Effect of Frozen Storage on Antioxidant Capacity, Polyphenol Oxidase Activity, and
Phenolic and Flavonoid Content and Color of Pawpaw 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
Gai Wang
August 2013
© 2013 Gai Wang. All Rights Reserved.
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This thesis titled
Effect of Frozen Storage on Antioxidant Capacity, Polyphenol Oxidase Activity, and
Phenolic and Flavonoid Content and Color of Pawpaw Pulp
by
GAI WANG
has been approved for
the School of Applied Health and Wellness
and the College of Health Sciences and Professions by
Robert G. Brannan
Associate Professor of Applied Health Science and Wellness
Randy Leite
Dean, College of Health Sciences and Professions 3
Abstract
WANG, GAI, M.S., August 2013, Food and Nutrition Sciences
Effect of Frozen Storage on Antioxidant Capacity, Polyphenol Oxidase Activity, and
Phenolic and Flavonoids Content and Color of Pawpaw Pulp
Director of Thesis: Robert G. Brannan
This thesis compared the concentration of total phenolics and total flavonoid of pulp extract from two different packaged pawpaw pulp samples, and also compared the antioxidant capacity of pawpaw pulp extract at two different packaged sample in three different assays 2,2-diphenyl-1-picrylhdrazyl (DPPH), ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC). The frozen pawpaw pulp was stored in vacuum packaging and in air packaging for 0, 2, 4, 6, 8, 10,
12 months, respectively, before it was put into frozen storage. Next, the concentration of total phenolics and total flavonoid with air packaged, ascorbic acid treatment with air package and N-acetylcysteine treatment with air packaged were compared.
Furthermore, the polyphenol oxidase (PPO) activity and color value (L*, a*, b*) at vacuum packaged, air packaged, ascorbic acid treatment with air packaged and N- acetylcysteine (NAC) treatment with air packaged were compared as well. Total phenolics were determined by Folin-Ciocalteu assay and reported as μmol gallic acid equivalent, and total flavonoids were determined spectrophotometrically using flavonoid rutin as a standard and reported as μmol rutin equivalent. From this research, both packaging and storage time could significantly affect total phenolics level, but only storage time not packaging could significantly affect total flavonoid level. In 4 addition, the study showed that both storage time and packaging could significantly affect antioxidant capacity at three different assays. Furthermore, the results obtained from the comparison of chemical treatment (ascorbic acid and NAC) of frozen pawpaw pulp and air storage frozen pawpaw pulp, showed that PPO has a significant effect on total phenolics, total flavonoids and color value (L*, a*, b*). In conclusion, these results indicated that antioxidant contents and antioxidant capacity are affected by packaging and storage, and PPO could affect antioxidant contents and color value as well.
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Acknowledgments
I want to thank all the individuals who have helped me to accomplish my master’s thesis in Food and Nutrition Sciences. Dr. Robert G. Brannan constantly helped me finish my program of study and thesis during these three years. He provided important support towards my research and thesis work.
I also want to thank the faculty and staff members in the School of Applied
Health Sciences and Wellness who have provided me a good atmosphere to conduct my research and finish my thesis work. I want to thank Dr. David Holben and Dr. Michael
Held for providing me beneficial suggestions for my thesis. Furthermore, I also want to thank Dr. Jennifer Horner for providing great help improving the format and style of my thesis. In addition, I want to thank all my friends that provided help to my study and research.
Lastly, I want to thank my family, especially my parents. They have provided endless support and have enabled me to overcome any difficulties throughout my 25 years of life. They gave me confidence when I encountered difficulty, they shared happiness with me when I overcame the difficulty. Thanks to everyone that provided help to me through my master’s degree process.
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Table of Contents
Page
Abstract ...... 3
Acknowledgments...... 5
List of Tables ...... 9
List of Figures ...... 10
Chapter 1: Introduction ...... 11
Pawpaw Background ...... 11
Statement of Problem ...... 11
Research Questions ...... 12
Chapter 2: Review of Literature ...... 15
Introduction to Pawpaw (Asimina triloba) ...... 15
Morphology of pawpaw...... 16
Pawpaw Ripening ...... 17
Color and firmness changes during pawpaw ripening...... 17
Pawpaw softening...... 18
Antioxidant changes during pawpaw ripening stage...... 22
Enzymatic Browning ...... 22
Polyphenol oxidase (PPO)...... 25
Control of enzymatic browning...... 26
Antioxidant Capacity Evaluation ...... 30
2,2-diphenyl-1-picrylhdrazyl (DPPH) assay...... 31 7
Ferric reducing antioxidant power (FRAP) assay...... 32
Oxygen radical absorbing capacity (ORAC) assay...... 33
Summary ...... 33
Chapter 3: Methodology ...... 35
Sample Preparation ...... 35
Treatments...... 35
Antioxidant Capacity Assays ...... 36
Total phenolics...... 36
Total flavonoids...... 36
Ferric reducing antioxidant power (FRAP)...... 37
Measurement of radical scavenging 2,2-diphenyl-1-picrylhdrazyl (DPPH)...... 37
Oxygen radical absorbing capacity (ORAC)...... 37
Quality indicators...... 38
Polyphenol oxidase (PPO) activity test...... 39
Statistical Analysis ...... 39
Chapter 4: Results ...... 40
Compounds from Pawpaw Pulp Extracts ...... 40
Total phenolic content...... 40
Total flavonoid content...... 41
Evaluation of Antioxidant Capacity of Pawpaw Pulp Extracts ...... 43
2,2-diphenyl-1-picrylhdeazyl (DPPH) radical scavenging ability of pawpaw pulp
extract...... 43 8
Reducing potential of pawpaw pulp extract...... 44
Oxygen radical absorbance capacity (ORAC) value of pawpaw pulp extract...... 45
Antioxidant Compounds in Untreated, Ascorbic Acid-Treated and N-acetylcysteine
(NAC)-Treated Samples ...... 47
Total phenolics...... 47
Total flavonoids...... 48
Pawpaw Pulp Color Change During Frozen Storage ...... 49
Lightness (L*) color value of chemically treated pawpaw pulp...... 49
Redness (a*) color value of chemical treated pawpaw pulp...... 50
Yellowness (b*) color value of chemically treated pawpaw pulp...... 51
Polyphenol oxidase (PPO) activity of chemically treated pawpaw pulp...... 52
Chapter 5: Discussion and Conclusion ...... 54
Antioxidant Activity in Pawpaw Pulp ...... 54
Color of Pawpaw Pulp ...... 59
Conclusion ...... 60
Future Recommendation ...... 61
References ...... 62
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List of Tables Page
Table 1: Comparison of Respiration Rates of Fruits From the Annonaceae Family ……22
Table 2: Polyphenol Oxidasse (PPO) Substrates in Selected Fruits…..…………………25
Table 3: Enzymatic Browning Inhibitors ………………..…...……………………….…27
Table 4: Ascorbic Acid Content in Annonaceae Family…...…………………………….28
Table 5: N-acetylcysteine (NAC) Content of Selected Fruits ..…………………….…...29
Table 6: Total Phenolics Concentration………..………..………………………….…....41
Table 7: Total Flavonoid Concentration.………………………………..…………….....42
Table 8: 2,2-diphenyl-1-picrylhdrazyl (DPPH) Radical Scavenging…………………....44
Table 9: Ferric Reducing Antioxidant Power (FRAP) Reducing Potential …………….45
Table 10: Oxygen Radical Absorbance Capacity (ORAC).…………….……………….46
Table 11: Mean Value for Lightness (L*)………..…………………………………...... 50
Table 12: Mean Value for Redness (a*)....…………………………………………..…..51
Table 13: Mean Value for Yellowness (b*)……..……………………..……….…….….52
Table 14: Mean Value for Polyphenol Oxidase (PPO) Activity ..………….…..…….….53
Table 15: Oxygen Radical Absorbance Capacity (ORAC) Value Comparison of Common
Fruits Including Pawpaw.……………………………….…………………………...…..58 10
List of Figures Page
Figure 1: Pawpaw fruit on trees…………………………………...... 17
Figure 2: Simplified ethylene biosynthesis mechanism ……….…...……………….…...20
Figure 3: Simplified enzymatic browning mechanism…………...……………………...23
Figure 4: Ascorbic acid structure……………….…………………………………...... 28
Figure 5: N-acetylcysteine (NAC) structure and cysteine...…………………….…...…..29
Figure 6: The structure of 2,2-diphenyl-1-picrylhdrazyl (DPPH) ………...…….……....32
Figure 7: Ferric reducing antioxidant power (FRAP) assay mechanism……………...... 33
Figure 8: Phenolic compounds comparison...………………………..………………..…47
Figure 9: Flavonoid compounds comparison……..……………………………………...48 11
Chapter 1: Introduction
Pawpaw Background
Pawpaw is a native fruit in the United States. It grows in 26 states in the eastern
United States, from as far south as Florida to as far north as Michigan, to as far west as
Oklahoma. Pawpaw is the only fruit in the Annonaceae family that grows at temperate climates (Galli, Archbold, & Pomper 2007). Pawpaw trees are understory trees that usually are found in well-drained fertile bottom lands and hilly upland habitats.
Furthermore, research has found that pawpaw has high genetic diversity among different genotypes (Pomper et al., 2010).
