EFFECTS OF EXTRACTION AND REFINING ON THE QUALITY AND COMPOSITION OF MUSCADINE GRAPE SEED OILS AND FLOUR OF THE NOBLE AND CARLOS

CULTIVARS

By

BRIAN K. WADA

A THESIS PRESENTED TO THE GRADUATE SCHOOL

OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2018

© 2018 Brian K. Wada

To my family, Jerry, Lisa, and Michelle, for supporting me throughout my endeavors

4

ACKNOWLEDGMENTS

I would like to thank Dr. Liwei Gu for giving me the opportunity to pursue my

master’s degree and for guiding and motivating me as I pursued an entirely new path in

my life. I would like to thank Dr. Yavuz for motivating me and guiding me through my

research. I would also like to thank Dr. Cheryl Rock for giving me the courage to move

across the country and pursue food science. I also greatly appreciate my lab mates and

friends Ruiqi Li, Mohammed Alrugaibah, Shaomin Zhao, Chi Gao,Ye Feng, and Jamie

Klaben. I would finally like to thank my girlfriend Jessica Lee.

I would like to acknowledge Muscadine Products Corporation and Lakeridge

Winery and Vineyard for providing me with muscadine pomace and seeds. I would also

like to thank the Florida Department of Agriculture & Consumer Sciences for providing

funding for this research

5

TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES ............................................................................................................ 7

LIST OF FIGURES .......................................................................................................... 8

LIST OF ABBREVIATIONS ............................................................................................. 9

ABSTRACT ................................................................................................................... 10

CHAPTER

1 INTRODUCTION .................................................................................................... 12

Muscadine Grape ................................................................................................... 12 Tocopherol and Tocotrienol .................................................................................... 16

Oil Extraction .......................................................................................................... 13 Oil Refining ............................................................................................................. 15 Phytochemicals in Muscadine Grapes .................................................................... 18

Summary ................................................................................................................ 18 Research Objectives ............................................................................................... 19

2 COMPARING EXTRACTIONS OF CRUDE AND REFINED OIL FROM MUSCADINE SEEDS FROM THE NOBLE AND CARLOS CULTIVARS ............... 23

Background ............................................................................................................. 23 Materials and Methods............................................................................................ 24

Chemicals ......................................................................................................... 24 Sample Preparation .......................................................................................... 24 Oil Extraction .................................................................................................... 25

Peroxide and Free Fatty Acid Content Analysis ............................................... 26 Determination of Fatty Acid Composition ......................................................... 27 Determination of Vitamin E Content ................................................................. 27

Oil Refining ....................................................................................................... 28 Statistical Analysis ............................................................................................ 28

Results and Discussion........................................................................................... 28 Oil Extraction .................................................................................................... 28 Peroxide Values and Free Fatty Acid Concentration ........................................ 30 Fatty Acid Composition .................................................................................... 32 Determination of Vitamin E Content ................................................................. 32

Refining Oil Yield .............................................................................................. 33 Peroxide and Free Fatty Acid Concentration .................................................... 35 Vitamin E Concentration After Refining ............................................................ 35

Summary ................................................................................................................ 37

6

3 MUSCADINE GRAPE SEED FLOUR ..................................................................... 52

Background ............................................................................................................. 52

Materials and Methods............................................................................................ 52 Chemicals ......................................................................................................... 52 Preparation of Flour .......................................................................................... 53 Proximate Analysis of Flour .............................................................................. 53 Phytochemical Extraction ................................................................................. 55

Total Phenolic Content ..................................................................................... 55 DPPH Assay ..................................................................................................... 56 HPLC Analysis of Muscadine Flour Phytochemicals ........................................ 56

Results and Discussions ......................................................................................... 57 Proximate Composition of Flour ....................................................................... 57

Total Phenolic Content and Antioxidant Capacity ............................................. 59 Characterization of Muscadine Flour Phytochemicals ...................................... 60

Summary ................................................................................................................ 62

4 CONCLUSIONS ..................................................................................................... 70

LIST OF REFERENCES ............................................................................................... 71

BIOGRAPHICAL SKETCH ............................................................................................ 77

7

LIST OF TABLES

Table page

2-1 Extraction yield of seed oil from Noble and Carlos muscadine seeds using different extraction methods ............................................................................... 39

2-2 Impact of extraction and refining on peroxide value and free fatty acids in Noble muscadine seed oil .................................................................................. 40

2-3 Impact of extraction and refining on peroxide and free fatty acid content of Carlos seed oil. ................................................................................................... 41

2-4 Fatty acid composition of Noble seed oil using different extraction methods. ..... 42

2-5 Fatty acid composition of Carlos seed oil using different extraction methods ..... 43

2-6 Impact of different extraction methods on the vitamin E concentration (mg/100 g) of crude Noble seed oil ..................................................................... 44

2-7 Impact of different extraction methods on the vitamin E concentration (mg/100 g) of refined Noble seed oil. .................................................................. 45

2-8 Impact of extraction methods on vitamin E concentration (mg/100 g) of crude Carlos seed oil. ................................................................................................... 46

2-9 Impact of extraction methods on vitamin E concentration (mg/100g) of refined Carlos seed oil .................................................................................................... 47

2-10 Refining yield (%, w/w) of seed oil from Noble and Carlos muscadine seeds at different steps of refining. ............................................................................... 48

3-1 Impact of extraction method on proximate analysis (%, w/w) of Noble seed flour and Carlos seed flour. ................................................................................ 64

3-2 Impact of extraction method on total phenols and antioxidant capacity of Noble seed flour and Carlos seed flour. ............................................................. 65

3-3 Impact of extraction method on phytochemical content (mg/100g) of Noble seed flour ............................................................................................................ 66

3-4 Impact of extraction method on phytochemical content (mg/100g) of Carlos seed flour ............................................................................................................ 67

8

LIST OF FIGURES

Figure page

1-1 Chemical Structure of tocopherol isomers ( and tocotrienol isomers

(. ........................................................................................................... 20

1-2 Chemical structure of flavonoid compounds, quercetin and kaempferol, in muscadine grape seeds. .................................................................................... 21

1-3 Anthocyanin compounds found in muscadine seeds .......................................... 22

2-1 Concentrations of tocopherol and tocotrienol in Noble seed oil after the refining process. Results are mean with error bars denoting standard deviation.. ........................................................................................................... 49

2-2 Concentrations of tocopherol and tocotrienol in Carlos seed oil after the refining process.. ................................................................................................ 50

2-3 Peroxide value in Carlos seed oil extracted using enzyme-assisted aqueous extraction with protease after treatment with bleaching earth clay of different concentrations.. .................................................................................................. 51

3-1 HPLC-DAD chromatogram of the anthocyanins in Noble grape seed flour at 520 nm.. ............................................................................................................. 68

3-2 HPLC-DAD chromatogram of the anthocyanins in Noble seed flour and Carlos seed flour at 360 nm. Peaks 1, 2, and 3 were myricetin, quercetin, and kaempferol, respectively. ............................................................................. 69

9

LIST OF ABBREVIATIONS

DPPH 2,2-diphenyl-1-picrylhydrazyl

FAME Fatty acid methyl esters

g Gram

g G-force

GAE Gallic acid equivalence

HPLC High performance liquid chromatography

L Liter

g Microgram

L MicroLiter

min Minute(s)

mL Milliliters

nm Nanometer

TE Trolox equivalence

Trolox 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid

UV Ultraviolet

V Volume

Vis Visible

w Weight

10

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science

EFFECTS OF EXTRACTION AND REFINING ON THE QUALITY AND COMPOSITION

OF MUSCADINE GRAPE SEED OILS AND FLOUR OF THE NOBLE AND CARLOS CULTIVARS

By

Brian K. Wada

May 2018

Chair: Liwei Gu Major: Food Science and Human Nutrition

Wine making or juicing using muscadine grapes results in large amounts of

discarded seeds in pomace. Muscadine seeds contain oil that consist predominantly of

polyunsaturated fatty acids and tocopherols. The objective of this research was to

investigate efficient extraction and refining technologies to produce muscadine seed oils

of good quality and characterize muscadine seed oils and flour. Seed oils were

extracted using a Soxhlet extractor by hexane, mechanical press at different moisture

and temperatures, or enzyme-assisted aqueous extraction using different enzymes.

Crude and refined oil was evaluated for peroxide value, free fatty acid content, fatty acid

composition and vitamin E content. Flour was analyzed for proximate composition,

antioxidant capacity, and phytochemicals. Hexane extracted oil resulted in significantly

higher yield for both Noble and Carlos seeds with the least amount of peroxides and

free fatty acids. Linoleic acid accounted for 72-79% total fatty acid in all oils.

Concentrations of tocopherols and tocotrienols in muscadine seed oil ranged from 11.8

to 53.8 mg/100 g and 25.3 to 44.3 mg/100 g, respectively. Oil refining removed

peroxides and free fatty acids but also removed 44-100% and 11-95% of tocopherols

11

and tocotrienols, respectively. Flours have up to 17.1% protein and 53.6% fiber. DPPH

values range from 101.3 to 183.3 Mol Trolox/ mg and total phenols range from 10.0 to

37.3 GAE (mg/mL). The predominant anthocyanin isomer and flavonal was malvidin 3,

5-glucoside (167 mg/100 g) and myricetin (235 mg/100 g). Muscadine seeds have the

potential to become a source of antioxidant rich oil and flour.

12

CHAPTER 1 INTRODUCTION

Muscadine Grape

The muscadine grape (Vitis rotundifolia) is a grape species indigenous to the

southeast United States and northern Mexico. Muscadine grapes have relatively large

berries with a thicker outer skin and two to six large seeds compared to the common

grape (Vitis vinifera). Muscadine grapes are resistant to disease, require little pesticides,

need little to no irrigation to grow on a farm, and thrive in warmer and humid climates

which is not suitable for Vitis vinifera (Olien, 1990). Grapes are sold in farmers’

markets, roadside stands, and “U-pick” operations, but a majority of muscadine grapes

are used for juice and wine production (Peter C. Anderson, 2013). Wine production

utilizing the muscadine grape in Florida totals 450,000 gallons of wine annually (Mueller,

2014). Extraction of the juice from the muscadine grapes gives a byproduct of pomace,

which consists of the leftover skin, stem, leaves, and seeds. Accounting for 10–20% of

the grapes weight, the pomace is typically put in landfills after the juice has been

extracted (Xu et al., 2014). Cell culture studies showed that phytochemicals of the

muscadine grapes inhibited proliferation of colon cancer cells and induced apoptosis in

them (Mertens-Talcott, Lee, Percival, & Talcott, 2006; Yi, Fischer, & Akoh, 2005).

Additionally, mice studies suggested that the phytochemicals of the muscadine grape

prevent obesity-associated metabolic complications and alleviate bowel inflammation

(Gourineni, Shay, Chung, Sandhu, & Gu, 2012; Li, Kim, Sandhu, Gao, & Gu, 2017; Li,

Sandhu, Gao, & Gu, 2016).

The muscadine grape seeds in pomace contain a large variety of antioxidant

phytochemicals. They are reported to contain hydroxybenzoic acid, quercetin

13

rhamnoside, hydrolysable tannins, ellagic acid, and flavan-3-ols and condensed tannins.

(Sandhu & Gu, 2010) The seeds contain 11-15% (w/w) edible oil. Although little

research has been done on the oil, it has been reported that the oil is predominantly

poly-unsaturated fatty acids (68.1-72.5%), and has a fatty acid profile comprised mostly

of linoleic acid (C18:2) (67.9-72.3%) (Zhao et al., 2015). Muscadine seed oil also

contains tocopherol and tocotrienol, classes of vitamin E (Zhao et al., 2015). Extracts of

the oil have been shown to attenuate obesity and exhibit dose-dependent inhibition of

cancer cell growth in vitro (Lutterodt, Slavin, Whent, Turner, & Yu, 2011; Zhao et al.,

2015). The goal of current research is to investigate extraction and refining

technologies to produce muscadine seeds oil of high quality and to characterize the

composition of seed oil and defatted seed flour. Comparison of different concentrations

of peroxide values, free fatty acid values, fatty acid composition, and vitamin E

concentration, will be done in oil before and after refining.

Oil Extraction

There are multiple technologies of oil extraction from lipid containing seeds but

the most common one is mechanical expression. The use of sheer pressure to destroy

oil bodies to release the oil has been a common practice throughout history. Much

larger, scaled up versions are used for industrial purposes and one of the most common

mill types is the continuous screw presses (Ajibola, Eniyemo, Fasina, & Adeeko, 1990).

Screw presses are common as they come in various sizes, are a continuous process,

and requires low maintenance. Screw presses are relatively easy to use as they do not

require chemicals and can be continuously fed. However, screw presses and all other

forms of expression crush oil bodies and extract about 60% of oil depending on

moisture, heat, and material (Ward, 1976). More commonly used in commercial

14

operations is solvent extraction using hexane because it is cheap and can retrieve oil at

a yield of 95% with about 95% solvent recovery (Rosenthal, Pyle, & Niranjan, 1996).

However, solvent disposal can be environmentally dangerous and expensive to

continually purchase.

An older form of oil extraction, aqueous extraction, utilizes hot water to release oil

from seeds and allow oils to separate based on the insolubility of oil in water. Recently,

studies have been conducted to determine whether the process can be made viable in

an industrial setting. The technique can be optimized through the uses of enzymes to

breakdown oil containing seeds. Enzymes facilitate release of oil without heat of

mechanical expression or use of hexane (Latif, Diosady, & Anwar, 2008). Yields of

aqueous extraction are typically lower than the other two forms of extraction. However,

since the lower heat preserves bioactive and heat-sensitive components of extracted oil,

there is still research interest in this method.

Previous research has been performed on cold-pressed muscadine seed oil and

hexane extracted muscadine seed oil, but neither considered the effect of extraction

method on the quality of oil (Lutterodt et al., 2011; Zhao et al., 2015). Additionally,

previous research on the enzyme-assisted aqueous extraction of oil from the seeds of

common table grapes has also been conducted, but never applied to the muscadine

seed (Passos, Yilmaz, Silva, & Coimbra, 2009). Muscadine seeds have lower oil

content (13-20%) compared to other seeds such as peanuts (50%). Developing an

efficient extraction method is crucial for muscadine seed oil due to low oil content but

high content of polyunsaturated fatty acids.

15

Oil Refining

The refining of edible oils is a process that requires both chemical reactions and

physical separation with the intention to remove oxidation products and other

components that cause off flavors or contribute to the rancidity of oil. There are three

important reactions in refining. They are hydration of phospholipids, neutralization of

free fatty acids, and removal of pigments (Wiedermann, 1981). These three reactions

occur in three separate refining steps: degumming, neutralizing, and bleaching.

Degumming is necessary to remove phospholipids in oil, by adding hot water to

hydrolyze and separate the phospholipids. If they are not removed they can emulsify the

oil or degrade and give the oil a dark color (Zufarov, Schmidt, & Sekretár, 2008).

