of the results obtained from deodorization of garlic breath by the use of the food rich in these phenolic compounds. Mint leaves which contains rosmarinic acid as its major phenolic compound, produced the highest deodorization of garlic odor of all the foods tested for most of the volatile compounds (Fig 11, Fig 14). The high deodorization produced by mint leaves may have been due to the presence of this particular phenolic compound. Therefore, the type of phenolic compound may play a role in the deodorization mechanism. The difference in deodorization level by the different phenolic compounds

118 may be due to differences in total antioxidant capacity of different phenolic compounds, where by some of the phenolic compounds such as rosmarinic acid have higher total antioxidant capacities than catechin and quercetin (Berker and others 2013; Peñarrieta and others 2008). Difference in total antioxidant capacity may results in differences in the oxidation reaction of the phenolic compounds that may in turn affect the deodorization process. The higher the total antioxidant capacity of the phenolic compounds the easier it is oxidized to form radicals that then react with the volatile compounds. Rosmarinic acid has a high antioxidant capacity that could be compared with antioxidants that are widely used by food industry (Preedy 2016; Vladimir-Knežević and others 2011). Rosmarinic acid has also been reported to have higher trolox equivalent antioxidant capacity (TEAC) than catechin and quercetin (Berker and others 2013) whereas quercetin was higher than catechin (Apak and others 2007). No one has reported different deodorization by the use of different phenolic compounds.

119

Diallyl disulfide Allyl mercaptan 800 a 3500 a a a a a a aaa b 700 a a 3000 a b bb a bb a bb b 600 a b bc bc bb c a a bb b 2500 b b c c a a b bb cc c a c 500 c bcc c 2000 b bbb c cc b 400 c d d d d b b c a c cc d bc b d d c 1500 bc c cb d d 300 d ccc cc c c

1000 200 Concentration (ppb) Water Concentration Concentration (ppb) PPO-CAT 500 100 PPO

0 0 0 20 40 60 0 20 Time (min) 40 60 Time (min)

Allyl methyl disulfide Allyl methyl sulfide 250 800 a a a 700 a b a b 200 a cc 600 b ab b cc a c a b 500 b c d 150 b c b a b 400 a b c b a d b c d e 100 300 b b d a b c b c a bb b d c c Concentration Concentration (ppb) b Concentration Concentration (ppb) 200 b c c b c a c cd b c 50 b a abab c bbb ba ab ccd d 100 a b cc cc d d bb b b c c c b 0 0 c 0 20 40 60 0 10 20 30 40 50 60 Time (min) Time (Min)

Figure 13: Effect of catechin, quercetin, rosmarinic acid, polyphenol oxidase and

polyphenol oxidase -catechin on diallyl disulfide, allyl mercaptan and allyl methyl

sulfide. Data points at the same time and same volatile bearing different letters are

significantly different (p ≤0.05)

120

4.3.3 Concentration of phenolic compounds in deodorization process

4.3.3.1 Polymerization of phenolic compounds by juicing

Juicing the food product and allowing it to stand at room temperature for 1 hour causes polymerization of the phenolic compounds resulting to no or lower levels of phenolic compounds compared to the non-juiced food (Niki and Noguchi 2000). Phenolic compounds are unstable and may exist as monomers or polymers (Friedman and Jurgens

2008; Zhu and others 2002; Abou El hassan and others 2000). Breaking the cell matrix of the food compounds exposes the phenolic compounds to oxidation particularly in the presence of PPO. Therefore, the juiced foods are expected to have a lower deodorizing capacity due to the reduced phenolic compounds compared to the whole foods. Mint leaves or apples were blended with water and filtered to form their respective juices. Mint juice and apple juice significantly reduced diallyl disulfide, allyl mercaptan and allyl methyl disulfide but they were not as effective as the raw apple and mint leaves (Fig 11; Table 17).

The deodorizing effect of the juices was lower compared to those of their respective non juiced foods. This implies that presence phenolic compounds have an effect on the deodorization processes and this is also supported by the deodorization achieved by pure phenolic compounds in figure 12. Apple juice had a greater deodorizing effect than mint juice. The differences in deodorizing level of mint juice and apple juice may be accounted for by the differences in the total antioxidant capacity of different phenolic compounds, where by rosmarinic has a higher deodorizing effect hence it may have reacted/polymerized faster than the phenolics in apples hence no more phenolics were left in mint juice for the deodorization reaction (Berker and others 2013; Peñarrieta and others

2008).

121

4.3.3.2 Total phenolic content of the different foods evaluated

To study further the contribution of the amount total phenolics in the deodorization process, the total phenolic content of the foods treatments was determined where mint leaves had the highest total phenolic content followed by, green tea, then raw apple and raw lettuce had the lowest total phenolics (Table 18). From this study, mint leaves had the highest deodorizing effect, which corresponded with its high total phenolic content (Fig 11,

Table 17) whereas green tea produced no deodorization in most of the volatile compounds

(fig 18). Furthermore, raw apple and raw lettuce showed a similar deodorizing effect in the first five minutes despite the differences in total phenolic content where raw apple had higher total phenolic than raw lettuce (Table 18). Therefore, from these results, it is possible to suggest that higher phenolic content does not result to higher deodorization.

Table 18: Total phenolic content found in the various food materials analyzed

Food Total phenolics (mg GAE/g) Mint leaves 1.48a Green tea 1.35b Heated apple 0.96c Raw apple 0.80d Heated lettuce 0.24e Raw lettuce 0.22e Values bearing different superscript letters are significantly different (p ≤0.05). GAE: gallic acid equivalent.

4.3.3.3 Deodorization by peppermint, spearmint and chocolate mint

To investigate the high deodorizing capacity of different concentration of rosmarinic acid, different mint varieties were tested. Different varieties of mint leaves are expected to differ in rosmarinic acid composition (Tahira and others 2011). Therefore, if rosmarinic acid is 122 the compound responsible for the deodorization process, we hypothesized that different mint varieties would possess different capacities in deodorization. Since rosmarinic acid also produced a higher deodorization than other phenolic compounds, the mint variety with the highest amount of rosmarinic acid was expected to produce a higher deodorizing effect than the other varieties. All the mint varieties produced a significant deodorization of most undesirable volatile compounds produced from garlic and their deodorization varied from one mint variety to another (Fig 13). The volatiles that exhibited a decrease with the different mint treatments included; allyl mercaptan, allyl methyl disulfide, diallyl disulfide, methyl mercaptan, diallyl trisulfide and in some cases allicin. All of these volatiles except diallyl trisulfide and allicin have been identified as volatile compounds important to garlic breath (Hansungrum and Barringer 2011). Peppermint and spearmint produced the highest deodorization for most of these volatile compounds and chocolate mint had the least deodorization in most cases. All the volatile levels increased with time for the control, while little to no changes were observed with the addition of all the three mint varieties except for allicin and methyl mercaptan. The increase of the volatiles over time indicates that the deodorization was not complete, and the volatiles were still being formed, though at a much lower rate than the control.

123

Allicin Allyl methyl disulfide 30 250 Control_garlic 25 Chocolatemint 200 Spearmint 20 Peppermint 150 15

10 100

5 Headspaceppbv conc.

50 Headspace Conc. Headspace pbbv 0 0 10 20 30 0 Time, min 0 10 Time, min 20 30

Diallyl disulfide Diallyl trisulfide 1400 80 70 1200 60 1000 50 800 40 600 30 400 20

Headspaceppbv conc. 200 10 Headspaceppbv conc. 0 0 0 10 20 30 0 10 20 30 Time, min Time, min

Allyl mercaptan Methyl mercaptan 35 200 180 30 160 25 140 120 20 100 15 80 60 10 40 ppbv conc. Headspace Headspaceppbv conc. 5 20 0 0 0 10 20 30 0 10 20 30 Time, min Time, min

Figure 14: Deodorization of garlic odor by use of peppermint, spearmint and chocolate mint 124

When garlic is cut or crushed, alliin and allinase come into contact and the action of allinase on alliin results in formation of allylsufenic that condenses to form allicin. Allicin is the first volatile compound formed after garlic has been cut or crushed (Block 1985;

Amagase and others 2001). This volatile was significantly decreased by spearmint whereas there was no significant decrease by peppermint and chocolate mint (Fig 13). The concentration of allicin is low because this volatile is rapidly converted to other volatiles.

Pure liquid allicin is unstable and it spontaneously transforms to other compounds such as diallyl disulfide, diallyl trisulfide and allyl methyl disulfide (Block 1985). These three volatiles were significantly lower than the control for all three mint treatments and the deodorization effect was much higher in these volatiles than for allicin.

Diallyl disulfide is reduced to form allyl mercaptan. Allyl mercaptan is the main volatile sulfur compound found in breath immediately after ingesting raw garlic (Laakso and others

1989; Minami and others 1989; Suarez and others 1999; Tamaki and Sonoki 1999; Tamaki and others 2008). This volatile compound was greatly reduced by the different mint treatments (Fig 13). Methyl mercaptan was the second most abundant volatile sulfur compound found in breath after ingestion of raw garlic (Suarez and others 1999; Tamaki and Sonoki 1999; Tamaki and others 2008) or heat-treated garlic (Tamaki and Sonoki

1999; Tamaki and others 2008). Similar to allyl mercaptan, there was a significant decrease in methyl mercaptan with all three mint treatments. There was no difference in deodorization levels between the three mint varieties on allyl mercaptan and methyl mercaptan.