Pawpaw fruit grows on trees. They are 2-6 inches (5-15 cm) in length and 1-3 inches (3-8 cm) in width. Pawpaw is one of the Annonaceae family fruit members along with cherimoya (Annona cherimola), sweetsop (Annona.squasoma) and soursop (Annona. muricata). Research has found that Annonaceae family fruit have high perishability
(Galli et al., 2007). However, the similarities among the Annonaceae family fruits have not been researched thoroughly. Pawpaw skin turns from green to yellow or brown when mature. Pawpaw pulp has a soft custard-like texture, and a distinctive flavor that tastes like a blend of banana, mango, and pineapple (Galli et al., 2007; Harris & Brannan, 2009).
In addition, pawpaw pulp is rich in antioxidants (Harris & Brannan, 2009).
Statement of Problem
Pawpaw currently has very limited commercial production because of the high perishability of pawpaw pulp. However, research has found that pawpaw is rich in antioxidants such as phenolics and flavonoids (Harris & Brannan, 2009). Pawpaw’s high 12 antioxidant value could increase its market value. On the other hand, pawpaw decays quickly after harvest (Galli et al., 2007). Because of the perishability of pawpaw, frozen storage and refrigerated storage is applied so that customers can have pawpaw out of the harvest season. However, some research has found the antioxidant content in pawpaw pulp declined as the refrigerated storage time increases (Galli, Archbold, & Pomper
2009). How the antioxidant and antioxidant capacity change during frozen storage needs more study. Therefore, the purpose of this research was to investigate the pawpaw pulp antioxidant content’s increase or decrease during frozen storage for up to 1 year and determine the effect of packaging on the antioxidant content and antioxidant capacity.
This research compared two types of packaging (vacuum packaging and air packaging) during frozen storage, to determine which type of packaging is better to store pawpaw and preserve the antioxidant content of pawpaw pulp. This research also compared antioxidant content between a control pawpaw pulp sample and chemically treated pawpaw pulp samples. In addition, the activity of polyphenol oxidase (PPO), which catalyzes enzymatic browning reaction, was evaluated in the project.
Research Questions
1. What are the dynamics of antioxidant content change of pawpaw pulp
during 12 months frozen storage? What are the changes induced by
packaging, and what are the changes induced by polyphenol oxidase
activity? 13
2. What are the dynamics of antioxidant capacity change of pawpaw pulp
during 12 months frozen storage, and what are the changes induced by
packaging?
3. How do pawpaw pulp color and antioxidant content change during 12
months frozen storage, and can these attributes be chemically controlled?
4. What are the dynamics of polyphenol oxidase (PPO) activity during 12
months frozen storage, and what are the changes of antioxidant content
induced by polyphenol oxidase activity.
Limitations
The pawpaw pulp samples used in the experiment were picked from one single
pawpaw tree; however, there are more than 40 varieties of pawpaw. Therefore, the
pawpaw sample used in this experiment may not be representative of other pawpaw
varieties, because the antioxidant contents, the enzymatic reactions, and the color value in
pawpaw pulp could be different across different varieties. In this project, total phenolics and total flavonoids were evaluated, but individual subclasses of flavonoids such as flavonol, flavones, flavanones, flavan-3ols were not evaluated in the project. Thus, in this project, the change in total phenolics, total flavonoids due to packaging, storage for up to
1 year, and chemical treatment were evaluated. PPO activity and antioxidant activity were evaluated as well, but these parameters could vary between in vivo and in vitro due to the variations in environment. A single assay for testing antioxidant capacity may not truly show the actual antioxidant capacity of pawpaw pulp. To minimize the bias of any single method of measurement, three different assays were used to test antioxidant 14 capacity of frozen pawpaw pulp extract, each of which quantified just one parameter of antioxidant capacity. The three assays, oxygen radical absorbance capacity (ORAC), 2,2- diphenyl-1-picrylhdrazyl (DPPH) and ferric ion reducing antioxidant power (FRAP), measure radical scavenging activity, antiradical activity, and reducing capacity of antioxidant compounds, respectively. However, each assay can only quantify one aspect of total antioxidant capacity; none of these assays can completely characterize the total antioxidant capacity of pawpaw pulp. 15
Chapter 2: Review of Literature
Introduction to Pawpaw (Asimina triloba)
Pawpaw is a native tree fruit in the Eastern United States, including Ohio (Galli et al., 2007). Pawpaw only can be grown in temperate growing zones even though pawpaw belongs to the tropical Annonaceae family. There are many fruits in Annonaceae family, including cherimoya (Annona cherimola) sweetsop (Annona squamosa), soursop
(Annona muricata), and custard apple (Annona atemoya). All fruits in Annonaceae family share some similarities with pawpaw (McGrath & Karahadian, 1994), but the pawpaw has some unique characteristics.
Pawpaw traditionally grows on understory trees or in dense aggregation shrub in temperate woodlands. Pawpaw is a challenging species to propagate (Geneve, Kester, &
Pomper, 2007), but researchers have developed grafting and budding methods to enhance pawpaw propagation. More than 40 cultivars of pawpaw are propagated by grafting and budding from superior trees onto common rootstock (Pomper, Crabtree, & Lowe, 2009).
However, there is little research about how grafting or budding affects antioxidant content.
Pawpaw’s high perishability is the biggest barrier to pawpaw commercialization.
Pawpaw’s high perishability was first noted in 1916 by the American Genetic
Association (Archbold, Koslanund, & Pomper, 2003). Research found that pawpaw firmness decreased significantly after harvest. Pawpaw’s rapid firmness decline is one of the major reasons for high perishability, and research has shown that decline in firmness could be caused by reactions catalyzed by cell-wall-degrading enzymes (Chisari, Silveira, 16
Barbagallo, Spagna, & Artes, 2009). Furthermore, pawpaw ethylene production triggers the ripening process and contributes to high perishability. However, research has found that refrigerated storage could effectively delay pawpaw ripening (Harris & Brannan,
2009).
To summarize, pawpaw is rich in antioxidants and has high commercial potential.
However pawpaw’s limited supply and highly perishability are limitations to its commercialization. Although pawpaw grows in the wild, several attempts had been made to commercially cultivate pawpaw in small orchards in the eastern United States. An example of this is a local company, Integration Acres (Albany, OH), which produces frozen pawpaw pulp and is the largest pawpaw processor in the United States.
Morphology of pawpaw. Pawpaw grows on a small tree which is usually 5 to 10 meters high, with dark green leaves and a straight stem. Pawpaw tree leaves are long, drooping, egg-shaped or oblong in shape with narrower ends (see Figure 1). Usually pawpaw leaves are 10 to 15 cm and 15 to 30 cm length. Unlike other species in
Annonaceae family, the texture of leaves of pawpaw is membranous.
Pawpaw is oblong and cylindrical in shape and 3 to 15 cm long, 3 to 10 cm wide, and weighs from 100 to 1000 grams. The pawpaw fruit is the largest native tree fruit in
North America.
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Figure 1. Pawpaw fruit on trees.
Pawpaw Ripening
Fruit ripening is an important consideration in postharvest research. Many fruits continue to ripen after harvest. As fruit become ripe and overripe, antioxidants such as chlorophylls and carotenoids are degraded, but antioxidants such as lycopene accumulate during ripening (Giovanelli, Lavelli, Peri, & Nobili, 2001; Roca & Minguez-Mosquera,
2001). In order to preserve antioxidant content, several postharvest techniques can be applied, such as refrigerated storage, frozen storage, vacuum atmosphere packaging, controlled atmosphere packaging, and modified atmosphere packaging (Hribar, Vidrih,
Simcic, & Plestenjak, 2002). In this study, two types of packaging were evaluated, vacuum packaging and air packaging. Vacuum packaging could preserve antioxidant contents by slowing respiration rate because oxygen is limited inside the packaging. The slower respiration rate could prevent antioxidant degradation.
Color and firmness changes during pawpaw ripening. The ripening stage of some fruits can be observed by their appearance. For example, three major maturity stages exist in the tomato: mature green (mature but green), half ripe (also called the breaker stage when the fruit turns to yellow), and full ripe (the edible stage), when the 18 tomato is red and soft (Moneruzzaman, Hossain, Sani, Saifuddin, & Alenazi, 2009).
Cherimoya is a climacteric fruit, and it belongs to Annonaceae family, and cherimoya turns from green to yellowish-green after harvest (Berger & Galletti, 2005). Skin color is not a reliable external indicator of pawpaw ripeness (McGrath & Karahadian, 1994), but pawpaw pulp color is a reliable indicator of pawpaw ripeness.
Besides color changes, fruit firmness also could be an indicator of fruit ripening.
The loss of firmness is caused by physiological reactions inside the fruit during the ripening stage. For example, the firmness of tomato and mango decrease rapidly during postharvest stage (Jarimopas & Kitthawee, 2007; Lana, Tijskens, & Kooten, 2005).
However, compared to color, firmness is not a reliable marker of ripeness for some commercial fruit (Valero, Crisosto, & Slaughter, 2007). For instance, some melon fruit ripeness cannot be determined by its firmness.
As for pawpaw, previous research has found that hardness value of preripe pawpaw ranges from 1-3 kg, while ripe pawpaw hardness value is around 0.5 kg
(McGrath & Karahadian, 1994). Research suggests that hydrolytic enzymes could be responsible for breaking down pawpaw cell-walls, and that hydrolytic enzyme activation is closely related with fruit harvest (Koslanund, Archbold, & Pomper, 2005b). This will be discussed in detail later in this thesis.
Pawpaw softening. Another important indicator of food ripening is softening.
