The Effects of High Pressure Processing, Browning Additives, and Storage Period on the

Inactivation of Polyphenol Oxidase in Nine Varieties of Pawpaw (Asimina Triloba L.)

Pulp

A thesis presented to

the faculty of

the College of Health Sciences and Professions of Ohio University

In partial fulfillment

of the requirements for the degree

Master of Science

Lin Zhang

August 2016

© 2016 Lin Zhang. All Rights Reserved. 2

This thesis titled

The Effects of High Pressure Processing, and Browning Additives, and Storage Period on the Inactivation of Polyphenol Oxidase in Nine Varieties of Pawpaw (Asimina Triloba L.)

Pulp

by

LIN ZHANG

has been approved for

the School of Applied Health Sciences and Wellness

and the College of Health Sciences and Professions by

Robert G. Brannan

Associate Professor of Applied Health Sciences and Wellness

Randy Leite

Dean, College of Health Sciences and Professions 3

Abstract

ZHANG, LIN, M.S., August 2016, Food and Nutrition Sciences

The Effects of High Pressure Processing, Browning Additives, and Storage Period on the

Inactivation of Polyphenol Oxidase in Nine Varieties of Pawpaw (Asimina Triloba L.)

Pulp

Director of Thesis: Robert G. Brannan

This study measured and compared the effects of high pressure processing (HPP), browning inhibitors (pasteurization, ascorbic acid, and steviosides), storage time (0 day,

15 days, 30 days, and 45 days at 4 °C, and fruit variety (Belle, Mitchell, PA-Golden,

Pickle, Seedling, Sunflower, Shenandoah, SAB OL, Wilson) on the polyphenol oxidase

(PPO) activity and the color of pulp of pawpaw fruit. In a related experiment, a descriptive sensory analysis was conducted using trained sensory panelists to determine the influence of the treatments on the sensory attributes of a single variety pawpaw

Shenandoah.

There were differences observed between the nine pawpaw varieties studied.

These varieties were ranked in sequence according to the PPO activity in the untreated samples as Seedling, Mitchell, Sunflower, Shenandoah, Wilson, PA-Golden, Pickle, SAB

OL, and Belle. HPP significantly decreased the PPO activity in all of the varieties except for Belle and Pickle. The browning inhibitors significantly inhibited PPO activity except that ascorbic acid and steviosides increased PPO activity in SAB OL and Shenandoah samples and pasteurization did not affect PPO activity in Mitchell and increased PPO

4 activity in Shenandoah. A general decreasing trend of PPO activity was detected along with the 45 days of storage period among all the HPP treated varieties.

Samples processed by HPP exhibited a darker, redder, and less yellow color, which is equivalent to more browning development. Because HPP is an excellent treatment for PPO inactivation but may cause undesirable browning of the pawpaw pulp, its commercialization potential is moderate but could be improved in combination with an effective browning inhibitor. Ascorbic acid, steviosides, and pasteurization all inhibited PPO activity. Ascorbic acid preserved the bright, less red, and yellow color of the fruits; therefore, its commercialization potential is high. Steviosides and pasteurization inhibited PPO activity but the pulps treated with steviosides exhibited increased redness and pasteurization led to darker and less yellow color change of the pulp. Hence, the commercialization potential of steviosides and pasteurization is medium.

Data from the descriptive sensory analysis revealed that HPP and pasteurization altered the color of the samples, with HPP promoting browning and pasteurization inhibiting browning. The samples treated with steviosides had a sweeter and more bitter taste than the untreated control. Among the treated samples, these findings suggest that ascorbic acid performed the best among the selected treatments to maintain the color and taste of Shenandoah pawpaw pulp.

Overall, ascorbic acid treatment had the highest commercial potential for pawpaw browning inhibition. The results of this study are expected to be useful to the food industry when selecting proper methods to prevent pawpaw or Annonaceae fruit browning, especially.

5 Preface

Chapter 3 contained within this thesis document serves as prepublication manuscript that has been submitted to Journal of Food Research. This thesis has been slightly reformatted to meet the guidelines set forth by both Journal of Food Research and Thesis and Dissertation Services at Ohio University.

6

Dedication

To my family. 7

Acknowledgements

I would like to give special thanks to Dr. Robert Brannan for his dedication to his students’ learning and his guidance to me during this entire research project, as well as throughout my 3 years of study in the master’s program of Food and Nutrition Sciences at Ohio University. I want to give my deep appreciation to the professors on my committee, Dr. Darlene Berryman and Dr. Diana Schwerha, as well as Dr. Jenifer Horner for their complete flexibility during my proposal and defense meeting arrangements, their constructive suggestions, and their valuable time editing my thesis. Besides, I am thankful to my colleagues, Jingyan Huang and Shun Dai, for their great help in the experiments and data collection. I also wound like to express my appreciation to Dr. Ron

Powell from Fox Paw Ridge Farm and Sandridge Food Cooperation for their generous donation of the pawpaw fruits and providing high pressure processing (HPP) treatment to the pawpaw samples for this experiment. Lastly, I would like to say “thank you very much” to all the faculty and staff in the College of Health Sciences and Professions, especially Professor Deborah Murray and Professor Jenifer Yoder, for always being so helpful and supportive to me. The College of Health Sciences and Professions at Ohio

University helped me discover my potential for excellence. Thank you for choosing me to be part of the college! 8

Table of Contents

Page

Abstract ...... 3

Preface ...... 5

Dedication ...... 6

Acknowledgements ...... 7

List of Tables ...... 12

List of Figures ...... 14

Chapter 1: Introduction ...... 15

1.1 Background ...... 15

1.2 Statement of the Problem ...... 17

1.3 Research Questions ...... 18

1.4 Significance of This Research ...... 19

1.5 Limitations ...... 20

1.6 Delimitations ...... 21

Chapter 2: Literature Review ...... 22

2.1 Pawpaw ...... 22

2.1.1 The taxonomy and basic characteristics of pawpaw ...... 22

2.1.2 Pawpaw history ...... 23

2.1.3 Pawpaw nutrition ...... 24

2.1.4 Pawpaw ripeness and quality ...... 28

2.1.4.1 Color ...... 28 9

2.1.4.2 Aroma ...... 30

2.1.4.3 Texture ...... 31

2.1.4.4 Sweetness ...... 32

2.1.4.5 Pawpaw flavor ...... 32

2.2 Browning...... 33

2.2.1 PPO ...... 34

2.2.2 PPO activity in fruit ...... 35

2.3 The Effect of Different Processing Factors on PPO Activity ...... 36

2.3.1 Variety ...... 36

2.3.2 pH ...... 37

2.3.3 Temperature ...... 39

2.3.4 HPP ...... 41

2.3.5 Chemical additives ...... 43

2.3.5.1 Ascorbic acid ...... 44

2.3.5.2 Stevia ...... 45

2.3.6 Storage period ...... 47

Chapter 3: The Effects of High Pressure Processing, Browning Additives, and Storage

Period on the Inactivation of Polyphenol Oxidase in Nine Varieties of Pawpaw (Asimina

Triloba L.) Pulp ...... 49

3.1 Abstract ...... 49

3.2 Introduction ...... 51

3.3 Materials and Methods ...... 53 10

3.3.1 Materials ...... 53

3.3.2 Experimental design and sample preparation ...... 54

3.3.3 HPP treatment...... 55

3.3.4 Post-treatment storage...... 56

3.3.5 Color measurement ...... 56

3.3.6 PPO activity determination...... 56

3.3.6.1 Crude enzyme extract preparation...... 57

3.3.6.2 Lowry protein assay...... 57

3.3.6.3 PPO enzyme assay ...... 58

3.3.7 Descriptive sensory evaluation ...... 58

3.3.8 Statistical analysis ...... 59

3.4 Results and Discussion ...... 60

3.4.1 The effect of the treatments in each of the nine pawpaw varieties ...... 62

3.4.1.1 Variety 1: Belle ...... 62

3.4.1.2 Variety 2:Mitchell ...... 66

3.4.1.3 Variety 3:PA-Golden ...... 70

3.4.1.4 Variety 4: Pickle ...... 74

3.4.1.5 Variety 5: Seedling ...... 78

3.4.1.6 Variety 6: Sunflower ...... 82

3.4.1.7 Variety 7: Shenandoah ...... 85

3.4.1.8 Variety 8: SAB OL ...... 89

3.4.1.9 Variety 9: Wilson ...... 92 11

3.4.2 Descriptive sensory analysis ...... 96

3.5 Conclusion ...... 99

3.5.1 Variety ...... 99

3.5.2 HPP ...... 100

3.5.3 Browning inhibitors ...... 100

3.5.4 Storage ...... 101

3.5.5 Sensory ...... 102

3.5.6 Summary and conclusion ...... 102

3.6 References ...... 104

Chapter 4: Summary and Conclusion ...... 111

4.1 HPP ...... 112

4.2 Browning inhibitors ...... 113

4.3 Sensory ...... 114

4.4 Summary ...... 114

References ...... 116

Appendix A: The First Official Documentation of the Nutritional Factors of Pawpaw

Published by the U.S. Department of Agriculture (USDA) in 1963 ...... 129

Appendix B: The Pawpaw Descriptive Lexicon That was Used in the Sensory

Analysis ...... 130 12

List of Tables

Page

Table 1: Research Questions and Hypothesis ...... 19

Table 2: Nutritional Facts of Edible Portion of Pawpaw Fruit ...... 27

Table 3: The Corresponding Headspace Volatiles to Selected Aromas Detected in Pawpaw

Pulp ...... 31

Table 4: The Independent Variables Utilized for the 9×4×3×2 Factorial Design in This

Study ...... 55

Table 5: Comparison of the PPO Activity of the HPP Versus NHPP Samples Under

Different Storage Time ...... 62

Table 6: The PPO Activity and L*, A*, B* Color Coordinates of Belle Pawpaw Pulp With or Without High Pressure Processing and With or Without the Addition of Browning

Inhibitors ...... 64

Table 7: The PPO Activity and L*, A*, B* Color Coordinates of Mitchell Pawpaw Pulp

With or Without High Pressure Processing and With or Without the Addition of Browning

Inhibitors ...... 68

Table 8: The PPO Activity and L*, A*, B* Color Coordinates of PA-Golden Pawpaw

Pulp With or Without High Pressure Processing and With or Without the Addition of

Browning Inhibitors ...... 72

Table 9: The PPO Activity and L*, A*, B* Color Coordinates of Pickle Pawpaw Pulp

With or Without High Pressure Processing and With or Without the Addition of Browning

Inhibitors ...... 76 13

Table 10: The PPO Activity and L*, A*, B* Color Coordinates of Seedling Pawpaw Pulp

With or Without High Pressure Processing and With or Without the Addition of Browning

Inhibitors ...... 80

Table 11: The PPO Activity and L*, A*, B* Color Coordinates of Sunflower Pawpaw

Pulp With or Without High Pressure Processing and With or Without the Addition of

Browning Inhibitors ...... 83

Table 12: The PPO Activity and L*, A*, B* Color Coordinates of Shenandoah Pawpaw

Pulp With or Without High Pressure Processing and With or Without the Addition of

Browning Inhibitors ...... 87

Table 13: The PPO Activity and L*, A*, B* Color Coordinates of SAB OL Pawpaw Pulp

With or Without High Pressure Processing and With or Without the Addition of Browning

Inhibitors ...... 90

Table 14: The PPO Activity and L*, A*, B* Color Coordinates of Wilson Pawpaw Pulp

With or Without High Pressure Processing and With or Without the Addition of Browning

Inhibitors ...... 94

Table 15: Mean Values of the Twelve Selected Sensory Attributes for Variety Shenandoah

Pawpaw Pulp With or Without High Pressure Processing and With the Addition of

Chemical Inhibitors ...... 98

Table 16: Trends of PPO Activity in Each of the Nine Varieties of Pawpaw Fruits After

Different Treatments ...... 112

Table 17: Overall Influences of Different Treatments/ Factors to the PPO Activity and CIE

Color (L*, A*, B*) in Pawpaw Pulp...... 114 14

List of Figures

Page

Figure 1: Pawpaw (Asimina triloba L.) ...... 23

Figure 2: CIELAB tri-coordinate model ...... 29

Figure 3: Effect of refrigerated storage (4 ℃) on the change of PPO activity in pawpaw pulp of variety Belle ...... 66

Figure 4: Effect of refrigerated storage (4 ℃) on the change of PPO activity in pawpaw pulp of variety Mitchell ...... 70

Figure 5: Effect of refrigerated storage (4 ℃) on the change of PPO activity in pawpaw pulp of variety PA-Golden ...... 74

Figure 6: Effect of refrigerated storage (4 ℃) on the change of PPO activity in pawpaw pulp of variety Pickle ...... 77

Figure 7: Effect of refrigerated storage (4 ℃) on the change of PPO activity in pawpaw pulp of variety Seedling ...... 81

Figure 8: Effect of refrigerated storage (4 ℃) on the change of PPO activity in pawpaw pulp of variety Sunflower ...... 84

Figure 9: Effect of refrigerated storage (4 ℃) on the change of PPO activity in pawpaw pulp of variety Shenandoah ...... 88

Figure 10: Effect of refrigerated storage (4 ℃) on the change of PPO activity in pawpaw pulp of variety SAB OL ...... 91

Figure 11: Effect of refrigerated storage (4 ℃) on the change of PPO activity in pawpaw pulp of variety Wilson ...... 95 15

Chapter 1: Introduction

1.1 Background

Pawpaw (Asimina triloba L.), a green “bean-shaped” fruit, is mainly cultivated in hardwood forests of the eastern North America, including the State of Ohio (Pomper &

Layne, 2010). It belongs to the tropical Annonacea fruit family even though it grows in temperate climate. The pawpaw fruit, which can grow up to 1 kg by weight, is reported to be the largest tree fruit native to the United States (Darrow, 1975). During the growing season, the pawpaw pulp has a whitish to light-green color that turns yellow to brown at maturity. The pulp of ripe fruit has the flavor of banana and mango, and the soft flesh surrounds two rows of large bean-shaped dark brown seeds (Brannan, Salabak, &

Holben, 2012; Wood & Peterson, 1999). Pawpaw is high in phenolic compounds and is a natural source of Vitamin C (9.3 mg/100 g ripe tissue) and flavonoids (0.32 umol rutin/100 g tissue), which are antioxidants that can relieve oxidative stress (Harris &

Brannan, 2009). The first documentation about pawpaw was written in 1541. Despite its long history, pawpaw is still a mysterious fruit that is not commonly recognized. A sensory study of tropical fruits shows that less than 10% of consumers who favored the pawpaw taste could identify it correctly from other tropical fruits (Brannan, Salabak, &

Holben, 2012).

Pawpaw fruit is highly perishable and usually is only available for purchase in local markets or from private gardeners’ backyards. The promotion of pawpaw to standardized markets is relatively difficult for it has weaknesses, one of which is its extreme perishability. The ripened pawpaw turns brown within 2 to 3 days after being

16 picked. Moreover, from the preliminary research, the color of cut pawpaw pulp changes to dark reddish brown within a couple hours. Though the fruit is still at the edible stage after turning brown, its appearance is not favorable; this can impact consumers’ perceptions of quality, freshness, palatability, and healthfulness (Boyd, 2015).

As a pawpaw ripens, its color changes from the cold spectrum to the warm spectrum and its sweetness and aroma reach its peak value. As pawpaw becomes overripe, its color, flavor and aroma deteriorate from consumers’ highly preferable range to less preferable. Therefore, customers may be more willing to purchase other varieties of fruit instead of a browned pawpaw or the pawpaw that immediately turns brown after being cut, because its unfavorable color conveys information of off-taste, poor nutritional value, and lack of freshness.

The undesired browning process of pawpaw is induced by a natural enzyme in the pulp, called polyphenol oxidase (PPO). The enzyme PPO is a copper-centered, protein- based enzyme that transfers oxygen and causes the browning reaction. Browning happens when the PPO presented in pulp is activated by oxygen when pawpaw is cut, bruised, or exposed to air. The oxygen activated PPO converts phenolic substrates (such as o- diphenol) into quinone compounds (o-quinones), which turn to dark melanins after polymerization (Gomes, Vieira, Fundo, & Almeida, 2014; Yoruk & Marshall, 2003) The continuous accumulation of the dark pigment results in browning of the pawpaw fruit. To delay or even cease the browning process and to extend the shelf life of pawpaw, the inhibition of the corresponding enzymatic browning reaction through PPO inactivation would be the first problem to be solved in this research.

17

Inhibiting the enzymatic reaction can be achieved by creating an environment that is not optimal for enzyme activity. In the food industry, pH adjustment, heat treatment

(thermalization), high pressure processing (HPP), and the use of additives are the four commonly used methods for the prevention of unwanted enzymatic reactions. Heat treatment ceases enzymatic reactions through denaturing the active enzymes. However, the applied heat in pasteurization may damage the heat sensitive nutrients and may change the texture as well as flavor of the food product while denaturing the unexpected enzymes. For heat-sensitive food products such as guacamole, HPP, which inactivates enzymatic reactions in food through elevated pressure force, might be a better choice.

HPP is also referred as high hydrostatic pressure processing (HHP) or ultra high-pressure processing (UHP). Through elevated pressures (between 100 and 800 MPa) under low- heat or no-heat conditions, with or without packaging, the enzymatic inactivation or alteration of the food attributes to consumer-desired qualities are achieved (Sulaiman &

Silva, 2013; Woolf et al., 2013). Besides PPO denaturation via physical methods

(pasteurization and HPP), the study of antibrowning additives, such as ascorbic acid and stevia extract, is also a trend to solve unwanted browning problems (Barba, Criado,

Belda-Galbis, Esteve, & Rodrigo, 2014; Ghidelli, Mateos, Rojas-Argudo, & Perez-Gago,

2014; Gomes et al., 2014; Shukla, Mehta, Bajpai, & Shukla, 2009; Tadhani, Patel, &

Subhash, 2007).