125

4.3.3.4 Rosmarinic acid content

Based on the HPLC chromatograms, rosmarinic acid was the dominant phenolic compound present in all three mint varieties (Fig 14). Chocolate mint had significantly higher rosmarinic acid content followed by spearmint and lastly peppermint (Table 19). The range of rosmarinic acid content obtained in this study (1-4mg/g) almost matched the values reported by other researchers of 0.32-3.62mg/g (Tahira and others 2011). The mint variety with the highest rosmarinic acid content was expected to have the highest deodorization level of the garlic volatiles but this was not the case. For some volatile compounds, higher deodorization level was produced by peppermint and spearmint whereas in other volatile compounds there was equal deodorization for all the three volatiles (Fig 13). The high deodorization capacity of peppermint and spearmint may be due to other phenolic or non- phenolic compounds present in mint. The concentration of rosmarinic acid may also be greater than a minimum threshold, so that further increasing of the phenolic compound has no effect on deodorization.

Rosmarinic acid exhibits high antioxidant properties due to its structure, which results in an intramolecular hydrogen bond effect after the abstraction of H (Cao and others 2005).

Therefore, its high antioxidant properties would result in faster formation of the radicals which then react with the volatile compounds to form odorless compounds or compounds with different odor. This high antioxidant property of rosmarinic acid may explain why mint is more effective for the deodorization process of garlic odor than other foods.

Rosmarinic acid was not detected when mint leaves and garlic were blended and then measured after 0min or 30min for any of the mint treatments. This is likely because the phenolics had been used up immediately.

126

Table 19: Rosmarinic acid content of different mint varieties

Type Rosmarinic acid (mg/g)

Pure Peppermint 1.14c

Pure Spearmint 2.62b

Pure Chocolate mint 4.16a

Peppermint + Garlic 0min ND

Peppermint + Garlic 30min ND

Spearmint + Garlic 0min ND

Spearmint + Garlic 30min ND

Chocolate mint + Garlic 0min ND

Chocolate Mint + Garlic 30min ND

Values in the same column bearing different superscript letters are significantly different (p ≤0.05). ND-Not Detected.

Rosmarinic acid

Chocolate mint

Spearmint

Peppermint Absorbance (328nm) Absorbance

Figure 15: HPLC Chromatograms of rosmarinic acid quantification of 3 different types of mint.

127

Some of the other volatile compounds demonstrated an increase with the mint treatments

(Table 20). These volatiles that increased with the mint treatments were also detected in pure peppermint leaves in levels that would lead to a significant increase. This may be because of the presence of conflicting compounds with similar masses as the actual sulfur compounds thus leading to false positive results. There will be no further discussion of these volatiles due to their high baseline values in all the mint varieties

Table 20: Garlic volatile compounds detected at 30 min

Treatment

Pure peppermint Peppermint + Volatile Compounds Garlic (ppb) (ppb) Garlic (ppb)

2-or 3-vinyl-4H-1 2or3-dithiin 2.34 4.24 3.84

Allyl methyl THS 3.19 3293 4121

Allyl methyl sulfide 10.5 8.53 23.3

Allyl methyl tetrasulfide 0.40 11.0 10.8

Allyl methyl trisulfide 9.28 49.0 103

Diallyl tetrasulfide 0.26 0.82 1.34

Dimethyl THS 1.29 16.5 38.8

Diallyl sulfide 12.2 7.16 10.7

Dimethyl disulfide 6.49 123 240

Dimethyl thioether 31.80 42.9 215

Dimethyl trisulfide 1.88 3.64 4.39

Blending garlic with 2 or 40mg of pure rosmarinic acid produced a decrease in most garlic volatiles in comparison to the control (Fig 15). Increasing the concentration of rosmarinic acid did not produce increased deodorization, implying that just a small concentration of

128 rosmarinic acid, 2mg or less is required for the deodorizing process. These findings support the findings of other researchers who suggest that deodorization of garlic volatiles is still possible in the presence of just phenolic compounds (Negishi and others 2002). However, a combination of rosmarinic acid (2mg) and peppermint (1g) was more effective in deodorization than either 2 or 40mg of pure rosmarinic acid. One gram of peppermint has approximately 1.14mg of rosmarinic acid (Table 19). Therefore, adding 2mg of rosmarinic acid is approximately doubling the amount of rosmarinic acid already present in 1g of peppermint. This clearly indicates that rosmarinic acid is not the main deodorizing compound for the garlic odor but other components present in mint may also contribute significantly to the deodorization process.

Pure rosmarinic acid did not deodorize methyl mercaptan (Fig 15). Although, when 2mg of rosmarinic acid was added to 1g of peppermint there was a significant deodorization of this volatile. The mechanism for deodorization of methyl mercaptan must therefore require other compounds present in the mint leaves. Deodorization of methyl mercaptan has been achieved by the use of peppermint and has been reported by other researchers (Negishi and

Negishi 1999; Negishi and others 2002).

129

Allicin Allyl methyl disulfide Control_garlic 30 250 25 2mg RA 200 20 40mg RA 150 15 2mg RA+1g peppermint 10 100

5 50 Headspace Conc, ppbv Headspace 0 ppbv HeadspaceConc. 0 10 20 30 0 0 10 20 30 Time, min Time, min

Diallyl disulfide Diallyl trisulfide 1400 80 1200 70 60 1000 50 800 40 600 30 400 20 200 10 Headspace conc. conc. Headspace ppbv 0 0 Conc., ppbv Headspace 0 10 20 30 0 10 20 30 Time, min Time, min

Allyl Mercaptan 200 40 Methyl mercaptan 180 35 160 140 30 120 25 100 20 80 15 60 40 10

Headspace Conc. Headspace ppbv 20 Headspace conc., conc., Headspace ppbv 5 0 0 0 10 20 30 0 10 20 30 Time, min Time,min

Figure 16: Effect of pure rosmarinic acid (RA) and a combination of RA and peppermint on the garlic headspace volatiles.

130

A mixture of 2 or 40mg of rosmarinic acid with 1or 10g of peppermint produced a significant decrease in levels of most of the garlic volatile compounds compared to the control, except for allicin (Fig 16). As also shown in the previous graphs, increasing the concentration of rosmarinic acid did not increase the deodorizing level of most of the volatiles, where 2mg RA+1g peppermint produced the same deodorizing effect as

40mg RA +1g peppermint and this was also true for 2mg RA+ 10g and 40mg RA +

10g peppermint. This may signify that only 2mg or less amount of rosmarinic acid is required for the deodorization processes. Adding 2 or 40mg rosmarinic acid to 10g of peppermint led to an increased headspace concentration of allicin. These treatments also had higher concentration of allyl methyl disulfide than when 2 or 40mg of rosmarinic acid in 1g of peppermint was used. Rosmarinic acid may be impacting the reactions that are responsible for the formation of compounds with the same masses as the increased volatile compounds hence the higher concentration.

131

Figure 17: Effect of rosmarinic acid (RA) in peppermint on the garlic headspace volatiles

132

4.3.3.4 Effect of mint quantity and rosmarinic acid on garlic odor deodorization

To assess the amount of mint required to cause deodorization, 1, 5 and 10g of peppermint were blended with garlic and the level of volatiles analyzed. All three amounts of peppermint produced a significant decrease in most of the garlic volatiles except allicin (Fig 17). Ten grams produced the greatest decrease in the volatiles compared to 1g and there was usually no difference between 1 and 5g or 5 and 10g of peppermint for most volatiles. Pure mint without garlic was lower or equal to the mint plus garlic treatments except for allicin. When the mint treatments were not different in volatile levels compared to pure peppermint, this signified a limit of detection for the volatile was attained and so complete deodorization may or may not have occurred.

133

Allicin 40 Allyl methyl disulfide 250 35 Control_garlic 30 200 Peppermint 1g Peppermint 5g 25 Peppermint 10g 150 20 Pure Peppermint 15 100 10 50 5

Headspace Conc. ppbv HeadspaceConc. 0 0 Headspace Conc. ppbv HeadspaceConc. 0 10 20 30 0 10 Time, min20 30 Time, min

Diallyl disulfide Diallyl trisulfide 1400 80 1200 70 1000 60 800 50 40 600 30 400 20 200 Headspace Conc. ppbv HeadspaceConc. 10

0 HeadspaceConc.ppbv 0 0 10 20 30 0 10 20 30 Time, min Time,min

Allyl mercaptan Methyl mercaptan 200 35 30 150 25 20 100 15 50 10

5 Headspace Conc. ppbv HeadspaceConc. Headspace Conc. ppbv HeadspaceConc. 0 0 0 10 20 30 0 10 20 30 Time, Min Time,min

Figure 18: Effect of different quantity of peppermint on garlic odor volatiles

134

4.3.4 Contribution of enzymes to the deodorization process of garlic breath

To assess the effect of enzymes on the deodorization process, lettuce and apples were

heated to deactivate enzymes present. Both the heated apple and heated lettuce

produced a significant reduction in diallyl disulfide, allyl mercaptan and allyl methyl

disulfide (Fig 18; Fig 11; Table 17) just as good as their respective raw foods. Since

polyphenol oxidase has been proposed to significantly contribute to the deodorizing

process of garlic breath any deodorization caused by these heated treatments does not

involve enzymes. Therefore, enzymes may not be the main deodorizing agents

involved in all cases.

8000 a a 8000 Water a 7000 a a 7000 a a Green tea Water 6000 a a 6000 Raw apple 5000 Heated apple 5000 Raw lettuce a a 4000 Heated lettuce b a 4000 Mint leaves d c 3000 d b a 3000 a b c d b a d c Concentration Concentration (ppb) 2000 b b a 2000 d e b a e c a bb a cd 1000 b a a d c a b a Concentration (ppb) 1000 c a a d c c a a 0 0 c 0 20 40 60 0 10 20 30 40 50 60 Time (min) Time (min)

Figure 19: Effect of heated apple, heated lettuce and green tea on diallyl disulfide.