There are two kinds of cell walls, the original cell wall and the secondary cell wall. The original cell wall mainly is composed of cellulose, pectin, and hemicellulose. The secondary cell wall, mostly composed of cellulose and lignin, is formed between the 19 original cell wall and the plasma membrane. Compared to the secondary cell wall, the primary cell wall contains more pectin and lignin. Softening is caused by cell-wall- degradation reactions that are catalyzed by cell-wall-degrading enzymes. There are many cell-wall-degrading enzymes, such as polygalacturonase (PG), endo-β-1,4-mannanase
(MAN), carboxymethyl-cellulase (CMCase). The activity of these enzymes significantly increases during the midripening period. Softening is one of the major factors in determining postharvest deterioration of fruit. The softening process could also be attributed to cell wall remodeling. Softening could affect fruit shelf life and pathogen infection of fruit.
The function of these cell-wall-degrading enzymes is similar across different fruits. The cell wall becomes increasingly hydrated because pectin gel changes during ripening. Because pectin holds the “cellulose-hemicellulose” network and acts as an adhesive to hold adjacent cells together, pectin modification can be an important factor that determines fruit texture and softening rate. Pawpaw is a dicot plant, as are strawberry, tomato and avocado. Dicot plant fruits share similar cell wall structure and composition, and exhibit a reduction in cell-to-cell adhesion at the early stage of ripening and rapid softening (Crookes & Grierson, 1983). Previous research has shown that cell- wall-degradation enzymes such as polygalacturonase (PG), endo-(1-4) beta-D-glucanase
(EGase) and endo-beta-1, 4-mannanase ( MAN ) can be found in pawpaw (Koslanund, et al., 2005b). Their enzymes activities are low in the preripening and early ripening stage but have a dramatic increase at the midripening stage (Koslanund, et al., 2005b).
Cherimoya has the same cell-wall-degrading enzymes as pawpaw. Research has found 20 that deactivation of cell-wall-degrading enzymes could slow down the softening process and delay cherimoya ripening (Alique & Zamorano, 2000).
Ethylene production and respiration rate. Ethylene production is one of the most important signals of fruit ripening. Ethylene plays the most important role in climacteric fruit ripening (Pech et al., 2002). As shown in Figure 2, Methionine and ATP combine to produce S-adenosyl-methionine (SAM), a reaction catalyzed by SAM synthetase. SAM is converted to 1-aminocyclopropane-1-carboxylic acid (ACC, see
Figure 2), catalyzed by ACC synthase. Ethylene is produced by the reaction of ACC with oxygen, catalyzed by ACC oxidase (Bleecker & Kende, 2000).
Figure 2. Simplified ethylene biosynthesis mechanism. SAM: S-adenosyl-methionine; ACC: 1-aminocyclopropane-1-carboxylic acid; ACS: 1-aminocyclopropane-1- carboxylic acid synthetase; ACO: 1-aminocyclopropane-1-carboxylic acid oxidase.
Previous research has shown that pawpaw displays an increase in ethylene production which peaks at room temperature about 3 days after harvest and that the ethylene climacteric peak and maximum activity of ACS and ACO occur simultaneously
(Koslanund, Archbold, & Pomper, 2005a). These authors speculate that ethylene production might be regulated by ACS and ACO (Koslanund et al. 2005a). Cherimoya displays two ethylene peaks, the first ethylene production peak arises 7 days after harvest, and the value of ethylene production can reach 46.2 µl kg-1 h-1; the other ethylene peak 21
(68.5 µl kg-1 h-1) arises two days after the first ethylene production peak. Unlike
cherimoya, pawpaw displays only one climacteric peak which is 14.4 µg kg-1h-1
(Archbold, 2003).
In order to regulate fruit ripeness, 1-methylcyclopropene (1-MCP) can be used to
control ethylene production. 1-MCP delays ripening by binding to the ethylene receptor.
Previous research found that apricots treated with 1-MCP exhibited significantly higher
antioxidant capacity compared to an untreated sample, and indicated that ripeness may be
inversely related to antioxidant capacity (Egea, Flores, Martinez-Madrid, Romojaro, &
Sanchez-Bel, 2010).
Respiration rate is another important indicator of fruit ripening. Fruits in the
Annonaceae family exhibit increased respiration rate after harvest. As mentioned earlier,
-1 -1 cherimoya exhibits a respiration peak of 90 ml CO2 kg h about 4 to 5 days after harvest.
-1 -1 Some species of cherimoya exhibit another respiration peak of 180 ml CO2 kg h 10 days after harvest. Unlike cherimoya, pawpaw exhibits only one respiration peak,
-1 -1 reaching a peak of 75 to 90 mg kg h of CO2. Respiration rates of fruit in Annonaceae family are shown in Table 1.
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Table 1
Comparison of Respiration Rates of Fruits from the Annonaceae Family
Fruit Peak respiration rate (CO2) Reference
Cherimoya 175 to 250 mg kg -1 h-1 Alique & Zamorano (2000)
Atemoya 90 to 400 mg kg -1 h-1 Brown, Wong, George, & Nissen (1988)
Sugar apple 50 to 180 mg kg -1 h-1 Brown, et al (1988)
Pawpaw 75 to 90 mg kg-1 h-1 Archbold (2003)
Antioxidant changes during pawpaw ripening stage. Fruit antioxidant contents
are different at different stages of ripening. For example, research had found that
anthocyanin synthesis in plum increased by 88% after harvest within 4-15 days. However,
carotenoid contents remained constant during this period (Puerta-Gomez & Cisneros-
Zevallos, 2011). Previous research also has shown that hydrophilic antioxidant activity of
tomato significantly decreased during the ripening stage (Ilahy, Hdider, Lenucci, Tlili, &
Dalessandro, 2011). Furthermore, no difference was found in total phenolics between
underripe and ripe pawpaw pulp, but total phenolics decreased in overripe pulp. However,
total flavonoids increased during ripening (Harris & Brannan, 2009).
Enzymatic Browning
Enzymatic browning, catalyzed by enzymes such as polyphenol oxidase and peroxidase, could cause undesired color changes to fruit. Dark melanin color pigment is generated due to polyphenol oxidase-catalyzed oxidation in the enzymatic browning 23 reaction (Pilizota & Subaric, 1998). Furthermore, polyphenol oxidase (PPO) catalyzes the conversion of phenolics to quinones, which are further converted to melanins (Oms-
Oliu, Aguilo-Aguayo, & Martin-Belloso, 2006), shown in Figure 3.
Figure 3. Simplified enzymatic browning mechanism.
Sometimes, the effects of enzymatic browning can be seen by the appearance of the fruit. For example, chlorogenic acid could generate black spots on banana and pear skin because of the enzymatic browning reaction (Pilizota & Subaric, 1998). Enzymatic browning could also affect pawpaw pulp. Two isoforms of PPO have been identified in pawpaw pulp and are speculated to be the major enzymes that catalyze enzymatic browning (Fang, Wang, Xiong, & Pomper, 2007).
There are many substances that are able to inhibit enzymatic browning. Some directly deactivate PPO or react with intermediates of the enzymatic browning reaction
(Pilizota & Subaric, 1998). Research conducted by Sayavedrasoto and Montgomery 24
(1986) found that sulfites, a potent enzymatic browning inhibitor, change PPO structure and therefore irreversibly inhibit PPO activity. However, sulfites are prohibited because of their toxicity so they are not an option to inhibit enzymatic browning in pawpaw pulp
(Timbo, Koehler, Wolyniak, & Klontz, 2004).
Many polyphenolic antioxidant compounds are substrates for enzymatic browning.
Phenolic compounds play an important role in food quality, appearance and flavor.
Phenolic compound degradation happens frequently in nature, is easily triggered by environmental factors, and is catalyzed by some oxidase enzymes. However, not all antioxidants and not all phenolic compounds are substrates for enzymatic browning.
Research has shown that total soluble phenolics and total anthocyanins do not show a significant correlation with enzymatic browning potential, but chlorogenic acid (a phenolic acid) shows a positive correlation with enzymatic browning (Cheng & Crisosto,
1995). Other antioxidant compounds that have been shown to be PPO substrates include catechin, flavonol, chlorogenic acid and caffeine acid (Richardforget, Rouetmayer,
Goupy, Philippon, & Nicolas, 1992). Some of the substrates for enzymatic browning in fruits are listed are listed in Table 2.
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Table 2
Polyphenol Oxidasse (PPO) Substrates in Selected Fruits
Fruits PPO substrate in fruits Reference
Apple Pyrogallol, catechol, Ni Eidhin, Murphy, catechin (peel) & O'Beirne (2006)
Apricot Catechol, L-dopa, and Arslan, Temur, & gallic acid Tozlu (1998)
Avocado 4-methyl catechol, Gomez-Lopez dopamine, catechol, (2002) chlorogenic acid, caffeic acid
Banana Dopamine Lewis, Fields, & Leucodelphinidin, Shaw, (1999);Yang leucocyanidin et al. (2004)
Grape Catechol and caftaric Yokotsuka & acid, caffeic acid Singleton (1997)
Mango Caffeic acid, catechol, Wang et al. (2007) catechin, chlorogenic acid, tyrosine
Pawpaw Catechol Fang et al. (2007)
Polyphenol oxidase (PPO). PPO is a copper-containing enzyme that can catalyze
reactions with polyphenols to generate O-quinone. Research shows that PPO is widely
distributed in fruits, usually located in the plastid and chloroplast tissue of the plant cell
(Murata, Tsurutani, Hagiwara, & Homma, 1997; Newman, Tantasawat, & Steffens,
2011). PPO is a tetramer containing four atoms of copper in each molecule. As shown in
Figure 3, two separate reactions can be catalyzed by polyphenol oxidase: The first
reaction is the transformation from monophenol to O-phenol by hydroxylation; the 26 second reaction is the oxidation of O-phenol to O-quinones (Tomas-Barberan & Espin,
2001). PPO activity is affected by the availability of substrates, environment pH, and temperature. PPO is inactive at pH < 5 or pH > 8 (Weemaes, Ludikhuyze, Van den
Broeck, & Hendrickx, 1998). PPO activity generally is highest between pH 7.5 and 7.6
(Gomez-Lopez, 2002), and in pawpaw its maximum activity has been reported to be between 6.5-7.0 (Fang, Wang, Xiong, & Pomper, 2007).