1.2 Statement of the Problem

Widespread pawpaw commercialization has not been achieved because pawpaw has high levels of the enzyme responsible for pulp browning (PPO), which causes quick

18 post-harvest browning. This affects the appearance, texture, and other attributes that may affect consumer perception of the quality of the fruit. The purpose of this research was to explore the effects of HPP associated with the addition of stevia and ascorbic acid and storage time (0 day, 15 days, 30 days, and 45 days) toward the inhibition of unwanted browning over different varieties of pawpaw pulps. The presence of active PPO usually causes the browning of freshly cut fruits, including pawpaw. To preserve the original characteristics of the fruits, like color, the effects of the six different processing measures on PPO inactivation in pawpaw pulps were studied and compared. The PPO content of different groups of pawpaw pulps under varied storage lengths will be analyzed so that the effects of the selected treatments are evaluated. The findings of this research are expected to be helpful in the designation of effective antienzymatic browning techniques, which may potentially make the pawpaw more preferable to consumers. This knowledge can be used by the pawpaw industry to promote the marketability of fresh pawpaw fruits.

1.3 Research Questions

As shown in Table 1, this study aims to determine the effect of HPP and the use of additives on PPO activity and sensory quality of the pulp. Further, these factors will be studied in different varieties of pawpaw pulp that is stored under refrigeration. 19 Table 1

Research Questions and Hypothesis

Research questions Hypothesis

1. Does the variety of pawpaw affect the The variety of pawpaw is an level of PPO activity in pawpaw pulp? important factor that affects the level of PPO activity in its pulp.

2. How does HPP with or without the HPP and the browning inhibitors will browning inhibitors (pasteurization, decrease PPO activity, which will be steviosides, or ascorbic acid) added to the further decreased in combination with pulp affect pawpaw fruit PPO activity? one another.

3. Is storage period (0 day, 15 days, Storage period is a vital factor that 30 days, and 45 days) a factor that significantly affects PPO activity in impacts the PPO activity in the pulp of pawpaw pulp. The longer the storage pawpaw fruit? time is, the higher the PPO activity would be.

4. In a single variety of pawpaw, does HPP HPP and the inhibitors and browning inhibitors (pasteurization, (pasteurization, stevia and ascorbic stevia and ascorbic acid) affect the acid) will alter sensory attribute of sensory attributes of pawpaw pulp? pawpaw pulp. Note. PPO-polyphenol oxidase; HPP-high pressure processing.

1.4 Significance of This Research

It is challenging to market pawpaw since the fruit cannot be transported long

distances due to discoloration. Not only pawpaw, much other fresh produce has this

problem as well. Therefore, research of antibrowning techniques is vital to the food-

marketing field broadly.

The effects of HPP combined with chemical additives (stevia or ascorbic acid) on

inhibiting the activation of PPO in different varieties of pawpaw fruit will be determined

in this research. The results of this research will show whether HPP is a processing 20 method that can prevent enzymatic browning on food products. At the same time, the effectiveness of stevia or ascorbic acid on assisting HPP to inactivate the PPO will be tested and compared. The findings of this research are expected to make a significant contribution to selecting a series of highly effective antienzymatic browning techniques for the current food industry. With the assistance of these techniques, more regional fresh produce, like pawpaw, could be spread world wide to increase the diversity of local food choices.

1.5 Limitations

The harvest season of the pawpaw is very short and the fruit do not ripen on the same tree at the same time. Therefore, the ripe fruits are usually picked manually and the pickers estimate the ripeness of them subjectively. The color, texture, and odor are factors that are used to deduct ripeness. An expert, Dr. Ron Powell from Fox Paw Ridge Farm, was consulted to determine ripeness but overall ripeness was not well controlled in the current research. Through visual observation, the degree ripeness of the pawpaw fruits is not strictly uniform so that bias can be caused. In order to minimize the errors caused by this factor, the pulp from each variety will be mixed and homogenized before processing.

The pawpaw fruits were harvested in Cincinnati, and it took 3.5 hr to transport them to Athens. During transportation, it was challenging to keep the fruits under optimal chilling conditions. Therefore, the unavoidable oxidation during harvest and transportation might have introduced biases.

The seeds and skin of the pawpaw fruits had to be manually removed, and the pulp was vacuum packed in plastic pouches before testing. Although precautions were 21 taken to keep the pulp temperature cold and sealed in plastic, the pulping and packing procedures were performed at room temperature with ambient oxygen exposure. The reactions that happened during transportation, pulping, and packing all could have caused inaccuracy of the results. The pawpaw pulps were stored in freezing conditions with a temperature of -18 °C to stop any enzymatic reaction after packing. It is nearly impossible to prevent further chemical reactions, but they were controlled to the acceptable range.

The pawpaw samples were processed at Sandridge Food Corporation in Medina,

Ohio. The high-pressure processor in this facility processes ready-to-eat salads and fruits at 600 Mega Pascal (MPa) at ambient temperature (4 °C) for 76 s, so these processing conditions were set. Only one HPP condition could be measured in this research.

1.6 Delimitations

Selection of pawpaw sample varieties for this study is based on the availability of pawpaw. As is mentioned earlier, over 80 varieties of pawpaw fruit have been currently discovered. This research tests the PPO activity of nine varieties (listed in Chapter 3).

These nine varieties of pawpaw were selected because they were the most ready for harvest at the farm in Cincinnati from where the pawpaw samples were gathered. Among these nine varieties, a sensory evaluation test was conducted on Shenandoah (SHEN) since the quantities of the other varieties was very limited.

22

Chapter 2: Literature review

2.1 Pawpaw

2.1.1 The Taxonomy and Basic Characteristics of Pawpaw

Pawpaw (Asimina triloba L.) is the only temperate member in the pantropical

Annonaceae family (Callaway, 1990). The plants of Annonaceae family, which is also known as custard apple family, are usually observed as tropical flowering trees or shrubs, with a few lianas (long stemmed wood vines) (Couvreur, Maas, Meinke, Johnson, &

Kessler, 2012). There are currently109 genera and over 2400 species of Annonaceae plants being recognized and documented (Callaway, 1990; Chatrou et al., 2012; Erkens,

Mennega, & Westra, 2012). Asimina is included as the only temperate climate genera in

Annonaceae family. Asimina (genera) triloba L. (species) is pawpaw. Over 80 varieties of pawpaw have been discovered. The pawpaw plant blooms in the early summer season with small, maroon or yellow color, yeasty, red wine, or baking bread scented flowers

(Goodrich, 2012). The fruit ripens in late summer and early fall and appears kidney shaped, light green to yellow, with the size varies from 5 cm to 20 cm long when ripe

(Brannan & Salabak, 2009; Callaway, 1990). Some species are recognized as the largest tree fruits native to North America and one individual fruit can weigh up to 1 kg (Layne,

1996). Ripe pawpaw turns from hard to custard-like texture within a few days and its inedible skin starts to brown quickly.

23

Figure.1 Pawpaw (Asimina triloba L.). Left image: “The Pawpaw Tree, Asimina triloba, Yields 3- to 5-Inch-Long Fruit, the Largest Fruit Native to the United States,” by S. Bauer, May 2006. Retrieved from the U.S. Department of Agriculture, Agricultural Research Services website: http://www.ars.usda.gov/is/graphics/photos/mar97/k7575- 8.htm. Right image:“Asimina Triloba Red Fern Farm,” by Clarknova, September 2004. Retrieved from https://commons.wikimedia.org/wiki/File: Asimina_triloba_red_fern_farm.jpg. rk

2.1.2 Pawpaw history

Pawpaw is indigenous to North America. Its fossils have been found in Colorado,

New Jersey, Texas, Mississippi, and Wyoming and date back to the Eocene epoch

(Peterson, 1990). Pawpaw was first documented in the Hernando De Soto expedition tracing back to 1541 (Layne, 1996). It was the major energy source for the Lewis and

Clark expedition in western Missouri during their return trip to the east in the fall of 1810 when they were running out of food supply (Peterson, 1990). Meanwhile, the native pioneers from Florida to the Mississippi River cultivated pawpaw for multiple uses. For instance, the local people consumed the fruit and made fishnets by its barks (Peterson,

1990). Nowadays, pawpaw cultivation is spread out worldwide. Countries like China,

Japan, Korea, Italy, and Belgium are all planting pawpaw trees for consumption or medical use (Coothankandaswamy et al., 2010). The United States is a dominant pawpaw growing country from the past until today. Its cultivation is mainly concentrated in the 24 southeast, such as Ohio, Kentucky, and Maryland (Callaway, 1990). Pawpaw fruit is currently consumed locally or used as ingredient of ice cream, pastry products, and beverages (Pomper et al., 2008; Wiese & Duffrin, 2003).

2.1.3 Pawpaw nutrition

The first official and systematic documentation of pawpaw nutrition is likely the

U.S. Department of Agriculture (USDA), 1963 Composition of Foods (Raw Processed

Prepared). This publication documented the nutritional factors of pawpaw and reported that 1 lb of pawpaw fruit contributed 289 Kcal and contained 17.7 g protein, 3.1 g fat, and 57.2 g carbohydrate (see Appendix A).

More recently, the major nutritional content of pawpaw (moisture, lipid, ash, protein, and carbohydrate by difference) of the pawpaw and its pulp at different stages of ripeness were measured (McGrath & Karahadian, 1994b). Based on the results, the macronutrients level varies during each stage of ripeness. Ripe fruit contained 77.0% moisture, 0.47% lipid, 0.74% ash, 0.81% protein, and 20.8% carbohydrate, and 22.5 ±

3.0 soluble sugar content (° Brix). This was less protein and lipid and more carbohydrate than reported in the 1963 USDA study, but consistent moisture content with the data

(71.9%-76.6%) in the study by (Brannan, Peters, & Talcott, 2015). Similar to what is found in McGrath and Karahadian (1994b), Kobayashi, Wang, and Pomper (2008) discovered that the soluble sugar (° Brix) content elevates during pawpaw ripening, and peaks when ripe. Whereas, the sugar levels from the research by McGrath and

Karahadian (1994b) are more varied in each ripening stage, and the sugar content (° Brix) 25 of ripe pawpaw from the study by McGrath and Karahadian (1994b) is close to that (19.9 to 28.0) in Brannan et al. (2015), but 25% higher than that in Kobayashi et al. (2008).

Ripeness and variety in pawpaw can affect not only the soluble sugar, but also the antioxidant nutrients and the antioxidant capacity. Phenolics and flavonoids are two of the commonly recognized antioxidant groups, which are excellent natural agents that promote human health, i.e., free radicals scavenging (Wada et al., 2007) and prevent oxidation in the meat industry (Brannan, 2008). The phenolic content decreases along pawpaw ripening, while antioxidant capacity maximizes at semiripe and minimize at ripe stage (Kobayashi et al., 2008). More recent pawpaw antioxidant studies observed the same change (Brannan et al., 2015; Harris & Brannan, 2009). The results indicate that the total phenolics and ascorbic acid content in pawpaw pulp reached its maximum when the fruit is ripe and will decrease significantly when overripe. The change of total phenolics along pawpaw ripening from the study by Harris and Brannan (2009) is consistent with the conclusion from Kobayashi et al. (2008). The total flavonoids content also peaked in unripe pawpaw pulp and decreased as the fruit ripens and becomes overripe (Brannan et al., 2015; Harris & Brannan, 2009). Besides the observation on ripeness, Brannan et al.

(2015) detected the significant difference of total phenolics, total flavonoids content and total reducing capacity due to variety by measuring nine varieties of pawpaw pulp.

Pawpaw varieties Zimmerman and Rebecca Gold contain the highest amount phenolics

(7.97 ± 0.06 µmol gallic acid equivalents per g pulp) and flavonoids (0.86 ± 0.01 µmol rutin equivalents per g pulp), respectively (Brannan et al., 2015).

26

The general nutritional facts of ripe pawpaw fruit pulp from the studies described above is collected and summarized in Table. 2 and standardized to 100 g of pawpaw pulp for ease of comparison. Since the present research studies the preservation and browning of the edible pawpaw, only the figures on ripe pawpaw are provided. This table is a collection of data based on multiple research results; the varieties of pawpaw that these studies tested might be different but the variance of the results are within an acceptable range.

27 Table 2

Nutritional Facts of Edible Portion of Pawpaw Fruit

Nutrient Content (per 100g References pulp)

Total Energy (Kcal) 64 kcal U.S. Department of Agriculture, 1963

Moisture 74.05 ± 2.95 % Brannan et al., 2015; McGrath & Karahadian, 1994b

Total carbohydrate 16.7 ± 4.1 g Brannan et al., 2015; Kobayashi et al., 2008; McGrath & Karahadian, 1994b; Soluble sugar 19.6 ± 8.4 U.S. Department of Agriculture, 1963 (° Brix)

Fat (g) 0.84 ± 0.37 g McGrath & Karahadian, 1994b; Peterson, Simmons, & Cherry, 1982; U.S. Department of Agriculture, 1963

Protein (g) 4.7 ± 3.9 g McGrath & Karahadian, 1994b; Peterson et al., 1982; U.S. Department of Agriculture, 1963

Ash 0.81 g McGrath & Karahadian, 1994b

Antioxidant Brannan et al., 2015; Harris & Brannan, 2009; Kobayashi et al., 2008 *Phenolic 0.92 ± 0.54 mmol

Ascorbic acid 4.98 ± 0.32 mg

Total flavonoid 0.81 ± 0.17 mg

Antioxidant capacity 1.63 ± 0.04 mmol Kobayashi et al., 2008 Note. Used Gallic acid molar mass =170.12 g/mol when converting unit mg GAE to mmol.

28

2.1.4 Pawpaw ripeness and quality

The quality of most fruits can be gleaned from four characteristics, which are color of the skin and the pulp, aroma, texture, and sweetness.

2.1.4.1 Color

According to research by McGrath & Karahadian (1994a), the external skin color of pawpaw is an indicator of ripeness. During ripening process, a continuous decline of hue angle (�), which indicates the color change from green to yellow and to red, is detected on the skins of different varieties of pawpaw (McGrath & Karahadian, 1994a).

Hue is the technical term of color appearance parameters. It measures the degree to which a stimulus can be described as red, green, blue, and yellow and expressed by angle degrees (Fairchild., 2004). The CIE model, which is abbreviated from the

International Commission on Illumination in French (Commission Internationale de l’Eclairage), applied the hue concept geometrically into a three-dimensional coordinate axis system (see Figure 2). The CIE model quantifies red (+ a* value), green (-a* value), yellow (+ b* value), and blue (– b* value) in positives versus negatives, and lightness (L* value) on a scale from 0 (dark) to 100 (light). Hue angle (�) is calculated based on a* and b* values obtained from CIE model by the equation below.

Hue angle (�) = ���!! !∗ !∗ 29

100

L -a * +b

-b +a

0

Figure 2. CIELAB tri-coordinate model.

As pawpaw fruits ripen, both a* and b* values of pawpaw skin increase from negative to positive, but no typical change of “L” value is observed (McGrath &

Karahadian, 1994a, 1994b). The skin of ripe pawpaw has L* value of 56.01 ± 2.96, a* of

+ 2.67 ± 2.37, b* of + 23.85 ± 1.10, and hue angle (�) of 95.20 ± 7.40 through CIE model measurement and calculation (McGrath & Karahadian, 1994a, 1994b). Thus, ripening of pawpaw skin shows a conversion from green to red and blue to yellow, with ripe pawpaw having a characteristic green-yellow colored external appearance.

30

The color of pawpaw pulp ranges from golden yellow to orange-red, which varies according to storage and varieties (Brannan et al., 2012; Duffrin & Pomper, 2006;

McGrath & Karahadian, 1994a, 1994b). A CIE quantitative color measurement study reported a L* value of 67.9 ± 0.1, a* of + 5.4 ± 0.6, b* of + 30.0 ± 1.8 of fresh pawpaw pulp and indicated that storage and air exposure led to darker, redder, but less yellow color change (Brannan et al., 2012). Besides, the hue angle (�) of fresh pawpaw pulps from four different cultivars vary from 75.5 to 87.8 (McGrath & Karahadian, 1994a).

2.1.4.2 Aroma

The intensity of aroma increases during pawpaw ripening process (McGrath &

Karahadian, 1994b). Even though the smell varies in varieties, the ripe pawpaw fruit carries a complex sweet, fruity, and slightly fermented aroma in general (Brannan et al.,

2012; Goodrich, Zjhra, Ley, & Raguso, 2006; McGrath & Karahadian, 1994b).

Descriptive terms like “tropical fruity, sweet, melon-like, and fermented” are used frequently by trained panelists in the odor evaluation of pawpaw pulp (Brannan et al.,

2012; Goodrich et al., 2006; McGrath & Karahadian, 1994b). The aromas that appear on pawpaw from different conditions are dependent on the prominence of specific headspace volatiles in the samples. See Table 3 for the commonly detected types of aromas in pawpaw and their corresponding headspace volatiles. 31

Table 3

The Corresponding Headspace Volatiles to Selected Aromas Detected in Pawpaw Pulp

Aroma descriptors Headspace volatile compound

Banana-like Ethyl acetate; Ethyl butanoate; Ethyl hexanoate

Pineapple-like Ethyl butanoate; Ethyl hexanoate; Methyl hexanoate

Orange-like Methyl octanoate.