Neutralization saponifies free fatty acids by using an alkali solution, removing them for

the oil in the form of soap. Free fatty acids are less stable than normal triglycerides and

are thus more likely to oxidize (Sanchez-Machado et al., 2015). Bleaching removes

color containing compounds like chlorophyll, residual phospholipids, and peroxyl

compounds.

Refining requires heating and chemical treatment to remove all undesirable

components that diminish the quality of the oil. However, the refining process also

removes desirable components of the oil, including tocopherol and tocotrienol. Previous

work showed the losses of vitamin E during refining of different types of oil with varying

results (Gogolewski, Nogala‐Kalucka, & Szeliga, 2000; Pestana-Bauer, Zambiazi,

Mendonça, Beneito-Cambra, & Ramis-Ramos, 2012; Puah, Choo, Ma, & Chuah, 2007;

Tasan & Demirci, 2005; Van Hoed et al., 2006). Gogolewski, Nogala-Kalucka et al.

observed that the refining of rapeseed oil had losses of 30% of total tocopherol from the

16

oil. In the work done by Puah, Choo et al. tocopherol concentrations were observed to

decrease at every step of the oil refining process.

Tocopherol and Tocotrienol

Muscadine grape seed oil is rich in tocopherol and tocotrienol with variances

among cultivars and growing seasons. The structure for -tocopherol and -tocotrienol

are shown in figure 1-1 and 1-2. Both tocopherol and tocotrienol occur naturally in four

isomers Among the isomers of tocopherol, -tocopherol has been the

main focus of previous research since tocopherol is the most predominant vitamin E

isomer in plasma and tissue of humans. Tocopherol’s structure allows it to interact

with the cell membrane and trap free radicals more efficiently than the other isomers,

making it the more preferentially absorbed form of vitamin E in humans (Booth &

Bradford, 1963). Research on tocopherol primarily notes its antioxidant capabilities

and anti-inflammatory capabilities (Reiter, Jiang, & Christen, 2007). In human studies,

tocopherol supplementation has been shown to improve indicators of immune

function including the increase of interleukin 2, a signaling molecule that regulates the

activity of white blood cells, and decrease prostaglandin E2, a pro-inflammatory

mediator (Meydani et al., 1990). Although tocopherol is the main isomer used in

vitamin E supplementation, tocopherol, a less predominant form of vitamin E, has

been shown to have the capability of having a bioavailability of up to 30-50% (Burton et

al., 1998). It is naturally found in corn and soybean oil, and in diets of the United states

where use of those oils are common it has an estimated 20% of total vitamin E activity

(Bieri & Evarts, 1974). Additionally, tocopherol has observed to inhibit

17

cyclooxygenase activity, meaning it also has anti-inflammatory properties (Jiang,

Christen, Shigenaga, & Ames, 2001).

Tocotrienols are structurally similar to tocopherol with a common chromanol head

and a side chain at the C2 position, but the two have different side chains (Theriault,

Chao, Wang, Gapor, & Adeli, 1999). Similarly, tocotrienols are primarily known for their

antioxidant capabilities, however they have other health promoting properties.

Tocotrienols have been reported to lower plasma cholesterol levels and other risk

factors of cardiovascular disease (Theriault et al., 1999). Recent studies suggest that

various forms of tocotrienol can protect against emphysema, obesity-related

inflammation, and oxidative stress (Goon, Azman, Ghani, Hamid, & Ngah, 2017; Peh et

al., 2017; Ramalingam et al., 2017).

Muscadine seed oil is a rich source of tocopherols and tocotrienols in

comparable to rice bran oil. In previous research, a tocotrienol-rich fraction of

muscadine seed oil extract was found to reduce adipocyte formation and adipogenesis

(Zhao et al., 2015). The treatment of human-adipose stem cells with the tocotrienol

fraction of muscadine seed oil showed significant reduction of transcription factors

responsible for adipogenesis. Another study done by Lutterodt et al. showed that

extracts from muscadine seed flour and oil inhibited the growth and proliferation of

cancer cells in vitro. Studies on the health benefits of muscadine seed oil typically used

oil extracted by hexane or cold pressing, however research on how different oil

extraction methods may impact the bioactive components of the oil have not been

conducted.

18

Phytochemicals in Muscadine Grapes

Phytochemicals are biologically-active, non-nutritive secondary metabolites in

plants that contribute to color and a bitter astringent flavor (Damodaran, Parkin, &

Fennema, 2007). Research regarding phytochemicals has demonstrated their ability to

reduce the risk of cancer, cardiovascular disease, Alzheimer’s, and other chronic

diseases associated with complication from oxidative stress (Liu, 2003). Muscadine

grapes possess a unique phytochemical profile compared to other member of the Vitis

species of grapes (Hudson et al., 2007). Muscadine grape seeds contain quercetin,

kaempferol, and anthocyanin 3,5-diglucosides (Figure 1-3 and 1-4) (Li et al., 2017).

Phytochemicals extracted from the muscadine grape have been shown to reduce

inflammation and metabolic complications and in vivo and adipogenesis in vitro

(Gourineni et al., 2012; Li et al., 2017; Zhao et al., 2015). Within the muscadine grape,

approximately 80% of the phytochemicals are located within the seeds (Sandhu & Gu,

2010).

Summary

Muscadine grape pomace is an underutilized byproduct of wine making. Seeds

and skin in pomace contain high concentration of polyphenols. In addition, seeds

possess oils consisting of predominantly polyunsaturated fatty acids and rich in

tocopherol and tocotrienol.

The extraction method of the oil is crucial as it dictates the yield and the quality of

the resulting oil. No studies have been conducted to determine the effect different

extraction technologies on the yield and quality of muscadine seed oil. Refining the oil

will also improve oxidative stability and ideally safe for consumption. It was unknown

how refining impacts the quality of oil and the content of vitamin E.

19

Extraction of oil from seeds results in defatted seeds, which can be ground and

sieved to make flour. Little previous research has been done on the defatted seed flour

of the muscadine grape seed.

Research Objectives

In this research, oils were extracted from muscadine seeds of two cultivars

(Noble and Carlos) using three oil extraction technologies. Noble is a purple grape

variety and Carlos is a bronze grape variety. Crude oils were refined to meet the quality

standards of edible oil. Quality and composition of the oils and seed flours were

characterized. The specific objectives of the research were:

1. To evaluate the yield of crude oil from Noble and Carlos seeds using Soxhlet

extraction with hexane, continuous screw pressings, and enzyme-assisted

aqueous extraction and to assess oxidative stability, fatty acid composition and

vitamin E concentration

2. To investigate the effects of refining on the oils oxidative stability, fatty acid

composition, and vitamin E concentration.

3. To assess the proximate composition, antioxidant capacity, and polyphenol

profiles in defatted seed flours.

20

-Tocopherol -Tocopherol l

-Tocopherol -Tocopherol

-Tocotrienol -Tocotrienol

-Tocotrienol -Tocotrienol

Figure 1-1. Chemical structure of tocopherol isomers ( and tocotrienol isomers

(.

21

Quercetin Kaempferol

Figure 1-2. Chemical structure of flavonoid compounds, quercetin and kaempferol, in muscadine grape seeds.

22

Anthocyanins R1 R2 R3 R4

Delphinidin 3,5-diglucoside OH OH glucose glucose

Cyanidin 3,5-diglucoside OH H glucose glucose

Petunidin 3,5-diglucoside OCH3 OH glucose glucose

Pelargonidin 3,5-diglucoside H H glucose glucose

Peonidin 3,5-diglucoside OCH3 H glucose glucose

Malvidin 3,5-diglucoside OCH3 OCH3 glucose glucose

Figure 1-3. Anthocyanin compounds found in muscadine seeds

23

CHAPTER 2 COMPARING EXTRACTIONS OF CRUDE AND REFINED OIL FROM MUSCADINE

SEEDS FROM THE NOBLE AND CARLOS CULTIVARS

Background

Over 90% of muscadine grapes are used for making wine, which requires

fermenting grapes or grape juicing and discarding the pomace. Each year

approximately 500 tons of pomace are discarded, 300 tons of which are seeds (Zhao et

al., 2015). The seeds contain 13-15% edible oil, rich in antioxidants. It is possible to

retrieve approximately 38 tons of muscadine seed oil and 250 tons of defatted seed

flour from this currently wasted material (Peter C. Anderson, 2013).

Muscadine seed oil is known to have many forms of tocopherol and tocotrienol.

These compounds are known for their antioxidant abilities and capacity to prevent

cancer, heart disease, and inflammation (Burton et al., 1998; Goon et al., 2017; Reiter

et al., 2007). Previous research supports that it is essential to choose the right

extraction method to obtain oil with high yield as well as preserve beneficial components

like vitamin E.

Refined edible oils are composed of triglycerides with little to no color, odor, or

oxidation products that cause off flavors and odors. Crude oil must undergo a multiple

step refining process to reach this state. The first step is degumming, where hot water is

added to crude oil to bind and aggregate phosphatides in order to separate them from

oil (Zufarov et al., 2008). Free fatty acids in a crude oil must then be neutralized by

alkali so they be removed as soaps from the oil (Sanchez-Machado et al., 2015). The

third necessary refining step is bleaching. Activated carbon or bleaching earth clay are

used to absorb colored compounds, residual soaps and phosphatides, and other

oxidation products (Rossi, Gianazza, Alamprese, & Stanga, 2001).

24

Refining steps remove the unwanted products in oil to make them more

appealing and pleasing to taste. However, it may also remove desirable lipids like

vitamin E. In this study, different oil extraction methods were used to obtain muscadine

seed oil from the seeds of two cultivars (Noble and Carlos). The objectives of this

research were to compare the extraction efficiency of different methods, determine

peroxide values and free fatty acids, analyze the fatty acid composition, determine

vitamin E concentrations, and evaluate effects of refining on oil quality.

Materials and Methods

Chemicals

N-Hexane, methanol, acetic acid, chloroform, saturated potassium iodide, starch,

ethanol, sodium hydroxide, 0.5 M sodium methylate in methanol, isooctane, sodium

bisulfate, Folin-Ciocalteu reagent, phenolphthalein, and formic acid were purchased

from Fischer Scientific (Pittsburg, PA, USA). Tocopherol and tocotrienol standards were

obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Enzymes cellulase,

protease, pectinase, and hemicellulase were purchased from Sigma-Aldrich (St. Louis,

MO, USA). Muscadine grape pomace of the Carlos cultivar and seeds of the Noble

cultivar were obtained from Lakeridge Winery and Vineyard (Clermont, FL, USA) and

dried muscadine seeds of the Noble and Carlos cultivar were obtained from Muscadine

Products Corporation (Wray, GA, USA).

Sample Preparation

Wet Carlos pomace and Noble seeds were obtained from Lakeridge Winery and

Vineyard (Clermont, FL, USA) and dry pomace was obtained from Muscadine Products

Corporation (Wray, GA, USA). Large twigs and skins were removed from wet Carlos

pomace by sifting through poultry netting with 1x1 inch openings. Noble seeds were

25

separated out after the fermenting process, where they were placed with juice and skins

to ferment. The sifted Carlos pomace and Noble seeds were placed in separate baking

pans and placed in an oven to dry at 77°C for 16 hours. Seeds were separated from the

rest of the dried pomace by wind sifting. Dried seeds were mixed with their respective

cultivar from Florida and Georgia in a 1:3 ratio (w:w) to obtain enough samples for all

methods of extraction. A portion of Noble and Carlos were separated and mixed with

deionized water, to moisten seeds to 13% and 16% (w/w). The seeds were mixed in a

bucket and sealed until the moisture was equilibrated. Prior to extractions seeds were

grinded using a RHH-1000 multi-function grinder (Enerburg, Guangzhou, China).

Oil Extraction

For mechanical expression, a Tokul Tarim Ekotok 1 continuous oil screw press

(Izmir, Turkey) was used. Pressing was conducted at 85° C, 90° C, 105° C, and 125° C

with initial moisture content of 9.83 ± 0.25 % and 10.3 ± 1.2 % for Noble and Carlos

seeds, respectively. Additional pressings were done at 90°C for at moisture contents of

13 % and 16%, wet basis. Soxhlet extractions were performed according to AOCS

official method Ba 3-38 (Firestone & American Oil Chemists, 2011). Seeds were grinded

using a RRH-1000 high-speed multifunction grinder (Enerburg, Guangzhou, China) and

approximately 50 g were placed in a fiberglass thimble and into the Soxhlet sample

holder. Samples were refluxed in Soxhlet extractors for 8 hours before hexane was

evaporated using a Buchi R-II rotary evaporator (Delaware, USA). A previously reported

enzyme-assisted aqueous extraction method was used with modification (Passos et al.,

2009). Seeds were grinded using the RRH-1000 high-speed multifunction grinder and

50 g were placed in 950 mL mason jars with the enzyme at 1% concentrations (v:w).

250 mL of citrate buffer with a pH of 5.0, 4.5, 4.0, 5.0, and 4.5 were used for cellulase,

26

hemicellulase, pectinase, protease, and a cocktail of all 4 enzymes, respectively. The

mixture was incubated in a reciprocal water bath shaker model R-76 (Edison, New

Jersey, USA) for 15 hours at 37° C, 40° C, 25° C, 45° C, and 37°C for cellulase,

hemicellulase, pectinase, protease, and a cocktail of all 4 enzymes, respectively. After

incubation, the mixture was placed in 500 mL polycarbonate centrifuge tubes and

centrifuged for 30 minutes at 1,450 g. The supernatant was mixed with 50 mL of hexane

in a centrifuge tube and centrifuged again. The upper hexane layer was collected in a

round bottom flask and evaporated using a Buchi R-II rotary evaporator to retrieve oil.

Oil yield was expressed as percentage of the weight of the oil obtained relative to the

dry weight of seeds extracted.

Peroxide and Free Fatty Acid Content Analysis

Peroxide values were determined using an AOAC official method 965.33

(International, 1995). Briefly, 5 g of oil were placed into a 250-mL Erlenmeyer flask and

mixed with 30 mL of acetic acid and chloroform (3:2, v/v). The solution was mixed

before adding 0.5 mL of saturated potassium iodide and stirring for one minute. After

that, 30 mL of water were added stirred with 0.5 mL of starch were added producing a

dark black color. The solution was then titrated using 0.10 M sodium thiosulfate until

clear. Titration volume was used to calculate peroxide content in mEq/kg.

Free fatty acid concentration was determine using an AOAC official method

940.28 (International, 1995). Briefly, five grams of oil were placed into a 250 mL

Erlenmeyer flask with 50 mL of ethanol. Phenolphthalein indicator was added to the

solution and titrated with 0.1M sodium hydroxide until a faint pink color was formed.