Control of enzymatic browning. Enzymatic browning could have a significant effect on both food quality and food nutrition value. Enzymatic browning often produces a color deemed undesirable and may reduce the antioxidant content by consuming polyphenolics. However, not all enzymatic browning is detrimental to food quality, as is the case in some preserved fruit. Nonetheless, control of enzymatic browning is a major focus of the fruit processing industry.
Some enzymatic inhibitors are able to inhibit PPO by forming a complex with the prosthetic group of PPO thus preventing enzymatic browning (Pilizota & Subaric, 1998).
These compounds are listed in Table 3. PPO activity can also be inhibited by limiting oxygen supply or heat treatment to denature the enzyme (Wakayama, 1995). Of the chemical compounds shown in Table 3, ascorbic acid and N-acetylcysteine are of importance to this study and will be described in detail.
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Table 3
Enzymatic Browning Inhibitors
Enzymatic inhibitors Reference
L-cysteine Toker & Bayindirli (2003)
Benzoic acid Sapers & Miller (1998)
Glutathione Billaud, Brun-Merimee, Louarme, & Nicolas (2004)
Cinnamic acid Sapers & Miller (1998)
Ascorbic acid Arias, Gonzalez, Oria, & Lopez-Buesa (2007)
N-acetylcysteine Demirkol, Adams, & Ercal (2004)
Sulfite Sims, Bates, & Mortensen (1991)
Ascorbic acid (vitamin C). Ascorbic acid is found in many fruits. Ascorbic acid
functions as an important enzymatic browning inhibitor. Ascorbic acid deactivates PPO
activity by chelating a copper atom, which is from the prosthetic group of PPO. The
structure of ascorbic acid is shown in Figure 4. Ascorbic acid also inhibits enzymatic
browning by reducing quinones back to phenols (Limbo & Piergiovanni, 2006). Fruits
from the Annonaceae family fruits are rich in ascorbic acid, as shown in Table 4. Sugar
apple processes the most ascorbic acid content compared to other fruits in Annonaceae
family. Although pawpaw pulp contains 4.98 mg ascorbic acid per 100 g (Harris &
Brannan, 2009), enzymatic browning is not completely inhibited (Fang et al., 2007).
28
Table 4
Ascorbic Acid Content in Annonaceae Family
Ascorbic acid content Fruit (mg/100g pulp) Reference
Sugar apple 37.38 ± 4.6 Pareek, Yahia, Pareek, & Kaushik (2011)
Custard apple 30.00 ± 0.0 Pareek, et al (2011)
Soursop 19.40 ± 3.0 Pareek, et al (2011)
Cherimoya 11.50 ± 5.5 Pareek, et al (2011)
Pawpaw 4.98 ± 0.3 Harris & Brannan (2009)
Figure 4. Ascorbic acid structure.
N-acetylcysteine (NAC). NAC is derivation of L-cysteine (see Figure 5). NAC is
a natural thiol-containing compound that could react with quinones at the initial stage of
the enzymatic browning reaction to reduce O-quinones to O-diphenols, producing colorless products (Demirkol et al., 2004). Naturally-occurring NAC is found in many 29 fruits, shown in Table 5. NAC content has not been measured in pawpaw yet.
Researchers have found that adding 0.75% of NAC could effectively prevent enzymatic browning on fresh-cut pears for up to 28 days at 4 ̊C (Oms-Oliu et al., 2006).
Table 5
N-Acetylcysteine (NAC) Content of Selected Fruits (Demirkol et al., 2004)
Fruits NAC content (nM/g Wet Weight)
Orange ND
Lemon 4
Grape 4
Mango ND
Papaya ND
Banana ND
Strawberry 5 Note. ND = not detected.
Figure 5. N-acetylcysteine (NAC) (left) and cysteine (right).
30
Co-inhibitor browning. Although NAC and ascorbic acid could effectively reduce enzymatic browning, multiple enzymatic inhibitors are often used together to reduce enzymatic browning. Research found that sulfur dioxide (Pilizota & Subaric,
1998), L-cysteine (Iyidogan & Bayindirli, 2004) and glutathione (Billaud, Brun-Merimee,
Louarme, & Nicolas, 2004) could effectively inhibit enzymatic browning. Research also found that combination of 1.5% NAC and 1.5% glutathione could better inhibit enzymatic browning than one enzymatic browning inhibitor NAC alone (Oms-Oliu et al.,
2006).
Antioxidant Capacity Evaluation
Antioxidant capacity is determined by quantifying the reaction between antioxidants and free radicals. In general, there are two underlying mechanisms that are used to test antioxidant capacity: hydrogen atom transfer (HAT) or single electron transfer (ET). HAT-based assays generally are composed of a free radical generator, an antioxidant, and a detector molecule (often referred to as a probe) that becomes oxidized.
The amount of oxidation of the probe molecule is detected and measures radical scavenging capacity (Huang, Ou & Prior, 2005). The ET-mechanism involves one redox reaction of an oxidant and evaluates antioxidant’s reducing capacity (Benzie & Strain,
1999).
However, total antioxidant capacity is a broad term that includes reducing capacity and reactive oxygen species (ROS) scavenging capacity. There is increasing concern that using a single assay for testing antioxidant capacity may not represent the actual antioxidant capacity (Bartosz, 2010). As an example, one very popular antioxidant 31 capacity assay (oxygen radical absorbance capacity, ORAC) only evaluates peroxyl radical scavenging capacity (Huang et al., 2005). Nevertheless, ORAC has been used widely by the food companies whose products have high ORAC values to assert that their products are healthier. For a time, the U.S. Department of Agriculture (USDA, 2012) maintained a database of ORAC values http://www.ars.usda.gov/Services/docs.htm?docid=15866), but this database was removed and the USDA declared, “Recently the USDA’s Nutrient Data Laboratory
(NDL) removed the USDA ORAC Database for Selected Foods from the NDL website due to mounting evidence that the values indicating antioxidant capacity have no relevance to the effects of specific bioactive compounds” (para. 1).
In this thesis, three different assays (DPPH, ORAC, FRAP) were used to test pawpaw pulp antioxidant capacity. Each assay quantified one parameter of antioxidant capacity.
2,2-diphenyl-1-picrylhdrazyl (DPPH) assay. DPPH is a dark-colored crystalline powder composed of stable free-radical molecules. Figure 6 shows the structure of DPPH.
DPPH’s stability is due to the space formed by its three-benzene structure. DPPH has a maximum absorption at wavelength of 515 nm. In a solution that contains DPPH and an antioxidant, oxidation causes the dark color of the solution to fade. The DPPH assay quantifies antioxidant capacity by measuring the absorbance decrease after oxidation.
Antioxidant capacity is determined by the ability of the antioxidant to retard color loss.
There are still controversies about the underlying mechanism of the DPPH assay.
Because DPPH is not a highly reactive peroxyl radical, most reactions between DPPH 32 and antioxidants are believed to involve the HAT mechanism; however, some research reports that the reaction may be more likely to occur through the ET mechanism (Foti,
Daquino, & Geraci, 2004).
Figure 6. The structure of DPPH.
Ferric reducing antioxidant power (FRAP) assay. The FRAP assay is an ET- based assay and measures an antioxidant’s ability to keep Fe (III) Tripyridyl Triazine
(TPTZ) in a reduced state. In other words, the FRAP assay measures reducing capacity by measuring the ability of an antioxidant to keep iron in a reduced state. In the FRAP assay, antioxidants reduce the Fe (III) Tripyridyl Triazine (TPTZ) complex to Fe (II) with
TPTZ. The complex compound formed by Fe (III) and TPTZ is a dark red solution which after the complex is reduced changes to dark blue. By measuring the absorbance level change within 4 minutes, the antioxidant capacity is calculated. A criticism of the assay is that some research suggests that absorbance is not stable after 4 minutes of reaction, and
2+ may increase slowly after a few hours. This could be because the [Fe(II)(TPTZ)2]
complex reacts with caffeic acid, tannic acid, or ascorbic acid to cause the absorbance
level to increase beyond 4 minutes (Pulido, Bravo, & Saura-Calixto, 2000). 33
Figure 7. FRAP assay mechanism.
Oxygen radical absorbing capacity (ORAC) assay. The ORAC assay is a typical HAT-mechanism-based method, because it employs a competitive reaction between antioxidants and a fluorescent probe, fluorescein. The peroxyl radical is generated by 2,2- azobis (2-amidinopropane) dihydrochloride (AAPH) (Huang, Ou,
Hampsch-Woodill, Flanagan, & Prior, 2002). Peroxyl radicals oxidation causes fluorescence to decrease. Antioxidants protect the fluorescein from degradation by peroxyl radicals (Roy et al., 2010). Trolox, a water-soluble vitamin E derivative, is used as the standard for comparison (Roy et al. 2010).