Fruity, tropical Ethyl acetate; Ethyl 2-butanoate; Ethyl 3-hydroxy-butanoate; Ethyl hexanoate; Methyl octanoate; Ethyl octanoate; Methyl dectanoate

Sweet � − hexalactone; Ethyl dectanoate

Fermented Ethyl acetate; ethanol; acetic acid Note. Information retrieved from (Goodrich et al., 2006; McGrath & Karahadian, 1994b).

2.1.4.3 Texture

The ripe pawpaw fruit usually has a soft texture (McGrath & Karahadian, 1994a).

As pawpaw ripens, the elevated production of hydrolytic enzyme breaks down cell wall and results in softness, causes fruit softening (Brady, 1987). The hardness of ripe whole fruit declines to less than half of the hardness of unripe fruit (McGrath & Karahadian,

1994a). The texture (or mouthfeel) of the pawpaw pulp is characterized by body and astringency (Brannan et al., 2012). The term “viscosity” measures if the consistency of the pulp is thin or thick; “surface” indicates whether the pulp is smooth or pulpy; and

“body” describes the sensation of firmness, cohesiveness, and denseness when the fruit is compressed between the tongue and palate of the panelist (Brannan et al., 2012; Pomper et al., 2008). Two conclusions are made based on the results: a) pawpaw pulp has a

32

“smooth, somewhat thick, and custard or avocado-like texture (Kral, 1960; Pomper et al.,

2008) and b) the body of pawpaw pulp varies based on storage conditions. The pawpaw samples that were exposed to air exhibited more body, which is interpreted as thicker, than the vacuum packaged samples (Kral, 1960; Pomper et al., 2008).

2.1.4.4 Sweetness

The soluble sugar content in pawpaw pulp increases as the fruit is ripe (McGrath

& Karahadian, 1994b). As is mentioned in the pawpaw nutrition section, the soluble sugar content (° Brix) peaks at value of 22.5 ± 3.0 when the fruit is defined as ripe

(McGrath & Karahadian, 1994b). Since the soluble sugar content can be considered as an indicator of sweetness, the ripe pulp would be sweetest compares to unripe and semiripe groups. No research study that compares the soluble sugar content of overripe fruit versus ripe fruit is found.

2.1.4.5 Pawpaw flavor

The most intense flavors of pawpaw pulp are “banana and mango.” Apple, melon, citrus fruits (orange, grapefruit, and tangerine), papaya, pineapple, blackberry, and estery flavors were also mentioned. Besides, a “sweet, bitter and astringent” aftertaste is detected (Barth, 2015; Brannan et al., 2012; Callaway, 1990; Duffrin & Pomper, 2006;

Peterson, 1990; Raloff, 1992). A preference test among mango, papaya, and pawpaw pulps indicates that pawpaw is more favored than papaya (14% of the panelists) but less favored than mango (70% of the panelists) by the panelists. However, only 26% of panelists who voted pawpaw as favorite and less than 14% of the panelists in total could identify pawpaw flavor (Brannan et al., 2012). 33

The extremely short postharvest shelf life of pawpaw can affect its flavor. When stored at ambient temperature, the whole pawpaw fruit reaches its climacteric peak within

3 days postharvest and deterioration begins (Harris & Brannan, 2009). Signs like increased volatile flavor (Harris & Brannan, 2009; McGrath & Karahadian, 1994a) and soluble solids, softening and discoloration of the fruit (Fang, Wang, Xiong, & Pomper,

2007; Harris & Brannan, 2009; McGrath & Karahadian, 1994b), as well as elevated rate of enzymatic activities (Fang et al., 2007) occur. These all may impact consumers’ sensory acceptance of this fruit significantly.

2.2 Browning

Browning is a process that turns the color of the food brown. This process can happen naturally (such as browning of cut apple) or through processing techniques (such as caramelization), through nonenzymatic or enzymatic reactions. Nonenzymatic browning, commonly seen in baking and cooking, induces a series of reactions without enzyme participation. Caramelization and Maillard reaction are the two major branches of nonenzymatic browning. Enzymatic browning is caused by the browning reactions associated with enzyme activities. There are hundreds of enzymes that participate in enzymatic browning. These enzymes usually present in food naturally in an inactivated stage and will be activated under oxygen exposure. Therefore, enzymatic browning can also be called oxidative browning. Usually enzymatic browning leads to customers’ perceptions of spoilage and over-ripening of the fruit (Boyd, 2015). Thus, the browning of a fruit directly causes the decline of its market value because of low customer preferences. However, in some cases, browning is desired in food, such as in coffee,

34 cocoa, and dried fruits. Since browning can be an important characteristic of some foods and even can be an economical impact in food industry, the control of browning process in food industry is vital.

2.2.1 PPO

Enzymatic browning occurs under the action of multiple oxidative enzymes, and

PPO is a major enzyme that induces the reactions. PPO naturally presents in a wide spectrum of plants, and sometimes may function during food processing. PPO from different food sources functions differently.

As is shown the reaction below, typical enzymatic browning in fruits and vegetables occurs when colorless o-diphenol react with oxygen from the air, resulting in the formation of o-quinones that generate dark melanin after polymerization (Yoruk &

Marshall, 2003). This reaction is mainly catalyzed by PPO with partial work of peroxidase (Gomes et al., 2014). Therefore, the rate of PPO activity decides the level of browning of food. High PPO activity stimulates frequent oxidation of colorless o- diphenol l brown colored o-quinone that the food becomes darker in color. The PPO extracted from most plants and fruits catalyzes o-diphenol, while the one found in mushrooms is capable of oxidizing both o-diphenol and mono-diphenol (Sanchez-Ferrer,

Bru, Cabanes, & Garcia-Carmona, 1988).

!"#$%!!"#$#!"#$%& !!" � − ���ℎ���� + ������ � − ������� (Colorless) (Brown)

35

2.2.2 PPO activity in fruit

PPO activity in fruits can be influenced by many factors, such as storage time and ripeness. Research on PPO activity of male papaya (Spanish Hermaphrodite) versus female papaya stated that the total PPO activity of female papaya meets its maximum at 1 day (green) of storage, declines to a minimum at storage day of 5 (green-mature), and then increases again at day 10 (ripe). The PPO activity in male papaya increased during storage until day 10, and then declined to its minimum at day 15 (overripe) (Cano, Lobo, de Ancos, & Galeazzi, 1996).

According to these “ripeness versus PPO activity” studies (Campos-Vargas et al.,

2008; Lima de Oliviera, Guerra, Sucupira Maciel, & Souza Livera, 1994), the PPO activity is high in immature fruits, including Chrimoya (Annona cherimola Mill.) and

Soursop (Annona muricata), decreases when fruits are mature but yet ripe, reaches to the peak when fruit is fully ripe, and declines to the bottom of the curve when overripe. Wu

(2000) found that the PPO activity of sugar apple (Annona squamosal L.) increases continuously as it matures, and reach to 100% when the fruit is overly soft.

Due to PPO activity, the pawpaw pulp, according to preliminary unpublished research, starts to change color immediately after air exposure, and turns to dark brown within 120 min at ambient temperature. The PPO activity of pawpaw varies based on factors such as genotype. In the 7 genotypes of pawpaw that are tested, GT20 has the highest PPO activity (≈1.05 s!! mg!!) and GT 6 has the lowest (≈ 0.17s!! mg!!)

(Fang et al., 2007). Opposite to the conclusion drawn on fresh papaya fruit (Carica papaya) (Cano et al., 1996), no correlation was found between storage period and PPO

36 activity of frozen pawpaw pulp (Asimia Triloba L.) (Wang, 2013). However, the samples in the research by Wang (2013) were tested after 0, 2, 4, 6, 8, 10, and 12 months of frozen storage (-18 ℃ in the first 2 months, and -40 ℃ in the following 10 months).

Hence, the temperature of storage condition might affect PPO activity.

2.3 The Effect of Different Processing Factors on PPO Activity

To solve the unwanted browning problem triggered by PPO, research was conducted in various ways to decrease PPO activity in foods. In this literature review, the effects of variety, pH, temperature, high pressure processing (HPP), chemical addition, and storage period on PPO activity inhibition are discussed.

2.3.1 Variety

Research that compared twelve types of tropical fruits reveals the PPO activity in different types of fruits is significantly varied. This study found that Mangosteen and lulo varieties showed the highest PPO activity and passion fruit has the lowest, while soursop, an Annonacea fruit, possesses the third highest PPO activity (but significantly lower than

Mangosteen and lulo) among these twelve fruits (Falguera et al, 2012). The effect of variety on PPO activity is controversial. Study on hermaphrodite versus female papaya indicated that even though the trends of PPO activity between these two varieties vary during ripening and storage, the specific PPO activity of these two at ripe stage were not significantly different (Cano et al., 1996). However, as is mentioned in “PPO activity of pawpaw,” pawpaw fruits from different genotypes demonstrate different PPO activity

(Fang et al., 2007). In the seven genotypes of pawpaw that are tested, GT20 had the highest PPO activity and GT 6 had the lowest.

37

2.3.2 pH

PPO activity is dependent on the pH (Cano et al., 1996). pH alters the function of

PPO, an enzyme that possesses the properties of protein, by changing its structure. The optimum pH of PPO activity varies in different plant sources, but the general range for

PPO to be active is from pH 4 to pH 8 (Yoruk & Marshall, 2003). A processing or storage environment away from this pH range may significantly decrease the activity of

PPO so that the unwanted browning phenomenon can be inhibited. Therefore, chemical additives that both alter pH of the product and affect its flavor the least are preferred.

Examples of these chemical additives are citric acid, ascorbic acid, phosphoric acid, potassium phosphate, etc.

The pH optimum range that activates PPO activity varies among different fruits.

The PPO activity of “Golden delicious apple” increases along the pH of 4.5 to 5.5, peaks at 5.5, decreases when pH is above 5.5, and activity ceases when at pH of 7.5. An environment with pH lower than 4.5 or greater than 7.5 inhibits PPO activity to less than

70% of its original (Soysal, 2009). With chlorogenic acid as a substrate, the PPO in pears

(Pyrus communis L. cv. Conferencia) is most active when its environment is within pH of

4 through 5 (peaks at 4.5), and no activity is detected when pH is less than 3.5 or greater than 5.5. (Arias, Lopez-Buesa, Oria, & Gonzalez, 2007). Similarly, the research on

“Rocha pear” found that under pH of 3.0, the PPO activity can be completely inhibited

(Gomes et al., 2014). Study on papaya fruits (Carica papaya Cv. Sunrise) states that under pH of 6.5, the PPO activity reaches to maximum, but when the pH is lower than 5.0, its activity reduces by 80% (Cano et al., 1996). The pH optimum of active PPO in Papaya

38

Fruits (Carica papaya Cv. Sunrise) is observed more neutral than that in apple and pears that was mentioned previously. In order to amplify PPO activity inhibition, other processing techniques under a pH-controlled environment are applied. However, the association of HPP and an environment with a pH of 3.5 is only able to decrease the PPO activity of mango puree by no more than 40% (Guerrero-Beltrán, Barbosa-Cánovas,

Moraga-Ballesteros, Moraga-Ballesteros, & Swanson, 2006). By comparing the PPO activity from different groups of fruits, the optimal pH condition selected to depress PPO activity in general is less than 3.5, and the depression is more efficient as pH decreases.

More neutral pH range (6.0-6.5) is observed to be best for maintaining PPO to be active in fruits from Annonaceae family, which may share more characteristics with pawpaw since they come from the same family. The study on atemoya (Annona cherimola Mill. ×Annona squamosal L.) shows the pH optimum for PPO activity of pH

7.0 when with catechol (Chaves, De Souza Ferreira, Da Silva, & Neves, 2011). The PPO activity peak in soursop fruit (Annona Muricata, L.) is observed at pH of 7.5 with 0.01 M catechol as substrate (Lima De et al., 1994). However, the pH that maintains the highest

PPO activity in Pawpaw (Asimina Triloba L.) is 6.5, which is more acidic than that of

Atemoya (Annona cherimola Mill. ×Annona squamosal L.) and Soursop fruit (Annona

Muricata, L.). At least 80% of PPO activity from Annonaceae fruits can be reduced when pH of the environment is no greater than 4.0 (Cano et al., 1996; Chaves et al., 2011; Fang et al., 2007; Guerrero-Beltrán et al., 2006; Lima de Oliviera et al., 1994). 39

2.3.3 Temperature

Temperature affects PPO activity in fruits, therefore, indirectly controls the browning rate. As mentioned previously, the browning process of fruits occurs due to the oxidation of colorless phenol compounds to brown colored quinone through PPO catalysis. The rate of this reaction is closely regulated by the activity of PPO that is naturally presented in the fruit. Temperature interferes with the speed of this reaction by affecting PPO activity. Based on theory, the rate of catalyst activity increases as temperature becomes higher, reaches its peak at optimum temperature, and the reaction ceases as temperature continues to elevate due to denaturation of the enzyme through break down of its three-dimensional structure. In addition, the solubility of oxygen, which is the major substrate in the corresponding enzymatic browning and further reaction, is also highly dependent on temperature (Yoruk & Marshall, 2003). Therefore, research strives to find the temperature optimum that both inhibits of PPO activity in its maximal effect and maintains the original property (including flavor, appearance, texture, etc.) of the fruit.

The temperature of heat treatment is negatively correlated with PPO activity reduction on green tea extract added to “Golden Delicious” apple. Results of the present study reflects that 80 ℃ heat treatment for at least 20 min associated with addition of green tea extract could reduce PPO activity by more than 90%; while heat treatment of 60

℃ or 70 ℃ for the same time length can only weaken the activity by at most 40%

(Soysal, 2009). Both ripe mango and its enzyme extract were tested to determine the inhibiting effect of PPO activity of two levels of thermalizations (50 ℃ or 60 ℃) for

40 varied time length (1 hr, 2 hr, and 4 hr). It is concluded that 4 hr of heat treatment under temperature of 60 ℃ reduces the PPO activity the most (nearly 85% in enzyme extract and 98% in ripe mango pulp) (Korbel, Servent, Billaud, & Brat, 2013). Results form these two heat treatment studies indicated that HTST (High Temperature Short Time) treatment (80 ℃ for at least 20 min) and LTLT (Low Temperature Long Time) treatment

(60 ℃ for 4 hr) both are able to deduct PPO activity of fruits by over 90% (Korbel et al.,

2013; Soysal, 2009).

Narrowing down the study subjects to Annonaceae fruit family, the results from the two available studies on Atemoya (Annona cherimola Mill. ×Annona squamosal L.) revealed a positive correlation between PPO activity and temperature before the temperature optimum is achieved. The PPO activity in Chaves et al. (2011) elevates from

10 ℃ to 30 ℃, meets maximum at 30 ℃ to 35 ℃, and decreases from 35 ℃ till 55 ℃. The

PPO activity of the control group (Torres, Sliva, Guaglianoni, & Neves, 2010) is moderately higher than the 20 ℃ heat-treated group, and the 40 ℃ heat-treated group is one-third lower than 20 ℃ heat-treated group. Unlike Chaves et al. (2011), the relationship of PPO activity and temperature is displayed as a downward linear trend in this study. Based on these two studies, a heat treatment with a temperature over 40 ℃ is suggested in order to receive the most effective PPO inactivation. The temperature PPO versus thermalization studies that conducted on Annonaceae fruits are very limited, and relevant research on pawpaw fruit is needed. 41

2.3.4 HPP

High pressure processing (HPP), also referred as high hydrostatic pressure processing or ultra high-pressure processing, is an increasingly popular processing technique used by fresh food manufactures today to prevent disfavored browning of products through the inactivation of the correlated enzymes, such as PPO. Through elevated pressures (between 100 and 800 MPa) under a low-heat or a no-heat condition, with or without package, the enzymatic inactivation or alteration of the food attributes to desired qualities, such as color preservation, are achieved by HPP application. Besides,

HPP can denature pathogenic organisms that are present in fresh food products without thermalizing them. Compared to heat-applied pasteurization, the nonthermal pasteurization methods are less promising for enzyme degradation and microbial inactivation, but they perform better on nutrients and flavor preservation (Chen, Yu, &

Rupasinghe, 2013).

As a new alternative to the traditional food processing and preservation methods that are mostly dependent on pasteurization, HPP can avoid damage to foods that are caused by high temperature through elevated pressure (Oey, Lille, Loey, & Hendrickx,

2008). Hence, the HPP processed products may be able to preserve more original status of the products, including their texture, flavor, and color due to the insignificant effect on the covalent bonds of low molecular-mass compounds that are used for color and flavor

(Oey et al., 2008). Since HPP is a relatively new technique, it has only been applied to

42 browning prevention and sterilization of certain products such as fresh juices, salsas, and soups to a very limited degree.

Researchers (Asaka & Hayashi, 1991; Barba et al., 2014; Dajanta,

Apichartsrangkoon, & Somsang, 2012; Guerrero-Beltran, Barbosa-Canovas, & Swanson;

Sulaiman & Silva, 2013; Woolf et al., 2013) have reported the effects of HPP on PPO activity inhibition on multiple types of fruits and have detected a significant reduction of activity when the pressure of HPP is over 500 Mpa. The PPO activity of Lychee in syrup

(Dajanta et al., 2012) and ‘Camarosa’ strawberry puree (Sulaiman & Silva, 2013) is reduced by 80% and 50-70%, respectively, under HPP with a pressure of 600MPa for 15 to 20 min. A 54% reduction of PPO activity is detected in the vacuum packed fresh-cut peaches processed by HPP (Garcia-Parra, Gonzalez-Cebrino, Cava, & Ramirez, 2014). In a study conducted on avocado slices, the effectiveness of PPO activity reduction by HPP under three different pressure levels (200MPa, 400MPa, and 600MPa) is tested. However, no activity reduction is found until the pressure reaches 600 MPa (Woolf et al., 2013).