27

Determination of Fatty Acid Composition

Fatty acid methyl esters (FAMEs) were prepared for both crude and refined oils

by basic methylation procedure. Briefly, 1 mL of sodium methylate in methanol (0.5 N)

was mixed with approximately 10 mg of extracted oil. The mixture was allowed to stand

in room temperature for 10 min, followed by the addition of 500 µL isooctane and 200

µL sodium bisulfate (5%).The mixture was centrifuged for 3 min at 14,500x g and the

upper isooctane layer containing the FAMEs was transferred to GC vials and stored at

−20°C until further analysis by gas chromatography (GC). A gas chromatography (GC)

(6890 Series, Agilent, and Wilmington DE) equipped with a flame ionization detector

using a DB-225MS column (30 mx0.25 mm x 0.25 m) at a flow rate of 0.8 mL/min was

used to separate 1 L of injected FAMEs. The oven temperature program began with an

initial temperature of 120 °C then temperature was increased at a rate of 4°C/min to a

final temperature of 220 °C with a hold time of 35 min. Fatty acids were identified by a

comparison of the retentions times with a standard mixture (Supleco® 37 component

Fatty Acid Methyl Ester Mix, Sigma-Aldrich, St. Louis, MO).

Determination of Vitamin E Content

Vitamin E content was determined for both crude and refined oils using a HPLC

system equipped with fluorescence detector and normal phase column (Luna 5 m

silica 100 Å, 250 x 4.6mm) as previously described.(Zhao et al., 2015) Briefly, seed oils

were weighed and dissolved in 10 mL of hexane. Separation and quantification was

conducted with a mobile phase consisting of hexane, isopropanol, ethyl acetate, and

acetic acid (97.6:0.8:0.8:0.8; v/v/v/v) at 1 mL/min. The wavelength was set at 270 nm for

excitation and 330 nm for emission.

28

Oil Refining

Degumming, neutralizing, and bleaching of the oil was done according to

Sanchez-Machdo et. al with modifications.(Sanchez-Machado et al., 2015) Crude

muscadine seed oil was heated in a water bath to 70 °C and boiling water was added

(20%, v/v). The mixture was stirred for 10 minutes before being placed in a 50 mL

centrifuge tube and oil was recovered by centrifugation at 1450x g for 15 minutes. Oil

was vacuum filtered with Whatman No. 1 filter paper and recovered. Neutralization was

done by adding a pre-determined amount of 20% sodium hydroxide and heated to 65°C

under reduced pressure. After 15 minutes the oil was washed with deionized water

(20%, v:v). After oil was separated from water and soap by centrifugation at 900 g for 15

minutes, the oil supernatant was then vacuum filtered through Whitman No. 1 filter

paper. Bleaching was performed with 10% bleaching earth clay (w/w) that was stirred

into oil and heated to 110°C for 10 minutes. After the oil was centrifuged and vacuum

filtered a final time. Collected refined oil was placed in screw top tubes and placed the

freezer after nitrogen flushing.

Statistical Analysis

Results are presented as mean ± standard error of the mean. One-way ANOVA

with Tukey’s multiple comparison tests were performed with JMP software (Version

13.0, SAS Institute Inc. Cary, NC). A difference with p < 0.05 was considered significant.

Results and Discussion

Oil Extraction

Soxhlet extraction using hexane was the most efficient method for obtaining oil in

both Noble and Carlos seeds (Table 1). Hexane is used to extract oil in industrial

settings because it can extract over 95% oils from seeds (Rosenthal et al., 1996). The

29

mechanical expression of Noble seeds had the highest yield at 85° at the initial

moisture content of 9.83% (wet basis). Expression of Noble seeds were only

significantly different among expression temperatures and conditions from the greatest

yield at 85°C (7.08%) and the lowest yield 125°C (6.11%). Expression at 85°C was

likely gave the highest yield among expressions of Noble seeds, because higher

temperatures can damage oil bodies and prevent them from releasing their oil.

Expression of Carlos seeds were at 90 °C and 125 °C with a higher oil yield than all

other expression extractions at 90 °C and at a moisture content of 13%. Higher moisture

contents prevent solvents from accessing oils in seeds, but a higher moisture content in

seed expression can bind the seeds during expression. This causes an increase in

pressure in the screw chamber, thus increasing the oil yield. This may be the case with

the Carlos seeds that were pressed at 90°C with a moisture content of 13% (Ajibola et

al., 1990).

Among enzyme-assisted aqueous extractions, the use of the enzyme cocktail

gave a higher oil yield (3.73 %) from Noble seeds than any individual enzyme. Carlos

seed oil yield utilizing the enzyme cocktail (9.31 %) was significantly greater than three

forms of mechanical expression. Oil extracted using aqueous extraction is surrounded

by buffer, that bind and aggregate phospholipids that would be considered in the yield of

crude Soxhlet oil. Hydrolytic enzymes are not as effective at penetrating lipid bodies and

obtaining oil as hexane, also resulting in a lower yield. Enzyme-assisted aqueous

extraction using a cocktail of all four enzymes (cellulase, hemicellulase, protease,

pectinase) obtained significantly higher yield than the use of a single enzyme for both

Noble and Carlos seeds. By using all four enzymes in a cocktail, the structural

30

polysaccharides making up the cell wall and proteins that form the lipid body

membranes were hydrolyzed, resulting in greater yield than a single enzyme (Passos et

al., 2009). In both oils, there was no significant difference in the yield of oils extracted

using one enzyme. Extraction using only one enzyme likely left either the

polysaccharide cell walls or lipid containing protein bodies intact, decreasing oil yield.

Noble seeds were not as responsive to the enzymes, likely due to a higher polyphenol

content than Carlos seeds (Sandhu & Gu, 2010). Similar polyphenols from other

sources have been reported to inhibit digestive enzymes including protease (Mcdougall

& Stewart, 2005).

Peroxide Values and Free Fatty Acid Concentration

Hydroperoxides are the primary products of lipid oxidation that eventually break

down into alcohols, aldehydes, free fatty acids, and ketones as secondary products

(Damodaran et al., 2007). Both peroxide values and free fatty acids are used to test the

extent of rancidity in oil samples. The peroxide values of grape seed oil extracted from

Noble and Carlos seeds are presented in Tables 2-2 and 2-3. Oil extracted from freeze-

dried seeds using a Soxhlet apparatus and hexane possessed lowest peroxide values

than all other oils collected, including seeds dried in an oven and extracted in the same

manner. This held true for both freeze dried Noble seed oil (2.33 mEq/kg) and Carlos

seed oil (6.33 mEq/kg). It suggests that drying the seeds in a conventional oven causes

oxidation of oil in seeds. Among seeds dried in the oven, Soxhlet extraction using

hexane had a significantly lower peroxide value than other forms of oil extraction

methods. Oils extracted using enzyme-assisted method had significantly higher

peroxide values than all other extractions for Noble seed oil and for all extractions

except expression at 125°C for Carlos seed oil. This data suggested that oil oxidation

31

occurred during the enzymatic incubation period. Similar observations were made in

previous studies on enzyme-assisted aqueous extraction (Embong & Jelen, 1977).

Screw expression exposes the seeds and oil temperatures of 85°C or more in

atmospheric conditions promoting the formation of hydroperoxides. Enzyme-assisted

aqueous extracted oil possessed even higher levels of peroxide formation, likely forming

during the 15 hour incubation period.

Free fatty acid content of muscadine grape seed oil from seeds of the Noble and

Carlos cultivars are presented in Tables 2-2 and 2-3. Soxhlet extractions utilizing

hexane had significantly less free fatty acid than other forms of extraction with 2.59%

and 0.99% for Noble and Carlos seed oil, respectively. Expression and enzyme-

assisted aqueous extraction had a significantly higher free fatty acid content.

Mechanical expression at 125°C had the greatest percentage for both cultivars, likely

since the high temperature and direct exposure to the oil promoted free fatty acid

formation. Free fatty acids content of the Noble and Carlos seed oil ranged from 2.59-

8.65% and 0.99-6.30%, respectively. Muscadine seed oil had lower free fatty acid

content than observations made in previous studies of hexane extracted grape seed oil

(Gómez, López, & de la Ossa, 1996). Thermal degradation is a common cause of free

fatty acid formation in oil. Long term exposure to incubation temperatures causes

significantly more free fatty acids to form in enzyme-assisted aqueous extracted oil.

Similarly, exposure to expression temperatures result in free fatty acid formation

(Nawar, 1969). Screw expressed oils showed patterns of thermal degradation, since

expression at higher temperatures resulted in higher free fatty acid content in both

Noble and Carlos seed oil.

32

Fatty Acid Composition

Fatty acid results (Tables 2-4 and 2-5) showed that polyunsaturated fatty acids

ranged from 72.9-74.0% and 73.3-79.3% for Noble and Carlos seed oil, respectively.

Monounsaturated fatty acids were the next most abundant for Noble seed oil (15.4-

16.6%) and Carlos seed oil (14.4-15.7%), followed by saturated fatty acids ranging from

9.35-10.8% and 10.94-12.31%, respectively. In Noble seed and Carlos seed oils,

linoleic acid (C18:2) is the most predominant (72.9-74.0% and 73.3-79.3%). The

following most abundant fatty acids are oleic (C18:1), palmitic (C16:0), and steric

(C18:0) acids, with ranges of 15.4-16.6% and 14.4-15.7%, 6.79-7.13% and 6.94-7.69%,

and 2.55-3.75% and 4.00-4.90% for Noble and Carlos seed oil, respectively. For oils

from the seeds of both cultivars, no significant differences were found among the fatty

acid percentages of different extraction methods. The results were similar to previously

reported fatty acid compositions (Lutterodt et al., 2011; Zhao et al., 2015). Fatty acid

profiles of Noble and Carlos seed oil suggest that they would be more susceptible to

oxidation. Linoleic acid is 10 times more susceptible to oxidation than oleic acid due to

its unconjugated double bonds. The oils susceptibility to oxidation could be responsible

for the oxidation that occurred during the drying process, screw expression, and

enzyme-assisted aqueous extraction.

Determination of Vitamin E Content

Concentrations of vitamin E in muscadine grape seed oil for both the Carlos and

Noble cultivars were analyzed by normal-phase HPLC (Tables 2-6, 2-7, 2-8, and 2-9).

Vitamin E in Noble seed oil were predominantly γ-tocopherol, ranging from 23.0-42.2

mg/100g. Noble seed oil had the highest concentration of γ-tocopherol when extracted

by the aqueous extraction using cellulase (42.2 mg/100g), followed by Soxhlet

33

extraction using hexane (39.8 mg/100g), and aqueous extraction using hemicellulase

(38.0 mg/100g). Noble seed oil extracted at 90°C at a moisture content of 16%

possessed significantly higher amounts of -tocopherol than any other oil extracted with

expression. Carlos seed oil was predominantly composed of α-tocotrienol, γ-

tocopherol, and γ-tocotrienol, with ranges of 9.35-18.9 mg/100g, 9.25-25.4 mg/100g,

and 15.8-19.7 mg/100g, respectively. The amount of γ-Tocotrienol in Carlos seed oil

showed no significant difference among various extraction methods. The highest

concentration of α-Tocotrienol was seen in the oil extracted by aqueous extraction with

assistance from hemicellulase (16.6 mg/100 g), protease (18.9 mg/100 g), and

pectinase (18.2 mg/100 g), and the combination of all four enzymes into a cocktail (15.1

mg/100 g). γ-Tocopherol had the highest concentration in oil extracted by the aqueous

method assisted by protease (25.4 mg/100 g) and pectinase (23.2 mg/100 g).

Tocopherol and tocotrienol concentrations were lower than previously reported values

from muscadine seed oil extracted by cold hexane (Zhao et al., 2015). This was likely

due to difference in extraction and analysis methods as well as vitamin E content in

seeds. This data suggests that enzymatic aqueous extraction is a better oil extraction

method for preserving tocopherol and tocotrienol in muscadine seed oil. Thermal

degradation is likely the cause of lower tocopherol and tocotrienol concentrations in the

oil, as enzyme-assisted aqueous extraction utilizes the least amount of heat.

Refining Oil Yield

Degumming is the first step of oil refining. It utilizes water to aggregate and

separate phospholipids from the oil.(Wiedermann, 1981) The “gum” removed from oil

contains water, phospholipids, and triglycerides. After degumming step, enzyme-

34

assisted aqueous extracted oil showed the highest yield for oils extracted from seeds of

both cultivars (Table 2-10). Enzyme-assisted aqueous extracted oil is separated from

the muscadine seeds by enzymatic hydrolysis in a citrate buffer. Water buffer

aggregated a majority of the phospholipids from the oil before the degumming step

(Latif et al., 2008). Data suggests that phospholipid content is higher than in the oil from

Vitis vinifera seeds (1%) (Ohnishi, Hirose, Kawaguchi, Ito, & Fujino, 1990). It is likely

that an emulsification of a portion of muscadine seed oil and water formed during

mixing, resulting in loss of triglycerides and low yield of oil after degumming. Based on

the data, muscadine grape seed oil could be a potential source of phospholipids, which

has been shown to benefit cognitive function and cardiovascular health (Muldoon et al.,

2010).

Neutralization removes free fatty acids by utilizing a low pressure heated

environment and an alkali solution (Sanchez-Machado et al., 2015). Neutralization

showed similar yields among oils of different extraction methods. However,

neutralization did remove more than the free fatty acids which accounted for 1-9% of the

oil. Losses of oil were likely due to excess alkali treatment being used during the

removal of free fatty acids and losses of oil sticking to the evaporation flask and being

absorbed by the filter paper.

Using bleaching earth clay, the oils are separated from hydroperoxides as well

as trace metals, and any remnant soaps and phosphatides (Rossi et al., 2001). Yield

after bleaching was only statistically significant between the two types of extraction

methods that required degumming (Soxhlet and screw press expression) and enzyme-

assisted aqueous extracted oil, for oil from both cultivars. Bleaching yields were similar

35

to previously reported work (Sanchez-Machado et al., 2015). Bleaching losses were

likely due to oil being absorbed by filter paper as well as the bleaching earth clay.

Peroxide and Free Fatty Acid Concentration

The refining process was effective in removing free fatty acids and peroxides

from the initial crude oil as seen in Tables 2-2 and 2-3. Bleaching earth clay effectively

removed peroxides in oil from seeds of both cultivar with values ranging from 0.00 to

0.06 (mEq/kg oil). Free fatty acids were also effectively removed ranging from 0.00 to

0.07 (% as oleic acid w/w). Utilizing caustic sodium hydroxide and a low-pressure

container with heat applied, free fatty acids were both neutralized and evaporated to be

removed from the oil (Sanchez-Machado et al., 2015). According to section 2 of the

codex standards, grape seed oil derived from grape seeds (Vitis vinifera), can have a

peroxide value of up to 10 mEq/ kg oil and 0.3% free fatty acids expressed as oleic acid

(Alimentarius, 1999). Refined muscadine seed oil from both the Noble and Carlos

cultivar are within quality standards. Less refining could be done to the oil to preserve

more tocopherol and tocotrienol, while still keeping the oil within standards. However, by

refining well below standards oil will remain oxidatively stable longer.