Summary
In summary, pawpaw belongs to Annonaceae family, and has a distinctive aroma and flavor. However, the parishability of pawpaw makes commercialization of pawpaw difficult. And the antioxidant (phenolics and flavonoid) content of pawpaw is regarded as its most valuable nutrient. Frozen storage is used to maintain antioxidant content and antioxidant capacity in pawpaw pulp. However, some research shows that PPO can catalyze enzymatic browning reactions that use antioxidants as substrates. Additionally, 34 previous research has shown that ascorbic acid and NAC can inhibit PPO activity. Thus, these two chemical compounds may be useful to inhibit enzymatic browning in pawpaw pulp. 35
Chapter 3: Methodology
Sample Preparation
Pawpaws were collected from a single tree in Athens, Ohio. Although these pawpaws are considered “wild,” this tree has consistently produced pawpaws that earned top honors in the “Best Pawpaw Contest” at the yearly Pawpaw Festival based on their weight, appearance, skin surface, aroma, skin thickness, flavor, texture, aftertaste, and number of seeds. The pawpaw pulp was separated from the skins and seeds and then all of the pulp was pooled and divided into 100 g portions. To some of the bags, ascorbic acid or NAC (a cysteine analog; see Figure 5) was added to achieve a final concentration of 1%. Once portioned, the pawpaw pulp was placed into polyethylene/nylon FoodSaver
(Jarden Corp., Rye, NY) 27.94-cm bags with an oxygen transmission rate of 6.7 cc/m2/24 h/23°C/0% RH. The bags were randomly selected prior to labeling. Once the bags were filled, they were either vacuum sealed (vacuum storage) or sealed without attempting to remove air prior to sealing (air storage), then immediately transferred into frozen storage at -18 ºC. At 2-month intervals, pawpaw samples were immediately transferred from -18
ºC to a freezer at -40 ºC to be analyzed at a later time.
Treatments
Pawpaw fruit pulp (5 g) was extracted in 25 ml of methanol as 1:5 (w/v) ratios using a Waring blender followed by agitation for 1 hour. Afterwards, methanol-extracted samples were filtered and stored in glass screw top vials.
Antioxidant capacity measurements (Research Questions #1 and #2) were performed on control samples packaged in either vacuum or air. Objective color 36 measurements (Research Question #3) were performed on control samples packaged in either vacuum or air, and on chemical treatment samples packaged in air. Polyphenol oxidase activity measurements were performed on the samples with and without 1% ascorbic acid or 1% NAC (Research Question #4). All of these measurements were taken at 2-month intervals during frozen storage.
Antioxidant Capacity Assays
Total phenolics. The measurement of total phenolics was determined spectrophotometrically by using the Folin–Ciocalteu (FC) assay. FC reagent was diluted with deionized water and the diluted reagent (750 μl) was mixed with aliquots of the pawpaw pulp extract (100 μl) and 7.5% bicarbonate solution (750 μl). After 120 minutes in the absence of light, absorbance was measured at 750 nm by using a Spectronic
Genesys 5 (Thermo Electric Corporation, Madison, WI). Total phenolics were quantified according to a standard curve prepared from gallic acid and expressed as μmol gallic acid equivalents.
Total flavonoids. Total flavonoids were measured spectrophotometrically.
Pawpaw extract (0.5 ml) was mixed with methanol (1.5 ml), to which 10% aluminum chloride (0.1 ml), 1 mol/l potassium acetate (0.1 ml), and deionized water (2.8 ml) was added. Samples were vortexed and allowed to sit for 40 minutes at 25 ºC, after which the absorbance was measured at 415 nm using a Spectronic Genesys 5 (Thermo Electric
Corporation, Madison, WI). The total flavonoids contents were quantified by comparing to the standard curve prepared from rutin (quercetin-3-O-rutinoside) and the 37 concentrations of total flavonoids were reported as μmol rutin equivalents (Bor, Chen, &
Yen, 2006).
Ferric reducing antioxidant power (FRAP). The ferric reducing capacity was measured using the FRAP assay (Benzie & Strain, 1996). The FRAP reagent (1.5 ml) consisted of sodium acetate buffer (pH = 3.6) mixed with 10 mmol/L (2,4,6,-tri(2- pyridyl)-S-triazine; TPTZ) in 40 mmol/L HCl and ferric chloride in a ratio of (10:1:1).
This buffer was mixed with 100 μl of pawpaw pulp extract. After 4 mintues at room temperature, absorbance was monitored at 593 nm using a Spectronic Genesys 5 (Thermo
Electric Corporation, Madison, WI). The reducing capacity was quantified according to a standard curve prepared from gallic acid and the concentrations were expressed in gallic acid equivalents.
Measurement of radical scavenging 2,2-diphenyl-1-picrylhdrazyl (DPPH).
The ability of pawpaw pulp extract to quench radicals was measured using the DPPH radical scavenging assay (Cheng, Moore, & Yu, 2006). Pawpaw pulp extract (50 μl) was added to 60 μM methanolic DPPH radical solutions. After 25 minutes of incubation in the absence of light, the absorbance at 517 nm was measured on a Spectronic Genesys 5
(Thermo Electric Corporation, Madison, WI). Blank samples containing water rather than pawpaw pulp were used as control sample for calculation. Radical scavenging was expressed as percent inhibition of the DPPH radical compared to a control sample which contained no pawpaw pulp extract.
Oxygen radical absorbing capacity (ORAC). The ORAC assay was conducted based on previously published methods (Ou, Hampsch-Woodill, & Prior, 2002; Roy et al., 38
2010). Briefly, pawpaw extracts were diluted (20 μl of extract with 5 μL of phosphate buffer, 75 mM pH 7.4) and 150 μl of sodium fluorescein (40 nM) were added to a black- walled 96-well plate. Next, the plate was placed into a microplate reader, set with an excitation filter of 485 nm and an emission filter of 535 nm, and was incubated for 15 mintues at 37 ̊C. Next, 25 μL AAPH (150 mM) was added to each well. Immediately after addition of AAPH, the plate was shaken and read every minute for 35 minutes until a 95% loss of fluorescent signal was reached. The first measurement was recorded as f0,
the min 1 measurement was recorded as f1 and so on. The AUC (area under curve) was
calculated by using the formula below.
AUC = 1+ f1/f0 + f2/f0 + f3/f0 + f4/f0 + ... + f34/f0 + f35/f0
Additionally, a standard curve for the ORAC assay was made by substituting 25
μL diluted sample with 25 μL trolox C solution at five different concentrations (1.25, 2.5,
5, 10 or 20 μM). The relative ORAC value (Trolox equivalents) was calculated as: (AUC
sample-AUCblank )/(AUCtrolox-AUC blank) *(molarity of trolox /molarity of sample). The
results were presented as mean ± standard deviation and an analysis of variance
(ANOVA) was performed for statistical analysis.
Quality indicators. Pulp color was characterized by using a Konica Minolta
Colorimeter based on the CIELAB system. This model provided three measures:
L*represented whiteness or blackness; +a* represented redness, –a* represented
greenness; +b* represented yellowness,–b* represented blueness. The color of the
samples was measured through plastic film by a Konica Minolta Colorimeter. 39
Polyphenol oxidase (PPO) activity test. PPO was determined according to the method of Soliva-Fortuny and others (Soliva-Fortuny, Biosca-Biosca, Grigelmo-Miguel,
& Martin-Belloso, 2002). Enzyme activity in crude PPO extract was assayed spectrophotometrically by adding 0.4 of 0.2 M catechol (Sigma-Aldrich) and 100 μL of extract to 5.5 mL phosphate buffer in a quartz cuvette of 1 cm path length. The changes in absorbance at 420 nm were recorded every 15s for 10 minutes from the time the enzyme extract was added using a Spectronic Genesys 5 (Thermo Electric Corporation,
Madison, WI). PPO activity was defined as the slope of the time course of catechol oxidation, i.e., the change in absorbance of per min per mg of pawpaw pulp.
Statistical Analysis
All measurements were generated based on three trials (n = 3). Data were analyzed statistically using SPSS version 16.0 for Windows Seattle, WA. Analysis of variance (ANOVA) was used to determine differences between the means. Significance was set at p < 0.05, and a post-hoc test, Duncan’s Multiple Range test, determined where significant differences occurred. The study was not replicated.
40
Chapter 4: Results
This study evaluated the impact of vacuum packaging versus non-vacuum packaging on the change in antioxidant content (Research Question #1) and antioxidant capacity (Research Question #2) during frozen storage of pawpaw pulp.
In addition to that, this study compared how two chemical preservatives, ascorbic acid and NAC, affected the antioxidant content and color (Research Question #3) and
PPO activity (Research Question #4) when added to nonvacuum packaged pulp.
Compounds from Pawpaw Pulp Extracts
Total phenolic content. The main effects of time of frozen storage (seven levels:
0, 2, 4, 6, 8, 10, 12 months) and packaging (two levels: vacuum, air) on total phenolics were analyzed. Total phenolics were significantly affected by storage time (p < 0.001), with phenolic levels significantly decreasing during frozen storage. Packaging also significantly affected the level of phenolics (p = 0.016). The concentration of phenolics was significantly higher in vacuum packaged pawpaw pulp than that stored in the presence of air. In other words, the main effect analysis indicated that phenolic concentration decreased during frozen storage regardless of the type of packaging, with vacuum packaging slowing the rate of phenolics degradation compared to samples stored in the presence of air.