Although most results have concluded that HPP has a significant effect on inhibiting PPO activity, higher PPO activity compared with the control was found in the study on avocado slices and peach puree when 200 MPa and 400 MPa pressure was given, respectively (p < 0.05) (Asaka & Hayashi, 1991; Woolf et al., 2013). Combining the microscope observation in the study by Woolf et al. (2013), it is speculated that the cell membrane breakdown after high pressure exposed the enzymatic residues from the cell, and triggers the elevated enzyme activity (Duong, Balaban, & Perera, 2015). No literature was found on HPP processed Anonnacea fruits or pawpaw (Garcia-Parra et al., 2014).

43

The application of chemical enhancer amplifies the effect of HPP on PPO inactivation. The research on ascorbic acid-treated peach puree associated with HPP under 517 MPa at 25 °C for 20 min was able to inactivate more than 95% of the PPO in the samples (Guerrero-Beltran et al., 2004). Another study used a 2.5% (w/v) stevia addition in a mixed papaya, mango and orange puree as a PPO inactivation enhancer. An

HPP of 500 MPa at 25 °C for 15 min was applied. Results demonstrated a 98% reduction of PPO activity in the puree (Barba et al., 2014). The conclusion can be made that the addition of proper amounts of ascorbic acid and stevia as HPP enhancers can significantly improve PPO inactivation of the food product.

2.3.5 Chemical additives

McEvily, Iyengar, and Otwell (1992) categorized PPO inhibiting chemicals that available in the food industry into six groups based on their mode of action:

1. Reducing agents (ascorbic acid and analogues, sulfates),

2. Chelating agents (ethylenediaminetetraacetate [EDTA], sodium diethyl

dithiocarbanate [DIECA], sodium azide,

3. Complexing agents (cyclodextrins, chitosan),

4. Acidulating agents (ascorbic acid, citric acid, malic acid, phosphoric acid),

5. Enzyme inhibitors (substrate analogs, halides), and

6. Enzyme treatments (proteases, o-methyltransferase).

These agents diminish or inhibit the PPO involvement in browning reaction by eliminating the active elements, such as enzymes and substrates, from the reaction

(Yoruk & Marshall, 2003). Though these compounds all disrupt the undesired browning

44 at different degrees, it can be determined whether they are appropriate to be applied to particular food processing procedures by multiple considerations, including the sensory impact of the inhibitors to the food, efficiency, cost, the impact of the chemical to the other processing steps, and the health impact on consumers. In this literature review, the effect of ascorbic acid and stevia is studied and discussed.

2.3.5.1 Ascorbic acid

Ascorbic acid, commonly recognized as vitamin C, has two functions in PPO inactivation. First, it is an active antioxidant in nature. Ascorbic acid has an unstable structure that allows it to react with oxygen first rather than PPO. For instance, a freshly cut apple would turn brown slower if its surface were covered by lemon juice, which contains large amount of ascorbic acid or vitamin C. This is because when oxygen in the air comes in contact with the surface of the apple, vitamin C reacts with those oxygen molecules faster and so there is no oxygen remaining for PPO in the apple. Once the vitamin C is used up, the apple scar starts to turn brown because the PPO that is waiting in line to have interaction with oxygen turns to quinones, which is the main substance that causes browning. By knowing the antioxidation capacity of ascorbic acid, part of this research is designed to study the effectiveness of ascorbic acid as a chemical enhancer of

HPP on PPO inactivation. In spite of this, ascorbic acid is also an acidulating agent, which alters pH of the food and the pH optimum of PPO activation can be avoided. This was already explained in detail in the “pH” section above.

Ascorbic acid is the one of the most developed browning inhibiting additive because it has been used in the food industry for a prolonged period. Its PPO inhibitory 45 ability is usually tested when it is added as antioxidant, pH adjuster, or as an HPP enhancer. Ascorbic acid is evaluated as a excellent inhibitor of the PPO extracted from

“ Rocha” pear (Chaves et al., 2011), bosc and red pear (Siddiq, Cash, Sinha, & Akhter,

1994), and lychee (Sun et al., 2008) because it is able to inhibit PPO activity by up to

98.1% (Chaves et al., 2011; Siddiq et al., 1994; Sun et al., 2008) . Regarding the impact of ascorbic acid on the PPO in Annonaceae fruits, study have shown that the addition of ascorbic acid in Atemoya fruit (Annona cherimola Mill. ×Annona squamosal L.) can inhibit PPO activity up to 75.2% after a lag phase of 5 min (Chaves et al., 2011).

Although few studies support this conclusion, ascorbic acid has less impact on PPO activity in Annonaceae fruits.

Second, ascorbic acid is an efficient HPP enhancer. Related research on ascorbic acid-treated apple extracts (Anese, Nicoli, Dallaglio, & Lerici, 1995) and peach puree with HPP (Guerrero-Beltran et al., 2004) conclude that the residue percentage of PPO at

414 MPa reduced by 75% and that at 537 MPa reduced by 100%, while the PPO production of the groups that were HPP processed with below 414 MPa all increased as pressure decreased. These results demonstrate that HPP at adequate pressure level associated with ascorbic acid addition is able to significantly suppress PPO activity.

2.3.5.2 Stevia

Stevia is commonly seen in the market as a natural noncaloric sweetener

(Gregersen, Jeppesen, Holst, & Hermansen, 2004). People recognize stevia as natural because it is extracted from Stevia Rebaudiana Bertoni, a leafy plant with over 150 different species (Abdulateef & Osman, 2011). The stevia leaves could be consumed

46 fresh or when dried and their sweetness can be released by infusing the leaves in tea.

Stevia is an ideal sweetener for people who are glucose intolerant since steviosides, the main component in stevia, are proved to have a positive effect on decreasing blood sugar in patients with type 2 diabetes and the blood pressure in patients with mild hypertension

(Gregersen et al., 2004; Jeppesen et al., 2003).

Research on diabetic mice reported that the stevioride in stevia passes through the digestive process directly without chemically breaking down, which makes it a safe sweetening substance for the people who want to regulate their blood glucose level

(Misra et al., 2011). It is also found that the leaf extracts of Stevia Rebaudiana, with the concentrations of 200 and 400 mg/kg base on the consumer’s body weight, decreased the blood glucose level significantly from 403 mg/dl on the third day of observation to 93 ml/dl on the tenth day of observation without causing hypoglycemia. Meanwhile, stevia causes less weight-loss for the patients with diabetes as compared to some of the commonly used drugs, such as glibenclamide (Misra et al., 2011). Therefore, stevia can be considered as beneficial to humans. In 2008, FDA began to allow stevia to be used as a sweetener and granted GRAS (Generally Regarded as Safe) status to it (Wood, 2011,

2015).

The sweet taste in stevia leaves is caused by eight kinds of glycosides. Stevioside, which is 250 to 300 times sweeter than a 0.4 % table sugar solution, is considered the sweetest of these eight glycosides (Lemus-Mondaca, Vega-Galvez, Zura-Bravo, & Ah-

Hen, 2012). Relevant research demonstrates that dry stevia leaves contain various types of minerals, vitamins, antioxidant compounds such as phenolic compounds and

47 flavonoids (Lemus-Mondaca et al., 2012; Tadhani et al, 2007). Stevia is recognized as having antimicrobial (Belda-Galbis et al., 2014; Tadhani & Subhash, 2007) and antioxidant properties (Barba et al., 2014; Muanda, Soulimani, Diop, & Dicko, 2011).

Besides the potential benefits on human health (Barba et al., 2014), it is also hypothesized that stevia can be a potential additive to inhibit the unexpected oxidation reactions in some of the fresh produce that turn brown quickly after oxygen exposure.

A comparison review of the antioxidant capacity of five medicinal plants indicates that among Pueraria mirifica, Curcuma longa Linn, Andrographis paniculata

(Burm.f.) Nees, and Cassia alata Linn, Stevia rebaudiana Bertoni has the highest Trolox equivalent antioxidant capacity, which explains the ability of the measured item combining with oxygen molecules in the air to prevent unexpected oxidation from happening (Phansawan & Poungbangpho, 2007).

Research showed that the addition of 2.5% (w/v) stevia rebaudiana Bertoni combined with HPP at 453 MPa for 5 min suppressed PPO activity in mixed papaya, mango and orange puree by approximately 99% (Barba et al., 2014). Different levels of

HPP and addition of varied amounts of stevia rebaudiana Bertoni concentrates from dry leaves infusion were conducted on a pureed orange, mango, and papaya pulp mixture

(Barba et al., 2014). The results suggested that a treatment of 453 MPa applied for 5 min combined with 2.5% (w/v) stevia maximized TPC content and antioxidant activity, while minimizing PPO activity (Barba et al., 2014). Since the use of stevia on PPO activity inhibition is innovative, the resources on “stevia as a PPO inhibitor (C)n fruits” are very limited.

48

2.3.6 Storage period

The relationship between storage period and PPO activity in fruits is unclear.

Study on Cherimoya (Annona cherimola Mill.) storage stated that the PPO activity of the fruit that stored under 0 ℃ refrigeration was lower in the twelfth day than in the sixth day of storage (Campos-Vargas et al., 2008). In other words, the PPO activity decreases as storage period increases. However, the research that compared the PPO activity among the pawpaw samples after 0, 2, 4 , 6, 8, 10, and 12 months of frozen storage (-18 ℃ in the first 2 months; -40 ℃ in the flowing 10 months) indicated that no correlation is found between storage period and PPO activity (Wang, 2013).

In conclusion, the pH of fruit’s environment, heat treatment, HPP, and browning inhibitors (this research focuses on ascorbic acid and steviosides), all are found to have varied effects on fruit’s browning prevention and PPO activity inhibition. Meanwhile, insufficient evidence is found on the correlation of variety and storage time with PPO activity of fruits. The comprehensive review of these factors is essential for the inspiration of how these technologies can be applied to prevent pawpaw browning and to inhibit the PPO activity in pawpaw.

49 Chapter 3: The Effects of High Pressure Processing, Browning Additives, and

Storage Period on the Inactivation of Polyphenol Oxidase in Nine Varieties of

Pawpaw (Asimina Triloba L.) Pulp1

3.1 Abstract

This study measured and compared the effects of high pressure processing (HPP), browning inhibitors (pasteurization, ascorbic acid, and steviosides), storage time (0 day,

15 days, 30 days, and 45 days at 4 °C), and pawpaw variety (Belle, Mitchell, PA-Golden,

Pickle, Seedling, Sunflower, Shenandoah, SAB OL, Wilson) on the polyphenol oxidase

(PPO) activity and the color of pulp from the pawpaw fruit. In a related experiment, a descriptive sensory analysis was conducted on a single variety of pawpaw fruit

(Shenandoah) using trained sensory panelists on pulp treated using the variables described above.

There were differences observed among the nine pawpaw varieties studied.

Before treatment, ranking for highest to lowest PPO activity was Seedling, Mitchell,

Sunflower, Shenandoah, Wilson, PA-Golden, Pickle, SAB OL, and Belle. HPP significantly decreased the PPO activity in all of the varieties except for Belle and Pickle.

Overall, the browning inhibitors significantly inhibited PPO activity. There were a few exceptions. Specifically, ascorbic acid and steviosides increased PPO activity in both

SAB OL and Shenandoah samples, and pasteurization did not affect PPO activity in

1 This chapter represents a prepublication manuscript submitted to Journal of Food Research, which has been adapted slightly to conform to Ohio University’s thesis format. Authors are Lin Zhang (School of Applied Health Sciences and Wellness, College of Health Sciences and Professions, Ohio University, Athens, OH) and Robert G. Brannan (School of Applied Health Sciences and Wellness, College of Health Sciences and Professions, Ohio University, Athens, OH). 50

Mitchell and increased PPO activity in Shenandoah. A general decreasing trend of PPO activity was detected with the greater storage period among all the HPP treated varieties.

Samples processed by HPP exhibited a darker, redder, and less yellow color, which is equivalent to more browning development. Because HPP is an excellent treatment for PPO inactivation but might cause undesirable browning of the pawpaw pulp, its commercialization potential is moderate but could be improved in combination with an effective browning inhibitor. Ascorbic acid, steviosides, and pasteurization all inhibited PPO activity. Ascorbic acid preserved the bright, less red, and yellow color of the fruits; therefore, its commercialization potential is high. Steviosides and pasteurization inhibited PPO activity but the pulps treated with steviosides exhibited increased redness and pasteurization, and led to darker and less yellow color change of the pulp. Therefore, the commercialization potential of steviosides and pasteurization is medium.

Data from the descriptive sensory analysis revealed that HPP and pasteurization altered the color of the samples, with HPP promoting browning and pasteurization inhibiting browning. The samples treated with steviosides had a sweeter and more bitter taste than the untreated control. Among the treated samples, these findings suggest that ascorbic acid performed the best among the selected treatments to maintain the color and taste of Shenandoah pawpaw pulp.

Overall, ascorbic acid treatment had the highest commercial potential for pawpaw browning inhibition. The results of this study are expected to be useful to the food 51 industry when selecting proper methods to prevent pawpaw or Annonaceae fruit browning, especially.

KEYWORDS: Pawpaw, Browning, Polyphenol oxidase (PPO), High pressure processing (HPP), pasteurization, ascorbic acid, Steviosides, storage, variety, descriptive sensory analysis

3.2 Introduction

The pawpaw (Asimina triloba L.) is a fruit that is cultivated in eastern North

America (Layne, 1996; Pomper & Layne, 2010). It belongs to the tropical Annonacea fruit family even though it grows in temperate climate (Callaway, 1990). The pawpaw fruit ripens in late summer and early fall and appears kidney shaped, light green to yellow, with a size that varies from 5 cm to 20 cm long (Brannan & Salabak, 2009; Callaway,

1990). The ripe fruit has a complex sweet, fruity, and slightly fermented aroma, and intense flavors of banana and mango (Brannan, Salabak, & Holben, 2012; Goodrich,

Zjhra, Ley, & Raguso, 2006; McGrath & Karahadian, 1994). The texture is described as soft, custard or avocado-like (Brannan et al., 2012; Kral, 1960; McGrath & Karahadian,

1994; Pomper et al., 2008). Studies on pawpaw nutrition show that 100 g pulp contains

64 kcal energy (U.S. Department of Agriculture, 1963), 74.1 ± 3.0 % moisture (Brannan et al., 2015; McGrath & Karahadian, 1994), 19.6 ± 8.4 (° Brix) soluble sugar content,

0.84 ± 0.37 g fat (McGrath & Karahadian, 1994b; Peterson, Simmons, & Cherry, 1982;

U.S. Department of Agriculture, 1963), and 4.7 ± 3.9 g protein (McGrath & Karahadian,

1994; Peterson et al., 1982; U.S. Department of Agriculture, 1963).

52

The pawpaw fruit is highly susceptible to enzymatic discoloration, which is mainly attributable to polyphenol oxidase (PPO) activity (Yoruk & Marshall, 2003). PPO catalyzes colorless o-diphenol in the pulp to the formation of o-quinones that generate dark melanin after polymerization (Yoruk & Marshall, 2003). Therefore, the level of PPO activity plays a role in the discoloration rate of food. The PPO activity in fruits can be influenced by multiple factors, including fruit variety ((Falguera et al, 2012; Fang, Wang,

Xiong, & Pomper, 2007), pH (Arias, Lopez-Buesa, Oria, & Gonzalez, 2007; Cano, Lobo, de Ancos, & Galeazzi, 1996; Chaves, Ferreira, Silva, & Neves, 2011; Fang et al., 2007;

Gomes, Vieira, Fundo, & Almeida, 2014; Guerrero-Beltrán, Barbosa-Cánovas, Moraga-

Ballesteros, Moraga-Ballesteros, & Swanson, 2006; Lima de Oliviera, Guerra, Sucupira

Maciel, & Souza Livera, 1994; Soysal, 2009; Yoruk & Marshall, 2003), processing techniques such as pasteurization (Torres, Sliva, Guaglianoni, & Neves, 2010; Soysal,

2009), addition of chemical inhibitors (Barba, Criado, Belda-Galbis, Esteve, & Rodrigo,

2014; Chaves et al., 2011; Guerrero-Beltran, Barbosa-Canovas, & Swanson, 2004;

Siddiq, Cash, Sinha, & Akhter, 1994; Sun et al., 2008), and storage time (Campos-Vargas et al., 2008).

High pressure processing (HPP), also referred as high hydrostatic pressure processing, is a nonthermal, pressure-applying technique used for preventing disfavored browning of fresh produce by inactivating the correlated enzymes, such as PPO. Research comparing HPP to thermal pasteurization suggests that HPP is better with respect to nutrient, flavor, and color preservation but less promising for enzyme degradation and microbial inactivation (Chen, Yu, & Rupasinghe, 2013; Oey, Lille, Loey, & Hendrickx, 53

2008). Research on fruits processed by HPP have shown a significant reduction of PPO activity when the pressure of HPP is over 500 Mpa (Asaka & Hayashi, 1991; Barba et al.,

2014; Dajanta, Apichartsrangkoon, & Somsang, 2012; Guerrero-Beltran et al., 2006;

Sulaiman & Silva, 2013; Woolf et al., 2013). PPO inactivation by HPP may be enhanced with the application of chemicals (Barba et al., 2014; Guerrero-Beltran et al., 2004). For example, peach puree treated with ascorbic acid before HPP (517 MPa at 25 °C for 20 min) treatment inactivates more than 95% of the PPO (Guerrero-Beltran et al., 2004).

