Author: Mandal, Mamta Effects of Different Extraction ...

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Author: Mandal, Mamta Title: Effects of Different Extraction Methods on the Chemical Properties of

Cranberry Seed Oils The accompanying research report is submitted to the University of Wisconsin-Stout, Graduate School in partial completion of the requirements for the Graduate Degree/ Major: MS Food & Nutritional Sciences Research Adviser: Eun Joo Lee, PhD Submission Term/Year: Fall, 2013 Number of Pages: 55 Style Manual Used: American Psychological Association, 6th edition

I understand that this research report must be officially approved by the Graduate School and that an electronic copy of the approved version will be made available through the University Library website

I attest that the research report is my original work (that any copyrightable materials have been used with the permission of the original authors), and as such, it is automatically protected by the laws, rules, and regulations of the U.S. Copyright Office.

My research adviser has approved the content and quality of this paper. STUDENT: NAME Mamta Mandal DATE: 12/19/2013 ADVISER: (Committee Chair if MS Plan B or EdS Thesis or Field Project/Problem): NAME Eun Joo Lee, PhD DATE: 12/19/2013 ---------------------------------------------------------------------------------------------------------------------- This section for MS Plan A Thesis or EdS Thesis/Field Project papers only Committee members (other than your adviser who is listed in the section above) 1. CMTE MEMBER’S NAME: DATE: 2. CMTE MEMBER’S NAME: DATE: 3. CMTE MEMBER’S NAME: DATE: ---------------------------------------------------------------------------------------------------------------------- This section to be completed by the Graduate School This final research report has been approved by the Graduate School. Director, Office of Graduate Studies: DATE:

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Mandal, M. Effects of Different Extraction Methods on the Chemical Properties of Cranberry

Seed Oils.

Abstract

Cranberry seed oil has an exceptional nutrient and antioxidant profile. It is the only edible oil

that has a natural occurring omega-6 to omega-3 ratio of 1:1. Extraction techniques have

significant effect on these natural phytochemicals present in the seed. Cold-pressed cranberry oil

from July (CP1), and September (CP2) harvested seeds, commercial cranberry seed oil (COM)

and accelerated solvent extracted cranberry seed oil (ASE) were evaluated for their extraction

yield, fatty acid profile, phytosterol content and antioxidant properties. All the fruit seed oils

content the significant level of essential fatty acids. The ratio of omega-6/omega-3 fatty acids

(1.2:1) and the ratio of polyunsaturated: saturated fatty acids (9.5:1) in all samples were very

ideal as a nutritional view point. ASE has lower α-tocopherols (84.70 ppm) and higher sterols

content. CP2 (273.55 mg/kg oil) contained the highest α-tocopherol content. Peroxide value

(P.V) ranged from 3.48±0.61 meq/kg (COM) to 10.89±0.50 meq/kg (ASE) of oil respectively.

TPC was higher observed in all samples (CP2-1.32 Gallic acid equivalents/g oil). It was found

that ASE showed good potential for the recovery than cold press whereas cold- pressed oils have

higher potential in improving quality and nutritional characteristics focusing on TPC,

tocopherols content, P.V compared to ASE.

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Acknowledgements

I would like to thank to all who supported and helped me in the completion of my

research experiment and thesis writing. First of all I would like to record my gratitude to Dr.

Eun Joo Lee, my research advisor, for her supervision, advice and guidance from the very

beginning of the research as well as throughout the work. Her support for the thesis editing in

limitless time made it possible to complete my research and thesis work. I would like to thank

Dr. Carol Seaborn, for her advice and guidance from the very first day of my graduate study until

the completion of my research work and thesis. I would like to thank Dr. Jennifer Grant for her

help and support during my research project. I wish to express my sincere gratitude to Connie

Galep and her assistants, who helped me in finding laboratory materials. I am also thankful to

Dr. Jonathan Smith for providing his facilities to make the cold press seed oil. Last but not the

least, I would like to thank my family members for their support and help me during my entire

graduate study at UW-Stout by financing, advice and love.