Vitamin E Concentration After Refining

Refining is an effective way to remove oxidative products and other unwanted

components from crude oil. However, the refining process also decreases the

concentration of vitamin E within the oil. Total tocopherol and tocotrienol concentrations

after refining are shown in Figures 2-1 and 2-2. Refined Noble seed oil extracted using

the Soxhlet method had a vitamin E retention of 45.8% of total tocopherol and

tocotrienol. After refining, -tocopherol was still the most predominant form of vitamin E

in the oil (18.2 mg/100 g), followed by the more bioactive forms -tocotrienol (9.80

36

mg/100 g) and -tocopherol (6.89 mg/100 g). Refined Noble seed oil extracted by the

Soxhlet method and screw press expression at 105°C had significantly greater retention

than all other oils of different extraction methods, followed by all other forms of

expression. Enzyme-assisted aqueous extracted oils had significantly lower vitamin E

retention than all other Noble seed oils.

Carlos seed oil had significantly greater vitamin E retention in refined Soxhlet

extracted oil. Carlos seed oil had greatest retention of tocopherol and tocotrienol in

Soxhlet extracted oil (70.8%) followed by enzyme assisted aqueous extracted oil using

an enzyme cocktail (40.5%) and continuous screw pressed oil at 105°C (26.5%). The

predominant form of vitamin E in refined Soxhlet extracted oil was -tocotrienol (13.8

mg/100g) followed by, -tocopherol (8.04 mg/100g), and -tocotrienol (7.42 mg/100g)

Although refined Soxhlet extracted oil had greater retention of vitamin E in Carlos seed

oil, it should be noted that Noble seed oil had higher initial concentrations. Refined

Soxhlet extracted oils likely had higher retentions of vitamin E, because they possessed

lower peroxide and free fatty acid concentrations. A lower peroxide concentration could

mean there was less oxidation of the oils to diminish vitamin E concentrations. Lower

free fatty acid concentrations would require less exposure to a caustic base during

refining; minimal exposure to a caustic base could increase retention of tocopherol and

tocotrienol. The bleaching process likely contributed to the loss of tocopherols and

tocotrienols. Bleaching earth clay in concentrations of 10% (w/w) were needed to

remove peroxides from the oil (Figure 2-3). Concentrations from 1-5% of activated

carbon or bleaching earth clay were used in the oil refining process in past work (Rossi

et al., 2001; Sanchez-Machado et al., 2015). Low concentrations of bleaching earth clay

37

have been reported to have a minor effect on tocopherol and tocotrienol concentrations,

but greater concentrations of bleaching earth clay have been shown to decrease

tocopherol and tocotrienol levels (Wei, May, Ngan, & Hock, 2004).These findings

support previous work that chemical and physical oil refining decrease tocopherol and

tocotrienol content in oil (Tasan & Demirci, 2005). Tocopherol and tocotrienol

concentrations are likely lower than previous findings, due to the initial oxidation of the

oil during seed drying and during the following oil extraction methods. Further loss of

tocopherol and tocotrienol likely occurred during neutralizing and bleaching, with caustic

base and bleaching earth clay altering and removing it from the oil.

Summary

Our results indicate that the oil extraction method and conditions affect yield,

peroxide and free fatty acid content, and vitamin E concentration. However, the type of

extraction does not produce significant changes in the fatty acid composition of the oil.

Soxhlet extraction using hexane was successful at obtaining the highest yield for both

Noble and Carlos seed oil while maintaining lower peroxide and free fatty acid

concentration. Additionally, vitamin E content was highest in oil extracted using a

Soxhlet apparatus and hexane. Refining was shown to use up a considerable amount of

oil, especially in the degumming step, but was able to successfully remove peroxides

and free fatty acids from the oil. Refining also proved to diminish vitamin E

concentrations to varying degrees in the muscadine seed oils of different extraction

methods. Muscadine seed oil has the potential to be a tocopherol and tocotrienol rich oil

for culinary or cosmetic purposes. Findings suggest that tocopherol and tocotrienol

concentrations could be preserved with optimized drying and extraction conditions.

38

Additionally, findings support that muscadine seed oil could be a potential source of

phospholipids.

39

Table 2-1. Extraction yield of seed oil from Noble and Carlos muscadine seeds using different extraction methods

Oil Extraction Methods Extraction yield (%, w/w dry base)

Noble Carlos

Soxhlet extraction

Hexane 11.1 ± 0.9a 13.9 ± 0.5a

Screw expression

85°C 7.08 ± 1.92b, AB 8.62 ± 0.45bc

90°C 7.21 ± 0.26b, A 9.52 ± 1.00bc

90°C at 13% Moisture* 5.27 ± 0.6bcd, AB 9.77 ± 0.68b 90°C at 16% Moisture 4.77 ± 0.42cd, B 7.69 ± 0.48c 105°C 5.22 ± 0.23cd, AB 8.30 ± 1.53bc

125°C 6.11 ± 0.25bcd, AB 10.0 ± 0.59b

Enzyme-assisted aqueous extraction Enzyme Cocktail 3.73 ± 0.25de,A 9.31 ± 0.39bc, A Cellulase 1.65 ± 0.08f, B 3.82 ± 0.44d, BC

Hemicellulase 2.27 ± 0.39ef, B 3.55 ± 0.21d, C

Pectinase 2.05 ± 0.34ef, B 4.13 ± 0.31d, BC Protease 1.56 ± 0.08f, B 4.65 ± 0.13d, B

All data represents means ± standard deviation (n=3). Oil yield was expressed as percentage of the weight of the oil obtained relative to the dry weight of seeds extracted. Noble and Carlos seeds were dried to an initial moisture content of 9.83 ± 0.25% and 10.3 ± 1.2%, respectively. Different lower-case superscripts denote difference across extraction types. Different upper case superscripts denote difference within the same extraction type at p ≤ 0.05 using Tukey's test.

40

Table 2-2. Impact of extraction and refining on peroxide value and free fatty acids in Noble muscadine seed oil

Oil extraction methods Peroxide value (mEq/kg oil) Free fatty acid (% as oleic acid w/w)

Unrefined oil Refined oil Unrefined oil Refined oil

Soxhlet using extraction hexane

Freeze dried seeds 2.33 ± 1.15h, B n/a n/a n/a

Air dried seeds 17.0 ± 0.0g, A 0 .00 ± 0.00 2.59 ± 0.08j 0.00 ± 0.00

Screw expression

85°C 29.3 ± 0.6d, A 0 .00 ± 0.00 4.09 ± 0.00i, F 0.00 ± 0.00

90°C 23.0 ± 2.0ef, BC 0.00 ± 0.00 6.49 ± 0.14f, D 0.00 ± 0.00

90°C at 13% Moisture 19.0 ± 0.0fg, D 0.00 ± 0.00 6.77 ± 0.00e, C 0.02 ± 0.04 90°C at 16% Moisture 25.7 ± 1.2de, B 0.03 ± 0.06 6.25 ± 0.08g, E 0.02 ± 0.04 105°C 19.7 ± 1.2fg, D 0.03 ± 0.06 7.47 ± 0.00d, B 0.05 ± 0.04 125°C 21.0 ± 0.0efg, CD 0.03 ± 0.06 8.65 ± 0.08a, A 0.02 ± 0.04 Enzyme-assisted aqueous extraction Enzyme Cocktail 52.3 ± 5.8b, B 0.00 ± 0.00 8.65 ± 0.08a, A 0.00 ± 0.00 Cellulase 43.0 ± 0.0c, C 0.03 ± 0.06 7.71 ± 0.08c, C 0.05 ± 0.04 Hemicellulase 61.7 ± 1.2a, A 0.03 ± 0.06 6.49 ± 0.00f, D 0.00 ± 0.00 Pectinase 63.7 ± 1.2a, A 0.03 ± 0.06 8.18 ± 0.00b, B 0.05 ± 0.04 Protease 59.0 ± 0.0a, AB 0.03 ± 0.06 5.97 ± 0.08h, E 0.00 ± 0.00

All data represent means ± standard deviation (n=3). Initial moisture content of dried Noble seeds was 9.83 ± 0.25%. Different lower-case superscripts denote difference across extraction types. Different upper case superscripts denote difference within the same extraction type at p ≤ 0.05 using Tukey's test. n/a is not applicable

41

Table 2-3. Impact of extraction and refining on peroxide and free fatty acid content of Carlos seed oil.

Oil extraction methods Peroxide value (mEq/kg oil) Free fatty acid (% as oleic acid w/w)

Unrefined oil Refined oil Unrefined oil Refined oil

Soxhlet extraction using hexane Freeze dried seeds 6.33 ± 1.15h, B n/a n/a n/a Air dried seeds 35.0 ± 0.0g, A 0.00 ± 0.00 0.99 ± 0.00i 0.00 ± 0.00 Screw press expression

85°C 47.7 ± 1.2f, E 0.00 ± 0.00 2.59 ± 0.08h, E 0.00 ± 0.00 90°C 79.7 ± 1.2c, B 0.06 ± 0.06 3.85 ± 0.16g, D 0.00 ± 0.00 90°C at 13% Moisture 75.0 ± 0.0d, C 0.03 ± 0.06 4.98 ± 0.08de, B 0.02 ± 0.04 90°C at 16% Moisture 69.0 ± 0.0e, D 0.00 ± 0.00 4.51 ± 0.00f, C 0.02 ± 0.04 105°C 73.0 ± 0.0d, C 0.03 ± 0.06 4.84 ± 0.08e, B 0.00 ± 0.00 125°C 86.3 ± 1.2b, A 0.06 ± 0.06 7.14 ± 0.16a, A 0.02 ± 0.04

Enzyme-assisted aqueous extraction

Enzyme Cocktail 86.3 ± 1.2b,B 0.00 ± 0.00 5.12 ± 0.08d, C 0.02 ± 0.04 Cellulase 81.7 ± 1.2c, C 0.00 ± 0.00 5.12 ± 0.08d, C 0.00 ± 0.00

Hemicellulase 85.0 ± 0.0b, B 0.00 ± 0.00 5.59 ± 0.08c, B 0.07 ± 0.05 Pectinase 81.0 ± 0.0c, C 0.00 ± 0.00 4.09 ± 0.00g, D 0.00 ± 0.00 Protease 89.0 ± 0.0a, A 0.00 ± 0.00 6.30 ± 0.08b, A 0.00 ± 0.00

All data represent means ± standard deviation (n=3). Initial moisture content of dried Carlos seeds was 10.3 ± 1.2%, respectively. Different lower-case superscripts denote difference across extraction types. Different upper-case superscripts denote difference within the same extraction type at p ≤ 0.05 using Tukey's test. n/a is not applicable.

42

Table 2-4. Fatty acid composition of Noble seed oil using different extraction methods. Oil extraction method Palmitic Acid Stearic Acid Oleic Acid Linoleic acid

Soxhlet extraction

Hexane 7.13 ± 1.92 3.25 ± 0.68 16.3 ± 5.7 72.9 ± 16.2 Screw expression 85°C 7.09 ± 2.31 3.01 ± 1.19 16.4 ± 5.6 73.1 ± 16.1

90°C 7.11 ± 2.39 2.85 ± 0.52 16.5 ± 6.3 73.1 ± 17.4 90°C at 13% moisture 7.11 ± 2.37 3.10 ± 0.83 16.5 ± 5.8 73.0 ± 14.1 90°C at 16% moisture 7.06 ± 1.64 3.05 ± 0.40 16.6 ± 4.1 72.9 ± 8.8 105°C 6.80 ± 1.41 2.55 ± 0.74 16.3 ± 3.5 74.0 ± 12.8 125°C 6.88 ± 0.34 3.07 ± 0.72 16.0 ± 1.5 73.7 ± 16.5 Enzyme-assisted aqueous extraction Enzyme Cocktail 6.98 ± 1.22 3.75 ± 0.57 15.4 ± 1.6 73.5 ± 4.9 Cellulase 6.90 ± 0.96 3.08 ± 0.85 15.9 ± 3.0 73.7 ± 19.7 Hemicellulase 6.90 ± 0.72 3.46 ± 0.70 15.7 ± 2.2 73.5 ± 11.4 Pectinase 6.79 ± 2.87 3.25 ± 1.45 16.3 ± 7.6 73.2 ± 24.1 Protease 7.10 ± 1.59 3.70 ± 1.46 15.6 ± 3.2 73.2 ± 12.1

All data represent means ± standard deviation (n=6) expressed as % of individual fatty acid to total fatty acids. Different superscripts denote significant differences in each extraction method at p ≤ 0.05 using Tukey's test.

43

Table 2-5. Fatty acid composition of Carlos seed oil using different extraction methods Oil extraction method Palmitic Acid Stearic Acid Oleic Acid Linoleic acid

Soxhlet extraction Hexane 7.50 ± 1.88 4.9 ± 1.31 15.1 ± 4.3 77.1 ± 15.8

Screw expression

85°C 6.94 ± 1.08 4.56 ± 1.00 14.5 ± 2.7 73.7 ± 16.7

90°C 7.05 ± 1.22 4.28 ± 0.37 14.4 ± 2.4 73.9 ± 3.9 90°C at 13% moisture 7.10 ± 1.03 4.56 ± 0.35 14.1 ± 1.0 73.9 ± 11.2 90°C at 16% moisture 7.07 ± 0.84 4.30 ± 0.40 14.8 ± 1.2 73.5 ± 7.7 105°C 7.00 ± 1.22 4.23 ± 0.61 14.5 ± 1.6 74.0 ± 13.4 125°C 7.69 ± 2.42 4.62 ± 1.06 15.7 ± 4.2 79.3 ± 20.0

Enzyme-assisted aqueous extraction Enzyme Cocktail 7.02 ± 2.02 4.34 ± 1.02 14.4 ± 3.3 73.9 ± 15.9

Cellulase 7.14 ± 3.33 4.20 ± 1.61 14.4 ± 5.9 74.0 ± 27.0 Hemicellulase 7.11 ± 1.54 4.34 ± 0.51 14.7 ± 3.0 73.5 ± 5.7 Pectinase 6.94 ± 1.12 4.00 ± 1.89 15.1 ± 1.9 73.6 ± 7.0 Protease 7.07 ± 2.46 4.18 ± 1.15 15.1 ± 4.9 73.3 ± 15.1

All data represent means ± standard deviation (n=6) expressed as % of total fatty acids. Different superscripts denote significant differences in each extraction method at p ≤ 0.05 using Tukey's test.

44

Table 2-6. Impact of different extraction methods on the vitamin E concentration (mg/100 g) of crude Noble seed oil

All data represent means ± standard deviation (n=6). Different lower-case superscripts denote difference across extraction types. Different upper-case superscripts denote difference within the same extraction type at p ≤ 0.05 using Tukey's test. n.d. is not detected.