Similarly, the addition of 2.5% (w/v) stevia addition before HPP (500 MPa at 25 °C for

15 min) resulted in a 98% reduction of PPO activity in a mixed papaya, mango and orange puree (Barba et al., 2014).

The objective of this study was to assess the effect of HPP, addition of chemical inhibitors (ascorbic acid and steviosides), and storage on PPO inhibition in nine varieties of pawpaw fruit.

3.3 Materials and Methods

3.3.1 Materials

Nine varieties of the pawpaw samples were donated and manually harvested from

Fox Paw Ridge Farm, Cincinnati, Ohio in September 2014. The nine pawpaw varieties, in alphabetical order, are Belle (B), Mitchell (MT), PA-Golden (PAG), Pickle (PC), SAB

OL (SAL), Seedling (SEED), Shenandoah (SHEN), Sunflower (SF), and Wilson (WIL).

The whole pawpaw fruits were labeled by variety, numbered, and were transported back to Athens in coolers on ice.

54

3.3.2 Experimental design and sample preparation

Immediately after harvest and transport, the weight, length, width, skin and pulp color (measured by CIE colorimeter, Konica Minolta, NJ), sugar content (measured by

B108 portable refractometer, E-Z Red), pH (measured by AB15 Plus pH meter, Fisher

Scientific), and hardness were measured for each individual pawpaw fruit. The skin and seeds were removed from the pulp manually. The pulp obtained from individual fruits from each variety was pooled and divided into forth portion. As described in detail below, one lot was treated with 0.18% (w/w = 0.18 g powder/ 100 g pawpaw pulp) steviosides (Hard Rhino U.S.A., Phoenix, AZ) based on previous research (Barba et al.,

2014), another lot treated with 400 parts per million (ppm) +99% L-ascorbic acid powder

(Fisher Scientific, PA), and one other lot received a 60 ℃ water bath for 30 min in sealed polyethylene/nylon FoodSaver 27.94-cm bags. The remaining pulp was chemically untreated. The pulp from each of the nine lots was assigned into one of 288 prelabeled bags (6 g of pulp in each bag), each representing a treatment group according to the 9 × 4

× 4 × 2 full-factorial design, which will be described later and is shown in Table 1. The bags, polyethylene/nylon FoodSaver 27.94-cm bags (Jarden Corp., Rye, NY) with an oxygen transmission rate of 6.7 cc/m2/24 h/23°C/0% RH, were sealed under vacuum using a VACMASTER vacuum sealer (Overland Park, KS). During all of the procedures described above, every precaution was taken to keep the pulp cold, including storing on ice or in the refrigerator. All 288 bags of pulp were stored at 4 °C for not longer than 12 hr until they were transported to the HPP facility where half of the samples were subjected to HPP as described below 55 Table 4

The Independent Variables Utilized for the 9 × 4 × 4 × 2 Factorial Design in this Study

Factors Levels

Factor category 1: Variety Nine varieties: Belle (B) Mitchell (MT) PA-Golden (PAG) Pickle (PC) Seedling (SEED) Sunflower (SF) Shenandoah (SHEN) SAB OL (SOL) Wilson (WIL)

Factor category 2: Browning Inhibitors None (C); 0.18% (w/w) Steviosides (S); 60 °C/ 30 mins Pasteurization (T); 400 ppm Ascorbic acid (AA).

Factor category 3: Processing methods HPP (HPP) (4 °C/ 600 MPa/ 76 secs); None-HPP (NHPP).

Note. Sample labeling codes of treatments are listed in parenthesis above.

3.3.3 HPP treatment

All 288 samples bags were transported to the HPP facility (Sandridge Food manufacture, Ohio, U.S.A.) on ice. At the facility, the 144 samples designated for HPP treatment were packaged into large bags and sealed using a MULTIVAC Thermosealer

(Switzerland) before being loaded into the pressure vessel. The remaining 144 samples remained in the cooler. The HPP samples were subjected to 600 MPa of pressure at ambient temperature (4 °C) for 76 s as recommended by previous research (Asaka &

Hayashi, 1991; Barba et al., 2014; Dajanta et al., 2012; Guerrero-Beltran et al., 2004;

Woolf et al., 2013). The pressure and time were controlled automatically during 56 processing. The samples sealed in the bags were transferred into iced water immediately after pressurization. All samples were transferred to the laboratory on ice (approximately

3 hr).

3.3.4 Post-treatment storage

Upon return from the HPP facility, samples designated as 0 day were immediately transferred to frozen storage (- 40 °C). The remaining samples were stored refrigerated

(4 °C) and transferred to frozen storage (- 40 °C) at their designated storage time, i.e., 15,

30, or 45 days.

3.3.5 Color measurement

A calibrated colorimeter (Konica Minolta, NJ) was used to determine C.I.E. L*, a*, and b* values. The pulp samples were placed into 2 oz disposable plastic sample cups, covered with plastic wrap such that air gaps between the wrap and the pulp surface were eliminated, and monitored for color by placing the colorimeter directly in contact with the plastic wrap. Means (± SD) were calculated using measurements from each individual fruit (skin and exposed pulp) or the triplicate values from each of the 288 sample treatments.

3.3.6 PPO activity determination

PPO activity was determined from crude enzyme extracts based on their protein content and the slope obtained from the PPO enzyme assay. Specific conditions for crude enzyme extract, protein content, and the PPO assay are described in detail below.

57

3.3.6.1 Crude enzyme extract preparation

Crude enzyme extracts were prepared in triplicate for each of the 288 samples according to published methods (Fang et al., 2007), as follows. Pawpaw pulp was thawed quickly and pulp (2 g) mixed with 3 mL of 0.2-M sodium phosphate

(Na2HPO4/NaH2PO4) buffer (pH 6.5) containing 5% (w/v) polyvinylpolypyrrolidone

(PVPP), 2% (w/v) Amberlite XAD-4 and 2% (v/v) Triton X100. After vortexing for 30 s at room temperature, the mixture was chilled in an ice water for 5 min and then centrifuged (IEC Model HN-S Centrifuge, Needham Heights, MA) at 3000 g at room temperature for 10 min. The supernatant (1 ml) from each sample was transferred to microcentrifuge tubes and centrifuged at 18,000 rpm (30065 × g, radius 8.3 cm) for 20 min at 4 °C using a Eppendorf 5417r refrigerated centrifuge (Hauppauge, NY). The supernatant, which contains the crude PPO, was kept at 0 °C before subjecting to protein and enzyme activity measurements, which were performed on the same day as extract preparation.

3.3.6.2 Lowry protein assay

Soluble protein in the supernatant was determined using the Lowry method. The

Lowry reagent was prepared by mixing 49 ml of 2% Na2CO3) in 0.1N NaOH solution with 0.5 ml of 1% CuSO4.5H2O and 0.5 ml of 2% KNaC4H4O6·4H2O. Three replicates for each crude enzyme extract was prepared by diluting 1:10 with distilled H2O to which

Lowry reagent (1 ml) was added. After 10 min, 50% Folin & Ciocalteu’s (FC) phenol reagent (100 ul) was added and the mixture was incubated at room temperature for 30 min. Absorbance was monitored using a Spectronic Genesys 5 Spectrophotometer 58

(Thermo Electric Corporation, Madison, WI) at 750 nm. Protein was quantified based on a standard curve prepared from pure albumin.

3.3.6.3 PPO enzyme assay

PPO activity was measured as the increase in absorbance over time when a PPO substrate (+99% catechol, Sigma-Aldrich) was added to the crude extracts. Pawpaw crude enzyme extract (33 ul) and 2 M phosphate buffer at pH = 6.5 (0.9 ml) was mixed for 5 min, after which catechol solution (0.4 ml), prepared by mixing 0.33 g catechol

(Sigma-Aldrich) with 15 ml distilled water, was added. Absorbance at 420 nm was monitored every 10 s for 60 s. The slope of the increase in absorbance was calculated and

PPO enzyme activity reported as ∆ABS/min/g protein.

3.3.7 Descriptive Sensory Evaluation

To protect the safety of human subjects, the completed protocol was viewed and approved by the Ohio University Institutional Review Board before the sensory analysis began. Nine participants who had been previously recruited and trained to perform descriptive tests were utilized to evaluate 12 sensory attributes of the Shenandoah (SHEN) pawpaw pulp. Variety Shenandoah was selected to among the nine variety pawpaw fruits because its volume was sufficient to serve to the panelists. Eight coded samples from the variety Shenandoah were presented to the panelists, which are categorized as a) No HPP, no chemical additive; b) No HPP, ascorbic acid-treated; c) No HPP, steviosides-treated; d)

No HPP, pasteurization-treated; e) HPP, no chemical additive; f) HPP, ascorbic acid- treated; g) HPP, steviosides-treated; h) HPP, pasteurization-treated. The nine trained panelists were given additional training focused on perception of flavors from the 59 complex flavor profile of the pawpaw (Brannan et al., 2012). The panel utilized a specialized pawpaw sensory lexicon (Brannan et al., 2012; McGrath & Karahadian,

1994) to describe the flavors that they detected. The pawpaw lexicon is referred from

Brannan et al. (2012). Panelists were presented with 25 g of each sample at room temperature (25 ℃) in small white plastics cups labeled with randomly coded with three- digit numbers. One sample was presented at a time. Panelists had access to purified water and unsalted saltine crackers between tastings. Responses were recorded by ballots with a

15-cm line scale anchored with standards.

3.3.8 Statistical Analysis

The 9 × 4 × 4 × 2 full-factorial experimental design shown in Table 4 was analyzed statistically using SPSS version 16.0 for Windows (Seattle, WA). All measurements were generated based on three trials (n = 3). Analysis of variance

(ANOVA) was used to determine differences between the means of the main effects at a significance level of p < 0.05. Means was separated using the post-hoc Duncan’s

Multiple Range test.

The large dataset was split by variety such that nine individual 4 x 4 x 2 full- factorial analyses were undertaken. The purpose of splitting the file by variety was to simplify the analysis. As before, Analysis of Variance (ANOVA) was used to determine differences between the means of the main effects at a significance level of p < 0.05.

Means was separated using the post-hoc Duncan’s Multiple Range test.

Sensory analysis was analyzed by ANOVA with the six group differences determined by Duncan’s Post Range test. 60

3.4 Results and Discussion

The main effect of processing (HPP versus non-HPP) showed that HPP significantly decreased PPO activity (p < 0.001) by 65%, from 16.9 ∆ABS/min/g protein to 6.1 ∆ABS/min/g protein, across all varieties and inhibitors. Previous research indicates that HPP has an effect on browning prevention and PPO inhibition in fruit (Barba et al.,

2014; Dajanta et al., 2012; Guerrero-Beltran et al., 2004; Sulaiman & Silva, 2013); however, the samples were browner as indicated by the fact that the samples treated with

HPP were significantly darker (lower L*), redder (higher a*), and less yellow (lower b*).

The same change is also detected in avocado slices and pear fruits (Asaka & Hayashi,

1991; Woolf et al., 2013).

The main effects of browning inhibitors (none, steviosides, pasteurization, ascorbic acid) were examined in relation to their potentially synergistic effect with HPP.

In the untreated control samples from each variety, HPP significantly reduced PPO activity (p < 0.001). Heat treatment to 60 ℃ (pasteurization) enhanced the PPO inhibiting effect of HPP, with heat-treated HPP samples exhibiting a 79% lower PPO activity than heat-treated samples that were not high pressure processed. Ascorbic acid, a known browning inhibitor, and steviosides were not as effective as the heat treatment at reducing

PPO activity in HPP-treated samples compared to non-HPP (NHPP) samples as both exhibited a 65% reduction in HPP-treated samples compared to NHPP treated samples. In addition, pasteurization caused darker (lower L*) and less yellow (lower b*) color change

(also observed in descriptive sensory evaluation), steviosides caused more redness 61

(higher a*), while ascorbic acid addition caused a lighter and yellower color change of the samples.

The two-way interaction of storage and HPP was examined. There were differences and similarities between the PPO activity of control and HPP samples during storage (see Table 5). The main difference was the fact that the overall magnitude of PPO activity values of the NHPP samples were three times higher than the HPP samples.

However, the trend of PPO activity during storage was similar. HPP samples exhibited a significant decrease (p < 0.001) from 11 ∆ABS/min/g protein at day 0 of storage to 5.07

∆ABS/min/g protein at day 15 of storage, and did not change significantly from day 15 to day 45 of storage (3.88 ∆ABS/min/g protein). Similarly, a significant decrease of PPO activity in the first 30 days of storage period was detected in the NHPP processed pawpaw samples (p < 0.001), after which the PPO activity decline of the NHPP group ceased at 30 days of storage instead of 15 days in the HPP group. Both trends implied that the PPO activity of pawpaw decreases in a decelerated rate along the storage time.

HPP stabilizes the PPO activity of pawpaw pulp in a faster rate compared to the control

(see Table 5). Similar to the conclusion on pawpaw color from previous research

(Brannan et al., 2012), prolonged storage (4 ℃) produced darker, redder, and less yellow color of the pawpaw pulp. 62

Table 5

Comparison of the PPO Activity of the HPP Versus NHPP Samples Under Different Storage Times

0 day 15 days 30 days 45 days

HPP 11 5.07 4.67 3.88

NHPP 25.21 20.62 10.13 11.85 Note. PPO activity = ∆ABS/min/g protein.

3.4.1 The effect of the treatments in each of the nine pawpaw varieties

Before the data was split by variety and analyzed, the main effect of variety was

examined. Variety influenced both the PPO activity (p < 0.001) and the color (p < 0.001)

of the pawpaw pulp (see Tables 6-14). Prior to treatments, Seedling possessed the highest

PPO activity and Belle had the lowest. The effects of the different treatments on color

and PPO activity within each variety are explained in detail below. Variety Mitchell

exhibited the darkest color, Belle pulp the reddest, and Mitchell the yellowest color.

3.4.1.1 Variety 1: Belle

The PPO activity and color of Belle pawpaw pulp under different processing

conditions are shown in Table 6.The application of both HPP (p < 0.001) and browning

inhibitors (p < 0.022) exhibited a significant effect on the PPO activity of Belle pawpaw

pulp. All the HPP processed samples, regardless of whether inhibitors were applied,

showed a significantly higher PPO activity than the NHPP ones. In other words, the HPP

procedure increased PPO activity in the pulp. This has been observed with pear and

avocado (Asaka & Hayashi, 1991; Woolf et al., 2013). A possible reason of this

phenomenon is the increase of the extractability of PPO activity after certain pressure is 63 applied (Woolf et al., 2013). However, all three inhibitors suppressed PPO activity in

HPP samples, especially pasteurization and ascorbic acid addition. In the NHPP groups, no significance of PPO activity was detected after treatments by the browning inhibitors.

No effect from HPP was observed on the color change, but ascorbic acid addition caused lighter (higher L*), less red (lower a*) color, and more yellow (higher b*) which may indicate a less browning effect. Of all the Belle samples, the HPP samples with steviosides or pasteurization were darker (lower L*), redder (higher a*), and less yellow

(b*) color, which indicates stronger browning compared to the other samples. In summary, HPP, steviosides and ascorbic acid all activated the PPO in Belle pawpaw, but the inhibitors all had various degrees of a PPO inhibiting effect when being utilized with

HPP treatment. Pasteurization without HPP inhibited the most PPO activity in Belle samples, but ascorbic acid addition had the best effect on browning prevention. 64

Table 6

The PPO Activity and L*, A*, B* Color Coordinates of Belle Pawpaw Pulp With or Without High Pressure Processing and Pulp With or Without the Addition of Browning Inhibitors

Treatment/ PPO Day 0 Day 15 Day 30 Day 45 Treatment activity1

L* a* b* L* a* b* L* a* b* L* a* b* HPP a ab bc a ab a b bcd ab bc c a e C 30.65 58.8 11.8 39.1 51.5 16.6 30.1 43.4 11.6 17.0 37.5 14.2 14.4

S 26.66ab 49.9c 19.0a 28.9ab 43.8bc 14.9ab 20.8cd 40.6d 11.6ab 14.8c 37.1c 9.7b 15.2e

T 6.50c 50.3c 19.7a 29.0b 41.6c 15.2a 18.3d 42.6cd 12.9ab 18.4bc 39.3c 13.5a 17.0de

AA 15.16bc 58.3ab 12.6bc 35.0ab 51.7ab 13.2bc 30.3b 50.2abc 13.6ab 24.4b 38.2c 13.0a 20.6cd NHPP C 4.26c 56.7ab 11.2bc 36.6a 47.7bc 14.8ab 26.2bcd 46.7abcd 9.6b 23.9b 47.7b 14.5a 28.5b

S 6.79c 54.3bc 13.9a 32.6ab 48.6bc 14.6ab 27.5bc 50.9ab 19.7a 23.0bc 46.1b 13.8a 24.6b

T 2.75c 54.3bc 8.8c 30.1b 50.5b 11.2d 27.4bc 44.8bcd 13.2ab 19.8bc 43.8b 13.6a 20.3cd c a bc a a cd a a b a a ab a AA 7.82 59.4 11.6 38.9 59.0 12.2 39.8 53.9 9.7 33.3 58.6 12.1 43.8 Note. 1PPO activity (∆ABS/min/g protein); KEY: no inhibitor (C), steviosides (S), pasteurization (T), ascorbic acid (AA). (Different superscripts within a column denote significant differences at p < 0.05.) 65 Storage time plays a significant role on the PPO activity of Belle pawpaw pulp (p

< 0.001). Despite the control NHPP group, the PPO activity of pulp in variety Belle showed a linear decreasing trend from day 0 to day 45 of storage (see Figure 3). In the absence of the inhibitors, the PPO activity of HPP samples was six-fold higher initially compared to the NHPP samples. Compared to initial PPO activity, half of the treated pawpaw groups exhibited a percent increase from initial and the other half exhibited a percent decrease from initial. However, by 30 days, all samples exhibited a reduction of

PPO activity compared to initial values except for the NHPP control, which may have been an outlier. At 30 days, all of the HPP samples exhibited a PPO reduction of greater than 93%. The color of the Belle pulp turned darker (less L*), and less yellow (lower b*) over storage period. 66

Figure 3. Effect of refrigerated storage (4 oC) on the change of PPO activity in pawpaw pulp of variety Belle. Samples were either high pressure processed (HPP) or not (NHPP) and contained no chemical browning inhibitor (C) or steviosides (S), ascorbic acid (AA), or was pasteurized to 60 oC (T).