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Table of Contents

Abstract ............................................................................................................................................2

List of Tables ...................................................................................................................................6

List of Figures ..................................................................................................................................7

Chapter I: Introduction .....................................................................................................................9

Statement of the Problem ...................................................................................................12

Statement of the Purpose ...................................................................................................12

Project Objectives ..............................................................................................................13

Assumptions .......................................................................................................................13

Limitations .........................................................................................................................13

Definition of Terms............................................................................................................14

Chapter II: Literature review .........................................................................................................15

Fruit Seed Oil .....................................................................................................................15

Cranberry Background .......................................................................................................16

Health Benefits of Cranberries...........................................................................................17

Antioxidant in Cranberries .................................................................................................19

Potential Value-Adding Components Detected in Cranberry Seeds .................................20

Extraction of Oil ................................................................................................................24

Chapter III: Methodology ..............................................................................................................27

Sample Collection and Preparation ....................................................................................27

Oil Extraction .....................................................................................................................27

Moisture Content Measurement .........................................................................................28

Antioxidant Activity Measurements ..................................................................................28

Lipid Oxidation Measurement (Peroxide Value) ...............................................................30

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Fatty Acid Analysis............................................................................................................30

Phytosterol Analysis ..........................................................................................................31

Data Analysis .....................................................................................................................32

Limitations .........................................................................................................................32

Chapter IV: Results and Discussion ..............................................................................................33

Effect of Different Extraction Method ...............................................................................33

Fatty Acid Profile ...............................................................................................................34

Phytosterol Profiles ............................................................................................................38

Antioxidant Property ..........................................................................................................42

Chapter V: Conclusions ................................................................................................................48

Recommendations for Further Study .................................................................................49

References ......................................................................................................................................50

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List of Tables

Table 1: Moisture Contents and Yields of Cranberry Seed Oil by Different Extraction Methods

and Harvesting Time ......................................................................................................... 34

Table 2: Fatty Acid Composition of Cranberry Seed Oils by Different Extraction Methods and

Harvesting Time................................................................................................................ 37

Table 3: Phytosterol Profiles of Cranberry Seed Oils by Different Extraction Methods and

Harvesting Time................................................................................................................ 40

Table 4: Antioxidant Properties and Lipid Peroxidation of Oil Extracts...................................... 43

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List of Figures

Figure 1: Percentage yield of cranberry seed oil by different extraction methods and harvesting

time (abc represents the statistical significant difference between different oils). ............. 34

Figure 2: Fatty acid profile of cranberry seed oil by different extraction methods and harvesting

time (abc represents the statistical significant difference between different oils). ............. 38

Figure 3: Comparison of α-tocopherol of cranberry seed oil by different extraction methods and

harvesting time (abc represents the statistical significant difference between different oils).

........................................................................................................................................... 41

Figure 4: Comparison of γ-tocotrienol of cranberry seed oil by different extraction methods and


........................................................................................................................................... 41

Figure 5: Comparison of stigmasterol of cranberry seed oil by different extraction methods and


........................................................................................................................................... 42

Figure 6: Comparison of β-sitosterol of cranberry seed oil by different extraction methods and

harvesting time (abc represents the statistical significant difference between different oils)

........................................................................................................................................... 42

Figure 7: Comparison of total phenol content (TPC) of cranberry seed oil by different extraction

methods and harvesting time (abc represents the statistical significant difference between

different oils) ..................................................................................................................... 44

Figure 8: Comparison of scavenging activities of cranberry seed oil by different extraction

methods and harvesting time (abc represents the statistical significant difference between

different oils). .................................................................................................................... 45

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Figure 9: Comparison of peroxide value of cranberry seed oil by different extraction methods

and harvesting time (abc represents the statistical significant difference between different

oils). .................................................................................................................................. 47

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Chapter I: Introduction

Cranberry, Vaccinium macocarpon, is one of the few fruits native to North America

(Cranberry Bogs & Marshes, 1955). Cranberries are grown throughout the northern part of the

United States - Wisconsin, Massachusetts, Oregon, New Jersey, and Washington primarily.