Oil extraction methods α-Tocopherol α-Tocotrienol γ-Tocopherol γ-Tocotrienol δ-Tocotrienol

Soxhlet

Hexane 10.8 ± 2.4abc 21.3 ± 0.4 39.8 ± 1.8ab 15.7 ±0.6abcd 2.95 ± 0.33ab

Screw expression

85°C 7.14 ± 1.03bc 21.3 ± 4.0 24.9 ± 2.1cd, B 14.1 ± 1.4d 2.55 ± 0.36b

90°C 7.57 ± 1.14abc 25.2 ± 2.8 23.6 ± 1.3d, B 15.8 ± 0.7abcd 2.73 ± 0.18ab

90°C at 13% moisture 6.48 ± 0.71c 25.8 ± 3.4 23.0 ± 1.2d, B 15.9 ± 1.4abcd 2.56 ± 0.36b

90°C at 16% moisture 8.74 ± 2.47abc 21.9 ± 3.3 32.1 ±4.4abcd, A 15.0 ± 1.1cd 2.93 ± 0.27ab

105°C 6.60 ±1.17c 23.5 ± 0.8 23.5 ±3.0d, B 15.5 ± 0.4bc 2.63 ± 0.38ab

125°C 6.84 ± 1.05bc 22.8 ± 1.8 23.2 ±2.8d, B 15.4 0.8c 2.94 ± 0.15ab

Enzyme-assisted aqueous extraction

Enzyme cocktail 9.18 ± 3.56abc 20.5 ± 4.3 30.4 ± 10.2bcd 17.5 ± 1.1a, A 2.92 ± 0.15ab

Cellulase 11.6 ± 1.1a 22.0 ± 1.3 42.2 ± 4.9a 15.4 ± 0.7c, B 2.92 ± 0.15ab

Hemicellulase 11.1 ± 3.5ab 22.4 ± 3.7 38.0 ± 6.9ab 16.7 ± 1.1abc, AB 3.23 ± 0.58a

Pectinase 9.16 ± 0.77abc 23.2 ± 2.1 34.3 ± 1.0abc 15.1 ± 0.5cd, B 2.95 ± 0.48ab Protease 10.1 ± 4.2abc 21.6 ±4.2 34.6 ± 10.3abc 17.2 ± 1.2ab, A 3.24 ± 0.25a

45

Table 2-7. Impact of different extraction methods on the vitamin E concentration (mg/100 g) of refined Noble seed oil.

All data represent means ± standard deviation (n=6). Different lower-case superscripts denote difference across extraction types. Different upper-case superscripts denote difference within the same extraction type at p ≤ 0.05 using Tukey's test.

Oil extraction methods α-Tocopherol α-Tocotrienol γ-Tocopherol γ-Tocotrienol δ-Tocotrienol

Soxhlet Hexane 6.89 ± 0.98a 9.80 ± 1.32a 18.2 ± 2.4a 6.67 ± 0.88ab n.d.

Screw expression

85°C 0.716 ± 0.361cd 2.73 ± 0.86cd 4.72 ± 2.58de, B 3.73 ± 2.05c, B n.d. 90°C 0.800 ± 0.030c 3.07 ± 0.03c 6.21 ± 1.10cd, AB 4.82 ± 1.48bc n.d. 90°C at 13% moisture 0.910 ± 0.100c 3.76 ± 0.54 8.03 ± 1.13bc, AB 6.46 ± 1.06ab, AB n.d. 90°C at 16% moisture 0.880 ± 0.190c 3.32 ± 1.29c 6.67 ± 3.23bcd, AB 5.11 ± 3.15bc, AB 0.163 ± 0.401 105°C 1.66 ± 0.35b 7.68 ± 1.78b 9.76 ± 1.78b, A 8.10 ± 1.44a, A 0.293 ± 0.454 125°C 0.530 ± 0.430cde 2.25 ± 0.65cde 7.29 ± 0.65bcd, AB 5.04 ± 1.34bc, AB 0.276 ± 0.428

Enzyme-assisted aqueous extraction

Enzyme cocktail 0.430 ± 0.030cde 1.11 ± 0.07de 1.81 ± 0.21ef, A 0.11 ± 0.28d n.d. Cellulase n.d. 1.10 ± 0.13de n.d. n.d. n.d. Hemicellulase n.d. 0.98 ± 0.21e 0.37 ± 0.41f, C n.d. n.d. Pectinase 0.709 ± 0.104cd 1.34 ± 0.22de 2.02 ± 0.25ef, A n.d. n.d. Protease 0.0565 ± 0.138de 1.03 ± 0.08e 1.18 ± 0.10f, B n.d. n.d.

46

Table 2-8. Impact of extraction methods on vitamin E concentration (mg/100 g) of crude Carlos seed oil. Oil extraction methods α-Tocopherol α-Tocotrienol γ-Tocopherol γ-Tocotrienol δ-Tocotrienol

Soxhlet

Hexane 2.94 ± 0.39 c 9.35 ± 1.23c 14.6 ± 1.4bc 15.8 ± 1.3 1.72 ± 0.16ab

Screw expression

85°C 3.12 ± 0.17 abc 12.5 ± 5.0abc 15.1 ± 4.0bc 18.0 ± 4.0 1.89 ± 0.30ab

90°C 2.39 ± 0.48 abc 12.0 ± 4.5abc 9.62 ± 0.50c 18.3 ± 3.6 1.73 ± 0.08ab

90°C at 13% moisture 4.35 ± 3.39 abc 13.5 ± 6.4abc 17.1 ± 1.0abc 16.6 ± 0.9 1.60 ± 0.16b

90°C at 16% moisture 2.77 ± 0.59bc 11.4 ± 4.9bc 12.6 ± 0.7c 17.4 ± 4.1 1.66 ± 0.05ab

105°C 2.71 ± 0.35abc 14.2 ± 3.6abc 9.60 ± 0.20c 19.1 ± 2.4 1.74 ± 0.12ab

125°C 2.53 ± 0.20abc 13.0 ± 2.7abc 9.25 ± 0.22c 18.8 ± 2.0 1.75 ± 0.17ab

Enzyme-assisted aqueous extraction

Enzyme cocktail 4.10 ± 0.41abc, B 15.1 ± 0.9abc, BC 14.7 ± 1.0bc, B 19.7 ± 0.7 1.76 ± 0.07ab, AB

Cellulase 3.84 ± 0.23 abc, B 14.0 ± 1.8abc, C 14.2 ± 1.8bc, B 18.6 ± 1.5 1.75 ± 0.15ab, AB

Hemicellulase 6.02 ± 2.25ab, AB 16.6 ± 3.4ab, ABC 16.9 ± 2.0abc, AB 19.3 ± 2.3 1.83 ± 0.17ab, AB

Pectinase 6.84 ± 1.53ab, A 18.2 ± 1.3ab, AB 23.2 ± 6.3ab, AB 17.9 ± 4.4 1.69 ± 0.06ab,

Protease 6.52 ± 1.28a, A 18.9 ± 1.0a, A 25.4 ± 10.4a, A 19.3 ± 4.4 1.92 ± 0.12a, AB

All data represent means ± standard deviation (n=6). Different lower-case superscripts denote difference across extraction types. Different upper-case superscripts denote difference within the same extraction type at p ≤ 0.05 using Tukey's test. n.d. is not detected.

47

Table 2-9. Impact of extraction methods on vitamin E concentration (mg/100g) of refined Carlos seed oil Oil extraction methods α-Tocopherol α-Tocotrienol γ-Tocopherol γ-Tocotrienol δ-Tocotrienol

Soxhlet Hexane 1.63 ± 0.20a 7.42 ± 0.64a 8.04 ± 0.68a 13.8 ± 1.3a 1.22 ± 0.01a Screw expression 85°C 0.269 ± 0.416b 1.71 ± 0.23c 0.566 ± 0.647b 1.52 ± 0.79cd 0.412 ± 0.217b 90°C n.d. 1.31 ± 0.19c 0.765 ± 0.660b 1.65 ± 1.15cd n.d. 90°C at 13% moisture n.d. 1.48 ± 0.15c 1.34 ± 0.46b 2.75 ± 1.23cd n.d. 90°C at 16% moisture n.d. 1.20 ± 0.23c n.d. 0.824 ± 0.061cd n.d. 105°C 0.573 ± 0.887b 3.56 ± 3.64bc 3.12 ± 4.58b 5.86 ± 7.01bc 0.391 ± 0.217b 125°C n.d. 1.33 ± 0.14c n.d. 1.05 ± 0.07cd n.d.

Enzyme-assisted aqueous extraction

Enzyme cocktail 1.91 ± 0.45a, A 5.63 ± 1.37ab, A 8.03 ± 1.89b, A 9.36 ± 2.24ab, A n.d. Cellulase n.d. 1.02 ± 0.11c, B n.d. 0.243 ± 0.376d, B n.d. Hemicellulase 0.432 ±0.67b, B 2.74 ± 2.42c, B 2.27 ± 3.52b, B 3.73 ± 5.78cd, B 0.407 ± 0.630b Protease 0.179 ± 0.430b, B 1.23 ± 0.06c, B n.d. n.d. n.d. Pectinase n.d. 1.03 ± 0.28c, B 0.805 ± 0.150b, B n.d. n.d.

All data represent means ± standard deviation (n=6). Different lower-case superscripts denote difference across extraction types. Different upper-case superscripts denote difference within the same extraction type at p ≤ 0.05 using Tukey's test. n.d. is not detected. n.d. is not detected

48

Table 2-10. Refining yield (%, w/w) of seed oil from Noble and Carlos muscadine seeds at different steps of refining.

Oil extraction method % Yield (w/w)

Noble Carlos

Degumming Neutralizing Bleaching Degumming Neutralizing Bleaching Soxhlet Hexane 64.8 ± 2.9b 47.4 ± 1.0c 41.1 ± 1.6b 84.7 ± 0.5c 64.7 ± 0.8 59.7 ± 1.0b Screw Expression 85°C 59.5 ± 1.5b 45.6 ± 2.1c 40.6 ± 0.1b 78.5 ± 0.9d 63.9 ± 2.6b 59.3 ± 1.9b 90°C 64.1 ± 1.2b 46.1 ± 1.3c 40.7 ± 0.7b 80.6 ± 0.4d 64.7 ± 0.7b 60.0 ± 0.7b 90°C at 13% moisture 59.8 ± 1.0b 46.2 ± 1.1c 39.1 ± 2.3b 78.5 ± 0.3d 64.4 ± 0.4b 59.1 ± 1.4b 90°C at 16% moisture 60.4 ± 1.0b 45.3 ± 1.7c 41.0 ± 1.0b 79.6 ± 0.8d 64.3 ± 1.1b 57.7 ± 0.7b 105°C 61.6 ± 2.1b 44.4 ± 2.2c 41.9 ± 1.0b 79.0 ± 1.0d 63.2 ± 1.2b 60.5 ± 0.6b 125°C 62.5 ± 0.6b 45.6 ± 0.4c 41.0 ±1.1b 75.9 ± 0.3e 62.9 ± 0.5b 59.7 ± 1.8b Enzyme-assisted aqueous extraction Cocktail 93.6 ± 3.7a 80.7 ± 1.6ab 72.7 ± 0.7b 99.2 ± 0.3a 81.2 ± 1.8a 72.5 ± 2.1a Cellulase 95.0 ± 1.6a 79.5 ± 1.0ab 71.6 ± 2.7b 97.4 ± 0.4a 80.0 ± 2.1a 72.9 ± 2.2a Hemicellulase 95.1 ± 1.6a 80.9 ± 2.9ab 72.4 ± 3.4b 97.4 ± 0.4a 81.6 ± 1.9a 70.4 ± 2.7a Pectinase 94.8 ± 0.6a 81.6 ± 2.0a 68.5 ± 2.9b 94.4 ± 1.7b 76.9 ± 3.1a 72.8 ± 2.5a Protease 94.8 ± 1.2a 76.2 ± 1.5b 72.2 ± 0.2b 94.8 ± 0.4b 80.2 ± 2.1a 69.5 ± 2.6a

All data represents means ± standard deviation (n=3). Oil yield was expressed as percentage of mass of oil remaining after refining step compared to initial mass. Different superscripts denote significant differences in each extraction method at p ≤ 0.05 using Tukey's test.

49

Figure 2-1. Concentrations of tocopherol and tocotrienol in Noble seed oil after the refining process. Results are mean

with error bars denoting standard deviation. Different lower-case superscripts denote difference across extraction types. Different upper-case superscripts denote difference within the same extraction type at p ≤ 0.05 using Tukey's test.

a

b b

bb

a

b

c, A

c, C c, BC c, Bc, A

0%

10%

20%

30%

40%

50%

60%

Hex

ane

85°C

90°C

90°C

at

13

% m

ois

ture

90°C

at

16

% m

ois

ture

105

°C

125

°C

Enzy

me

co

ckta

il

Cel

lula

se

Hem

ice

llula

se

Pe

ctin

ase

Pro

teas

e

Soxhlet Screw expression Enzyme-assisted aqueous

Re

ten

tio

n⍺-Tocopherol 𝞬-Tocopherol ⍺-Tocotrienol 𝞬-Tocotrienol δ-Tocotrienol

50

Figure 2-2. Concentrations of tocopherol and tocotrienol in Carlos seed oil after the refining process. Results are mean

with error bars denoting standard deviation. Different lower-case superscripts denote difference across extraction types. Different upper-case superscripts denote difference within the same extraction type at p ≤ 0.05 using Tukey's test.

a

cd cdcd

cd

bc

cd

b, A

d, B

cd, B

d, B d, B

0%

10%

20%

30%

40%

50%

60%

70%

80%

Hex

ane

85°C

90°C

90°C

at

13

% m

ois

ture

90°C

at

16

% m

ois

ture

10

5°C

12

5°C

Enzy

me

co

ckta

il

Cel

lula

se

Hem

ice

llula

se

Pe

ctin

ase

Pro

teas

e

Soxhlet Screw expression Enzyme-assisted aqueous

Re

ten

tio

n⍺-Tocopherol 𝞬-Tocopherol ⍺-Tocotrienol 𝞬-Tocotrienol δ-Tocotrienol

51

Figure 2-3. Peroxide value in Carlos seed oil extracted using enzyme-assisted aqueous extraction with protease after

treatment with bleaching earth clay of different concentrations. Results are mean with error bars denoting standard deviation. Carlos seed oil extracted with assistance of protease had an initial peroxide value of 89.0 mEq/kg oil.

y = 3776.6x2 - 1347x + 94.752R² = 0.9874

0

10

20

30

40

50

60

70

80

90

0.0% 2.0% 4.0% 6.0% 8.0% 10.0% 12.0%

Pero

xid

e v

laue (

mE

q/k

g o

il)

Bleaching earth clay oncentration (%, w/w)

52

CHAPTER 3 MUSCADINE GRAPE SEED FLOUR

Background

Muscadine seeds are a byproduct of juice pressing and wine making. Of the 300

tons of seeds that are disposed of or used as cattle feed, 250 tons could potentially be

utilized as muscadine grape seed flour (Husmann & Dearing, 1916). The muscadine

grape seeds possess unique phytochemicals including hydrolyzable tannins, flavon-3-

ols, condensed tannins, ellagic acid derivatives, and quercetin rhamnoside (Sandhu &

Gu, 2010). Extracts of muscadine seeds showed antioxidant function as well as

antimicrobial activity against E. coli O-157:H7 and E.Sakazakii (Kim et al., 2009; Kim et

al., 2008; Sandhu & Gu, 2010). Additionally, phytochemical extracts of the cold pressed

muscadine seed flour reduced the proliferation of human colon cancer cells (Lutterodt et

al., 2011). Muscadine seed flour was added in baked products to enhance antioxidant

capacity (Hoye Jr & Ross, 2011; Ross, Hoye Jr, & Fernandez‐Plotka, 2011), suggesting

it could be used as a potential replacement for other flours. The aim of this study was to

observe the effects of different oil extraction methods on the proximate composition,

phenolic content, antioxidant capacity, and phytochemical profile of defatted muscadine

seed flour.