Overall, PPO activity in variety Belle was stimulated by HPP as exhibited by the higher PPO activity values for all HPP treatments (see Table 6). However, by 30 days of storage, PPO activity in the HPP samples was reduced by at least 93% (see Figure 3), which is not the case in the NHPP samples. What is not known is which of these observations is more relevant as it relates to pawpaw pulp quality; i.e., the initial level of

PPO activity or the inhibition during storage.

3.4.1.2 Variety 2: Mitchell

The PPO activity and color of Mitchell pawpaw pulp under different processing conditions are shown in Table 7. HPP (p < 0.001), but not the steviosides and pasteurization (p = 0.172), exhibited a significant impact on the PPO activity of Mitchell pawpaw pulp. All the HPP processed samples, regardless of whether inhibitors were 67 applied, showed a significantly lower PPO activity than the NHPP ones. Therefore, as hypothesized, HPP is able to inhibit the PPO activity in Mitchell pawpaw pulps.

Nevertheless, no significant influence of the browning inhibitors (except for ascorbic acid) was observed on the PPO activity in both HPP and NHPP Mitchell samples.

No effect from the browning inhibitors was observed on the color change, but the average color of HPP samples was darker, less red, and less yellow than the NHPP samples. In summary, HPP inhibited the PPO activity and caused a darker less red, and less yellow color change in the Mitchell pawpaw. However, the browning inhibitors showed no effect on the PPO activity and the color of the Mitchell pawpaw. 68

Table 7

The PPO Activity and L*, A*, B* Color Coordinates of Mitchell Pawpaw Pulp With or Without High Pressure Processing and With or Without the Addition of Browning Inhibitors

Treatment/ PPO Day 0 Day 15 Day 30 Day 45 Treatment activity1

L* a* b* L* a* b* L* a* b* L* a* b* HPP C 6.24c 47.6cd 7.4abc 24.2de 42.6c 6.3b 15.0e 39.9b 7.5ab 13.3b 40.6c 10.0a 15.1d

S 3.83c 46.3d 5.9bc 21.7e 39.9d 6.4b 16.7de 39.3b 8.1ab 16.0b 43.0d 7.9c 19.9c

T 4.96c 49.9bc 9.1ab 28.0cd 42.0cd 9.3a 19.3cd 39.4b 8.9a 14.6b 41.3c 9.9ab 17.8cd

AA 1.81c 51.0abc 5.4bc 27.5 cd 42.2cd 9.0a 23.1c 40.2b 5.7b 16.5b 37.8c 5.3d 17.5cd NHPP C 35.37ab 50.5abc 4.7c 31.7bc 47.7b 7.2ab 28.5b 47.6a 7.3ab 17.9b 40.8c 9.6ab 24.2b

S 45.77a 51.5ab 8.4abc 33.3b 48.6b 6.3b 27.3b 46.9a 8.0ab 25.4a 46.5b 7.7c 28.9a

T 38.52ab 50.8abc 7.0abc 29.1bc 43.2c 9.3a 19.7cd 39.3b 9.3a 14.9b 41.4c 8.7abc 19.3cd b a a a a b a a a a a bc a AA 28.48 53.9 10.8 38.4 53.2 5.5 35.6 49.2 8.9 28.6 50.4 8.3 32.1 Note. 1PPO activity (∆ABS/min/g protein); KEY: no inhibitor (C), steviosides (S), pasteurization (T), ascorbic acid (AA). (Different superscripts within a column denote significant differences at p < 0.05.) 69 Storage time played a significant role on the PPO activity of Mitchell pawpaw pulp (p < 0.001). Generally, the PPO activity of pulp in variety Mitchell showed a linear decreasing trend from day 0 to day 45 of storage (see Figure 4). However the samples treated with ascorbic acid and HPP showed an increase of PPO activity at day 30, which is also observed in “ Yali” Pears (Cheng et al., 2015). Compared to initial PPO activity, half of the HPP treated pawpaw groups exhibited a percent increase from initial and the other half as well as all of the NHPP samples exhibited a percent decrease from initial.

However, by 45 days, all samples exhibited a reduction of PPO activity compared to initial values except for the HPP ascorbic acid-treated group and the HPP steviosides- treated group, which may have been outliers. At 45 days, the HPP samples and the NHPP samples exhibited a PPO reduction of 70% and 20%, respectively. The color of the

Mitchell pulp, in general, turned darker (less L*) and less yellow (lower b*) over storage period. 70

Figure 4. Effect of refrigerated storage (4 °C) on the change of PPO activity in pawpaw pulp of variety Mitchell. Samples were either high pressure processed (HPP) or not (NHPP) and contained no chemical browning inhibitor (C) or steviosides (S), ascorbic acid (AA), or was pasteurized to 60 °C (T).

Overall, HPP was able to inhibit the PPO activity in variety Mitchell by up to

80% and caused a darker and less yellow color change to the pulp (see Table 7). By 45 days of storage, PPO activity in the HPP samples was reduced by at least 70% (see

Figure 4), which is not the case in the NHPP samples. The browning inhibitors did not have a significant effect on the PPO levels of the samples.

3.4.1.3 Variety 3: PA-Golden

The PPO activity and color of PA-Golden pawpaw pulp under different processing conditions are shown in Table 8. Both HPP and browning inhibitors exhibited significant impacts (p < 0.001) on the PPO activity of PA-Golden. Nearly all of the HPP- treated samples showed a significantly lower PPO activity than the NHPP ones. The 71 application of the three inhibitors, compared to the control, all exhibited varying decreases of PPO activity in the NHPP samples, of which ascorbic acid addition exhibited the most reduction and pasteurization the least. No difference on PPO activity was detected among the HPP samples treated by different inhibitors.

HPP caused a darker, redder, and less yellow color of the PA-Golden pulp. The use of ascorbic acid alone increased the lightness (higher L*), decreased redness (lower a*) and yellowness (lower b*) of the PA-Golden pawpaw; stevoisides treatment triggered the samples to become redder. In conclusion, both HPP alone and ascorbic acid addition only offered the best effect on PPO activity inhibition in PA-Golden pawpaw, but ascorbic acid addition may be preferred when taking the color change of the pulp into consideration.

72

Table 8

The PPO Activity and L*, A*, B* Color Coordinates of PA-Golden Pawpaw Pulp With or Without High Pressure Processing and With or Without the Addition of Browning Inhibitors

Treatment/ PPO Day 0 Day 15 Day 30 Day 45 Treatment activity1

L* a* b* L* a* b* L* a* b* L* a* b* HPP C 5.92b 56.7b 8.7b 36.9ab 46.5c 10.0abcd 25.5cd 43.4bc 11.6a 17.0bcd 43.9cd 11.2a 23.0c

S 6.21b 54.8bc 11.2a 35.2bc 49.5c 10.3abc 27.6bc 40.6bc 11.6a 14.8cd 43.4cd 10.5a 26.6c

T 5.69b 56.1b 7.0c 30.6e 46.2c 6.2e 20.8e 42.6bc 12.9a 18.4bcd 44.9cd 9.7a 23.8c

AA 6.64b 52.3cd 7.5bc 32.2de 47.4c 10.7ab 25.4cd 50.2ab 13.6a 24.4b 48.1bc 10.0a 26.2c NHPP C 17.30a 51.2d 10.4a 35.6bc 49.9c 10.8a 29.9b 33.2c 11.2a 17.1bcd 55.0ab 7.4a 35.1b

S 9.98b 55.1bc 6.6c 33.8cd 55.7b 7.7bcde 35.8a 43.5bc 9.6ab 23.0bc 49.7bc 10.1a 26.2c

T 12.07ab 54.9bc 2.4d 30.8e 50.7c 7.4cde 23.8d 34.6c 9.3ab 10.1d 39.0d 9.8a 14.2d b a d a a de a a b a a a a AA 5.50 63.3 3.9 39.0 63.4 7.0 38.8 55.8 6.2 34.6 60.9 8.0 43.1 Note. 1PPO activity (∆ABS/min/g protein); KEY: no inhibitor (C), steviosides (S), pasteurization (T), ascorbic acid (AA). (Different superscripts within a column denote significant differences at p < 0.05) 73 Storage time played a significant role on the PPO activity of PA-Golden pawpaw pulp (p < 0.001). Generally, the PPO activity of pulp in variety PA-Golden showed a decrease at day 15 and day 45, but an increase at day 30 of storage (see Figure 5). This observation is similar to the findings on “Yali” pear (Cheng et al., 2015). Compared to initial PPO activity, all the HPP-treated pawpaw groups and half of the NHPP-treated groups exhibited a significant decrease at day 45. However, by 45 days, the NHPP ascorbic acid-treated group showed an increase of PPO activity, which may have been an outlier. At 45 days, the HPP samples and the NHPP samples exhibited nearly 100% and greater than 70% PPO reductions, respectively. The color of the PA-Golden pulp, in general, turned darker, redder, less yellower color with along with storage time, especially at day 30. 74

Figure 5. Effect refrigerated storage (4 °C) on the change of PPO activity in pawpaw pulp of variety PA-Golden. Samples were either high pressure processed (HPP) or not (NHPP) and contained no chemical browning inhibitor (C) or steviosides (S), ascorbic acid (AA), or was pasteurized to 60 °C (T).

Overall, HPP was able to inhibit the PPO activity in variety PA-Golden by up to

80% and caused a darker and less yellow color change to the pulp (see Table 8). By 45 days of storage, PPO activity in the HPP samples was reduced by nearly 100%, while greater than 70% PPO activity reduction was achieved in the NHPP samples (see Figure

5), The browning inhibitors did not have a significant effect on the PPO levels of the samples. Ascorbic acid addition provided less brown color change in PA-Golden pawpaw.

3.4.1.4 Variety 4: Pickle

The PPO activity and color of Pickle pawpaw pulp under different processing conditions are shown in Table 9. HPP in general had no effect (p = 0.066), but the 75 browning inhibitors had significant effect (p < 0.001) on the PPO activity inhibition of variety Pickle. The application of pasteurization, compared to the control, exhibited the most decrease of PPO activity in the both NHPP and HPP samples. Steviosides was able to reduce the PPO activity of the HPP-treated group by 40%, but had no significant effect in the NHPP group. Ascorbic acid treatment decreased the PPO activity in NHPP samples by 68% but increased the PPO activity in HPP samples by 33%.

No L* (p = 0.837) and a* (p = 0.3) color difference was detected by CIE measurement between HPP and NHPP samples, but HPP led to less yellow color change

(lower b*) of the Pickle pulp. Pasteurization induced a redder and less yellow color change of the pulp. In conclusion, pasteurization offered the best effect on PPO activity inhibition in Pickle pawpaw, but this treatment also caused a redder and less yellow color change of the pulp. 76

Table 9

The PPO Activity and L*, A*, B* Color Coordinates of Pickle Pawpaw Pulp With or Without High Pressure Processing and With or Without the Addition of Browning Inhibitors

Treatment/ PPO Day 0 Day 15 Day 30 Day 45 Treatment activity1

L* a* b* L* a* b* L* a* b* L* a* b* HPP C 8.17bc 65.0a 8.3bc 33.0ab 45.3c 8.5c 21.6d 43.4cd 11.6ab 17.0cd 41.7e 11.1cd 19.5d

S 4.98cd 59.3cd 9.3b 33.2ab 61.9a 8.8c 20.4d 40.6d 11.6ab 14.8d 41.8e 11.2cd 16.4e

T 1.37d 58.1d 13.2a 33.5ab 43.2c 9.6bc 18.9d 42.6d 12.9ab 18.4bcd 44.5e 13.3bcd 19.5d

AA 10.89ab 55.5e 14.6a 31.5b 54.5b 9.5bc 31.2ab 51.2b 13.6ab 24.4b 50.7bc 12.0cd 27.4bc NHPP C 15.02a 61.3bc 7.2bc 36.8ab 56.6b 11.4b 31.4ab 47.7b 15.7a 21.4bc 52.3b 16.3a 28.9b

S 12.17ab 61.7bc 6.9bc 34.7ab 57.9ab 7.8cd 29.5bc 48.9b 10.2ab 22.6bc 48.9bc 13.9b 25.7c

T 3.27cd 62.9ab 5.5c 32.9ab 54.0b 17.4a 26.3c 47.1bc 10.7ab 17.1cd 47.1cd 17.5a 21.0d cd bc bc a a d a a b a a c a AA 4.87 61.7 8.1 37.4 60.7 5.8 34.3 57.1 7.5 32.6 58.5 10.2 32.1 Note. 1PPO activity (∆ABS/min/g protein); KEY: no inhibitor (C), steviosides (S), pasteurization (T), ascorbic acid (AA). (Different superscripts within a column denote significant differences at p < 0.05.) 77 Storage time played a significant role on the PPO activity of Pickle pawpaw pulp

(p < 0.001). The PPO activity of pulp in NHPP variety Pickle showed a linear decrease from day 0 to day 30, and an increase at day 45 of storage (see Figure 6). Compared to initial PPO activity, all of the Pickle pawpaw groups exhibited a significant decrease at day 45. However, at 15 days, the HPP-treated control group showed a 200% increase of

PPO activity, which may have been an outlier. At 45 days, both the HPP samples and the

NHPP samples exhibited approximately 50% PPO reduction. No correlation was found between the storage time and lightness (L*) of the Pickle pulp, but a redder and less yellow color change was detected as storage went longer.

Figure 6. Effect of refrigerated storage (4 °C) on the change of PPO activity in pawpaw pulp of variety Pickle. Samples were either high pressure processed (HPP) or not (NHPP) and contained no chemical browning inhibitor (C) or steviosides (S), ascorbic acid (AA), or was pasteurized to 60 °C (T). 78

Overall, HPP showed no significant impact on PPO activity inhibition, and pasteurization was able to decrease PPO activity by up to 83% (see Table 9). Meanwhile, the pasteurization-treated samples showed a redder and less yellow color change. By 45 days of storage, the PPO activity in all of the Pickle samples was significantly decreased compared to the initial day of storage (see Figure 6). A redder and less yellow color change was observed when longer storage time was given.

3.4.1.5 Variety 5: Seedling

The PPO activity and color of Seedling pawpaw pulp under different processing conditions are shown in Table 10. HPP and browning inhibitors both exhibited significant effects on the PPO activity inhibition of the Seedling samples (p < 0.001). The HPP- treated groups showed greater than 50% PPO activity reduction than the NHPP groups.

Pasteurization was able to reduce the PPO activity of the HPP-treated group and NHPP group by over 50% and 55%, respectively. Steviosides reduced the PPO activity of the

NHPP-treated group by nearly 50%, but had no significant effect in the HPP group. No effect on the PPO activity was observed after ascorbic acid treatment in the Seedling samples.

The HPP-treated Seedling samples were yellower compared to the untreated samples, but no difference was detected in its lightness (p = 0.732) and redness (p =

0.131). No significant difference of lightness (p = 0.215) was detected among the browning inhibitors in general, but ascorbic acid caused less redness and more yellowness to the Seedling pulp. In conclusion, HPP, pasteurization, and steviosides all 79 offered excellent effect on PPO activity inhibition, but ascorbic acid treatment showed less brown color change in the Seedling pawpaw pulp. 80

Table 10

The PPO Activity and L*, A*, B* Color Coordinates of Seedling Pawpaw Pulp With or Without High Pressure Processing and With or Without the Addition of Browning Inhibitors

Treatment/ PPO Day 0 Day 15 Day 30 Day 45 Treatment activity1

L* a* b* L* a* b* L* a* b* L* a* b* HPP C 30.58b 55.5b 11.1abc 36.0ab 44.6b 12.9ab 22.7de 38.8d 10.9ab 17.3d 38.6e 10.1b 17.3d

S 22.25b 62.2a 11.6abc 38.2a 53.3ab 12.7abc 32.4abcd 40.7d 11.9a 19.8cd 42.3cd 12.3b 21.2cd

T 15.25b 56.1b 12.1ab 37.6ab 46.0b 11.7abc 24.5cde 40.9d 10.7ab 21.2cd 40.1de 11.2b 18.4cd

AA 25.41b 57.9b 9.8c 37.0ab 51.5ab 9.6bc 21.5e 41.5d 9.5ab 22.2cd 43.6c 11.3b 17.3cd NHPP C 63.19a 56.3b 12.6a 38.9a 52.1ab 12.9ab 32.9abc 46.0c 12.9a 25.0bc 44.5c 15.0a 27.0bc

S 32.73b 57.9b 10.6bc 37.3ab 56.6a 12.0abc 35.0ab 50.5b 12.6a 28.0b 54.7a 15.4a 35.5a

T 28.44b 54.5b 5.5d 32.0b 50.6ab 15.4a 27.0bcde 47.8bc 7.1b 20.4cd 48.7b 11.2a 20.3cd a ab bc a a c a a c a a c ab AA 55.86 58.6 10.6 38.7 58.4 8.7 37.0 56.4 3.3 33.9 55.5 4.6 34.0 Note. 1PPO activity (∆ABS/min/g protein); KEY: no inhibitor (C), steviosides (S), pasteurization (T), ascorbic acid (AA). (Different superscripts within a column denote significant differences at p < 0.05.) 81 Storage time played a significant role on the PPO activity of Seedling pawpaw pulp (p < 0.001). All of the HPP-treated groups exhibited nearly100% reduction of PPO activity at day 15 and day 45 of storage (see Figure 7). Compared to initial PPO activity in the NHPP groups, all of the Seedling pawpaw groups exhibited a significant decrease at day 45 except for the steviosides-treated NHPP group, which may have been an outlier.