Data points at the same time bearing different letters are significantly different (p

≤0.05)

Further analyses on effect of PPO in the deodorization process of garlic odor, pure

PPO and a combination of PPO and catechin (PPO-CAT) were blended with garlic and

135 the headspace volatiles analyzed. PPO and PPO-CAT significantly reduced most volatiles in comparison to water (Fig 13). However, there was an increase of allyl mercaptan in the presence of PPO and PPO-CAT. Comparing the PPO-CAT results to those of catechin alone, addition of PPO resulted in no difference on the levels of dially disulfide, allyl methyl disulfide and allyl methyl sulfide with the control but there was an increase on the levels of allyl mercaptan. Garlic contains PPO (Kim and others 1981) therefore; addition of this enzyme may not have had an effect on allyl mercaptan. PPO also significantly decreased allyl methyl disulfide whereas a combination of PPO and catechin exhibited no decrease except for allyl methyl sulfide

(Fig 13). From this study presence of PPO-CAT showed a slight or no deodorization.

Garlic contains PPO that may be sufficient for the reaction, therefore adding more

PPO would not have any effect.

4.3.5 Other possible deodorization mechanism by non-phenolic compounds

From the results obtained in this study, the presence of PPO and phenolic content may not be the only compounds involved in garlic odor or garlic breath deodorization. For example, high deodorization was achieved by heated apple and heated lettuce (Fig 18).

Heated apple with no enzymatic activity causes a deodorization of diallyl disulfide.

This may be due to the presence of phenolic compounds that are also able to oxidize in the absence of polyphenol oxidase (Yasuda and Arakawa 1995) and other phenolic compounds present in apples. Furthermore, these results suggest that PPO may not be the major deodorizing agent involved in the deodorization process. On the other hand, heated lettuce containing low total phenolic content and no enzyme was able to

136 deodorize most of the garlic breath volatiles just as effectively as heated apple or raw apple which has relatively high concentration of phenolic compounds and enzymes in the raw apple (Fig 18). This suggests that there are non-phenolic compounds that may be involved in the deodorization process. Other researchers have reported reduction of diallyl disulfide by food due to what they called capturing activity (Negishi and others

2002). This is a physical and chemical interaction between the sulfur compounds and foods by an affinity to molecules (hydrophobicity) or by a trapping to porous polymers contained in foods (Negishi and others 2002). The capturing activity of lettuce against diallyl disulfide was 70% compared to that of apple, which was only 14% (Negishi and others 2002). Green tea had no effect on the levels of diallyl disulfide despite its high level of phenolic compounds (Fig 12, Table 17). Green tea lacks enzymes and due to its liquid form capturing activity (which involves trapping by porous polymer) is less likely. However, green tea powder has demonstrated the ability to deodorize the volatile sulfur compounds hydrogen sulfide and methyl mercaptan on breath in another study (Lodhia and others 2008).

The deodorization capacity of the juiced foods was significantly lowered compared to the non-juiced food (Fig 11). It is important to note that by juicing and filtering, other components involved in the deodorization process may also been filtered away with the waste hence reducing the deodorization capacity of the juices.

Additionally, a combination of peppermint and pure rosmarinic acid had a higher deodorization than when pure rosmarinic acid was used alone (Fig 15). Conversely,

137 increasing the amount of peppermint from 1g to 10g led to an increased deodorization

(Fig 17) but there was no difference between 1 and 5g or 5 and 10g of peppermint.

This may be explained by the fact that increasing the amount of peppermint had a significant deodorization up to a certatin point after which no more deodorization took place even by adding more peppermint.

Different food products have different composition of macronutrients that may significantly affect flavor release. For example, food with higher lipid content is expected to retain more of the hydrophobic volatile compounds resulting to a lower concentration of these volatiles in the headspace (Belitz and others 2009). Proteins have also been reported to bind with some volatile compounds that in turn affect their release. The protein binding mechanism of flavor in high moisture foods involve the interaction of nonpolar ligands with hydrophobic patches or cavities on the protein surface (Belitz and others 2009) or flavor compounds with polar head groups, such as hydroxyl groups may interact with proteins through hydrogen bonding and electrostatic interaction.

A possible interaction between the amino acids residues and garlic volatile compounds is the formation of disulfide bonds. Disulfide bonds in proteins can occur both inter- molecularly and intra-molecularly (Damodaran 2008). Therefore, formation of disulfide bonds between the amino acids and some of garlic volatile compounds is a possible means of deodorization for the garlic odor. When two sulfur residues, in this case cysteine residue and the thiol gases (allyl mercaptan and methyl mercaptan), are brought into proximity with proper orientation, oxidation of the sulfhydryl groups by

138 molecular oxygen results to the formation of disulfide bonds (Damodaran 2008).

Disulfide exchange is also a possible reaction, where by the cysteine residue that is available for reaction will react with diallyl disulfide via disulfide exchange-reaction

(Fig 20) (Borlinghaus and others 2014). The positioning of the amino acid residue is essential, for example in figure 20 the cysteine residue that is sterically blocked will not react with diallyl disulfide (Borlinghaus and others 2014).

Figure 20: Interaction between diallyl disulfide and cysteine residue

4.4 Conclusion

Different foods had different deodorization effect. Raw apple, raw lettuce and mint leaves containing different types of phenolic compounds produced a significant decrease of all the garlic breath volatiles in vivo except allyl methyl sulfide. Mint leaves had the highest deodorization of all the foods tested. Similarly, different types of phenolics compound produced different deodorization. Rosmarinic acid, catechin and quercetin significantly decreased all garlic breath volatiles in vitro where rosmarinic acid which is a major phenolic compound in mint leaves produced the

139 highest deodorization. Therefore, the type of phenolic compound may play an important role in the deosorization process.

The amount of phenolic compounds may not be a major factor affecting deodorization.

Apple juice and mint juice also had a deodorizing effect on most of the garlic volatiles but were generally not as effective as the raw food, probably due to polymerization of most phenolic compounds. All three mint varieties produced a significant decrease for some of the volatiles where peppermint and spearmint tended to produce a higher deodorization than chocolate mint. Chocolate mint with higher rosmarinic acid content had lower deodorization of most volatile compounds. Pure rosmarinic acid at 2 and

40mg also decreased some volatile compounds but there was no significant difference in deodorizing level between 2 and 40mg of rosmarinic acid.

Enzyme activity may not majorly affect deodorization process where both heated apple and heated lettuce produced a significant reduction of diallyl disulfide and allyl mercaptan. Also, PPO and PPO -Catechin were slightly effective on all volatile compounds and the deodorization by pure PPO and PPO-Catechin was not as good as phenolic compounds.

Other non-phenolic compounds such as proteins may be significantly affecting the deodorization process whereby addition of 1g of peppermint to 2mg of rosmarinic acid produced a greater deodorizing effect than just 2mg of pure rosmarinic acid. The non- phenolic compound may also explain why deodorization was achieved in the heated food product.

140

References

Abou El Hassan MAI, Touw DJ, Wilhelm AJ, Bast A, van der Vijgh WJF 2000. Stability

of monoHER in an aqueous formulation for i.v. administration. Int J Pharmaceutics.

211(1-2):51-56a.

Amagase H, Petesch BL, Matsuura H, Kasuga S, Itakura Y (2001). Intake of garlic and its

bioactive components. J Nutri, 131(3s): 955S–962S.

Apak R, Güçlü K, Demirata B, Özyürek M, Çelik SE, Bektaşoğlu B, Berker K, Özyurt D.

2007. Comparative Evaluation of Various Total Antioxidant Capacity Assays Applied

to Phenolic Compounds with the CUPRAC Assay. J Mol 12:1496-1547

Barrett DM, Weakley C, Diaz JV, Watnik M. 2007. Qualitative and Nutritional Differences

in Processing Tomatoes Grown under Commercial Organic and Conventional

Production Systems. J Food Sci. 72(9):441-451

Belitz HD, Grosch W, Schieberle P. 2009. Food Chemistry. Springer. Heidelberg, Berlin.

Berker KI, Ozdemir Olgun FA, OzyurtD, Demirata B, and Apak R 2013. Modified

Folin−Ciocalteu Antioxidant Capacity Assay for Measuring Lipophilic Antioxidants.

J Agric Food Chem. 61(4783-4791)

Block E. 1985. The chemistry of garlic and onions. Sci. Am. 252(3):114-119.

141

Borlinghaus J, Albrecht F, Gruhlke MCH, Nwachukwu ID, Slusarenko AJ. 2014. Allicin:

Chemistry and Biological properties. Molecule 19(8):12591- 12618

Cao H, Cheng W, Li C, Pan X, Xie X, Li T 2005. DFT study on the antioxidant activity of

rosmarinic acid. J. Mol Sci 719:177 183.

Challier B, Perarnau JM, Viel JF. Garlic, onion and cereal fiber as protective factors for

breast cancer: A French case-control study. European Journal of Epidemiology 1998;

14(8):737–747.

Damodaran S. 2008. Food Chemistry: Amino acids, peptides and proteins. Taylor and

Francis group. New York.

Fleischauer AT, Arab L. 2001. Garlic and cancer: A critical review of the epidemiologic

literature. J Nutrition; 131(3s):1032S–1040S.

Friedman M, Jürgens HS. 2000. Effect of pH on the Stability of Plant Phenolic

Compounds. J Agric Food Chem 48(6): 2101-2110.

Hansanugrum A, Barringer SA. 2010. Effect of milk on the deodorization of malodorous

breath after garlic ingestion. J Food Sci 75(6):549–58.

Heimdal H, Larsen LM, Poll L. 1994. Characterization of polyphenol oxidase from photosynthetic and vascular lettuce tissues (Lactucu saliva). J. Agric. Food. Chem. 42,

1428-1433

Holderbaum DF, Kon T, Kudo T and Guerra MP 2010. Enzymatic browning polyphenol

oxidase activity, and polyphenols in four apple cultivars: Dynamics during fruit

development. HortScience 45(8): 1150-1154.

142

Hsing AW, Chokkalingam AP, Gao YT, et al. Allium vegetables and risk of prostate

cancer: A population-based study. Journal of the National Cancer Institute 2002;

94(21):1648–1651.