Wisconsin grows almost 57% of the cranberries in the U.S. while Massachusetts accounts for

another 28% (Cranberry Marketing Committee, 2013). A report by United States Department of

Agriculture (USDA) in the year 2007 showed that the United States cranberry total production

was 7.61 million barrels in the year 2008, up by 16% than the previous year. About 20% of the

total cranberries produced are consumed during thanksgiving week, and the rest are consumed

throughout the year in juice, as sweetened and dried products, and as ingredients (Cranberry

Bogs & Marshes, 1955).

Cranberries have been associated with healthy living. According to Joe Vinson

(Williams, 2013), a research chemist at the University of Scranton, cranberries contain the most

antioxidant phenol compared to 19 commonly eaten fruits. All these polyphenols give cranberry

an anti-adhesion property that inhibits bacteria related to urinary tract infection, gum diseases,

and stomach ulcers (Health Research, 2013). Recent scientific research showed that cranberries

and cranberry products are even helpful in protecting against heart diseases, cancer, and other

diseases (Blumberg et al., 2013)

Due to associated health benefits and disease fighting capabilities, cranberries are

continually increasing in popularity. Traditionally cranberries were consumed as a whole fruit or

processed to juice only. However, many new cranberry products are emerging in the market.

Some of the newer cranberry food items include cranberry crackers, ice cream topping, and

pancake mix while non-food items such as cranberry seed soap and body lotions are also

available (Rindt, 2008). With the increase in cranberry production and processing, it is also

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equally true that the by-products (seed, skin, and pomace) of the cranberry are also increasing.

These by-products have been considered a waste and dumped or used as animal feed. However, a

recent study has revealed that these by-products are the most powerful nutrients in the cranberry

(Fruit Essentials, 2006).

Cranberry seed oil has an exceptional nutrient and antioxidant profile. It is the only edible

oil that has a natural occurring omega-6 to omega-3 ratio of 1:1, and contains all eight isomers of

vitamin E, plant sterols, phospholipids, and flavonoids. The beneficial ingredients that are found

in cranberry seed oil are highly concentrated (Eno, 2007). It is a rich source of essential fatty

acids, containing between 35 and 44% linoleic acid (18:2n-6) and 23-35% α-linolenic acid, along

with significant levels of β- sitosterol and R- and γ- tocopherols (Parry et al., 2005). Cranberry

seed oil extract shows significant radical scavenging activities against 2, 2-diphenyl-1-

picrylhydrazyl (DPPH) and 2, 2’-azino-bis 3-ethylbenzthiazoline -6- sulphonic acid (ABTS),

protected protein from oxygen radical attack, and suppressed lipid peroxidation in human low

density lipoprotein (LDL). These data suggest that fruit seed oils might serve as potential dietary

sources for natural antioxidants and other phytochemicals. Further investigation on chemical

compositions and other properties of fruit seed oils is required to evaluate the potential of fruit

seed oils as sources of quality oil for food applications.

Following this recent discovery, food engineers have started to extract the oil from

cranberry seeds. Seed oil extraction is traditionally based on either the use of a mechanical press

or organic solvents. Both of these methods produce a product that has to be further processed to

yield an oil of required purity. Mechanical pressing leaves large amounts of oil residues in press

cake which needs to be solvent extracted again. And for solvent extraction, the extract consists of

a mixture of edible oil and organic solvent which needs to be separated by different separation

techniques. Importantly, concerns are the negative effects of the heat generated during pressing

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on the phytochemicals of the oil. Even for the solvent extraction (Soxhlet method), there has

been concern regarding the volume of organic solvent used (with the associated human

exposure), along with increased purchase and disposal cost. These have emphasized the need for

a more efficient sample extraction method (Luthria et al., 2004).