Materials and Methods

Chemicals

Methanol (HPLC grade), n-Hexane, 2,2,-Diphenyl-1-picryhydryazyl (DPPH), 6-

Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox), potassium sulfate,

Kjeltabs, Kjelsorb, sulfuric acid, sodium hydroxide, formic acid (HPLC grade), and gallic

acid were purchased, fiber glass thimbles from Fisher Scientific Co. Pittsburgh, PA,

53

USA) Quecetin, kaempferol, and myricetin were purchased from Sigma Aldrich (St.

Louis, MO, USA). Anthocyanin standards: delphinidin 3,5-diglucoside, malvidin 3-

5,diglucoside, cyanidin 3,5 diglucoside, petunidin 3,5-diglucoside, pelargonidin 3,5-

diglucoside, and peonidin 3,5-diglucoside were purchased from Phytolab

(Vestenbergreuth,Germany). Filter bags, heat sealer, digestion flasks, and a dessicant

were purchased from Ankom Technology (Macedon, NY, USA)

Preparation of Flour

Defatted seeds were obtained for Soxhlet extraction using hexane and

pressed cake was obtained from expression using a continuous screw press. Pressed

cake contains residual oil, so it was placed in a RRH-A1000 high speed multi-function

grinder and grinded to a fine power. Pressed cake powder was placed in fiber glass

thimbles and into a Soxhlet apparatus. Pressed cake powder was washed with n-

hexane in the Soxhlet apparatus for 8 hours to obtain defatted press cake. Defatted

press cake and defatted seeds for Soxhlet extraction were placed in the RRH-A100 high

speed multi-function grinder to be ground. Once ground defatted seeds from Soxhlet

extraction and ground defatted press cake were run through a No.60 sieve (250 m).

Resulting powder was collected as muscadine flour.

Proximate Analysis of Flour

Ash in flour was determined by a previously described by AOAC official method

923.03 (International, 1995). In summary, 3-5 grams of muscadine seed flour were

placed into an ashing dish that had been ignited, cooled in a desiccator, and weighed

after reaching room temperature. Samples were placed in a furnace at 550°C until it

was a light gray ash. Samples and dishes were placed in a desiccator and weighed

54

after reaching room temperature. Finals weights were compared to initial weights of

sample to receive ash content.

Protein in flour was determined using the Kjeldahl method with modifications to

the one described by AOAC official method 920.87 (International, 1995). One gram of

muscadine seed flour was placed in a digestion flask with one Kjeltab and 10 mL of

sulfuric acid. Flasks were placed in an inclined position and heated gently. Flasks were

allowed to digest for 30 minutes after the solution became clear. Clear contents were

poured into the Labconco Rapidstill I (Labconco Corp, Kansas City, MO, USA) micro-

Kjeldahl distillation unit. Distillation flasks were washed with distilled water and also

poured into the distillation unit.

During the digestion, the micro-Kjeldahl distillation unit was preheated. A cup

containing 30 mL of Kjelsorb was placed near the distillation unit. After digested

samples were placed into the distillation unit, concentrated sodium hydroxide (50%) was

slowly added. Kjelsorb was placed at the end of the distillation unit with swirling to

prevent the initial backflow. Distillation was allowed to run for approximately 10 minutes

or until the final volume was around 70-80 mL.

The Kjelsorb solution was then titrated using 0.05 M hydrochloric acid. Volume of

hydrochloric acid was used to determine nitrogen content. Protein was calculated by

using a conversion factor of 5.3 as described in previously published work (Kamel,

Dawson, & Kakuda, 1985).

Neutral detergent fiber content was analyzed by an external laboratory (Central

Analytical Laboratory, Fayetteville, AR, USA). Neutral detergent fiber was measured

using a filter bag technique (Ankon, 2011).The mass of each filter bag is recorded and

55

0.45 – 0.50 g of prepared flour were placed into each bag. Each bag was sealed closed

and placed into an acetone bath with 20 g of sodium sulfite. Bags were heated in the

solution for 75 minutes. After the bags were rinsed in hot water for five minutes and

placed into a room temperature acetone bath. After, bags were removed, air-dried, and

then dried in an oven at 102°C. After being completely dried, each bag was placed in a

desiccant pouch and cooled to room temperature before being weighed. Weight of the

sample before and after treatment were used to calculate percent neutral detergent

fiber.

Phytochemical Extraction

Phytochemicals in muscadine seed flour were extracted using a previously

described method with modification (Sandhu, Gray, Lu, & Gu, 2011). Briefly, one gram

of seed flour and 10 mL of methanol/water/acetic acid (85:15:0.5; v/v/v) were placed in

a 50 mL conical centrifuge tube. The mixture was vortexed for 30 seconds then

sonicated for five minutes and left in the sonicator to rest for 20 minutes. After the

mixture was vortexed for 30 seconds and centrifuged at 1450 g for eight minutes. The

supernatant was stored at -20°C until used.

Total Phenolic Content

Folin-Ciocalteu Reagent purchased from Fisher Scientific (Pittsburg, PA, USA)

was diluted with deionized water 1:9 ratio (v/v) to create a working solution. In a test

tube one mL of Folin-Ciocalteu working solution and 100 mL of deionized water as a

blank. A standard was prepared using seven test tubes with 100 L of gallic acid

solution with concentrations of 0, 100, 200, 300, 400, 500, and 600 mg/mL. A final test

tube contains 100 L of the phytochemical extraction. One mL of was added to each

56

tube before a solution of 15% sodium carbonate was added and vortexed. The mixture

was incubated for 30 minutes before absorbance was read at 765 nm.

DPPH Assay

The DPPH scavenging activity of muscadine seed flour was determined using a

previously published method (Brand-Williams, Cuvelier, & Berset, 1995). A DPPH stock

solution was made by dissolving 20 mg of DPPH into 100 mL of methanol and stored at

-20°C. A DPPH working solution was made by mixing 2.8 mL of DPPH stock solution

and 7.2 mL of methanol. 50 L of methanol was placed in the 96-well plate as a blank,

along with the extract from the muscadine seed flour, and trolox standards with

concentrations of 50,100, 200, 300, 400, and 500 mol/mL. 200 L of DPPH working

solution were put into each well and incubated in the dark at room temperature for 30

minutes. Absorbance was measured at 515 nm on a microplate reader (SPECTRAMax

190, Molecular Devices, Sunnyvale, CA). Results of the DPPH assay were expressed

as micromoles of trolox equivalent (mol trolox/g of flour)

HPLC Analysis of Muscadine Flour Phytochemicals

High-performance liquid chromatography (HPLC) analyses of muscadine seed

flour extract was performed on 1200 system equipped with a binary pump, an

autosampler/injector, and a diode array detector (Agilent Technologies, Palo Alto, CA,

USA). Separation was conducted using a Zorbax-SB C18 column (4.6 mm X 250 mm,

5m, Agilent Technologies). UV vis spectra were scanned from 220 to 600 nm on a

diode array detector with detection wavelengths of 280, 360, and 520 nm. Anthocyanin

analysis quantification was determined using a previously published method (Li et al.,

2017). Muscadine flour extracts were filtered before injection. The mobile phases

57

consisted of methanol (phase A) and 5% formic acid in water (phase B). The linear

gradient was as follows: 0-10 min, 95-85% B; 10-30 min, 85-70% B; 30-45 min, 70% B;

47 min, 30% B; with a flow rate of 0.8 mL/min.

To quantify other phytochemicals including kaempferol and myricetin a previously

published method was used (Sandhu & Gu, 2010). Acid hydrolysis of samples was

done before HPLC analysis. 1.2 M hydrochloric acid was added to muscadine seed

flour methanol extract to create a 50% solution and sonicated for five minutes.

Hydrolysis was conducted in a precision water bath (Thermo Fisher scientific, Waltham,

MA) for 80 minutes at 90°C. The mobile phases consisted of 0.5% aqueous formic acid

solution (phase A) and methanol (phase B). The linear gradient was as follows: 0-2 min,

5% B; 2-10 min, 5-20% B; 10-15 min, 30-35% B; 60-65 min, 80-85% B; 65-70 min, 85-

5% B; with a flow rate of 1 mL/min.

Results and Discussions

Proximate Composition of Flour

Ash content of Noble seed flour and Carlos seed flour are shown in Table 3-1.

The ash content of Noble seed flour ranged from 6.11 to 13.4 g/100 g. Different

methods of extraction, as well as different temperatures of screw press expression, did

not show significant effects on the ash content of Noble seed flour. However, there were

significant differences in the ash content of Carlos seed flour. Flour obtained from

expression at 90 °C with a Carlos seed moisture content of 16% (w/w) had significantly

higher ash content than all other flours (17.2 g/100 g). Muscadine grape seed flour

possessed more ash content than previously reported grape seed flour (Özvural &

Vural, 2011). This data suggests that muscadine seed flour is a greater source of

minerals than grape seed flour than other grapes of the Vitis genus.

58

Table 3-1 displays the protein content of the flours obtained from seven different

extraction methods. Noble seed flour protein content was the highest in Soxhlet defatted

Noble seed flour and expression defatting at 90°C, 105°C, and 125°C Noble seed flour.

Carlos seed flour contained the highest protein content after being expressed at 125°C.

Protein content of both cultivars ranged from 14.3 to 17.8 %. Muscadine seed flour

contained higher levels of protein than previously reported data on Vitis Vinifera grape

seed flour (Kamel et al., 1985). However similar values have been reported in whole

wheat flour (Davis, Cain, Peters, Le Tourneau, & McGinnis, 1981). Differences in

protein content among flours are likely due to thermal treatment differences in each oil

extraction method. This data suggests that muscadine seed flour could provide as much

protein as whole wheat flour, but unlike whole wheat flour, muscadine seed flour does

not contain gluten. Muscadine seed flour has the potential to be a gluten free substitute

for wheat flour.

Noble seed flour had a carbohydrate range from 60.3 to 72.6% and 55.0 to

70.1% for Noble and Carlos, respectively. Among the Noble seed flours, there was

significant difference among the carbohydrate content, with hexane defatted seed flour

possessing the highest carbohydrate content. Among the Carlos seed flours, flour that

underwent expression at 90°C had the highest carbohydrate content. Muscadine seed

flour fiber content ranged from 42.0 to 49.5 and 46.3 to 51.7% for Noble and Carlos,

respectively. Both Noble and Carlos seed flour displayed no significant difference in

fiber content among different defatting conditions to obtain the flour. Muscadine seed

flour possessed similar carbohydrate content to those found in wheat in previous

literature, and more than flour from various Vitis vinifera seeds (Davis et al., 1981;

59

Kamel et al., 1985). Fiber found in Noble and Carlos seed flour is neutral detergent

fiber, which consists of lignin, hemicellulose, and cellulose, components of insoluble

fiber. These results demonstrate that Noble and Carlos seed flours are comparable

sources of carbohydrates and insoluble fibers to that found in wheat flours and are a

greater source than that found in grape seed flour from Vitis vinifera grape seeds.

Lipid content in flour largely depended on the efficiency of oil extraction. Soxhlet

defatted flour of the Noble and Carlos seed flour possessed the least amount of lipids

because they were removed by the hexane. The least efficient oil extraction method for

noble seeds, with pressing conditions of 90°C at 16% moisture, had the Noble seed

flour with the highest lipid content. High moisture resulted in oil expression being less

effective resulting in a higher lipid content flour. Expression of Carlos seed oil was also

less effective than Soxhlet extraction using hexane. As a result, the muscadine seed

flours that underwent expression possessed significantly higher lipid content in flour

than flour exposed to Soxhlet extraction using hexane. This data suggests that,

extraction method plays a significant role in lipid content in flour. Higher lipid

concentrations in flour can result in greater risk of oxidation and rancidity (Galliard,

1986).

Total Phenolic Content and Antioxidant Capacity

Table 3-2 shows the total phenolic content and antioxidant capacity of the

muscadine seed flours defatted by different extraction methods and conditions. Based

on defatting method, Soxhlet extracted Muscadine grape seed flour possessed the

highest phenols expressed in gallic acid equivalence (32.8 mg Gallic acid/mL). Similarly,

Carlos seed flour obtained after a Soxhlet and hexane treatment possessed the most

total phenols (22.4 mg gallic acid/mL). The antioxidant capacity of Noble seed flour was

60

found to be the highest in the flour that underwent oil extraction using expression at

90°C; however, there was no significant difference among the antioxidant capacity of

Noble seed flour. Carlos seed flour displayed significantly higher antioxidant capacity in

seed flour defatted with Soxhlet extraction (151 mol Trolox/100g), and expression at

85°C (184 mol Trolox/100g), 105°C (183 mol Trolox/100g), and 125°C (129 mol

Trolox/100g). Carlos seed flours with the oil extracted using the Soxhlet extraction and

expression at 85°C , 105°C, and 125°C were significantly more effective in neutralizing

DPPH free radicals. Total phenolic content and antioxidant capacity of Noble and Carlos

flour were less than previously reported data (Sandhu & Gu, 2010). The data

demonstrates that Noble and Carlos seed flour experienced losses of total phenolic

content and antioxidant capacity that could likely be attributed to oxidation and thermal

degradation during the oil extraction process. Findings support Soxhlet extraction using

hexane more efficient at preserving phenolic compounds in Noble and Carlos seed flour

than screw expression.

Characterization of Muscadine Flour Phytochemicals

Phytochemical composition of Noble and Carlos seed flour are shown in Tables

3-3 and 3-4, respectively. Phytochemical profiles show similarity to identified

compounds in previous literature (Li et al., 2017). HPLC chromatograms of Noble seed

flour and Carlos seed flour were recorded at 360 and 520 nm (Figures 3-1 and 3-2).