Henry JG, Gehr R. 1980. Odor control: an operator’s guide. J Water Pollut Control Fed

52(10):2523–37. Kim SR, Jung YR, An HJ, Kim DH, Jang EJ, Choi YJ. 2013. Anti-

Wrinkle and Anti-Inflammatory Effects of Active Garlic Components and the

Inhibition of MMPs via NF-κB Signaling. PLoS ONE 8(9): e73877.

doi:10.1371/journal.pone.0073877.

Iciek M, Kwiecien I, Wlodek L. 2009. Biological Properties of Garlic and Garlic-Derived

Organosulfur Compounds. Eviron. Mol. Mutagen. 50: 247-265.

Kim SR, Jung YR, An HJ, Kim DH, Jang EJ, Choi YJ. 2013. Anti-Wrinkle and Anti-

Inflammatory Effects of Active Garlic Components and the Inhibition of MMPs via

NF-κB Signaling. PLoS ONE 8(9): e73877. doi:10.1371/journal.pone.0073877

Kim DY, Rhee CO, Kim YB.1981. Characteristics of polyphenol oxidase from garlic

(Allium sativum L.). Food and Agriculture Organization of the United Nations 24(3):

167-173.

Laakso I, Seppänen-Laakso T, Hiltunen R, Müller B, Jansen H, Knobloch K. 1989.

Volatile Garlic Odor Components: Gas Phases and Adsorbed Exhaled Air Analysed

by Headspace Gas Chromatography-Mass Spectrometry. Planta Med. 55(3):257-261.

Lawson LD. 1998. Garlic: a review of its medicinal effects and indicated active

compounds. In: Lawson LD, Bauer R, editors. Phytomedicines of Europe: chemistry

and biological activity. Washington DC: American Chemical Society. p 176–209.

143

Lee K, Kim Y, Kim D, Lee H, Lee C 2003. Major phenolics in apple and their contribution

to the total antioxidant capacity. J Agric Food Chem, 51:6516–6520.

Lodhia P, Yaegaki K, Khakbaznejad A, Imai T, Sato T, Tanaka T, Murata T, Kamoda T.

2008. Effect of green tea on volatiles sulfur compounds in mouth air. J. Nutrition Sci

Vitaminol.

Lu S, Luo Y, Feng H. 2006. Inhibition of apple polyphenol oxidase activity by sodium

chlorite.

Minami T, Boku T, Inada K, Morita M, Okazaki Y. 1989. Odor components of human

breath after the ingestion of grated raw garlic. Journal of Food Science 54(3): 763-

765.

Mirondo R, Barringer S 2016. Deodorization of garlic breath by the use of food materials,

polyphenol oxidase and phenolic compounds. J Food Chem.

Munch R, Barringer S.A. 2014.Deodorization of garlic breath volatiles by food ad food

components. J. Food Sci. 79(4):526-533

Nagata Y, Takeuchi N. 1990. Determination of odor threshold value by triangle odor bag

method. Bull Japan Environ Sanit Center 17:77–89.

Nagao T, Hase T, Tokimitsu I, 2007. A green tea extract high in catechins reduces body fat

and cardiovascular risks in humans. Obesity 15(6):1473–1483

Negishi O. and Negishi Y. 1999. Enzymatic deodorization with raw fruits, vegetables and

mushrooms. Food Sci. Technol. Res 5(2):176-80.

Negishi O, Negishi Y, Ozwa T 2002. Effects of food materials on removal of Allium-

specific sulfur compounds.J. Agric. Food Chem.50: 3856-3861 144

Negishi O, Negishi Y, Yamaguchi F, Sugahara T. 2004. Deodorization with ku-ding-cha

containing a large amount of caffeoyl quicin and acid dervatives. J. Agric. Food

Chem 52:5513-8

Negishi O. Ozawa T. 1997. Effect of polyphenol oxidase on deodorization. Biosci.,

Biotechnol., Biochem. 61, 2080- 2084.

Neurath H, Bailey K. 1954. The Proteins: chemistry., Biological activity and methods.

Academic Press, New York. pp534

Niki E, Noguchi, N. 2000. Evaluation of Antioxidant Capacity. What Capacity is Being

Measured by Which Method? IUBMB Life 50: 323-329.

Peñarrieta, J. M. Alvarado JA, Åkesson B, Bergenståhl B. 2008. Total antioxidant capacity

and content of flavonoids and other phenolic compounds in canihua (Chenopodium

pallidicaule): An Andean pseudocereal. Mol. Nutr. Food Res. 52(6): 1613-4133.

Preedy VR, 2016. Essential Oils in Food Preservation, Flavor and Safety. Elsevier,

London, UK. P. 558

Rocha AMCN and Morais AMMB. 2001. Poly- phenoloxidase activity and total phenolic

content as related to browning of minimally processed ‘Jona- gored’ apple. J. Sci

Food Agric, 82 (1): 120-126.

Sarkaki A, Valipour Chehardacheric S, Farbood Y, Mansouri SMT, Naghizadeh , Basirian

E. (2013). Effects of fresh aged and cooked garlic extracts on short- and long-term

memory in diabetic rats. Avicenna Journal of Phytomedicine, 3(1): 45–55.

145

Spanel P, Smith D. 1998. Selected ion flow tube studies of the reactions of H3O+, NO+,

and O2+ with some organosulfur molecules. International Journal of Mass

Spectrometry 176: 167-176.

Suarez F, Springfield J, Furne J, Levitt M. 1999. Differentiation of mouth versus gut as site

of origin of odoriferous breath gases after garlic ingestion. Am J Physiol 276:G425–

30.

Suarez F, Springfield J, Furne J, Levitt M. 1999. Differentiation of mouth versus gut as site

of origin of odoriferous breath gases after garlic ingestion. Am. J. Physiol. 276:

G425-G430.

Tahira R, Naeemullah M, Akbar F, Masood M 2011. Major phenolic cids of local and

exotic mint germplasm grown in Islamabad. Pak. J. Bot 43:151-154.

Tamaki T, Sonoki S. 1999. Volatile sulfur compounds in human expiration after eating raw

or heat-treated garlic. J Nutr Sci Vitaminol 45: 213-222.

Tamaki K, Sonoki S, Tamaki T, Ehara K. 2008. Measuremet of odour after in vitro or in

vivo ingestion of raw or heated garlic, using electronic nose, gas chromatography

and sensory analysis. Intl J Food Sci Technol 43:130

Thomson M, Al-Qattan KK, Bordia T, Ali M 2006. Including garlic in the diet may help

lower blood glucose, cholesterol and triglycerides. J. Nutrition; 136 (3): 800S-802S

Vladimir-Knežević S, Blažeković B, Štefan MB, Alegro A, Kőszegi T, Petrik J. 2011.

Antioxidant Activities and Polyphenolic Contents of Three Selected Micromeria

Species from Croatia. J Mol 16: 1454-1470

146

Yasuda H, Arakawa T. 1995. Deodorizing mechanism of (–) epigallocatechin gallate

against methyl mercaptan. Biosci Biotech Biochem 59:1232–6.

Zhu QY, Holt RR, Lazarus SA. (2002). Stability of the Flavan-3-ols epicatechin and

catechin and related dimeric procyanidins derived from cocoa. J Agric Food Chem

50:1700-1705.

147

General conclusion and recommendation

This study covered the formation and destruction of volatile compounds in food that are either enhanced or inhibited by compounds present in food. Peels had a significant contribution to the levels of most important volatile compounds. Mango puree made with peel had lower levels of the lipoxygenase derived volatiles compared to mango puree made without peel. This was probably due to the inhibition of the lipoxygenase enzyme by a phenolic compound known as quercetin. On the other hand, tomato juice made with peel had higher levels of lipoxygenase derived volatiles probably due to higher levels of free fatty acids that are used as precursors for the formation of these volatile compounds. Thus, components in food can be used to inhibit or enhance the formation of some important volatiles. Understanding the inhibition, enhancing or destruction mechanisms of important volatile compounds may allow processors to include part of a fruit that are highly nutritious in processing and in turn inhibit, enhance or destroy the desirable or undesirable volatile compounds that are a result of the parts of the fruit added.

Different food components have achieved deodorization of undesirable garlic breath. A better understanding of the deodorization mechanism of undesirable garlic breath may be applied in the deodorization of undesirable volatile compounds in other foods and food products. This study analyzed the contribution of phenolic compounds and enzymes in the deodorization process as proposed by previous researchers. Phenolic compounds may contribute to the deodorization process but they may not be the only deodorizing agents

148 involved, whereas enzymes produced trivial or no contibuion to the deodorization process.

Other non-phenolic compounds may take part in the deodorization process. Therefore, more study needs to be done on the contribution of non-phenolics in the deodorization of undesirable volatile compounds.

149

Bibliography

Abou El Hassan MAI, Touw DJ, Wilhelm AJ, Bast A, van der Vijgh WJF 2000. Stability

of monoHER in an aqueous formulation for i.v. administration. Int J Pharmaceutics.

211(1-2):51-56a.

Aguilar-Rosas SF, Ballinas-Casarrubias ML, Nevarez-Moorillon GV, Martin-Belloso O,

Ortega-Rivas E. 2007. Thermal and pulsed electric fields pasteurization of apple

juice: Effects on physicochemical properties and flavour compounds. J Food Eng.

83(1):41-46

Ahmed J, Shivhare US, Kaur M. 2002. Thermal colour degradation kinetics of mango

puree. Int J. Food Properties 5(2): 359–366.

Ajila CM, Bhat SG, Prasada Rao UJS. 2007. Valuable components of raw and ripe peels

from two Indian mango varieties. J Food Chem. 102(4): 1006-1011.

Ajila CM, Rao UJS. 2013. Mango peel dietary fiber: Composition and associated bound

phenolics. J Functional Food. 5(1):444-450

Akhtar S, Riaz, M, Ahmad A, Nisar A. 2010. Physico-chemical, microbiological and

sensory stability of chemically preserved mango pulp. Pakistan Journal. Bot. 42(2):

853-862.