Many new efficient methods have been suggested as an alternative to the conventional

method of oil extraction, however the most commonly used and studied methods are the cold

press method and accelerated solvent extraction method.

Cold pressing is a seed oil extraction process that does not involve chemicals or heat

prior to or during the procedure (Parry et al., 2005). The absence of heat allows minimum loss of

nutrient and quality profile of the cranberry seed oil. The process retains more phytochemicals

including natural antioxidants.

In the accelerated solvent extraction (ASE) method, the same aqueous and organic

solvent as traditional Soxhlet solvent extraction is used, but an elevated temperature and

pressures. Increasing temperature improves the kinetics of the process, resulting in more efficient

extractions (faster and using less solvent) compared with traditional approaches (Luthria et al.,

2004).

Parry et al. (2005) used cold press method for the extraction of oil from marionberry,

boysenberry, red raspberry, and blueberry seeds. Accelerated fluid extraction method on the

other hand, has been mostly used in the extraction of environmental and some food samples.

Even though there are numerous reports on various extraction of oil from various seeds,

the literature is lacking information on the extraction of cranberry seed. The present study is

conducted to compare the recovery of cranberry seed oil by two methods: cold press method and

accelerated solvent extraction. The recovered oils will be tested for chemical composition and

antioxidant properties. The information obtained from this study can evaluate effects of

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extraction techniques and parameters on recovery of cranberry oil, on its chemical and

bioactivity properties, and thus recommend the best method of extraction of cranberry seed oil

for analytical or industrial production purposes.

Statement of the Problem

About 5% of all cranberries produced in USA are packaged and distributed to the market

as fresh fruit. The remaining 95% are processed into juice drinks, sauces, or sweetened dried

products (Eno, 2007). These processing of cranberry produces large amount of highly nutrient

packed by-product like seed, skin and pomace. Despite the hype of the nutrient benefits

associated with cranberry seed oil, currently it has only been sold in the form of a supplement to

aid in everyday nutrition and not as edible oil in food formulation due to the lack of sufficient

study and efficient extraction techniques. Edible oil is one of the most commonly consumed

products in the food industry. Recent studies make it clear that consumed oils have a tremendous

effect on human physiology, including lipid metabolism, development of chronic disease, and

overall well-being (Kaufman & Wiesman, 2007). Although conventional edible oils, such as

soybean, corn, and canola have their own importance, there are more rare and unfamiliar oils

having unique characteristics and health-promoting traits.

Statement of the Purpose

Cranberry seed oil is a rich source of balanced essential fatty acids, heart-healthy

phytosterols, powerful antioxidant polyphenols, phospholipids and other healthy phytonutrient

(Fruit Essentials, 2006 and Parry, 2006). Extraction techniques can cause adversity on these

natural phytochemicals present in the seed. Among the different extraction techniques, cranberry

seed will be extracted using cold press and accelerated solvent extraction methods. This study

will compare the two extraction methods by assessing the yield, antioxidant activity, fatty acid

profile, phytosterol profile and lipid oxidation of the extracted oil. The main focus is to achieve a

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comprehensive and detailed profile of the different components of cranberry seed oil. The fatty

acid profile and phytosterol composition will be determined by gas chromatography (GC).

Project Objectives

The research had the following two objectives.

1. Evaluate the recovery yield of oil from cranberry seed by different extraction methods,

cold pressing and accelerated solvent extraction methods.

2. Determine the functional properties of cranberry seed oils including fatty acid profile,

phytosterol profile, antioxidant activities and lipid oxidation.