Flavonals had a maximum absorption at 360 nm and anthocyanins at 520 nm. Of the

two flours, only Noble seed flour was shown to possess any anthocyanins. This could

be explained by the wine-making process, in which red Noble grapes are crushed and

seeds and skins are fermented together with juices (Talcott & Lee, 2002). Wine

61

fermentation may also be responsible for concentrating other flavonoids in the seeds, as

Noble seed flour possessed myricetin and kaempferol and Carlos seed flour was only

possessed myricetin. The predominant anthocyanin in the Noble seed flour was

malvidin 3,5-diglucoside. Among fat extraction methods, Noble seed flour that had

undergone only Soxhlet extraction using hexane for defatting contained the highest

malvidin 3,5-diglucoside content (167 mg/100g). Noble seed flour that was defatted

using a Soxhlet extraction also had significantly higher concentrations of delphinidin 3,5-

diglucoside (31.0 mg/100g), cyaniding 3,5-diglucoside (76.9 mg/100g), petunidin 3,5-

diglucoside (36.0 mg/100g), and peonidin 3,5-diglucoside (26.8 mg/100g). Soxhlet

defatted Noble seed flour likely has higher anthocyanin content as it only has to be

defatted once, while pressed flour experiences the pressing temperatures as well as

Soxhlet extraction to remove as much oil from the seeds as possible before the flour

process. High temperature pressings and heated solvent extraction likely diminish the

anthocyanin content of the Noble seed flour. The predominant flavonoid in Noble seed

flour was myricetin, ranging from 211 to 235 mg/100g, followed by kaempferol ranging

from 39.7 to 44.7 mg/100g, with no detection of quercetin. There was no significant

difference in concentration of myricetin among Noble seed flour from different oil

extraction methods. Kaempferol concentrations were highest in Noble seed flour that

underwent expression at 125°C but only significantly higher than Noble seed flour that

underwent expression at 85°C. Similar phytochemical compositions were reported in

previous work (Li et al., 2017). Phytochemical concentrations were higher in Noble seed

flour than Carlos seed flour. This data suggests that the fermentation process, where

seeds and juice are fermented together, concentrates anthocyanins and flavonoids in

62

the Noble seeds. Additionally, the data suggests that Soxhlet extraction using hexane is

the best method for preserving phytochemicals within Noble seed flour. Anthocyanin

and flavonoid concentrations were lower than previously reported values for muscadine

seeds, possibly from losses experienced throughout the oil extraction process.

Carlos seed flour obtained from all oil extraction methods had no detectable

anthocyanins that were found in Noble seed flour. Additionally, Carlos seed flour did not

show any kaempferol, a flavonoid that was found in Noble seed flour. Carlos seed flour

did, however, show similar levels of myricetin, the predominant flavonoid in the Noble

seed flour. Myricetin concentrations in Carlos seed flour ranged from 165 to 234 mg/100

g. Myricetin concentrations in Carlos seed flour showed no difference among seed flour

that underwent different defatting conditions. Carlos seed flour had lower reported

myricetin levels than previous work (Pastrana-Bonilla, Akoh, Sellappan, & Krewer,

2003). Differences in Carlos seed flour myricetin levels could likely be attributed to

oxidation and thermal degradation throughout the oil extraction process that effected all

Carlos seed flours.

Summary

Our results indicate that muscadine seed flour from Noble and Carlos seeds

contain a proximate composition similar to whole-wheat flour but without gluten proteins.

In Carlos seed flour, expression of the seed at 125°C resulted in flour with the highest

amount of protein. Carlos seed flour with lower temperature expressions (85°C, 90°C,

90°C at 13% moisture, and 90°C at 16% moisture) possessed higher amounts of

carbohydrates. Fiber content showed no significant difference among flour treated with

different oil extraction treatments. However, Carlos seed flour did on average possess

more fiber than Noble seed flour. Similarly, ash content was not significantly different

63

among the various flours. Both Noble and Carlos seed flour defatted by a Soxhlet

apparatus and hexane possessed the highest total phenols. Noble seed flour showed

no significant difference in antioxidant capacity despite possessing a difference in

phenolic content. Carlos seed flour possessed significantly different antioxidant

capacities that did not correspond directly with higher phenolic contents in the flour.

Noble seed flour was shown to have a wider variety of phytochemicals possessing

multiple anthocyanins and kaempferol unlike the Carlos seed flour.

64

Table 3-1. Impact of extraction method on proximate analysis (%, w/w) of Noble seed flour and Carlos seed flour.

Oil extraction method Moisture Ash Protein Lipids NDF Fiber Total Carbohydrates

Noble seed flour

Soxhlet extraction

Hexane 4.92 ± 0.13abc 6.11 ± 0.84 16.4 ± 0.1ab 0.00 ± 0.90c 44.8 ± 5.3 72.6 ± 2.0a

Screw expression

85°C 4.65 ± 0.40bc 8.09 ± 3.32 16.1 ± 0.2b 3.76 ± 1.85ab 44.8 ± 1.0 67.4 ± 0.9ab

90°C 3.93 ± 0.29bc 12.3 ± 3.5 16.6 ± 0.2a 3.58 ± 0.25b 46.3 ± 1.4 63.5 ± 3.3b

13% at 90°C 5.17 ± 0.87ab 10.2 ± 3.7 16.4 ± 0.3ab 5.51 ± 0.63ab 46.0 ± 0.3 60.3 ± 3.0b

16% at 90°C 6.03 ± 0.27a 13.4 ± 3.6 16.8 ± 0.0ab 6.04 ± 0.39a 42.0 ± 2.6 64.0 ± 3.4b

105°C 3.68 ± 0.54c 12.6 ± 1.7 16.4 ± 0.1a 5.49 ± 0.21ab 49.5 ± 0.3 64.2 ± 3.1b

125°C 3.62 ± 0.44c 9.56 ± 3.20 14.3 ± 0.1c 4.62 ± 0.22ab 48.0 ± 0.3 61.5 ± 3.5b

Carlos seed flour

Soxhlet extraction

Hexane 5.17 ± 0.60 13.8 ± 1.6 16.3 ± 0.1c 0.00 ± 0.57b 46.3 ± 0.8 64.7 ± 1.5ab

Screw expression

85°C 5.07 ± 0.20 9.43 ± 2.60 17.0 ± 0.2b 5.02 ± 0.43a 53.6 ± 1.1 64.7 ± 4.4ab

90°C 5.31 ± 0.72 4.66 ± 1.21 16.3 ± 0.2c 4.17 ± 1.01a 51.2 ± 0.6 70.1 ± 0.7a

13% at 90°C 3.73 ± 0.19 8.11 ± 0.70 17.1 ± 0.3b 3.79 ± 0.68a 51.2 ± 3.9 62.3 ± 2.5bc

16% at 90°C 5.02 ± 0.60 12.9 ± 2.2 17.8 ± 0.0a 5.90 ± 0.48a 51.7 ± 0.0 55.0 ± 3.2c

105°C 4.58 ± 0.72 12.2 ± 2.9 16.3 ± 0.1c 5.27 ± 1.52a 51.7 ± 4.5 64.7 ± 1.5ab

125°C 5.11 ± 1.15 17.22 ± 2.8 16.7 ± 0.1bc 3.70 ± 0.46a 48.6 ± 1.5 64.7 ± 1.9ab

All data represent means ± standard deviation (n=3). Different superscripts denote significant difference in each extraction method at p< 0.05 using Tukey’s test. Total carbohydrates were calculated by difference. NDF stands for neutral detergent fiber (lignin, hemicellulose, cellulose).

65

Table 3-2. Impact of extraction method on total phenols and antioxidant capacity of Noble seed flour and Carlos seed flour.

Oil extraction methods

Noble Carlos

Total phenols (mg

Gallic acid /mL)

Antioxidant capacity (µmol

Trolox/ 100 g)

Total phenols (mg

Gallic acid /mL)

Antioxidant capacity

(µmol Trolox/ 100 g)

Soxhlet extraction

Hexane 32.8 ± 1.3a 162 ± 20 22.4 ± 1.3a 151 ± 5ab

Screw expression

85°C 25.1 ± 1.4bcd 169 ± 23 7.92 ± 0.54e 148 ± 29ab

90°C 22.9 ± 1.3d 171 ± 22 11.4 ± 1.2cd 119 ± 23b

90°C at 13% moisture 23.7 ± 0.5cd 160 ± 33 8.20 ± 1.51c 101 ± 14b

90°C at 16% moisture 23.8 ± 0.3cd 122 ± 10 9.96 ± 0.71b 113 ± 26b

105°C 27.5 ± 0.4b 114 ± 7 13.1 ± 1.6de 183 ± 31a

125°C 25.9 ± 0.1bc 168 ± 42 18.4 ± 0.6cde 129 ± 10ab

All data represent means ± standard deviation (n=3). Different superscripts denote significant difference in each extraction method at p< 0.05 using Tukey’s test.

66

Table 3-3. Impact of extraction method on phytochemical content (mg/100g) of Noble seed flour

Phytochemicals

Soxhlet Screw expression

Hexane 85°C 90°C 90°C at 13%

moisture

90°C at 16%

moisture 105°C 125°C

Anthocyanin

Delphinidin 3,5-

diglucoside 31.0 ± 0.4a 28.8 ± 0.5b 28.7 ± 0.2b 28.9 ± 0.5b 29.2 ± 0.2b 28.4 ± 0.6b 28.9 ± 0.5b

Malvidin 3,5-

diglucoside 167 ± 1a 117 ± 5c 116 ± 3c 124 ± 8c 140 ± 4b 110 ± 9c 121 ± 4c

Cyanidin 3,5-

diglucoside 76.9 ± 0.4ab 68.8 ± 1.8b 77.8 ± 4.1ab 77.0 ± 2.5ab 82.3 ± 3.5a 73.8 ± 5.6ab 74.1 ± 2.0ab

Petunidin 3,5-

diglucoside 36.0 ± 0.1a 34.7 ± 0.3c 34.9 ± 0.2bc 34.6 ± 0.2c 35.8 ± 0.3ab 34.8 ± 0.3c 35.2 ± 0.7abc

Pelargonidin 3,5-

diglucoside 17.7 ± 0.1 17.4 ± 0.1 17.4 ± 0.1 17.4 ± 0.2 17.8 ± 0.1 17.7 ± 0.3 17.4 ± 0.1

Peonidin 3,5-

diglucoside 26.8 ± 0.5a 25.3 ± 0.2bc 25.0 ± 0.2bc 24.3 ± 0.5c 24.9 ± 0.1bc 25.0 ± 0.6bc 25.6 ± 0.3b

Flavonoid

Myricetin 221 ± 46 235 ± 5 215 ± 5 214 ± 4 221 ± 7 211 ± 10 218 ± 1

Kaempferol 42.7 ± 0.2ab 39.7 ± 0.7b 43.0 ± 1.5ab 41.5 ± 2.2ab 41.6 ± 0.8ab 42.7 ± 0.9ab 44.7 ± 1.3a

Quercetin n.d. n.d. n.d. n.d. n.d. n.d. n.d.

All data represent means ± standard deviation (n=3). Different superscripts denote significant difference in each extraction method at p< 0.05 using Tukey’s test.

67

Table 3-4. Impact of extraction method on phytochemical content (mg/100g) of Carlos seed flour

Phytochemicals

Soxhlet Screw expression

Hexane 85°C 90°C 90°C at 13%

moisture

90°C at 16%

moisture 105°C 125°C

Anthocyanin

Delphinidin 3,5-diglucoside n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Malvidin 3,5-diglucoside n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Cyanidin 3,5-diglucoside n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Petunidin 3,5-diglucoside n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Pelargonidin 3,5-diglucoside n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Peonidin 3,5-diglucoside n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Flavonoid

Myricetin 181 ± 19 199 ± 25 234 ± 33 214 ± 44 223 ± 42 171 ± 24.3 165 ± 36

Kaempferol n.d. n.d. n.d. n.d. n.d. n.d. n.d.

Quercetin n.d. n.d. n.d. n.d. n.d. n.d. n.d.

All data represent means ± standard deviation (n=3). Different superscripts denote significant difference in each extraction method at p< 0.05 using Tukey’s test.

68

Figure 3-1. HPLC-DAD chromatogram of the anthocyanins in Noble grape seed flour at 520 nm. Peaks 1, 2, 3, 4, and 5

were delphinidin 3,5-diglucoside, cyaniding 3,5-diglucoside, petunidin 3,5-diglucoside, pelargonidin 3,5-diglucoside, and malvidin 3,5-diglucoside, respectively.

min5 10 15 20 25 30 35 40

mAU

0

10

20

30

40

50

601

2

3

4

5

6

69

Figure 3-2. HPLC-DAD chromatogram of the anthocyanins in Noble seed flour and Carlos seed flour at 360 nm. Peaks 1,

2, and 3 were myricetin, quercetin, and kaempferol, respectively.

min 10 20 30 40 50 60 70

mAU

0

10

20

30

40

50

60

1

2

3

70

CHAPTER 4 CONCLUSIONS

Muscadine grape seed oil are high in polyunsaturated fatty acids primarily linoleic

(18:2) and possess vitamin E in the form of tocopherol and tocotrienol. Additionally, the

resulting defatted seed flour can possess a wide variety of phytochemicals including

anthocyanin and flavonoids. Different oil extraction methods were shown to have a

significant impact on the quality as well as quantity of oil and resulting flour. Results

suggest that muscadine grape seeds, the byproduct of the wine making industry, could

be utilized in the culinary arts or cosmetics for its high polyunsaturated fatty acids and

antioxidant properties.

The major limitation of this study is the accessibility to the grape pomace and

seeds after pressing. Carlos pomace was only able to be retrieved after pressing at

least a day later and Noble pomace after the fermentation process. Oil seeds are

typically steam treated to denature any oxidative enzymes. In attempting to dry our

seeds faster and denature oxidative enzymes, muscadine seeds were dried in

convection ovens that had no ventilation for moisture, slowing down the drying process

and causing further oxidation in the seeds.

Potential future research areas that can be explored are proper drying methods

for the seeds before extraction, to see if proper drying methods reduce oxidation and

increase vitamin E concentration. Additionally, super-critical fluid extraction could be

another extraction method to explore, as it is used in industry to extract nutraceutical

components and high quality oils.

71

LIST OF REFERENCES

Ajibola, O., Eniyemo, S., Fasina, O., & Adeeko, K. (1990). Mechanical expression of oil from melon seeds. Journal of Agricultural Engineering Research, 45, 45-53.

Alimentarius, C. (1999). Codex standard for named vegetable oils. Codex Stan, 210, 1-13.

Ankon, T. (2011). Neutral detergent fiber in feeds, filter bag technique: Method.

Bieri, J. G., & Evarts, R. P. (1974). Gamma tocopherol: metabolism, biological activity and significance in human vitamin E nutrition. The American journal of clinical nutrition, 27(9), 980-986.