Amagase H, Petesch BL, Matsuura H, Kasuga S, Itakura Y. 2001. Intake of garlic and its

bioactive components. Journal of Nutrition; 131(3s):955S–962S.

Anthon GE, Barrett D M. 2008. Combined enzymatic and colorimetric method for

determining the methanol and uronic acid content of pectins application to tomato 150 products. J Food Chem110: 239–247. AOAC 1995. Official Methods of Analysis, 16th

edition, Association of Official Analytical Chemists, Washington D.C, 138pp. Apak

R, Güçlü K, Demirata B, Özyürek M, Çelik SE, Bektaşoğlu B, Berker K, Özyurt D.

2007. Comparative Evaluation of Various Total Antioxidant Capacity Assays Applied

to Phenolic Compounds with the CUPRAC Assay. J Mol 12:1496-1547

Arctander S.1969. Perfume and flavor chemicals; Arctander: Montclair, NJ, 1969

Barrett DM, Weakley C, Diaz JV, Watnik M. 2007. Qualitative and Nutritional Differences

in Processing Tomatoes Grown under Commercial Organic and Conventional

Production Systems. J Food Sci. 72(9):441-451

Berker KI, Ozdemir Olgun FA, OzyurtD, Demirata B, and Apak R 2013. Modified

Folin−Ciocalteu Antioxidant Capacity Assay for Measuring Lipophilic Antioxidants.

J Agric Food Chem. 61(4783-4791). Belitz HD, Grosch W, Schieberle P. 2009. Food

Chemistry. Spinger. Heidelberg, Berlin.

Bhardwaj, R.L. and Mukherjee, S. 2011. Effects of fruit juice blending ratios on kinnow

juice preservation at ambient storage condition. African Journal of Food Science 5(5):

281 - 286.

Block E. 1985. The chemistry of garlic and onions. Sci. Am. 252(3):114-119.

Borlinghaus J, Albrecht F, Gruhlke MCH, Nwachukwu ID, Slusarenko AJ. 2014. Allicin :

Chemistry and Biological properties. Molecule 19(8):12591- 12618

Bouwmeester HJ, Gershenzon J, Konings MC, Croteau R. 1998. Biosynthesis of the

monoterpenes limonene and carvone in the fruit of caraway. I. Demonstration Of

enzyme activities and their changes with development. J Plant Physiol 117(3): 901-

912.

151

Buettner A, Montse M, Fischer A, Guasch J, Schieberle P. 2003. Evaluation of most odor-

active compounds in the peel of clements (citrus recticulate blanco cv. Celmentine)

Eur. Food Res. Technol, 216 (1),11-14.

Buttery RG, Ling LC. 1993. Enzymatic production of volatiles in tomatoes. In: Schreir P,

Winterhalter P, editors. Flavor precursors. Wheaton IL: Allured Publications. p 137-

146

Byarugaba-Biaruzake, G. W. 2008. Effect of Enzymatic Processing on Banana Juice and

Wine. Dissertation for award of PhD at Stellenbosch University, South Africa, 110pp.

Cabrera SG, Jang JH, Kim St, Lee YR, Lee HJ, Chung HS, Moon KD. 2009. Effects of

processing time and temperature on thequality components of campbell grape juice. J

Food Processing and Preservation 33(1):347-360

Cao YH, Cao RH. 1999. Angiogenesis inhobited by drinking tea. Nature, 6726, 381-398.

Cao H, Cheng W, Li C, Pan X, Xie X, Li T 2005. DFT study on the antioxidant activity of

rosmarinic acid. J. Mol Sci 719:177 183.

Challier B, Perarnau JM, Viel JF. 1998. Garlic, onion and cereal fibre as protective factors

for breast cancer: A French case-control study. European J. Epidemiology;

14(8):737–747.

Charles-Rodrı´guez AV, Neva´rez-Moorillo GV, Zhang QH, Ortega-Rivas E. 2007.

Comparison of thermal processing and pulsed electric fields treatment in

pasteurization of apple juice. J Chem Eng 85(2): 93-97

Correa MIC, Chaves JBP, Jham GN, Ramos AM, Minim VPR, Yokota SRC. 2010.

Changes in guava (Psidiumguajava L. var. Paluma) nectar volatile compounds

152

concentration due to thermal processing and storage. Ciência e Tecnologia de

Alimentos 30(4): 1061-1068.

Corey KA, Schlimme DV, Frey BC. 1986. Peel removal by high pressure steam from

processing tomatoes with yellow shoulder disorder. J. Food Sci. 51: 388–390, 394.

Croft KPC, Juttner F, Slusarenko AJ. 2003. Volatile products of the lipoxygenase pathway

evolved from Phaseolus vulgaris (L.) leaves inoculated with Pseudomonas syringae

pv phaseolicola. Plant Physiol. 1993; 101:13–24.

Dak M, Verma RC, Sharma GP., 2006. Flow characteristics of juice of ‘‘Totapuri’’

mangoes. J. Food Eng 76 (1) 557–561

Dak M, Verma RC, Jaafrey SNA. 2007. Effect of temperature and concentration on

Rheological properties of ‘‘Kesar’’ mango juice. J. Food Eng 80 (2007) 1011–1015

Damodaran S. 2008. Food Chemistry: Amino acids, peptides and proteins. Taylor and

Francis group. New York.

Darrigues A, Hall J, Knaap EV, Francis MD. 2008. Tomato Analyzer-color Test: A New

Tool for Efficient Digital Phenotyping. J. Amer. Soc. Hort. Sci. 133(4):579–586.

Del Valle M, Cámara M, Torija ME. 2007. The nutritional and functional potential of

tomato by-products. Acta Hort. (ISHS) 758:165-172.

De Pascual TS, Johnston KC, Dupont MS. 2004. Quecertin metabolic downregulates

cyclooxygenase 2 transcrition in human lymphocytes ex vivo but not invivo. J.

Nutri.3:552-557

El Hadi MAM, Zhang F, Wu F, Zhou C, Tao J. 2013. Advances in fruit aroma volatile

research. J. Molecules 18: 8200-8229

153

E1-Nemr SE, Ismail IA. 1988. Aroma changes in mango juice during processing and

storage. J Food Chem 30: 269-275

Engel, K.-H.; Flath, R. A.; Buttery, R. G.; Mon, T. R.; Ramming, D. W.; Teranishi,

R.1988. Investigation of volatile constituents of aroma components in some nectarine

cultivars. J. Agric. Food Chem. 36, 549-553.

Fito PJ, Clemente G, Sanz, FJ. 1983. Rheological behavior of tomato concentrates (Hot

break and Cold break). J Food Eng. 2: 51-62.

Fisher, C.; Scott, T. T.1997. Food Flavours: Biology and Chemistry; The Royal Society of

Chemistry: Hunt Valley, MD,

Fleischauer AT, Arab L. 2001. Garlic and cancer: A critical review of the epidemiologic

literature. J Nutri; 131(3s):1032S–1040S.

Friedman M, Jürgens HS. 2000. Effect of pH on the Stability of Plant Phenolic

Compounds. J Agric Food Chem 48(6): 2101-2110.

Forget, F. L.; Ggauillard, F.; Rigal, D. Changes in the carotenoid content of apricot

(Prunus armeniaca, var. Bergeron) during enzymatic browning: β-carotene inhibition of chlorogenic acid degradation. J. Sci. Food Agric. 2000, 80,763-768.

Galliard T, Matthew JA, Wright AJ, Fishwick MJ. 1977. The enzymic breakdown of lipids

to volatile and non-volatile carbonyl fragments in disrupted tomato fruits. J Sci Food

Agric 28:863-868.

Garcia E, Barrett D M. 2006. Peel ability and yield of processing tomatoes by steam or lye.

J Food Process Pres 30 (10): 3–14

Goodman CL, Fawcett S, Barringer SA. 2002. Flavor, viscosity, and color analyses of hot

and cold break tomato juices. J Food Sci 67(1): 404-408.

154

Iagher F, Reicher F, Ganter JL. 2002. Structural and rheological properties of

polysaccharides from mango (Mangifera indica L.) pulp". Int. J. Biol Macromol.

31(1-3): 9-17.

Hal PH, Bosschaart C, Twisk C, Verkerk R, Dekker M. 2012. Kinetics of thermal

degradation of vitamin C in marula fruit (Sclerocarya birrea subsp. caffra) as

compared to other selected tropical fruits. J Food Sci Technol 49(1): 188-191

Hansanugrum A, Barringer SA. 2010. Effect of milk on the deodorization of malodorous

breath after garlic ingestion. J Food Sci 75(6):549–58.

Harding GJ. 2001. Peeler for fruits and vegetables, U. S. patent No. 6324969. U. S. patent

and trade mark office, Washington, D.C.

Hatanaka A, Kajiwara T, Sekiya J. 1987. Biosynthesis pathway for C6 aldehyde formation

from linolenic acid in green leaves. Chem. Phys. Lipids, 44:341-361.

Hayes WA, Smith PG, Morris AEJ. 1998: The Production and Quality of Tomato

Concentrates, Critical Reviews in Food Science and Nutrition, 38(7): 537-564.

Heimdal H, Larsen LM, Poll L. 1994. Characterization of polyphenol oxidase from

photosynthetic and vascular lettuce tissues (Lactucu saliva). J. Agric. Food. Chem.

42, 1428-1433

Henry JG, Gehr R. 1980. Odor control: an operator’s guide. J Water Pollut Control Fed

52(10):2523–37. Kim SR, Jung YR, An HJ, Kim DH, Jang EJ, Choi YJ. 2013. Anti-

Wrinkle and Anti-Inflammatory Effects of Active Garlic Components and the

Inhibition of MMPs via NF-κB Signaling. PLoS ONE 8(9): e73877.

doi:10.1371/journal.pone.0073877.