Assumptions

The following are possible assumptions:

The seeds of many berries are very small in size and contain small amount of oil. It is assumed

that solvent extraction method will have higher extraction of oil than the traditional methods of

extraction (mechanical cold pressing). Out of the two methods (solvent extraction and cold

pressed method), the accelerated solvent extracted method will give a higher yield than cold

press extraction; however, since accelerate method use higher pressure and temperature, the

bioactivity of the oil might be lower than the cold press method.

Limitations

The limitation of the study is that the cranberry seed will be obtained from a cranberry

processing plant. The bioactivity of nutrients present in the seed may have been already affected

by the processing condition of cranberry product manufacturing and seed separation process.

Another major limitation of the study is accelerated solvent extraction method is very expensive

methods, thus unless a very high nutrient and bioactivity value oil is obtained, the use of such

methods is not justifiable.

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Definition of Terms

For clarity of understanding definitions are provided below. These definitions provide a

more depth description and will be used throughout the paper.

Antioxidant. Type of phytochemical that defends against oxidative stress.

Essential Fatty Acids. Fats that cannot be synthesized in the body and must be

consumed in the diet.

Extraction. Removal of lipid from lipid containing matrix.

Flavonoids. Sub class of polyphenolic compounds that are distributed in plants.

Polyphenols. Secondary metabolite of plants involved in body’s defense mechanism.

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Chapter II: Literature review

This chapter will cover general fruit seed oil, background and health benefits of

cranberry. This will also include different antioxidants found in cranberry, and finally conclude

with effects of different extraction methods on the chemical properties of cranberry seed oils and

their potential use in food.

Fruit Seed Oil

Fruit seeds are a major byproduct from the manufacture of fruit juice. Recent studies

have documented encouraging findings of beneficial components and physicochemical

properties of fruit seed oils (Parry, 2006). Reported sources for berry seed oils are red raspberry,

black raspberry, boysenberry, Marion blackberry, evergreen blackberry, blueberry, strawberry

and cranberry (Hoed et al., 2009). All berry seed oils have a high content of polyunsaturated

fatty acid (PUFAs) in common, providing essential fatty acids (EFAs). Berry seed oils on the

other hand have a favorable omega -6 and omega-3(n-6/n-3) FA ratio compared with some other

vegetable oils (Parker et al., 2003; Parry, 2006). Moreover, these seed oils are also rich in

various antioxidants, which are related to a protective effect against cardiovascular lipid

oxidation and antitumor activities (Hoed et al., 2009). A number of edible oils from fruit seeds

have been shown to contain high levels of unsaturated fatty acids and other biologically active

phytochemicals such as tocopherols and phytosterols (Bakowska-Barczak, 2009). The fruit seed

oils might serve as potential dietary sources for natural antioxidants and other phytochemicals.

Further investigation of the chemical compositions of fruit seed oils is required to evaluate the

potential of fruit seed lipids as sources of valuable oil for food applications (Bakowska-Barczak,

2009). Similarly other potential berry seed oil found in market is grape seed oil, pomegranates

seed oil, seabuckthorn berries seed oil, wolfberry seed oil, acai berry seed oil. Cranberry and red

raspberry are the most stable oils that possess a longer shelf life than other berries seed oils. This

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stability can be attributed to the high content of natural antioxidants including tocopherols and

tocotrienols (Parkers, 2003).

Cranberry Background

Cranberries, along with blueberries and grapes, are one of the three fruits that are native

to North America. Native Americans were the first to use the fruit in the human diet (McNamee,

2007). The majority of cranberries that are harvested in the United States are from the species of

Vacciunium macrocarpon. This species of cranberries are known as the American cranberry or

bearberry (U.S Department of Agriculture, 2007). Cranberry requires a very popular growing

condition. An acidic peat soil base, a top layer of sand and an abundant fresh water supply are

the keys to a successful cranberry harvest (Ocean Spray, 2007). The only way cranberries can

survive and flourish is with the proper combination of soils and water. The beds are irrigated

throughout the year in order for the soil to maintain proper moisture levels (Ocean Spray, 2007).