Booth, V., & Bradford, M. (1963). Tocopherol contents of vegetables and fruits. British Journal of Nutrition, 17(1), 575-581.

Brand-Williams, W., Cuvelier, M.-E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LWT-Food science and Technology, 28(1), 25-30.

Burton, G. W., Traber, M. G., Acuff, R. V., Walters, D. N., Kayden, H., Hughes, L., & Ingold, K. U. (1998). Human plasma and tissue alpha-tocopherol concentrations in response to supplementation with deuterated natural and synthetic vitamin E. The American journal of clinical nutrition, 67(4), 669-684.

Damodaran, S., Parkin, K. L., & Fennema, O. R. (2007). Fennema's food chemistry: CRC press.

Davis, K., Cain, R., Peters, L., Le Tourneau, D., & McGinnis, J. (1981). Evaluation of the nutrient composition of wheat. II. Proximate analysis, thiamin, riboflavin, niacin and pyridoxine. Cereal Chem, 58(2), 116-120.

Embong, M., & Jelen, P. (1977). Technical feasibility of aqueous extraction of rapeseed oil-a laboratory study. Canadian Institute of Food Science and Technology Journal, 10(4), 239-243.

Firestone, D., & American Oil Chemists, S. (2011). Official methods and recommended practices of the AOCS (Vol. 6th, 2nd print.). Urbana, Ill: AOCS.

Galliard, T. (1986). Hydrolytic and oxidative degradation of lipids during storage of wholemeal flour: Effects of bran and germ components. Journal of Cereal Science, 4(2), 179-192.

Gogolewski, M., Nogala‐Kalucka, M., & Szeliga, M. (2000). Changes of the tocopherol

and fatty acid contents in rapeseed oil during refining. European journal of lipid science and technology, 102(10), 618-623.

72

Gómez, A. M., López, C. P., & de la Ossa, E. M. (1996). Recovery of grape seed oil by liquid and supercritical carbon dioxide extraction: a comparison with conventional solvent extraction. The Chemical Engineering Journal and the Biochemical Engineering Journal, 61(3), 227-231.

Goon, J. A., Azman, N. H. E. N., Ghani, S. M. A., Hamid, Z., & Ngah, W. Z. W. (2017). Comparing palm oil tocotrienol rich fraction with α-tocopherol supplementation on oxidative stress in healthy older adults. Clinical Nutrition ESPEN, 21, 1-12.

Gourineni, V., Shay, N. F., Chung, S., Sandhu, A. K., & Gu, L. W. (2012). Muscadine Grape (Vitis rotundifolia) and Wine Phytochemicals Prevented Obesity-Associated Metabolic Complications in C57BL/6J Mice. Journal of agricultural and food chemistry, 60(31), 7674-7681. doi:10.1021/jf3013663

Hoye Jr, C., & Ross, C. F. (2011). Total phenolic content, consumer acceptance, and instrumental analysis of bread made with grape seed flour. Journal of food science, 76(7).

Hudson, T. S., Hartle, D. K., Hursting, S. D., Nunez, N. P., Wang, T. T., Young, H. A., . . . Green, J. E. (2007). Inhibition of prostate cancer growth by muscadine grape skin extract and resveratrol through distinct mechanisms. Cancer Research, 67(17), 8396-8405.

Husmann, G. C., & Dearing, C. (1916). Muscadine grapes: US Dept. of Agriculture.

International, A. (1995). Official methods of analysis of AOAC International(Journal, Electronic). Retrieved from http://ufl.summon.serialssolutions.com/2.0.0/link/0/eLvHCXMwdV1NawIxEB2EUmrpw

aqlWoX8gS3ZZOMmx3VrWOhBhHqWuJv0UlRs-_-

biXFpS3sJIYd8M5kMb94D4OyRJr9sApe1UkzabZ5mLM8bf_CU-89HI2rrTIr5zusqX-

mZfsqqDpxFHT9dy-

Po6ogFOqDFCqB45BRkdK2DCrj39JEVNOUCBRye56qNtFAhaRbUihFPlyDHHT4k7u

3bQ6J7cBk9wFvo2F0frovXY2S_sH24Ks_yawNgy0juQE4iz-

9k74iJFCJYL5ZFSX4E9IYw0YuXskr8qJu4ok07bXYHNwbx7LuPkPfW3ANRVig3S61

kSma-NJJZroxCeh7vIogRDP_sa_xP-wN0T8nYGDyYwIXzl9tOwzZ8ASCLc_w

Jiang, Q., Christen, S., Shigenaga, M. K., & Ames, B. N. (2001). γ-Tocopherol, the major form of vitamin E in the US diet, deserves more attention–. The American journal of clinical nutrition, 74(6), 714-722.

Kamel, B. S., Dawson, H., & Kakuda, Y. (1985). Characteristics and composition of melon and grape seed oils and cakes. Journal of the American Oil Chemists' Society, 62(5), 881-883.

Kim, T., Silva, J., Weng, W., Chen, W., Corbitt, M., Jung, Y., & Chen, Y. (2009). Inactivation of Enterobacter sakazakii by water-soluble muscadine seed extracts. International journal of food microbiology, 129(3), 295-299.

73

Kim, T., Weng, W., Stojanovic, J., Lu, Y., Jung, Y., & Silva, J. (2008). Antimicrobial effect of water-soluble muscadine seed extracts on Escherichia coli O157: H7. Journal of food protection, 71(7), 1465-1468.

Latif, S., Diosady, L. L., & Anwar, F. (2008). Enzyme‐assisted aqueous extraction of oil

and protein from canola (Brassica napus L.) seeds. European journal of lipid science and technology, 110(10), 887-892.

Li, R., Kim, M.-H., Sandhu, A. K., Gao, C., & Gu, L. (2017). Muscadine Grape (Vitis rotundifolia) or Wine Phytochemicals Reduce Intestinal Inflammation in Mice with Dextran Sulfate Sodium-Induced Colitis. Journal of agricultural and food chemistry, 65(4), 769-776.

Li, R., Sandhu, A. K., Gao, C., & Gu, L. (2016). Muscadine Grape (Vitis rotundifolia) and Wine Phytochemicals Mitigates Dextran Sulfate Sodium-induced Colitis in Mice. The FASEB Journal, 30(1 Supplement), 1158.1153-1158.1153.

Liu, R. H. (2003). Health benefits of fruit and vegetables are from additive and synergistic combinations of phytochemicals. The American journal of clinical nutrition, 78(3), 517S-520S.

Lutterodt, H., Slavin, M., Whent, M., Turner, E., & Yu, L. L. (2011). Fatty acid composition, oxidative stability, antioxidant and antiproliferative properties of selected cold-pressed grape seed oils and flours. Food Chemistry, 128(2), 391-399.

Mcdougall, G. J., & Stewart, D. (2005). The inhibitory effects of berry polyphenols on digestive enzymes. Biofactors, 23(4), 189-195.

Mertens-Talcott, S. U., Lee, J.-H., Percival, S. S., & Talcott, S. T. (2006). Induction of cell death in Caco-2 human colon carcinoma cells by ellagic acid rich fractions from muscadine grapes (Vitis rotundifolia). Journal of agricultural and food chemistry, 54(15), 5336-5343.

Meydani, S. N., Barklund, M. P., Liu, S., Meydani, M., Miller, R. A., Cannon, J. G., . . . Blumberg, J. B. (1990). Vitamin E supplementation enhances cell-mediated immunity in healthy elderly subjects. The American journal of clinical nutrition, 52(3), 557-563.

Mueller, K. (2014). A Tribute to Florida's Mighty Muscadine. Tallahasee Magazine, July-August 2014.

Muldoon, M. F., Ryan, C. M., Sheu, L., Yao, J. K., Conklin, S. M., & Manuck, S. B. (2010). Serum Phospholipid Docosahexaenonic Acid Is Associated with Cognitive Functioning during Middle Adulthood, 2. The Journal of nutrition, 140(4), 848-853.

74

Nawar, W. W. (1969). Thermal degradation of lipids. Journal of agricultural and food chemistry, 17(1), 18-21.

Ohnishi, M., Hirose, S., Kawaguchi, M., Ito, S., & Fujino, Y. (1990). Chemical composition of lipids, especially triacylglycerol, in grape seeds. Agricultural and Biological Chemistry, 54(4), 1035-1042.

Olien, W. C. (1990). The muscadine grape: botany, viticulture, history, and current industry. HortScience 25 (7) 830-831.

Özvural, E. B., & Vural, H. (2011). Grape seed flour is a viable ingredient to improve the nutritional profile and reduce lipid oxidation of frankfurters. Meat science, 88(1), 179-183.

Passos, C. P., Yilmaz, S., Silva, C. M., & Coimbra, M. A. (2009). Enhancement of grape seed oil extraction using a cell wall degrading enzyme cocktail. Food Chemistry, 115(1), 48-53. doi:10.1016/j.foodchem.2008.11.064

Pastrana-Bonilla, E., Akoh, C. C., Sellappan, S., & Krewer, G. (2003). Phenolic content and antioxidant capacity of muscadine grapes. Journal of agricultural and food chemistry, 51(18), 5497-5503.

Peh, H. Y., Tan, W. D., Chan, T. K., Pow, C. W., Foster, P. S., & Wong, W. F. (2017). Vitamin E isoform γ-tocotrienol protects against emphysema in cigarette smoke-induced COPD. Free Radical Biology and Medicine, 110, 332-344.

Pestana-Bauer, V. R., Zambiazi, R. C., Mendonça, C. R., Beneito-Cambra, M., & Ramis-Ramos, G. (2012). γ-Oryzanol and tocopherol contents in residues of rice bran oil refining. Food Chemistry, 134(3), 1479-1483.

Peter C. Anderson, T. E. C., and Jacque Breman. (2013). The Muscadine Grape. Horticultural Sciences Department, UF/IFAS Extension, HS763.

Puah, C., Choo, Y., Ma, A., & Chuah, C. (2007). The effect of physical refining on palm vitamin E (tocopherol, tocotrienol and tocomonoenol). American Journal of Applied Sciences, 4(6), 374-377.

Ramalingam, L., Menikdiwela, K., Scoggin, S., Shen, C.-L., Tomison, M. D., Kaur, G., . . . Moustaid-Moussa, N. (2017). Effects of delta-tocotrienol on obesity-related adipocyte hypertrophy, inflammation and hepatic steatosis in high-fat-fed mice. The Journal of nutritional biochemistry, 48, 128-137.

Reiter, E., Jiang, Q., & Christen, S. (2007). Anti-inflammatory properties of α-and γ-tocopherol. Molecular aspects of medicine, 28(5), 668-691.

Rosenthal, A., Pyle, D., & Niranjan, K. (1996). Aqueous and enzymatic processes for edible oil extraction. Enzyme and Microbial Technology, 19(6), 402-420.

75

Ross, C. F., Hoye Jr, C., & Fernandez‐Plotka, V. C. (2011). Influence of heating on the

polyphenolic content and antioxidant activity of grape seed flour. Journal of food science, 76(6), C884-C890.

Rossi, M., Gianazza, M., Alamprese, C., & Stanga, F. (2001). The effect of bleaching and physical refining on color and minor components of palm oil. Journal of the American Oil Chemists' Society, 78(10), 1051-1055.

Sanchez-Machado, D. I., Lopez-Cervantes, J., Nunez-Gastelum, J. A., de la Mora-Lopez, G. S., Lopez-Hernandez, J., & Paseiro-Losada, P. (2015). Effect of the refining process on Moringa oleifera seed oil quality. Food Chemistry, 187, 53-57. doi:10.1016/j.foodchem.2015.04.031

Sandhu, A. K., Gray, D. J., Lu, J., & Gu, L. (2011). Effects of exogenous abscisic acid on antioxidant capacities, anthocyanins, and flavonol contents of muscadine grape (Vitis rotundifolia) skins. Food Chemistry, 126(3), 982-988.

Sandhu, A. K., & Gu, L. (2010). Antioxidant capacity, phenolic content, and profiling of phenolic compounds in the seeds, skin, and pulp of Vitis rotundifolia (muscadine grapes) as determined by HPLC-DAD-ESI-MS n. Journal of agricultural and food chemistry, 58(8), 4681-4692.

Talcott, S. T., & Lee, J.-H. (2002). Ellagic acid and flavonoid antioxidant content of muscadine wine and juice. Journal of agricultural and food chemistry, 50(11), 3186-3192.

Tasan, M., & Demirci, M. (2005). Total and individual tocopherol contents of sunflower oil at different steps of refining. European food research and technology, 220(3-4), 251-254.

Theriault, A., Chao, J.-T., Wang, Q., Gapor, A., & Adeli, K. (1999). Tocotrienol: a review of its therapeutic potential. Clinical biochemistry, 32(5), 309-319.

Van Hoed, V., Depaemelaere, G., Ayala, J. V., Santiwattana, P., Verhé, R., & De Greyt, W. (2006). Influence of chemical refining on the major and minor components of rice brain oil. Journal of the American Oil Chemists' Society, 83(4), 315-321.

Ward, J. (1976). Processing high oil content seeds in continuous screw presses. Journal of the American Oil Chemists’ Society, 53(6), 261-264.

Wei, P. C., May, C. Y., Ngan, M. A., & Hock, C. C. (2004). Degumming and bleaching: effect on selected constituents of palm oil. Journal of oil palm research, 16(2), 57-63.

Wiedermann, L. (1981). Degumming, refining and bleaching soybean oil. Journal of the American Oil Chemists' Society, 58(3), 159-166.

76

Xu, C., Yagiz, Y., Borejsza-Wysocki, W., Lu, J., Gu, L., Ramírez-Rodrigues, M. M., & Marshall, M. R. (2014). Enzyme release of phenolics from muscadine grape (Vitis rotundifolia Michx.) skins and seeds. Food Chemistry, 157, 20-29.

Yi, W., Fischer, J., & Akoh, C. C. (2005). Study of anticancer activities of muscadine grape phenolics in vitro. Journal of agricultural and food chemistry, 53(22), 8804-8812.

Zhao, L., Yagiz, Y., Xu, C., Lu, J., Chung, S., & Marshall, M. R. (2015). Muscadine grape seed oil as a novel source of tocotrienols to reduce adipogenesis and adipocyte inflammation. FOOD & FUNCTION, 6(7), 2293-2232. doi:10.1039/c5fo00261c

Zufarov, O., Schmidt, S., & Sekretár, S. (2008). Degumming of rapeseed and sunflower oils. Acta Chimica Slovaca, 1(1), 321-328.

77

BIOGRAPHICAL SKETCH

Brian Wada graduated from California State University of Long Beach with a

Bachelor of Science in nutrition and dietetics in May of 2015. After taking a year off to

work Brian was offered a research assistantship to pursue his education in food science

under the advisement of Dr. Liwei Gu at the University of Florida. Brian graduated in

May 2018 with a master’s in food science and human nutrition.