155

Holderbaum DF, Kon T, Kudo T and Guerra MP 2010. Enzymatic browning polyphenol

oxidase activity, and polyphenols in four apple cultivars: Dynamics during fruit

development. HortScience 45(8): 1150-1154.

Hsing AW, Chokkalingam AP, Gao YT. 2002. Allium vegetables and risk of prostate

cancer: A population-based study. J of the National Cancer Institute; 94(21): 1648–

1651.

Hsu KC. 2008. Evaluation of processing qualities of tomato juice induced by thermal and

pressure processing. LWT, 41; 450-459.

Hui YH, Evranuz EO. 2012. Hand Book of plant base fermented food and beverage

technology.2nd ed. New York: Taylor and Francis. 802p.

Iciek M, Kwiecien I, Wlodek L. 2009. Biological Properties of Garlic and Garlic-Derived

Organosulfur Compounds. Eviron. Mol. Mutagen. 50: 247-265.

Ikegwu OJ, Ekwu FC. 2009. Thermal and physical properties of some tropical fruits and

their juice in Nigeria. J Food Techn 7(2): 38-42

Kaur D, Sharma R, Wani A, Gill BS, Sogi DS. 2006. Physicochemical changes in seven

tomato (Lycopersicon esculentum) of different cultivars during rippening in short

term storage. Int J Food Protect 9:747-757.

Kim HJY, Moon D, Kim M, Lee H. Cho YS, Choi AK, Cho SK. 2010. Antioxidant and

antiproliferative activities of mango( (mangifera indica L.) flesh and peel. J Food

Chem, 121:429-436

Knoblich M, Anderson B, Latshaw D. 2005. Analyses of tomato peel and seed byproducts

and their use as a source of carotenoids. J Sci Food Agric 85(10):1166–1170.

156

Kim SR, Jung YR, An HJ, Kim DH, Jang EJ, Choi YJ. 2013. Anti-Wrinkle and Anti-

Inflammatory Effects of Active Garlic Components and the Inhibition of MMPs via

NF-κB Signaling. PLoS ONE 8(9): e73877. doi:10.1371/journal.pone.0073877

Kim DY, Rhee CO, Kim YB.1981. Characteristics of polyphenol oxidase from garlic

(Allium sativum L.). Food and Agriculture Organization of the United Nations 24(3):

167-173.

Kratchanova M, Benemou C, Kratchanov C. 1991. On the pectic substances of mango

fruits. J Carbohydrate Polymers 15(3):271-282

Laakso I, Seppänen-Laakso T, Hiltunen R, Müller B, Jansen H, Knobloch K. 1989.

Volatile Garlic Odor Components: Gas Phases and Adsorbed Exhaled Air Analysed

by Headspace Gas Chromatography-Mass Spectrometry. Planta Med. 55(3):257-261.

Lakshminarayana S,Subhadra NV, Subramanyan H. 1970.Some aspects of developmental

physiology of mango fruit. J Hort Sci 45:133-42

Lawless HT and Heymann N. 2010. Sensory evaluation of food. 2nd ed. New York:

Springer. 88p

Lawson LD. 1998. Garlic: a review of its medicinal effects and indicated active

compounds. In: Lawson LD, Bauer R, editors. Phytomedicines of Europe: chemistry

and biological activity. Washington DC: American Chemical Society. p 176–209.

Lee K, Kim Y, Kim D, Lee H, Lee C 2003. Major phenolics in apple and their contribution

to the total antioxidant capacity. J Agric Food Chem, 51:6516–6520.

Lewinsohn E, Sitrit S, Bar E, Azulay Y, Ibdah M, Meir A, Yosef E, Zamir D and Tadmor

Y. 2005. ‘Not just colors-carotenoid degradation as a link between pigmentation and

aroma in tomato and watermelon fruit. Trends Food Sci. Technol 16:407-415.

157

Lindsay RC. 2007. Flavors. Fennema OR, Damodaran S, Parkin KL. 2007. Food

Chemistry. New York: Taylor and Francis Group. 597p.

Lodhia P, Yaegaki K, Khakbaznejad A, Imai T, Sato T, Tanaka T, Murata T, Kamoda T.

2008. Effect of green tea on volatiles sulfur compounds in mouth air. J. Nutrition Sci

Vitaminol.

Lopes, D. C.; Fraga, S. R.; Rezende, C. M. 1999. Aroma impact substances on commercial

Brazilian mangoes by HRGC-OAEDA- MS. Quim 22, 31-36.

Lu S, Luo Y, Feng H. 2006. Inhibition of apple polyphenol oxidase activity by sodium

chlorite.

Macleod AJ, Troconis NG. 1982. Volatile flavour components of mango fruit. J

Phytochemistry, 21(10): 2523-2526

Mahattanatawee K, Rouseff R, Valim MF, Naim M. 2005. Identification and aroma impact

of norisoprenoids in orange juice. J Agric Food Chem 53(2):393–7.

Manohar B, Ramakrishna P, Ramteke RS. 1990. Effect of pectin content on flow properties

of mango pulp concentrates J of Texture Studies 21: 179-190

Markovic K, Panjkota KI, Krpan M, Bicanic D, Vahcic N. 2010. The lycopene content in

pulp and peel of five fresh tomato cultivars. Acta Alimentaria 39(1):90-98.

Marsili R. 2007 (ed). Sensory directed flavor analysis. Florida: Taylor and Francis group

256p.

Masibo M, He Q. 2008. Major Mango Polyphenols and Their Potential Significance to

Human Health. J Food Sci and Food Safety 7(4):309-319.

Medlicott PA, Thompson KA. 2006. Analysis of sugar and organic acids in ripening mango

fruit (Mangifera indica L.var Keitt). J Sci Food Agric. 36(7):561-566

158

Min S, Jin Z T, Zhang QH. 2003. Commercial scale pulsed electric field processing of

tomato juice. J Agric Food Chem 51 (11): 3338–3344.

Minami T, Boku T, Inada K, Morita M, Okazaki Y. 1989. Odor components of human

breath after the ingestion of grated raw garlic. J Food Sci 54 (3): 763-765.

Mirondo R, Barringer S 2016. Deodorization of garlic breath by the use of food materials,

polyphenol oxidase and phenolic compounds. J Food Chem.

Molwane, S. D. and Gunjal, B. B. 1985. Viscometric Characteristics of Cold-break and

Hot-break Extracted Tomato Juice Concentrates. J Food Sci. Tech., 22: 353-357.

Munch R, Barringer S.A. 2014.Deodorization of garlic breath volatiles by food ad food

components. J Food Sci. 79(4):526-533

Moneruzzaman, K.M., Al-Saif, A.M., Alebidi, A.I., Hossain, A.B., Normaniza, O. and

Nasrulhaq, B.A. 2011. An evaluation of the nutritional quality evaluation of three

cultivars of Syzygium samarangense under Malaysian conditions. African Journal of

Agricultural Research 6(3): 545-552.

Moore J P. 2003. Carotenoid synthesis and retention in mango (mangifera indica) fruit and

puree as influenced by postharvest and processing treatments. Thesis presentation,

University of Florida

Nagata Y, Takeuchi N. 1990. Determination of odor threshold value by triangle odor bag

method. Bull Japan Environ Sanit Center 17:77–89.

Nagao T, Hase T, Tokimitsu I, 2007. A green tea extract high in catechins reduces body fat

and cardiovascular risks in humans. Obesity 15(6):1473–1483

Negishi O. and Negishi Y. 1999. Enzymatic deodorization with raw fruits, vegetables and

mushrooms. Food Sci. Technol. Res 5(2):176-80.

159

Negishi O, Negishi Y, Ozwa T 2002. Effects of food materials on removal of Allium-

specific sulfur compounds. J. Agric. Food Chem.50:3856-3861

Negishi O, Negishi Y, Yamaguchi F, Sugahara T. 2004. Deodorization with ku-ding-cha

containing a large amount of caffeoyl quicin and acid dervatives. J. Agric. Food

Chem 52:5513-8

Neurath H, Bailey K. 1954. The Proteins: chemistry., Biological activity and methods.

Academic Press, New York. pp534

Niki E, Noguchi, N. 2000. Evaluation of Antioxidant Capacity. What Capacity is Being

Measured by Which Method? IUBMB Life 50: 323-329.

Pandit SS, Chidley HG, Kulkarni RS, Pujari KH, Giri AP, Gupta VS. 2009. Cultivar

relationships in mango based on fruit volatile profile. J Food Chem 114:363–372.

Peñarrieta, J. M. Alvarado JA, Åkesson B, Bergenståhl B. 2008. Total antioxidant capacity

and content of flavonoids and other phenolic compounds in canihua (Chenopodium

pallidicaule): An Andean pseudocereal. Mol. Nutr. Food Res. 52(6): 1613-4133.

Petro-Turza M. 1987. Flavor of tomato and tomato products. Food Rev Int 2(3):309–51.

Pino JA, Mesa J. 2006. Contribution of volatile compounds to mango (Mangifera indica L.)

aroma. J. Flav. Frag., 21(2): 207–213.

Pino JA, Mesa J, Munoz Y, Martia MP, Marbot R. 2005. Volatile components from mango

(Mangiferaindica L.) cultivars. J Agric Food Chem. 53(6): 2213-2223.

Preedy VR, 2016. Essential Oils in Food Preservation, Flavor and Safety. Elsevier,

London, UK. P. 558

160

Qiao Y, Xie BJ, Zhang Y, Zhang Y, Fan G, Yao XL, Pan SY. 2008. Characterization of

aroma active compounds in fruit juice and peel oil of jinchen sweet orange fruit

(Citrus sinensis(L) Osbeck) by GC-MC and GC-O. Molecules. 13:1334-1334

Reboul E , Borel P , Mikail C , Abou L , Charbonnier M , Caris-Veyrat C , Goupy P ,

Portugal H , Lairon D , Amiot M. 1996 Enrichment of Tomato Paste with 6% Tomato

Peel Increases Lycopene and β-Carotene Bioavailability in Men. J Nutr. vol. 135(4):

790-794

Rocha AMCN and Morais AMMB. 2001. “Poly- phenoloxidase activity and total phenolic

content as related to browning of minimally processed ‘Jona- gored’ Apple.” J Sci

Food Agric, 82 (1): 120-126.