A redder and yellower color change was detected of the Seedling pulp as storage went longer.

Figure 7. Effect refrigerated storage (4 °C) on the change of PPO activity in pawpaw pulp of variety Seedling. Samples were either high pressure processed (HPP) or not (NHPP) and contained no chemical browning inhibitor (C) or steviosides (S), ascorbic acid (AA), or was pasteurized to 60 °C (T).

Overall, HPP treatment reduced the PPO activity in variety Seedling by over 50%.

All the browning inhibitors treatments showed significant effect on PPO activity 82 reduction, and pasteurization and steviosides addition had better effort. By 45 days of storage, the PPO activity in nearly all of the Seedling samples was significantly decreased compared to the initial day of storage (see Figure 7). A redder and yellower color change was observed when longer storage time was given.

3.4.1.6 Variety 6: Sunflower

The PPO activity and color of Sunflower pawpaw pulp under different processing conditions are shown in Table 11. HPP and browning inhibitors both exhibited significant effects on the PPO activity inhibition of the Sunflower samples (p < 0.001 and p = 0.041 respectively). The HPP-treated groups showed 65% PPO activity reduction than the

NHPP groups. Steviosides was able to reduce the PPO activity of the NHPP group by over 85%, but had no significant effect in the HPP group. Ascorbic acid caused 56% PPO activity inhibition in the HPP group but increased the PPO activity in the NHPP-treated group by nearly 25%. Pasteurization caused 34% PPO activity inhibition in the NHPP group but increased the PPO activity in the HPP group.

The HPP-treated Sunflower pulps showed a darker, redder, and less yellow color compared to the untreated samples. The pasteurization-treated samples were darker and less yellow; while ascorbic acid-treated samples were lighter and yellower. In conclusion,

HPP and steviosides offered the excellent effect on PPO activity inhibition, but HPP treatment may cause more brown color change in the Sunflower pawpaw pulp.

83

Table 11

The PPO Activity and L*, A*, B* Color Coordinates of Sunflower Pawpaw Pulp With or Without High Pressure Processing and With or Without the Addition of Browning Inhibitors

Treatment/ PPO Day 0 Day 15 Day 30 Day 45 Treatment activity1

L* a* b* L* a* b* L* a* b* L* a* b* HPP C 9.31c 45.2e 9.7a 23.1c 48.1c 12.3a 22.4c 53.1c 19.2a 27.7abc 45.4d 11.5a 24.9cd

S 8.54c 60.6c 7.9bc 39.9a 47.2c 7.2ab 24.8bc 50.9c 10.5bc 29.2ab 47.1d 8.9ab 24.8cd

T 11.27bc 55.3d 8.8ab 32.4b 45.7c 9.7ab 22.6c 53.0c 14.6b 25.3bc 46.2d 9.6a 23.8d

AA 4.14c 65.3b 4.1ef 40.1a 60.8b 8.3ab 36.7a 65.9ab 15.3b 33.3ab 48.6cd 7.9a 26.2cd NHPP C 30.18ab 72.6a 6.9cd 38.5a 69.6a 5.3ab 36.3a 71.2a 5.3c 37.1a 70.5a 8.7a 41.8a

S 4.54c 68.4 b 5.3de 38.3a 67.4a 4.6b 37.3a 58.8bc 11.2bc 29.6ab 54.9b 7.7a 28.7c

T 19.23abc 64.3bc 2.7f 34.7ab 58.5b 6.2ab 28.3b 54.6c 5.0c 18.1c 51.8b 12.2a 23.4d a b d a a ab a ab b ab a a b AA 37.86 68.3 5.9 39.3 68.6 5.0 37.8 63.5 6.4 31.1 66.9 9.3 37.9 Note. 1PPO activity (∆ABS/min/g protein); KEY: no inhibitor (C), steviosides (S), pasteurization (T), ascorbic acid (AA). (Different superscripts within a column denote significant differences at p < 0.05.) 84 Storage time played a significant role on the PPO activity of Sunflower pawpaw pulp (p < 0.001). The HPP-treated groups exhibited a linear decrease of PPO activity from 100% at day 0 to 0% at day 45 of storage. The NHPP groups showed a general linear decrease of PPO activity from day 0 to day 30, and a slight increase at day 45 of storage. Compared to initial PPO activity in the NHPP groups, nearly all of the Sunflower pawpaw groups exhibited varying degrees of decrease at day 45 except for the control

NHPP group, which may have been an outlier. The Sunflower pulp turned darker and less yellow along with storage period.

Figure 8. Effect of refrigerated storage (4 °C) on the change of PPO activity in pawpaw pulp of variety Sunflower. Samples were either high pressure processed (HPP) or not (NHPP) and contained no chemical browning inhibitor (C) or steviosides (S), ascorbic acid (AA), or was pasteurized to 60 °C (T). 85

Overall, HPP reduced the PPO activity in variety Sunflower by 65%. The ascorbic acid-treated HPP group and steviosides-treated NHPP group exhibited the most PPO activity reduction compare to the others. Pasteurization and ascorbic acid, when being used as single treatments, caused significant rise of PPO activity in the samples. Along 45 days of storage time, most NHPP samples displayed an increase of PPO activity at day 15 and 45 and a decrease at day 30. The HPP samples all exhibited a continuous decline and a complete inhibition at day 45.

3.4.1.7 Variety 7: Shenandoah

The PPO activity and color of Shenandoah pawpaw pulp under different processing conditions are shown in Table 12. HPP and browning inhibitors showed significant impact on the PPO activity of Shenandoah pawpaw samples (p < 0.001). HPP treatment alone was able to decrease the PPO activity of Shenandoah samples by 86%, which was more effective than the combination of HPP with the browning inhibitors. The

PPO activity of the HPP with steviosides or pasteurization treatments were 1.5 times higher than HPP control samples, while the ascorbic acid-treated HPP samples was 6.8 times higher than the HPP control. All the browning inhibitors promoted PPO activity in

NHPP groups, and ascorbic acid increased the PPO activity by more than 5 times of the

NHPP control, which is adverse to the results from other varieties of pawpaw and the findings on Chinese Toon in precious research (Wang, Zhang, Zhang, & Cheng, 2013).

HPP samples, in general, were darker and redder than the NHPP samples, which is consistent with the sensory analysis results shown later. Ascorbic acid treatment led to a brighter and less red pulp appearance, and steviosides combined with HPP led to more 86 red appearance. In conclusion, HPP treatment exhibited the greatest effect on PPO activity inhibition, but caused stronger brownness of the Shenandoah pulp. 87

Table 12

The PPO Activity and L*, A*, B* Color Coordinates of Shenandoah Pawpaw Pulp With or Without High Pressure Processing and With or Without the Addition of Browning Inhibitors

Treatment/ PPO Day 0 Day 15 Day 30 Day 45 Treatment activity1

L* a* b* L* a* b* L* a* b* L* a* b* HPP C 3.42c 62.0a 3.3de 39.6a 56.4b 12.8a 30.6c 44.7f 13.8b 19.3d 41.4e 15.2a 19.6e

S 6.45bc 61.3a 12.8a 34.7bc 46.2c 14.5a 23.1d 55.4cd 13.6b 32.4b 50.1d 11.5ab 30.0d

T 6.22bc 59.4a 10.7ab 33.3bc 50.1c 13.2a 23.1d 52.9de 11.0c 24.4c 48.2d 8.9bc 22.5e

AA 23.36bc 64.0a 7.6bc 37.5ab 59.6b 13.9a 33.5bc 51.6e 16.2a 28.8bc 51.9d 15.0a 27.3d NHPP C 24.02bc 64.3a 6.3cd 39.8a 66.2a 7.2b 41.0a 58.1bc 10.2c 33.5b 55.9bc 15.7a 34.4b

S 25.14bc 66.6a 4.9cde 35.2bc 67.1a 4.9b 37.7ab 58.7b 13.5b 33.6b 61.2ab 14.8a 36.1ab

T 32.07b 59.2a 3.1de 31.9c 57.1b 1.8c 29.3c 59.0b 7.3d 27.1bc 60.6b 6.8c 33.2bc a a e c a b ab a e a a d a AA 104.28 57.7 2.1 31.3 66.9 5.0 38.4 69.0 4.0 42.1 66.3 2.7 38.8 Note. 1PPO activity (∆ABS/min/g protein); KEY: no inhibitor (C), steviosides (S), pasteurization (T), ascorbic acid (AA). (Different superscripts within a column denote significant differences at p < 0.05.) 88 Storage time played a significant role on the PPO activity of Shenandoah pawpaw pulp (p < 0.001). All the samples showed a general linear decrease of PPO activity from day 0 to day 30, and a slight increase at day 45 of storage (see Figure 9). The activity of

HPP groups remained stable but varying amount of increases appeared in NHPP groups at day 45. Compared to initial PPO activity in the Shenandoah samples, all of the groups exhibited varying degrees of decrease at day 45, in which the combination of HPP with ascorbic acid or pasteurization showed the greatest effort. The Shenandoah pulp turned darker and redder along with storage time.

Figure 9. Effect of refrigerated storage (4 °C) on the change of PPO activity in pawpaw pulp of variety Shenandoah. Samples were either high pressure processed (HPP) or not (NHPP) and contained no chemical browning inhibitor (C) or steviosides (S), ascorbic acid (AA), or was pasteurized to 60 °C (T).

Overall, HPP exhibited the greatest effect on PPO activity reduction but darker and redder color of the Shenandoah pulp. All the inhibitors, especially ascorbic acid, 89 caused different levels of increase of PPO activity in variety Shenandoah. All of the

Shenandoah groups displayed a decrease of PPO activity along 45 days of storage. The overall PPO was least active at day 15 and day 30 of storage (see Figure 9). The pasteurized and ascorbic acid-treated HPP groups revealed complete inhibition of PPO activity at day 15, and remained stable afterwards.

3.4.1.8 Variety 8: SAB OL

The PPO activity and color of SAB OL pawpaw pulp under different processing conditions are shown in Table 13. Both HPP (p < 0.001) and browning inhibitors (p =

0.002) showed significant impact on the PPO activity of SAB OL pawpaw samples (p <

0.001). All the HPP groups had significantly lower PPO activity than the corresponding

NHPP groups, and HPP alone was able to inhibit the PPO activity of NHPP control by

60%. The samples treated with browning inhibitors, excluding the pasteurized HPP ones, showed adverse effects on PPO inhibition. The combined treatment of HPP and pasteurization decreased the PPO activity of SAB OL pawpaw by 80%.

The use of steviosides and ascorbic acid caused the HPP pulps to become darker and redder, and pasteurization increased the lightness of the HPP samples. The color change of the NHPP group could not be compared due to incidental loss of the NHPP samples before color measurement. In conclusion, HPP treatment alone and HPP combined with pasteurization both exhibited the excellent effect on PPO activity inhibition. 90

Table 13

The PPO Activity and L*, A*, B* Color Coordinates of SAB OL Pawpaw Pulp With or Without High Pressure Processing and With or Without the Addition of Browning Inhibitors

Treatment/ PPO Day 0 Day 15 Day 30 Day 45 Treatment activity1

L* a* b* L* a* b* L* a* b* L* a* b* HPP C 4.27cd 68.2a 5.6d 34.6b 56.1a 14.9ab 30.6ab 52.6a 15.3a 28.1a 53.3a 16.0a 11.9a

S 4.62cd 62.4b 10.6b 34.3b 53.9a 13.3b 29.9b 43.2b 14.3b 20.0b 44.3bc 12.9a 21.9a

T 1.21d 72.3a 7.1c 42.9a 57.7a 16.9a 33.0a 42.0b 12.8c 17.1b 47.8b 16.4a 24.3a

AA 7.12cd 57.7c 13.6a 32.9b 45.6b 15.2ab 22.3c 41.8b 12.5c 16.5b 41.3a 13.8a 18.3a NHPP C 13.35bc

S 18.59ab No Data

T 26.03a No Data ab AA 20.42 Note. 1PPO activity (∆ABS/min/g protein); KEY: no inhibitor (C), steviosides (S), pasteurization (T), ascorbic acid (AA). (Different superscripts within a column denote significant differences at p < 0.05.) 91 Storage time played a significant role on the PPO activity of SAB OL pawpaw pulp (p < 0.001). The general PPO activity showed a declining trend along the storage period with one exception (ascorbic acid-treated HPP) at day 15, which may have been an outlier (see Figure 10). All the samples exhibited the lowest PPO activity at day 30 of storage, and the steviosides-treated HPP and NHPP groups, the pasteurization-treated

NHPP group, and the ascorbic acid-treated NHPP group were able to inhibit the activity by nearly 100%. At day 45, the PPO of pasteurized HPP samples was 100% inactivated

(see Figure 8). The HPP-treated SAB OL pulp turned darker, redder, and less yellow color along with storage time.

Figure 10. Effect of refrigerated storage (4 °C) on the change of PPO activity in pawpaw pulp of variety SAB OL. Samples were either high pressure processed (HPP) or not (NHPP) and contained no chemical browning inhibitor (C) or steviosides (S), ascorbic acid (AA), or was pasteurized to 60 °C (T). 92

Overall, HPP exhibited the significant effect on PPO reduction in the SAB OL samples. The use of browning inhibitors alone and the combination of HPP with ascorbic acid or steviosides all caused varying degrees of PPO activity increase. Nearly all the samples exhibited decreases of PPO activity after day 0, and received the least PPO activity at day 30 of storage. Further CIE colorimetric data of the NHPP SAB OL samples is needed to compare the impact of HPP and storage on the color of the SAB OL pulp.

3.4.1.9 Variety 9: Wilson

The PPO activity and color of Wilson pawpaw pulp under different processing conditions are shown in Table 14. HPP treatment (p < 0.001) and browning inhibitors (p

< 0.001) significantly decreased PPO activity in the Wilson pawpaw. The PPO activity of the HPP-treated groups 53% lower than the NHPP groups despite that HPP treatment alone slightly increased the PPO activity in the control group. Although HPP alone activated PPO activity of the Wilson samples, it decreased the PPO activity of the pulp by up to 76% when combined with browning inhibitors. Among the three inhibitors, pasteurization caused decreases of PPO activity in both the HPP group and the NHPP group by 76% and 29% respectively; steviosides reduced the PPO activity of the HPP group by over 59%, but increased the PPO activity in the NHPP group; ascorbic acid caused 51% reduction of PPO activity in the HPP group but 112% increase of the activity in the NHPP group.

The CIE results revealed that ascorbic acid increased the lightness, steviosides and pasteurization increased the redness, and pasteurization and ascorbic acid increased 93 the yellowness of the HPP-treated Wilson pulps. The effect of HPP to the color change of the Wilson pawpaw samples was unknown since we incidentally lost the NHPP samples before color measurement. 94

Table 14

The PPO Activity and L*, A*, B* Color Coordinates of Wilson Pawpaw Pulp With or Without High Pressure Processing and With or Without the Addition of Browning Inhibitors

Treatment/ PPO Day 0 Day 15 Day 30 Day 45 Treatment activity1

L* a* b* L* a* b* L* a* b* L* a* b* HPP C 19.31a 52.2b 4.9b 29.7ab 45.3b 8.5a 21.6b 41.4b 6.7b 15.2c 41.7a 7.1a 18.2a

S 7.97c 50.5b 11.8a 27.3b 44.8b 8.8a 20.4b 41.3b 7.5ab 17.3b 43.7a 10.4a 19.8a

T 4.60c 56.1ab 9.9a 36.3a 43.2b 9.6a 18.9b 42.3b 9.7a 17.5b 42.7a 6.2a 12.6a

AA 9.47bc 60.2a 6.1b 36.3a 54.5a 9.5a 31.2a 45.1a 9.9a 20.6a 38.2a 9.1a 18.5a NHPP C 17.68bc

S 22.58b No Data

T 12.55bc No Data a AA 35.00 Note. 1PPO activity (∆ABS/min/g protein); KEY: no inhibitor (C), steviosides (S), pasteurization (T), ascorbic acid (AA). (Different superscripts within a column denote significant differences at p < 0.05.) 95 There was no significance found on the effect of storage time to PPO activity of

Wilson pawpaw in general (p = 0.058). However, differences (p < 0.001) were detected when the samples were split to HPP versus NHPP groups. All the samples within HPP group displayed the lowest PPO activity at day 15 of storage, and gradual increases afterwards. The browning inhibitors applied NHPP groups all exhibited PPO activity peaks at day 15 and linear decreases from day 15 to day 30 (see Figure 11). The utilization of browning inhibitors caused PPO activity increase in the NHPP samples. The

HPP-treated Wilson pulp turned darker and less yellow color along with storage time.