In late September, or early October, cranberries are harvested when they reach a deep red color.

There are two different harvesting process wet harvesting and dry harvesting. The first step in the

wet harvesting process is to flood the bog. The floating cranberries are then collected and

pumped or conveyed out of the bogs into waiting trucks (Burlington County Library System,

2007). Dry harvesting of cranberries may also be done. In dry harvesting the cranberries are

taken off of their vines by the use of a mechanized picking machine (Burlington County Library

System, 2007). The majority of harvested cranberries are further processed to make fruit juice

and other cranberry food products, 35% are processed into sauce products and 60% are

processed into various fruit drinks (Vattem, Ghaedian, & Shetty, 2005). When cranberries are

processed to make cranberry juice, 85% of the total cranberry is used. The other 15% of the

cranberry that is removed consists of the skin, seeds, and pomace (Fruit Essentials, 2006). This

by-product of pomace is mainly composed of the skin, flesh and seeds of the fruit. Traditionally

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the pomace has been used in animal feed; however, recent evidence shows that the pomace has

the potential to be a cheap source of natural antioxidants. Fruit pomace has been shown to

contain especially rich levels of disease fighting phenolic (Vattem, Ghaedian, & Shetty, 2005).

So the portion that was frequently thought of as waste product may actually be the most

nutritious part of the fruit. It is packed with powerful antioxidants that have the potential to help

fight heart disease (Fruit Essentials, 2006).

Health Benefits of Cranberries

It was these foods that shaped the genetic nutritional requirements of the modem human.

Cereal grains became a major staple in the human diet about 10,000 years ago with the

Agricultural Revolution. Consumption of cereal grains has greatly affected the health of humans,

introducing a diet high in carbohydrates and omega-6 fatty acids and low in omega-3 fatty acids

and antioxidants. This type of diet can increase insulin resistance and hyperinsulinemia leading

to the increased risk of coronary heart disease (CHD), diabetes, obesity, and hypertension. This

increase in consumption of omega-6 fatty acids has increased even more in the last 100 years

with the growing technology in the vegetable oil industry (Simopoulos, 1999). Processing and

refining techniques allowed for the increased production of oils for cooking. With the

introduction of hydrogenation, the ALA that was in these oils was reduced leaving a high

concentration of linoleic acid. This was not the only cause for increase in omega-6 consumption.

Modem agriculture put emphasis on grain feeds, high in omega-6 fatty acid, for livestock. This

increase in consumption of omega-6 fatty acids in domestic livestock increased the amount of

these fatty acids present in their bodies. In turn, when humans consume these animals or by-

products of these animals, they are consuming increased amounts of omega-6 fatty acids. Even

fish, a well-known source of omega-3 fatty acids, are becoming less likely to contain the levels

of omega-3 fatty acids expected. Fish contain omega-3 fatty acids because of the plants they

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consume. Commercially raised fish are fed food low in omega-3 fatty acids and high in omega-6

fatty acids causing the balance to change (Simopoulos, 2002).

Cranberries and their products have been associated historically with many positive

benefits on human health. Recently, many studies have been done to show the exact mechanism

of cranberry nutrients that can be used in the human body (Burger et al., 2002; Manach et al.,

2004; Reeds, 2002). Heart disease is the leading cause of death in the United States (Center of

Diseases Control and Prevention, n.d.). Eating a heart healthy diet is one of the important steps in

the prevention of heart diseases. Cranberries are part of heart healthy diets. They are a good

source of dietary fiber, flavonoids, polyphenols, essential fatty acids, and other phytochemicals

that offer wide range of health benefits. Variable and complex mixtures of hydroxycinnamic

acids, anthocyanins, flavonols, and proanthocyanidin make cranberries the greatest potential as

functional foods to improve cardiovascular health in human (Reeds, 2002).