Saeed A, Shahid M, Haider S J, Ijaz A, Shaukat U. 2012. Quality evaluation of different

brands of Tetra Pak mango juices available in market. PAK. J. Food SCI., 22(2),

2012:96-100.

Sahin S, Sumnu GS. 2006. Physical properties of foods. New York: Springer. 257p

Sandoval IS, Hernández AS, Valdez GV, Cruz-López L.2007. Volatiles of Mango var.

Ataulfo Characterized by SPME and Capillary GC/MS Spectroscopy. J. Mex. Chem.

Soc. 51(3): 145-147

Sarkaki A, Valipour Chehardacheric S, Farbood Y, Mansouri SMT, Naghizadeh , Basirian

E. (2013). Effects of fresh aged and cooked garlic extracts on short- and long-term

memory in diabetic rats. Avicenna Journal of Phytomedicine, 3(1): 45–55.

Sawamura M, Tu NTM, Onishi Y, Ogawa E, Choi HS. 2004. Characteristic odor

components of citrus reticulate blanco (ponkan) cold-pressed oil. Biosci Biotechnol

Biochem. 68(8):1690-1697.

161

Schieber A, Ullrich W, Carle R. 2000. Characterization of polyphenols in mango puree

concentrate by HPLC with diode array and mass spectrometric detection. Innovative

Food Science and Emerging Technologies 1:161-166.

Schlimme DV, Corey KA, Frey BC. 1984. Evaluation of lye and steam peeling using four

processing tomato cultivars. J. Food Sci. 49: 1415–1418.

Schwartz SJ, Von Elbe JH, Giusti MM. 2007. Colorants. Fennema OR, Damodaran S,

Parkin KL. 2007. Food Chemistry. New York: Taylor and Francis Group. 597p.

Sieso V., Crouzet J. 1977. Tomato volatile components: effect of processing. J Food Chem

2: 241-252

Singh' NI, Eipeson WE. 2000. Rheological behaviour of clarified mango juice

concentrates. J Texture Studies, 31: 287-295.

Sharma SK, Le Maguer M. 1996. Lycopene in tomatoes and tomato pulp fractions. Italian J Food Sci 8(2): 107- 113

Spanel P, Smith D. 1998. Selected ion flow tube studies of the reactions of H3O+, NO+,

and O2+ with some organosulfur molecules. International Journal of Mass

Spectrometry 176: 167-176.

Suarez F, Springfield J, Furne J, Levitt M. 1999. Differentiation of mouth versus gut as site

of origin of odoriferous breath gases after garlic ingestion. Am J Physiol 276:G425–

30.

Tahira R, Naeemullah M, Akbar F, Masood MS. 2011. Major phenolic acids of local and

exotic mint germplasm grown in Islamabad. Pak. J. Bot., 43: 151-154.

Tamaki T, Sonoki S. 1999. Volatile sulfur compounds in human expiration after eating raw

or heat-treated garlic. J Nutr Sci Vitaminol 45: 213-222.

162

Tamaki K, Sonoki S, Tamaki T, Ehara K. 2008. Measuremet of odour after in vitro or in

vivo ingestion of raw or heated garlic, using electronic nose, gas chromatography and

sensory analysis. Intl J Food Sci Technol 43:130–9.

Tandon KS, Baldwin EA, Scott JW, Shewfelt RL. 2003. Linking sensory description to

volatile and non volatile componets of fresh tomato flavor. J Food Sci 68(7):2366-71.

Tasnim, F, Anwar HM, Nusrath S , Kamal HM, Lopa D, Formuzul HKM. 2010. Quality

assessment of industrially processed fruit juices available in Dhaka City, Bangladesh.

J Mal Nutr 16 (3): 431-438.

Taucher J, Hansel A, Jordan A, Lindinger W. 1996. Analysis of Compounds in Human

Breath after Ingestion of Garlic Using Proton-Transfer-Reaction Mass Spectrometry.

J. Agric. Food Chem. 44: 3778-3782.

Ties P, Barringer S. 2012. Influence of lipid content and lipoxygenase on flavor volatiles in

the tomato peel and flesh. J Food Sci 77(7): 830-837pp.

Thomson M, Al-Qattan KK, Bordia T, Ali M 2006. Including garlic in the diet may help

lower blood glucose, cholesterol and triglycerides. J. Nutrition; 136 (3): 800S-802S

Threlfall RT, Morris JR, Howard LR, Brownmille RCR, Walker TL. 2005. Pressing effects

on the yield, quality and nutraceutical content of juice, seeds and skins from Black

Beauty and Sunbelt grapes. J. Food Sci. 70: 167–171.

Tongnuanchan P. Benjakul S. Prodpran T. 2010. Roles of lipid oxidation and pH on

properties and yellow discolouration of red tilapia muscle based film. Food

Innovation Asia Conference: Indigenous Food Research and Development to Global

Market, June 17-18, 2010, BITEC, Bangkok, Thailand.

163

Rivera, A.P. and Cabornida, I.M.B. 2008, Development of ready-to-drink green mango

juice. USM R & D J 16(1): 71-77.

Veeranjaneya RL, Sarath VRO. 2009. Production, optimization and characterization of

wine from mango (Mangifera indica Linn.). Natural Product Radiance, Research

paper 8(4): 426-435.

Vallverdú-Queralt A, Bendini A, Barbieri S, Di Lecce G, Martin-Belloso O, Gallina Toschi

T. 2013. Volatile profile and sensory evaluation of tomato juices treated with pulsed

electric fields. J Agric Food Chem 61: 1977−1984.

Vladimir-Knežević S, Blažeković B, Štefan MB, Alegro A, Kőszegi T, Petrik J. 2011.

Antioxidant Activities and Polyphenolic Contents of Three Selected Micromeria

Species from Croatia. J Mol 16: 1454-1470.

Yasuda H, Arakawa T. 1995. Deodorizing mechanism of (–) epigallocatechin gallate

against methyl mercaptan. Biosci Biotech Biochem 59:1232–6.

Xu S, Shoemaker CF, Luh BS. 1986. Effect of break temperature on rheological properties

and microstructure of tomato juices and pastes. J. Food Scienc 51(2)-399.

Xu Y, Barringer S. 2009. Effect of temperature on lipid-related volatile production in

tomato puree. J Agric Food Chem. 57(19): 9108–9113.

Zhu QY, Holt RR, Lazarus SA. (2002). Stability of the Flavan-3-ols epicatechin and

catechin and related dimeric procyanidins derived from cocoa. J Agric Food Chem

50:1700-1705.

164

Appendix: Tables, figures and questionnaires

Table 21: The concentration on different volatile compounds present in unpeeled and peeled tomato juice and pulp.

Analyte Peeled Peeled Unpeeled Unpeeled (ppb) microwaved (ppb) microwaved (ppb) (ppb)

(E)-2-heptenal 22.67 20.25 20.61 19.52 (E)-2-hexenal H3099 718.15 1618.11 935.00 1964.53 (E)-2-hexenal NO71 713.69 1030.09 784.48 1696.64 (E)-2-hexenal NO97 596.45 1365.67 726.74 1504.91 (E)-2-nonenal 3.61 Ru8y2.97 2.79 3.10 (E)-2-octenal 18.68 9.99 18.35 7.37 (E)-2-pentenal 133.08 131.55 138.48 203.22 (E,E)-2,4-decadienal 9.14 6.81 7.64 7.36 (E,Z)-2,6-nonadienal 3.38 5.16 3.46 4.54 (Z)-3-hexen-1-ol 72.69 63.52 76.98 106.36 (Z)-3-hexenal H3o81 287.21 163.96 327.43 181.94 (Z)-3-hexenal NO 70 278.44 127.17 294.51 141.62 (Z)-3-hexenal NO69 606.83 437.88 645.48 509.60 1-butanol 284.19 159.25 177.06 159.30 1-hexanol 84.68 36.54 117.25 49.71 1-octen-3-ol 16.92 16.81 16.91 22.28 1-penten-3-one 90.52 117.84 96.98 121.10 1-propanol 59.82 54.77 48.05 54.68 2,3-butanediol 19.73 26.82 18.36 41.92 2,3-butanedione 74.24 94.76 68.87 105.95 2-isobutylthiazole 5.14 3.87 3.43 4.11 2-methylpropanal 131.23 189.40 144.24 311.96 2-pentanol 173.23 164.89 157.88 183.23 2-pentanone 21.65 19.82 48.87 30.72 2-pentylfuran 24.99 16.89 21.08 16.41 3-methylbutanal 277.94 279.95 242.96 351.21 6-methyl-5-hepten-2-one 44.94 43.14 40.10 42.35 acetaldehyde 1242.03 1199.01 1396.56 1186.79