Table 6 shows the mean values of total phenolics for two-way interactions between storage time and packaging. Compared to the control, levels of phenolics in vacuum packaged pawpaw pulp did not significantly decline until after 6 months of frozen storage. This result agrees with previous work (Harris & Brannan, 2009). Unlike 41 vacuum packaged samples, total phenolics in pawpaw pulp stored in air exhibited an immediate decline in phenolic compounds, as evidenced by the fact that compared to the control samples, significantly fewer total phenolics content were detected at 2 months of storage and every subsequent month. It should be noted that after 6 months of frozen storage, there were no differences observed in total phenolics between vacuum packaged and air packaged samples.
Table 6
Total Phenolics Concentration
Total phenolics
Month Vacuum Air
0 9.78 ± 0.26bcd 15.32 ± 1.38a
2 12.37 ± 2.24ab 4.61 ± 0.36g
4 8.30 ± 0.73cde 7.19 ± 0.15defg
6 10.31 ± 2.68bc 7.78 ± 0.17cdef
8 5.74 ± 1.07efg 6.51 ± 0.50efg
10 6.51 ± 0.43efg 4.77 ± 0.68fg
12 5.96 ± 1.05efg 4.41 ± 3.84g Note. Mean values (± standard deviation) of the two-way interactions (month of storage and packaging method) for total phenolics (µmol gallic acid equivalents/g pawpaw pulp) in frozen stored pawpaw pulp (p < 0.05).
Total flavonoid content. The main effects of frozen storage time (seven levels: 0,
2, 4, 6, 8, 10, 12 months) and packaging (two levels: vacuum, air) for total flavonoids
were analyzed. Flavonoids were significantly affected by storage time (p < 0.001) but not 42 packaging (p = 0.215 > 0.05). Total flavonoids increased at 2 months of frozen storage, and then returned to baseline (0 month) values thereafter.
Table 7 shows the mean values ± standard deviation of total flavonoid content and the results of a two-way ANOVA for effects of months of frozen storage and packaging.
These results are difficult to interpret, as flavonoid values in vacuum packaged samples exhibited their minimum value at 0 month whereas air packaged samples exhibited their maximum value at 0 month. Previous research on vacuum packaged, frozen stored pawpaw pulp agrees with this finding, as flavonoid values initially increased then remained constant during 6 months of frozen storage (Brannan & Salabak, 2009).
Table 7
Total Flavonoids Concentration
Total flavonoids
Month Vacuum Air
0 1.36 ± 0.11f 4.75 ± 0.21a
2 5.02 ± 0.61a 2.7 ± 0.06bc
4 2.52 ± 0.08bc 2.26 ± 0.10cde
6 2.52 ± 0.45bc 2.68 ± 0.26bc
8 1.99 ± 0.21cdef 1.75 ± 0.34def
10 3.11 ± 0.57b 2.11 ± 0.06cde
12 2.33 ± 0.71cd 1.55 ± 0.36ef Note. Mean values (± standard deviation) of the two-way interactions (month of storage and packaging method) for total flavonoids (µmol rutin equivalents/g pawpaw pulp) in frozen stored pawpaw pulp. (p < 0.05).
43
Evaluation of Antioxidant Capacity of Pawpaw Pulp Extracts
2,2-diphenyl-1-picrylhdeazyl (DPPH) radical scavenging ability of pawpaw pulp extract. The main effects of time of frozen storage (seven levels: 0, 2, 4, 6, 8, 10,
12 months) and packaging method (two levels: vacuum, air) on DPPH radical scavenging ability were analyzed. DPPH radical scavenging ability was significantly affected by storage time (p = 0.004) and packaging (p = 0.015). The DPPH radical scavenging ability in pawpaw pulp in vacuum packaging is significantly higher than pawpaw pulp stored in the presence of air. In other words, vacuum packaging preserved the radical scavenging ability of the pulp compared to samples stored in the presence of air. Table 8 shows the two-way interactions for DPPH radical scavenging among storage time and packaging. Compared to the control, DPPH radical scavenging ability was not significantly different for either vacuum or air stored samples until after 10 months of storage. It should be noted that vacuum stored samples at month 0 exhibited significantly higher DPPH radical scavenging than air stored samples.
44
Table 8
2,2-diphenyl-1-picrylhdrazyl (DPPH) Radical Scavenging
DPPH radical scavenging
Month Vacuum Air
0 66.67 ± 10ab 45.37 ± 10cd
2 73.86 ± 02a N/A
4 56.83 ± 08bc 52.58 ± 30bcd
6 54.26 ± 10bcd 53.08 ± 10bcd
8 60.60 ± 11abc 53.66 ± 03bcd
10 60.09 ± 06abc 59.35 ± 04abc
12 46.88 ± 11cd 41.02 ± 03d Note. N/A = not analyzed. Mean values (± standard deviation) of the two-way interactions (month of storage and packaging method) for DPPH radical scavenging (%) in frozen stored pawpaw pulp. Radical scavenging denotes a significant difference at p < 0.05.
Reducing potential of pawpaw pulp extract. The main effects of frozen storage
time (seven levels: 0, 2, 4, 6, 8, 10, 12 months) and packaging (two levels: vacuum, air)
on reducing potential (FRAP) were analyzed. Although reducing potential was significantly affected by storage time (p = 0.002) and packaging (p = 0.025), no clear
trends were observed for storage time effect. Table 9 shows the two-way interactions for reducing potential comparing months of frozen storage and packaging. The results for the two-way ANOVA were similar to the main effects. Generally, there was no increase or decrease in reducing potential throughout the storage period regardless of packaging method. 45
Table 9
Ferric Reducing Antioxidant Power (FRAP) Reducing Potential
Reducing potential (FRAP)
Month Vacuum Air
0 2.23 ± 0.32bcd 2.80 ± 0.17ab
2 2.05 ± 0.31cd N/A
4 3.09 ± 0.30a 2.95 ± 0.22a
6 2.73 ± 0.30ab 1.85 ± 0.27d
8 2.87 ± 0.43ab 2.71 ± 0.03ab
10 3.11 ± 0.58a 2.63 ± 0.13abc
12 2.73 ± 0.16ab 2.52 ± 0.08abc Note. N/A = not analyzed. Mean values (± standard deviation) of the two-way interactions (month of storage and packaging method) for reducing potential (µmol gallic acid equivalents/g pawpaw pulp) in frozen stored pawpaw pulp. Reducing potential denotes a significant difference at p < 0.05.
Oxygen radical absorbance capacity (ORAC) value of pawpaw pulp extract.
The main effects of month of frozen storage (seven levels: 0, 2, 4, 6, 8, 10, 12 months)
and packaging (two levels: vacuum, air) on ORAC value were analyzed. ORAC measures
the ability of pawpaw extract to scavenge free radicals. ORAC value was significantly
affected by storage time (p < 0.001) and packaging (p < 0.001). The ORAC value in
pawpaw pulp stored frozen in vacuum packaging is significantly lower than pawpaw held
in the presence of air. It is difficult to interpret the trend of ORAC value by storage time,
however, we can conclude that air package protected the free radical scavenging ability
compared to samples stored in the vacuum packaging. 46
Table 10 shows the two-way interactions for ORAC values comparing months of frozen storage and packaging. Air stored samples exhibited a significantly higher ORAC value than vacuum packaged samples at every month of storage. ORAC values in samples stored in either vacuum packaging or in the presence of air exhibited an increase then decline to baseline values at 12 - month of storage.
Table 10
Oxygen Radical Absorbance Capacity (ORAC)
Oxygen radical absorbance capacity (ORAC)
Month Vacuum Air
0 2.96 ± 0.02e 2.95 ± 0.02b
2 2.90 ± 0.04de N/A
4 2.93 ± 0.03bc 3.03 ± 0.02a
6 2.91 ± 0.03cd 3.03 ± 0.02a
8 2.83 ± 0.02f 3.03 ± 0.02a
10 2.87 ± 0.02e 3.02 ± 0.05a
12 2.87 ± 0.01e 2.92 ± 0.02bc Note. N/A = not analyzed. Mean values (± standard deviation) of the two-way interactions (month of storage and packaging method) for ORAC (µmol trolox equivalents/g pawpaw pulp) in frozen stored pawpaw pulp. ORAC value denotes a significant difference at p < 0.05.
47
Antioxidant Compounds in Untreated, Ascorbic Acid-Treated and N-acetylcysteine
(NAC)-Treated Samples
Total phenolics. Figure 8 shows the two-way interactions for total phenolics between packaging and storage. Whereas a significant decline in phenolics was observed for air stored samples by 2 months of storage and vacuum stored samples after 6 months of storage, no decline in total phenolics throughout the storage period was observed for pawpaw pulp to which ascorbic or NAC was added. By 12 months of storage, samples that contained ascorbic acid or NAC exhibited significantly higher phenolics content that either the vacuum stored or air stored samples.