Figure 11. Effect of refrigerated storage (4 °C) on the change of PPO activity in pawpaw pulp of variety Wilson. Samples were either high pressure processed (HPP) or not (NHPP) and contained no chemical browning inhibitor (C) or steviosides (S), ascorbic acid (AA), or was pasteurized to 60 °C (T). 96

Overall, HPP treatment alone increased PPO activity of the control samples, but it showed significant PPO activity decreasing effect when used in combination with the three browning inhibitors. Though no impact of storage time was observed towards the

PPO inactivation in Wilson pawpaw, the HPP samples showed continuous 100% PPO activity inhibition from day 15 to day 45 of storage. Further CIE colorimetric data of the

NHPP Wilson samples is needed to compare the impact of HPP and storage on the color of the pulp.

3.4.2 Descriptive sensory analysis

The Shenandoah fresh pawpaw was selected to conduct a descriptive sensory analysis due to the sufficient quantity of the pulp. The sensory analysis aimed to measure and predict the influence of the treatments to the flavor, taste, appearance and mouthfeel to the samples. The results from PPO activity and CIE color measurements implied that

HPP significantly reduced PPO activity but caused a redder color of the Shenandoah pulp.

All the inhibitors, especially ascorbic acid, caused different levels of increase of PPO activity in variety Shenandoah. However, no significant color change was observed due to the applications of different browning inhibitors (see Table 12).

This sensory study used a highly trained panel to describe twelve sensory attributes that used a pawpaw lexicon developed by previous research (Brannan et al.,

2012). These attributes include five flavors (banana, melon, mango, papaya, tropical), two basic tastes (sweet, sour), two mouthfeels (body, astringent), and two aftertaste

(rindy, bitter), and color (Brannan et al., 2012). Training and calibration are required to avoid a panelist effect (N'Kouka, Klein, & Lee, 2004). A panelist effect was detected in 97 all the sensory attributes (p < 0.001) except for bitterness (p = 0.078). The panelist effect is an indicator that the panelists may have been using their scales differently to measure their perspectives even though these scales were anchored to standards.

Similar to the conclusion from previous research (Brannan et al., 2012), the most intense flavors in Shenandoah pawpaw pulp were banana, mango, melon, and papaya

(see Table 15). The overall sweetness of the pulp is 2 times stronger than its sourness.

Statistical results suggested that HPP treatment affected the color (p = 0.023) attribute of Shenandoah pulp. According to the nine panelists, the color of the HPP samples in general were 18% browner than the NHPP samples. This fact was also observed in the color measurement in the parameter of lightness (L*) and redness (a*), shown in Table 12. The use of inhibitors altered the color (p = 0.017), sweetness (p =

0.002) and bitterness (p = 0.003) of the samples. The color of the samples treated by pasteurization was 28% and 21% less brown than the average and the control, respectively (see Table 15). The 0.18% steviosides-treated samples (both HPP and NHPP) were 46% sweeter and 57% more bitter than the other samples. The intensity of the other sensory attributes is similar within the samples with different treatments. 98

Table 15

Mean Values of the Twelve Selected Sensory Attributes for Variety Shenandoah Pawpaw Pulp With or Without High Pressure Processing and With the Addition of Chemical Inhibitors Pasteurization Ascorbic acid Attributor Control (C) (T) (AA) Steviosides (S)

HPP NHPP HPP NHPP HPP NHPP HPP NHPP Color 3.00ab 3.00b 2.77b 2.53b 4.23a 3.17b 3.46ab 3.00b

Body 5.24b 4.69b 6.88ab 5.72a 7.70a 6.31ab 5.49ab 5.87ab

Sweet 1.89c 2.36abc 1.92c 2.25abc 2.15bc 2.19abc 3.31a 3.24ab

Sour 1.21ab 1.38ab 1.52ab 1.05ab 1.76ab 0.93b 2.28a 1.83ab

Banana 2.48a 2.45a 2.48a 2.75a 2.23a 2.75a 2.78a 2.19a

Melon 2.66a 2.22a 3.15a 2.28a 2.34a 2.11a 2.34a 3.15a

Mango 2.78a 2.70a 2.59a 2.37a 2.54a 2.11a 2.23a 2.69a

Papaya 1.88a 2.99a 2.76a 2.55a 2.65a 1.97a 1.92a 1.83a

Tropical 1.39a 1.30a 1.52a 1.68a 1.65a 1.52a 1.65a 1.69a

Astrin- 3.19a 3.77a 2.38a 2.65a 2.84a 2.45a 3.60a 3.09a gent

Rindy 3.89a 2.65a 3.38a 2.99a 4.02a 3.68a 4.40a 3.13a

Bitter 4.16ab 2.83b 2.92b 3.06b 3.18b 3.94b 6.76a 5.46ab 3.1 Note. Different superscripts within a column denote significant differences at p < 0.05. Key: High pressure Processing (HPP); No high pressure processing (NHPP). 99

3.5 Conclusion

The purpose of this research was to determine the effect of HPP, browning inhibitors (steviosides, ascorbic acid, pasteurization), variety, and storage period on the

PPO activity and color of pawpaw fruits.

3.5.1 Variety

There were differences observed between the nine pawpaw varieties studied, so

PPO can be added the list of characteristics in pawpaw that vary by variety, such as size, color, sugar content, phytochemical content (Brannan et al., 2015), and antioxidant capability (unpublished observations). Among the nine varieties that were selected in this study, Seedling exhibited the highest PPO activity (= 63.19 ∆ABS/min/g protein) whereas Belle had the lowest (= 4.26 ∆ABS/min/g protein). The activity among varieties were ranked as: as Seedling > Mitchell > Sunflower > Shenandoah > Wilson > PA-

Golden > Pickle > SAB OL > Belle (see Tables 6-14). If a variety with low PPO activity, such as Belle, produced fruit that do not brown as fast, it may have more commercial value compared to varieties that have high PPO activity. However, what is not known conclusively is the relationship between the activity of PPO and the color of the pulp. In this study, pulp from a majority of the varieties in the untreated samples became browner

(i.e., darker, redder and less yellow) over storage. These results also suggest that the influence of HPP and the browning inhibitors on PPO inactivation varied among pawpaw varieties. 100

3.5.2 HPP

HPP significantly decreased the PPO activity in all of the varieties except for

Belle and Pickle. Previous research (Asaka & Hayashi, 1991; Woolf et al., 2013) indicated that the PPO activity in fruits could increase during HPP unless sufficient pressure is applied to inactivate the enzyme, and this may be due to the increase of extractability of PPO under certain pressure (Woolf et al., 2013). In this study, our conditions (600 MPa for 76 s) appear to be sufficient to reduce PPO in all varieties except

Belle and Pickle. This suggests that a higher pressure might be required in these two varieties. Considering that variety Belle has a low PPO activity in unprocessed fruit which increases when HPP is applied, the conclusion reached earlier—that Belle might be a variety to be commercially promoted due to its low PPO activity—should be qualified to suggest that Belle might be a variety to be promoted in unprocessed fruit.

However, a variety such as PA-Golden, for which HPP reduced PPO activity by 66%, which was further reduced during storage, may be a starting point for the identification of a variety with potential for HPP processing. Overall, the samples processed by HPP exhibited a darker, redder, and less yellow color, which is equivalent to more browning development according to the definition of brown. Hence, HPP is an excellent treatment for PPO inactivation but might cause undesirable browning of the pawpaw pulp.

3.5.3 Browning inhibitors

The browning inhibitors showed mixed results. Pasteurization significantly inhibited PPO activity in all varieties except for Mitchell and Shenendoah, whereas the ascorbic acid and steviosides increased PPO activity. The inhibitors suppressed the brown 101 color development (L*, a*, b*) of the pawpaw to various degrees. The ascorbic acid and control groups preserved the lightest, least red, and yellowest color of the fruits, and steviosides increased the redness of the pulps significantly. Pasteurization was the most effective PPO inhibitor, which is not surprising because heat inactivates enzymes, but led to darker and less yellow color development of the pulps. This discoloration might be caused by the Millard reaction when temperature was applied to the samples. Similar to the findings in other fruits (Anese, Nicoli, Dallaglio, & Lerici, 1995; Chaves et al., 2011;

Siddiq et al., 1994; Sun et al., 2008), ascorbic acid was the most effective treatment for color preservation.

What is clear is that PPO activity is not the only determinative factor of browning of the pawpaw fruit. There could have been other enzymatic or nonenzymatic factors that affect browning, such as catechol oxidase and Millard reaction. In order to prevent the unexpected browning development (caused by enzymatic reaction or processing techniques) during storage, ascorbic acid treatment over HPP, pasteurization, and steviosides is suggested to be applied on pawpaw even though it is less effective on inhibiting the PPO activity of this fruit.

3.5.4 Storage

A continuous decreasing trend of PPO activity was detected during the 45 days of storage period among majority of the HPP treated varieties. This is consistent with findings on Cherimoya fruit that was processed by HPP (Campos-Vargas et al., 2008).

By contrast, the control samples followed the same decreasing trend from day 0 to day 30 but had an increase of PPO activity at day 45 of storage. Ascorbic acid treatment 102 significantly inhibited the browning development (less L* and a* decrease, more b* increase) in the control groups during storage. These results suggest that HPP inhibited

PPO activity during storage of pawpaw pulp and ascorbic acid preserved the fresh color of the pulp throughout storage period.

3.5.5 Sensory

Data from the descriptive sensory analysis revealed three important findings. First, he most intense flavors detected in Shenandoah pulp were banana, mango, melon, and papaya, consistent with previous research (Brannan et al., 2012). Second, HPP and pasteurization altered the color of the Shenandoah samples, with HPP promoting browning and pasteurization inhibiting browning. Third, the samples treated with steviosides had a sweeter and more bitter taste than the untreated control. Taken together, these findings suggest that ascorbic acid performed the best among the selected treatments to maintain the original color and taste of Shenandoah pawpaw pulp.

3.5.6 Summary and Conclusion

The results suggest that HPP, steviosides, pasteurization, and ascorbic acid treatment inhibit PPO, but the effect is not consistent among all the pawpaw varieties.

Pasteurization and HPP showed the most effectiveness on PPO activity inhibition in most of the pawpaw varieties, but they caused browning to the pulp in certain instances.

Steviosides and ascorbic acid increased PPO activity. All the pawpaw samples showed continuous decrease of PPO activity from day 15 to day 30 of the storage period, but the control samples exhibited increased PPO activity at day 45, which was also observed in

“Yali” Pears (Cheng et al., 2015). This phenomenon might be caused by the reactivation 103 of the remaining PPO. Based on these findings, the following recommendations can be made:

• Belle is recommended for further study if NHPP is required.

• PA-Golden is a suitable variety for further testing in HPP samples.

• According to the PPO stabilization trend during storage (see Table 5), the optimal

refrigerated storage time of the unprocessed pawpaw is 30 days, which can be

extended to 45 days in HPP samples.

• HPP suppressed PPO during storage.

• Ascorbic acid treatment preserved the fresh color of the pulp during storage.

The results of this study are expected to be useful to the food industry when selecting proper methods to prevent pawpaw or Annonaceae fruit browning. Future work on substrate specificity for pawpaw PPO could lead to the replacement of catechol with another mono-, di-, or tri-phenol because there is evidence that different phenolic substrates may have higher efficiency than catechol (Wang et al., 2013). This study was not able to compare the effectiveness of different levels or concentrations of the selected variables due to time and resource limitations. Further research should focus on optimizing ascorbic acid concentration as an antibrowning processing aide in these nine pawpaw varieties. Also, studies analyzing the antibrowning effectiveness of ascorbic acid with other antibrowning agents (e.g., calcium lactate), vacuum packaging (Perez-Cabrera,

Chafer, Chiralt, & Gonzalez-Martinez, 2011), or frozen storage would be useful

(Sulaiman & Silva, 2013; Wang, 2013). 104

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The present study measured PPO activity and color of nine varieties of pawpaw fruits and compared unprocessed and HPP treatment, three browning inhibitors, and the effect of refrigerated storage. There were differences observed between the nine pawpaw varieties studied, so PPO can be added the list of characteristics in pawpaw that vary by variety, such as size, color, sugar content, phytochemical content (Brannan et al., 2015), and antioxidant capability (unpublished observations). The activity among varieties were ranked as Belle < SAB OL = Pickle = Wilson < Shenandoah < Sunflower < Mitchell <

Seedling (see Tables 6-14). Table 15 summarizes the effect on PPO activity of the independent variables (processing, browning inhibitors, storage) for each variety. 112 Table 16

Trends of PPO Activity in Each of the Nine Varieties of Pawpaw Fruits After Different Treatments

Refrigerated Processing Browning inhibitors storage

Effect of Pasteurization Ascorbic Storage Variety HPP (60oC) Steviosides Acid time

Belle ↑ ↓ ↓ ↓ ↓

Mitchell ↓ = = ↓ ↓

= HPP/ = HPP/ = HPP/ PA-Golden ↓ ↓ NHPP ↓ NHPP ↓ NHPP ↓

↓ HPP/ Pickle = ↓ ↓ ↑ HNPP ↓

Seedling ↓ ↓ ↓ ↓ ↓

↑ HPP/ ↑ HPP/ Sunflower ↓ ↓ NHPP ↓ ↓ NHPP ↓

Shenandoah ↓ ↑ ↑ ↑ ↓ /↑ 45

SAB OL ↓ ↓ ↑ ↑ ↓/↑ 45

↓ HPP/ ↓ HPP/ Wilson ↓ ↓ ↑ HNPP ↑ HNPP ↑/↓ 45 Note. “↑” represents increased level compared to control; “↓” represents decreased level compared to control; “=” represents no effect.

4.1 HPP

HPP significantly decreased the PPO activity in all of the varieties except for

Belle and Pickle (see Table 16). Previous research (Asaka & Hayashi, 1991; Woolf et al.,

2013) indicated that the PPO activity in fruits could increase during HPP unless sufficient pressure is applied to inactivate the enzyme. In this study, our conditions (600 MPa for 113

76 s) appear to be sufficient to reduce PPO in all varieties except Belle and Pickle. This suggests that a higher pressure might be required in these two varieties. The browning inhibitors significantly inhibited PPO activity except that ascorbic acid and steviosides increased PPO activity SAB OL and Shenandoah samples and pasteurization did not affect PPO activity in Mitchell and increased PPO activity in Shenandoah (see Table 15).

A general decreasing trend of PPO activity was detected along with the 45 days of storage period among all the HPP treated varieties (see Table 16).

4.2 Browning Inhibitors

Table 17 summarizes the effect on PPO activity and color (L*, a*, b*) of the independent variables (processing, browning inhibitors, storage) across all of the varieties.

Presented this way, Table 5 shows that the samples processed by HPP exhibited a darker, redder, and less yellow color (see Table 17), which is equivalent to more browning development. Because HPP is an excellent treatment for PPO inactivation but might cause undesirable browning of the pawpaw pulp, its commercialization potential is moderate but could be improved in combination with an effective browning inhibitor.

Ascorbic acid, steviosides, and pasteurization all inhibited PPO activity. Ascorbic acid and control groups preserved the lightest, least red, and yellowest color of the fruits, so its commercialization potential is high. Steviosides and pasteurization inhibited PPO activity but pulps treated with steviosides exhibited increased redness and pasteurization received inhibited PPO activity to the highest degree, but led to darker and less yellow color change of the pulp. Therefore, the commercialization potential of steviosides and pasteurization is medium. 114 Table 17

Overall Influences of Different Treatments/ Factors to the PPO Activity and CIE Color (L*, a*, b*) in Pawpaw Pulp

Potential as Treatment PPO activity L* a* b* Commercial Treatment

HPP ↓ ↓ ↑ ↓ Moderate

Pasteurization ↓ ↓ = ↓ Moderate

Steviosides ↓ = ↑ ↓ Moderate

Ascorbic acid ↓ ↑ = ↑ High Note. “↑” represents decreased level “↓” represents decreased level; “=” represents no effect.

4.3 Sensory

Data from the descriptive sensory analysis revealed three important findings.

First, the most intense flavors detected in Shenandoah pulp were banana, mango, melon, and papaya, consistent with previous research (Brannan et al., 2012). Second, HPP and pasteurization altered the color of the samples, with HPP promoting browning and pasteurization inhibiting browning. The samples treated with steviosides had a sweeter and more bitter taste than the untreated control. Third, among the treated samples, these findings suggest that ascorbic acid performed the best among the selected treatments to maintain the color and taste of Shenandoah pawpaw pulp.

4.4 Summary

The results of this study are expected to be useful to the food industry when selecting proper methods to prevent pawpaw or Annonaceae fruit browning, especially.

This study was not able to compare the effectiveness of different levels or concentrations 115 of the selected variables due to time and resource limitations. Further research should focus on optimizing ascorbic acid concentration as an antibrowning processing aide in these nine pawpaw varieties. Also, studies analyzing the antibrowning effectiveness of ascorbic acid with other antibrowning agents (e.g., calcium lactate), vacuum packaging

(Perez-Cabrera, Chafer, Chiralt, & Gonzalez-Martinez, 2011), or frozen storage would be useful (Sulaiman & Silva, 2013; Wang, 2013). 116

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