Continued

165

Table 22 Continued

Analyte Peeled Peeled Unpeeled (ppb) Unpeeled (ppb) microwaved microwaved (ppb) (ppb)

acetone 290.10 405.20 258.07 480.37 benzaldehyde 191.42 198.77 174.50 236.45 benzene ethanol 7.20 9.74 6.96 9.86 benzyl alcohol 13.89 20.61 13.08 37.25 beta-ionone 1.73 2.72 1.68 2.17 Citral 35.08 26.16 29.34 28.27 cyclic terpenes 21.75 19.95 19.37 21.81 decanal 4.26 4.35 3.65 5.92 dimethyl disulfide 7.40 6.03 10.87 10.11 dimethyl sulfide 426.14 966.26 538.08 1403.51 dodecanal {SB} 3.66 5.14 3.86 6.89 ethanol 661.47 191.59 674.72 267.38 ethyl acetate 38.65 39.26 39.43 43.09 eugenol 2.23 1.82 1.93 2.73 furfural 27.97 34.43 28.46 32.92 guaiacol 47.76 64.84 52.44 84.51 hexanal 2927.80 3050.29 3283.47 2875.82 hexanoic acid 22.79 17.56 17.61 17.01 hexyl acetate 10.93 9.01 9.94 9.79 isobutanal 131.23 189.40 144.24 311.96 isobutyl alcohol 260.51 145.98 162.31 146.03 21532.1 methanol 7 29601.98 29716.93 40537.20 methional 74.04 103.94 111.33 143.17 methyl benzoate 397.01 412.27 361.92 490.42 methyl hexanoate {SB} 20.65 14.54 16.91 15.47 methyl salicylate 8.20 10.88 8.63 15.40 methylbutanoic acid 28.07 25.16 24.01 29.33 nonanal 10.36 22.64 9.01 20.78 octanal 25.19 17.90 26.88 20.29 phenylacetaldehyde 37.20 36.71 41.29 42.82 propanal 278.61 289.02 259.38 420.40 propanoic acid 29.03 25.05 34.81 31.32

166

Majority of the volatiles seemed not to be significantly different for both peeled and the unpeeled juice and pulp, but there was some volatile that showed difference between the four treatments. (E)-2 hexenal, Z-3-Hexanal and hexanal, derived from lipoxygenase activity were all higher in peeled than unpeeled juice and pulp. These results are different from previous reports (Ties and Barringer 2012) whereby they reported higher volatile levels in the peels than the pulp in most of the cultivars.

Difference in cultivar would be one of the reasons of the higher volatile levels in the peeled than the unpeeled, another possible reason would be, not enough time was provided for lipoxygenase activity in the unpeeled juice and pulp. Dimethyl sulfide and methanol were higher in unpeeled juice than in the peeled juice. These results were in agreement with other researchers (Ties and Barringer 2012). Other volatiles that were higher in peeled juice than unpeeled were ethanol and acetaldehyde; these volatiles enhance the perception of sweetness in juice (Baldwin and others 1998).

167

2500.00 Peeled (ppb)

2000.00 Peeled microwaved (ppb) 1500.00 Unpeeled (ppb)

1000.00 Unpeeled microwaved (ppb) 500.00

0.00

Figure 21: Differences in volatile compounds present in peeled and tomato unpeeled juice and pulp

200 a 180 160 b 140 c 120 100 80 cde cd 60

Concentration (pb) Concentration 40 def def ef f f f 20 f 0

Treatments

168

250 a 200 b c 150 100 d d e ef ef 50 f f f f

0 Concentration (ppb) Concentration

8 a 7 b bc 6 bc cd 5 de ef 4 fg fg fg 3 fg g 2

1 Concetration(ppb) 0

Treatments

169

16 a 14 ab 12 bc cd 10 de de de de 8 e 6 f 4 f f

Concentration (ppb) Concentration 2 0

Treatments

0.8 a 0.7 ab ab 0.6 ab ab ab 0.5 ab ab b b 0.4 b b 0.3 0.2 0.1

0.0 Concentration (ppb) Concentration

170

2.5 a ab ab 2.0 bc bcd 1.5 cde def def ef def 1.0 ef f

0.5 Concentration (ppb) Concentration 0.0

Treatments Figure 27: The concentration of methional in tomato and sauces at different heat treatments. Values in the same row bearing different superscript letters are significantly different (p ˂ 0.05)

350 a 300 b 250 c 200 150 d 100 ef ef de f 50 g g g g

0 Concentration (ppb) Concentration

Treatments Figure 28: The concentration of dimethyl sulfide in hot and cold break juices and sauces and at different thermal treatments. Values in the same row bearing different superscript letters are significantly different (p ˂ 0.05).

171

6 a 5 4 b 3 bc bc de cd ef fg ef 2 h h 1 gh

Concentration (ppb) Concentration 0

Treatment

Figure 29: The concentration of dimethyl disulfide in hot and cold break juices and sauces and at different thermal treatments. Values in the same row bearing different superscript letters are significantly different (p ˂ 0.05)

3.5 a 3 2.5 b bc 2 cde bcd 1.5 def def def ef ef ef 1 f 0.5

0 Concentration (ppb) Concentration

Treatments

Figure 30: The concentration of furfural in hot and cold break juices and sauces and at different thermal treatments. Values in the same row bearing different superscript letters are significantly different (p ˂ 0.05)

172

7000000

6000000

5000000

4000000

3000000 AUC (328 nm)

AUC (328 nm) AUC (328 2000000 Linear (AUC (328 nm))

1000000 y = 63609x + 66174 0 R² = 0.9921 0 20 40 60 80 100 120 Concentration (mg/100g)

Figure 31: Standard Curve for rosmarinic acid content

0.5 0.45 y = 0.0077x + 0.0571 0.4 R² = 0.9981 0.35 0.3 0.25 0.2

Absorbance 765 Absorbance 0.15 0.1 0.05 0 0 10 20 30 40 50 mg/100g

Figure 32: Standard curve for total phenolics for different mint varieties

173

Table 22: Rosmarinic acid content and total phenolic content of different mint varieties

Rosmarinic acid Total phenolics (mg (mg/100g) GAE/100g)

Pure Peppermint 5.84c 1.74c

Pure Spearmint 9.18b 1.89c

Pure Chocolate mint 23.1a 4.35a

Peppermint + Garlic 0min ND 2.15b

Peppermint + Garlic 30min ND 1.65c

Spearmint + Garlic 0min ND 1.4cd

Spearmint + Garlic 30min ND 1.00e

Chocolate mint + Garlic 0min ND 1.07e

Chocolate Mint + Garlic 30min ND 0.92e

Values in the same column bearing different superscript letters are significantly different (p ≤0.05).

14000 Water 12000 Raw apple 10000 Raw lettuce

8000 Mint leaves

6000

4000 Concentration (ppb) Concentration

2000

0 0 10 20 30 40 50 60 Time (min)

Figure 33: Effect of raw apple, raw lettuce and mint leave on allyl mercaptan. Data points at the same time bearing different letters are significantly different (p ≤0.05) 174

14000 a a Water 12000 b a a Mint juice 10000 b Apple juice b 8000 a c

6000 c b a 4000

Concentarion (ppb) Concentarion c ab a 2000 b a a a a a a a a 0 0 10 20 30 40 50 60 Time (Min)

Figure 34: Effect of mint juice and apple juice on allyl mercaptan. Data points at the same time bearing different letters are significantly different (p ≤0.05)

16000 a a a 14000 Water a Green tea 12000 a b a Heated apple 10000 Heated letuce 8000 b a b a 6000 c b c a c ac Concentration (ppb) Concentration 4000 a c a a 2000 a a b b a a a 0 a 0 10 20 30 40 50 60 Time (Min)

Figure 35: Effect of green tea, heated apple and heated lettuce on allyl mercaptan. Data points at the same time bearing different letters are significantly different (p ≤0.05)

175

1200

1000 Water Raw apple 800 Raw lettuce

600 Mint leaves

400 Concetration (ppb) Concetration 200

0 0 10 20 30 40 50 60 Time (Min)

Figure 36: Effect of raw apple, raw lettuce and mint leaves on allyl methyl disulfide Data points at the same time bearing different letters are significantly different (p ≤0.05)

1200 a a Water 1000 a Mint juice 800 Apple juice

600 a b b b 400

Concentration (ppb) Concentration c a 200 ab a a a a aa a a b a a a 0 a 0 10 20 30 40 50 60 Time (Min)

Figure 37: Effect of Mint juice and apple juice on allyl methyl disulfide Data points at the same time bearing different letters are significantly different (p ≤0.05)

176

3000 a Water 2500 Heated apple a 2000 Heated lettuce a Green tea 1500 b a b a 1000 b a c c a 500 c b a Concentration(ppb) b b b b ba 0 0 10 20 30 40 50 60

Time (Min)

Figure 38: Effect of heated apple, heated lettuce and green tea on allyl methyl disulfide Data points at the same time bearing different letters are significantly different (p ≤0.05)

450 a Control 400 a a a a b a a Mint juice 350 a b b b b a a Apple juice 300 b a b 250 a b b 200 b 150

Concentration (ppb) Concentration 100 50 0 0 10 20 30 40 50 60 Time (min

Figure 39: Effect of different food materials on allyl methyl sulfide. Data points at the same time bearing different letters are significantly different (p ≤0.05) Continued

177

800 Control 700 a a a a a Raw apple a a 600 Raw lettuce a 500 b Mint leaves b b b c b 400 b b b b c b b c bc bc 300 c bc c c c c c Concentration(ppb) 200 c 100 0 0 10 20 30 40 50 60 Time (min)

3500 a Control 3000 a Green tea

2500 a Heated apple Heated lettuce 2000 a 1500 a 1000 Concentration (ppb) Concentration a b b b b b b ba ba a 500 b a ba

0 0 20 40 60 Time (min)

Figure 40 continued: Effect of different food materials on allyl methyl sulfide. Data points at the same time bearing different letters are significantly different (p ≤0.05)

178

Figure 41: HPLC Chromatograms of rosmarinic acid quantification of 3 different types of mint blended with garlic at 0 and 30min.

179

Questionnaire

Sensory Analysis on Tomato Juice

Gender

a) Female b) Male

What is your age?

a) 18-30 b) 31-40 c) 41-50 d) 51-65 e) 66 and over

1. How often do you consume tomato juice? a) Always (3 times a week) b) Weekly c) Monthly d) Once in 3 months e) Rarely (once in 1 or 2 years) f) Never

You are provided with two sample of tomato juice, sample 232 and 415

2. Look at the product and rank how you like the “Color”.