18 air only
16 air + ascorbic acid
14 air + NAC
12
10
8
6
4
2
Total Phenolics 0 024681012 Month of Frozen Storage
Figure 8. Phenolics compounds comparison. Total phenolic content (gallic acid equivalents/g pawpaw tissue) of pawpaw pulp that was stored vacuum packaged or in the presence of air with or without ascorbic acid and n-acetylcysteine (NAC) during frozen storage. 48
Total flavonoids. Figure 9 shows the two-way interactions for total flavonoids between packaging and storage. Total flavonoid concentration was observed to increase throughout storage. Total flavonoid concentration in air stored samples was observed to decrease by 2 months of storage. However, no significant change in flavonoid was observed for ascorbic acid treated samples. NAC treated samples exhibited a decline in total flavonoids after 2 months of storage. For each month of storage, samples that contained ascorbic acid or NAC exhibited significantly lower total flavonoids content that either vacuum stored or air stored sample.
6 air only
5 air + ascorbic acid
air+ NAC 4
3
2
1
Total Flavonoid 0 1234567 Month of Frozen Storage
Figure 9. Flavonoid compounds comparison. Total flavonoid content (rutin acid equivalents/g pawpaw tissue) of pawpaw pulp that was stored vacuum packaged or in the 49 presence of air with or without ascorbic acid and n-acetylcysteine (NAC) during frozen storage.
Pawpaw Pulp Color Change During Frozen Storage
Lightness (L*) color value of chemically treated pawpaw pulp. Table 11 shows the two-way interactions for lightness (L*) between packaging and storage.
Whereas the vacuum packaged samples exhibited a significant decline in lightness after 8 months of storage, air stored samples did not exhibit lower lightness values until after 10 months of storage. Lightness value remained constant in samples that contained ascorbic acid and NAC throughout storage. Compared to vacuum packaging, treatment with ascorbic acid and NAC prevented the loss of lightness associated with all air stored samples and vacuum stored samples after 8 months of storage.
50
Table 11
Mean Value for Lightness
Month Vacuum Control Ascorbic acid NAC
0 65.77 ± 0.57ab 52.13 ± 0.40ef 70.70 ± 0.46a 66.60 ± 0.44ab
2 66.43 ± 0.40ab 53.20 ± 1.80ef 55.20 ± 18.79def 64.90 ± 1.47abc
4 61.73 ± 0.21bcd 58.50 ± 1.15cde 66.33 ± 0.64ab 64.97 ± 1.50abc
6 67.23 ± 0.96ab 57.27 ± 0.40cde 62.27 ± 4.65abcd 66.77 ± 0.35ab
8 61.73 ± 0.50bcd 54.30 ± 0.98ef 65.67 ± 0.51ab 67.00 ± 0.36ab
10 56.33 ± 1.90def 58.37 ± 0.95cde 65.40 ± 0.66abc 66.97 ± 0.59ab
12 57.13 ± 1.12def 50.37 ± 0.15f 65.93 ± 0.55ab 66.30 ± 1.15ab Note. NAC = N-acetylcysteine. Mean L*(Lightness) values (± standard deviation) for two-way interactions between months of storage and chemical treatment (vacuum, air and ascorbic acid and NAC). Different superscripts within L* value, groups which do not share a common superscript letter within L* value denote significant difference at p < 0.05.
Redness (a*) color value of chemical treated pawpaw pulp. Table 12 shows
the mean value for redness (a*). Positive a* values identify the intensity of red color in
the samples, whereas negative a* values would have indicated the intensity of green color
in the samples. Redness was unaffected by vacuum packaging and air packaging during
frozen storage however redness decreased after 2 months of frozen storage then remained
constant thereafter in ascorbic acid and NAC treated sampled.
51
Table 12
Mean Value for Redness
Month Vacuum Control Ascorbic acid NAC
0 5.93 ± 0.15ghij 6.90 ± 0.30efg 7.13 ± 0.72defg 5.90 ± 0.20ghij
2 5.83 ± 0.15ghij 10.73 ± 0.55a 6.30 ± 0.53ghi 4.90 ± 0.70jkl
4 6.23 ± 0.95ghij 6.93 ± 1.75efg 5.10 ± 0.62hijk 3.60 ± 0.26lmn
6 9.33 ± 0.86bc 8.00 ± 0.00de 4.20 ± 0.10klm 3.87 ± 0.15klmn
8 6.67 ± 0.35fg 10.33 ± 0.15ab 5.00 ± 0.66ijk 5.00 ± 0.20ijk
10 6.40 ± 2.40gh 7.87 ± 0.06def 4.97 ± 0.06ijk 3.27 ± 0.12mn
12 8.37 ± 0.45cd 8.17 ± 0.12cde 4.27 ± 0.32klm 2.80 ± 0.92n Note. NAC = N-acetylcysteine. Mean a*(redness) values (± standard deviation) for two- way interactions between chemical treatment (vacuum, air, ascorbic acid and NAC) and month of storage; Different superscripts within a* value, groups which do not share a common superscript letter within a* value denote significant difference at p < 0.05.
Yellowness (b*) color value of chemically treated pawpaw pulp. Table 13
shows the mean value for yellowness (b*). Positive b* values identify the intensity of
yellow color in the samples, whereas negative b* values would have indicated the
intensity of blue color in the samples. These results are hard to interpret since b* value
exhibited no obvious trends. The b* value of ascorbic acid and NAC treated samples
generally remained constant throughout storage.
52
Table 13
Mean Value for Yellowness
Month Vacuum Control Ascorbic acid NAC
0 29.30 ± 0.10fg 19.27 ± 2.00j 44.13 ± 2.28a 40.17 ± 0.32bc
2 29.83 ± 1.57efg 28.83 ± 1.58fgh 39.20 ± 0.20bcd 37.97 ± 1.72bcd
4 29.03 ± 1.66fgh 25.80 ± 1.57h 36.77 ± 2.21cd 36.03 ± 3.41d
6 40.00 ± 0.50bc 19.30 ± 0.66j 32.83 ± 4.25e 37.83 ± 1.17bcd
8 37.27 ± 0.67bcd 27.30 ± 0.56gh 37.37 ± 3.11bcd 40.60 ± 0.62b
10 31.90 ± 0.75ef 28.40 ± 0.62gh 37.53 ± 0.95bcd 38.50 ± 0.00bcd
12 28.40 ± 2.20gh 22.43 ± 0.06i 39.17 ± 1.53bcd 36.10 ± 3.54d Note. NAC = N-acetylcysteine. Mean b* (yellowness) values (± standard deviation) for two-way interactions between chemical treatment (vacuum, air, ascorbic acid and NAC) and month of storage; Different superscripts within b* value, groups which do not share a common superscript letter within b* value denote significant difference at p <0.05.
Polyphenol oxidase (PPO) activity of chemically treated pawpaw pulp. Table
14 shows the mean value of PPO activity and the effects of storage time and packaging.
For the samples that were not treated chemically, i.e., vacuum packaged and air stored
samples, PPO activity remained constant during storage. Ascorbic acid and NAC
treatment dramatically reduced PPO activity.
53
Table 14
Mean Value for Polyphenol Oxidase (PPO) Activity
Month Vacuum Control Ascorbic acid NAC
0 0.12 ± 0.01a 0.09 ± 0.004abcd 0.040 ± 0.010efg 0.000 ± 0.000h
2 0.08 ± 0.01abcd 0.06 ± 0.002bcde 0.086 ± 0.100abcd 0.003 ± 0.002gh
4 0.09 ± 0.01abcd 0.09 ± 0.002abcd 0.035 ± 0.006fgh 0.002 ± 0.002gh
6 0.06 ± 0.00def 0.11 ± 0.01ab 0.020 ± 0.009gh 0.001 ± 0.001g
8 0.08 ± 0.00abcd 0.07 ± 0.004bcde 0.006 ± 0.010gh 0.000 ± 0.000h
10 0.11 ± 0.00a 0.07 ± 0.003cdef 0.006 ± 0.01gh 0.008 ± 0.014gh
12 0.10 ± 0.00abc 0.09 ± 0.018abcd 0.000 ± 0.00h 0.000 ± 0.000h Note. NAC = N-acetylcysteine. Mean value (± standard deviation) for PPO activity (ABS/(s*mg)) for two-way interactions between chemical treatment and month of storage; Values that do not share a common superscript letter denote significant difference at p < 0.05.
54
Chapter 5: Discussion and Conclusion
Antioxidant Activity in Pawpaw Pulp
As noted in Table 6, total phenolics in pawpaw pulp were significantly affected by the presence of air during storage. The mean value of total phenolics decreased by 39% in vacuum packed pawpaw pulp over 12 months of frozen storage. However, total phenolics decreased by 71% while stored for 12 months in the presence of air. Research has shown that the effect of freezing on the level of antioxidant compounds in fruit pulp depends on the species. Total phenolic content was reduced by 28% in frozen cambuci
(176 mg/100g) compared to fresh cambuci (246 mg/100g) (Genovese, Pinto, Goncalves,
& Lajolo, 2008). However, the antioxidant content (3.2 µmol BHT equiv/g fresh weight) of guava that was stored frozen for several months was 60% higher than the antioxidant value of fresh guava pulp (2.0 µmol BHT equiv/g fresh weight) (Hassimotto, Genovese,
& Lajolo, 2009). In pawpaws, pulp stored frozen for 300 days exhibited four times more total phenolics and flavonoids than fresh pulp (Harris & Brannan, 2009). The results reported in this thesis suggest that the oxidative degradation of total phenolics in pawpaw pulp was enhanced by the presence of air. Other researchers have speculated that polymerization of phenolics during storage causes a decline in the level of phenolics that can be detected using the Folin Calcitaue assay (Harris & Brannan, 2009); however, it is not known if such a mechanism exists in vivo. The results shown in Table 6 also indicate that the decline observed in total flavonoid levels of pawpaw pulp were not significantly affected by the presence of air in the package during 12 months of frozen storage. This 55 could mean that the flavonoid fraction, which is a subset of the total phenolic fraction, is not as susceptible to air-induced oxidation as other phenolic compounds.
One possible reason for the degradation of phenolic compounds in pawpaw pulp could be the activity of PPO, which was inhibited in pawpaw pulp to which ascorbic acid and NAC were added (shown in Table 12). Research has shown that PPO can catalyze oxidative reactions that use phenolic compounds as substrates, causing a significant decrease of total phenolics (De Leonardis & Macciola, 2011). Research also has shown that PPO can cause the oxidative degradation of flavonoids such as catechol, chlorogenic acid (Kader, Nicolas, & Metche, 1999), and quercetin (Makris & Rossiter, 2000).
Quercetin decline was especially pronounced in the presence of air (Makris & Rossiter,
2001). Catechol and quercetin have been shown to be found in pawpaw pulp (Ahn, 2011;
Fang et al., 2007). Quercetin is one of the substrates that can serve as a substrate for polymerization of phenolics, which could lead to the decreased level of total phenolics detected by the Folin Calcitaue assay. However, polyphenol oxidase is not the only enzyme that can cause polymerization. Perhaps there are other enzymes (i.e., peroxide) that could catalyze the polymerization reactions that use other pawpaw pulp phenolics as substrate. More research on enzymatic polymerization is needed in order to further investigate the cause of total phenolic loss.
It should be noted that the addition of ascorbic acid and NAC to pawpaw pulp inhibited PPO activity. Thus, both ascorbic acid and NAC could protect flavonoids.
However, the results show that the level of flavonoids in pawpaw pulp treated with ascorbic acid and NAC is significantly less than that detected in the air packaging and 56 vacuum packaging samples. This suggests oxidation or polymerization of flavonoids that is occurring via another mechanism. In addition to that, research shows that flavonoid compounds are mostly found in the vacuole of cells (Winkel-Shirley, 2001), while phenolic compounds can be found in vacuole or the apoplastic compartments between the plasma membrane and the cell wall (Bagniewska-Zadworna, Zenkteler, Karolewski, &
Zadworny, 2008). Tiny ice crystals may have formed during frozen storage that could impale the cell wall, thus causing phenolic compound and flavonoid compound loss from the cell.
The impact the presence of air has on antioxidant capacity measurements (FRAP,
DPPH, ORAC) in pawpaw pulp during frozen storage appears to be independent of the level of phenolics and flavonoids. As shown in Table 7, both radical scavenging (DPPH and ORAC) and reducing potential were not affected by 12 months of frozen storage.
However, differences were observed between DPPH values and ORAC values during frozen storage. Specifically, samples held in a vacuum exhibited significantly higher initial DPPH values than those stored in the presence of air, while the reverse was true for
ORAC values. Regardless of the magnitude of initial DPPH or ORAC values, little change in these values was observed during storage. The reason for that could be that the three assays (DPPH, FRAP and ORAC) each measure a different aspects of antioxidant capacity. FRAP assay is an electron-transfer based assay that measures antioxidant reducing capacity, while DPPH assay and ORAC assay are hydrogen atom transfer-based assays that quantify hydrogen atom donation capacity (Huang, Ou, & Prior, 2005).
However, there is still controversy about whether the DPPH assay is a hydrogen atom- 57 transfer based assay or an electron-transfer based assay. Measurement of different aspects of antioxidant capacity could result in different trends.
These data from this thesis disagree with previous research which showed that total phenolic level in pawpaw is highly correlated with radical scavenging ability (R2 =
0.92) (Harris & Brannan, 2009). In this thesis, no statistically significant correlation was found between total phenolics and radical scavenging (data not shown).
The ORAC assay is the most frequently used HAT method to test antioxidant capacity. The ORAC method measures the decrease in fluorescence as a result of oxidative damage caused by a source of peroxyl radicals. It used to be the standard test of antioxidant capacity, but is no longer (see discussion in Chapter 2). Nonetheless, ORAC does allow for data comparison between different fruits and different laboratories. The
ORAC values of the edible part of some common fruits are listed in Table 15.
58
Table 15
Oxygen Radical Absorbance Capacity (ORAC) Value Comparison of Common Fruits
Including Pawpaw
ORAC value (µmol Fruits T.E./g) Reference
Plum 50.00 U.S. Department of Agriculture (2012)
Peach 6.80 Stockham, Paimin, Orbell, Adorno, & Buddhadasa (2011)
Cherry 40.00 Stockham et al. (2011)
Cranberry 16.00 Stockham et al. (2011)
Blueberry 48.50 Stockham et al. (2011)
Mango 12.00 Stockham et al. (2011)
Pawpaw 2.93 This thesis
As seen in Table 15, the results of previous research have shown that the ORAC
values in blueberry and cherry are significantly higher than in other common fruits.
However, the ORAC value of pawpaw pulp is significantly lower than other fruits. There
could be multiple reasons for this result. First, the antioxidant contents that account for
ORAC values might not be completely extracted by methanol only. Second, the ORAC
assay is a pH sensitive method, different pH environments of fruit could result in
different ORAC results. Third, the results from ORAC assays could be quite different
across different laboratories or equipment. Considering ORAC values in the context of 59 other antioxidant capacity measurements may give a better picture of pawpaw antioxidant capacity than results gleaned from ORAC alone.
Color of Pawpaw Pulp
Shown in T8, 9 and 10, the color of pawpaw pulp is affected by the presence of air, ascorbic acid and NAC during 12 months of frozen storage. Pawpaw pulp stored for
12 months was significantly darker (lower L*), redder (more positive a*), and less yellow
(less positive b*) in vacuum and air stored samples. This agrees with a previous study of sensory analysis of pawpaw (Salabak & Brannan, 2012). One possible reason for this could be the formation of color pigment via enzymatic browning reaction catalyzed by
PPO. Thus the discoloration reaction is catalyzed in the presence of air and further leads to the formation of dark color pigments. However, the color value of vacuum storage pawpaw pulp is also significantly different from that of pawpaw pulp added with ascorbic acid and NAC. One possible explanation is that a polymerization reaction occured in air storage of pawpaw pulp but was inhibited by the addition of ascorbic acid and NAC.
Considering the results shown in Tables 8, 9, 10 and 12, PPO activity could be a factor that affects the color value of pawpaw pulp. One possible reason for this could also be that PPO causes the enzymatic browning that further generates dark pigments inside the pawpaw pulp and affects the color value of pawpaw pulp. This study showed that pawpaw pulp preserved with ascorbic acid or NAC was lighter, less red and more yellow.
This could be due to PPO activity being inhibited in these samples, because ascorbic acid and NAC can both prevent diphenol from forming O-quinone. Without PPO inhibitors added to pawpaw pulp, browning pigment (O-quinone) accumulation in vacuum 60 packaged and air packaged pulp was significantly higher than pawpaw pulp containing ascorbic acid or NAC. These results agree with the research conducted by Ciou, Lin,
Chiang, Wang, and Charles (2011), which showed that L* value decreases due to PPO’s effect. Other research showed that ascorbic acid (Jang & Moon, 2011) and NAC (Garcia-
Molina, Penalver, Rodriguez-Lopez, Garcia-Canovas, & Tudela, 2005) can inhibit PPO activity.
Packaging also affected the color value of pawpaw pulp. The pawpaw pulp packaged in air processes significantly lower L* value, higher a* value and lower b* value compared to that packaged in a vacuum. Further, as discussed above in relation to antioxidant compounds that are affected by PPO, the color value (L*, a*, b*) could be affected by other enzymes as well. For example, research has shown that peroxidase could affect the CIE color system in pumpkin (Goncalves, Pinheiro, Abreu, Brandao, &
Silva, 2007) and apples (Nicolas, Richardforget, Goupy, Amiot, & Aubert, 1994).
However, peroxidase has not identified in pawpaw pulp. In order to better understand the reasons why color value is affected by frozen storage, further studies of enzymes such as peroxidase will be needed.
Conclusion
Results showed that phenolic compounds and flavonoid compounds significantly decreased during frozen storage without addition of ascorbic acid and NAC. However,
PPO was inactive in pawpaw pulp samples preserved with ascorbic acid or NAC, leading to an increase of phenolic but not flavonoid compounds after 6 months of storage.
Additionally, pawpaw pulp tended to be lighter, less red, and more yellow in vacuum 61 packed storage or with the addition of ascorbic acid or NAC. In addition, antioxidant capacity was not affected by months of frozen storage regardless of packaging. Finally, antioxidant capacity was closely correlated with phenolic compounds in frozen, stored pawpaw pulp.
Future Recommendation
The results of this research show a need for more research on each aspect investigated in this study. For antioxidant compounds, an unknown peak of phenolic compound content in samples stored for 2 months and unknown peak of flavonoid compounds in the 0 month sample need to be further studied. In addition, individual flavonoid compounds in pawpaw pulp need to be further studied to understand the substrates that could participate in polymerization reactions. Additionally, PPO may not be the only enzyme that catalyzes the polymerization reaction that causes both phenolic compound decreases and color value change during frozen storage. Further investigation into other enzymes that catalyze the polymerization reaction could help us to better understand antioxidant compound loss and color change. Also, further investigation into specific flavonoid compounds and phenolic compound will also help us further understand the reason for antioxidant compounds loss. Finally, further characterization of antioxidant capacity by different assays may offer additional insights.
62
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