Cranberries are also known for their ability to both prevent and treat urinary tract

infections, which are most commonly known as bladder and kidney infections (Dales & Dales,

2000). Cranberries contain proanthocyanidins (PACs), which inhibit the fimbrial adhesion

of bacteria, including Escherichia coli, to the urinary tract epithelium and hence the subsequent

reproduction required for infection (Health Research, 2013). Cranberries also contain significant

amounts of flavonoids and polyphenolic compounds that have been demonstrated to inhibit low

density lipoprotein oxidation and thus alleviate or minimize the risk of atherosclerosis. Burger et

al. (2002) revealed that cranberries are beneficial in the prevention of peptic ulcers through

inhibition of bacteria adhesion in gastric mucus and stomach epithelium. Cranberry and

cranberry extracts have also shown anticancer activity (Vattem, Ghaedian, & Shetty, 2005). Low

and high molecular weight components (condensed and hydrolysable tannins) from cranberry are

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thought to have anti-viral properties because of the ability of tannins and other polyphenols to

form non-infectious complexes with viruses.

Cranberry seed oil, obtained from the extraction of juice press residue, also has an

exceptional nutrient and antioxidant profile. It is the only edible oil found to have a natural

occurring 1:1 ratio of omega-6 polyunsaturated fatty acids (n-6 PUFA) to omega-3

polyunsaturated fatty acids (n-3 PUFA) (Fruit Essentials, 2006). The seed oil extract shows

significant radical scavenging activities against 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) and

azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), protected protein from oxygen

radical attack, and suppressed lipid peroxidation in human LDL (Parry et al., 2005).

Antioxidant in Cranberries

Antioxidants are substances that protect cells from oxidative stress and the effects of free

radicals. Our bodies produce antioxidants endogenously in order to protect ourselves against free

radicals. An epidemiological study conducted by Manach et al. (2004) indicated that diets rich in

fruits and vegetables are associated with lower incidences of oxidation-linked diseases such as

cancer, CVD, and diabetes. These protective effects of fruits and vegetables are now linked to

the presence of antioxidant vitamins, and phenolic phytochemicals having antioxidant activity,

which support the body’s antioxidant defense system.

According to Halvorsen et al. (2006), cranberries are among the top five foods containing

the highest antioxidant content per serving. Antioxidants found in cranberries are vitamins C and

E, polyphenol, proanthocyanins, and anthocyanins.

Polyphenols are secondary metabolites of plants and are generally involved in defense

against ultraviolet radiation or aggression by pathogens (Manach et al., 2004). In cranberries two

distinct types of polyphenols namely phenolic acids and flavonoids are found. Hydroxycinnamic

acids, a form of phenolic acid in cranberries, contribute to sensory and nutritional qualities of

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cranberries (Eno, 2007). Cranberry flavonoids were found to be associated with decreased risk of

cardiovascular diseases (Reeds, 2002). They are members of three groups: anthocyanins,

flavonols, and proanthocyanins.

Anthocyanins are responsible for pigment of cranberries and have very good health

implications. They protect the heart by maintaining good blood flow by reduction of oxidative

stress, anti-inflammatory action, and platelet aggregation (Zafra-Stone et al., 2007). Flavonol

quercetin is found in cranberries and has been shown to have a very potent anti-oxidant activity.

Proanthocyanins, also known as condensed tannins, cause the astringency in cranberries

(Manach et al., 2004). They are thought to have up to 50 or more sub-units attached to their

flavonoid ring structure. This increased degree of polymerization has been linked to a greater

ability to inhibit LDL oxidation (Eno, 2007).

In 2006, fresh cranberry was examined for total phenolic content, total anthocyanin’s,

and effects on human MCF-7 breast cancer including cell proliferation, apoptosis, and cell cycle

analysis (Sun and Liu, 2005). The fresh cranberry fruit was extracted with 80 % acetone,

evaporated and brought back into 100% H2O for analyses. The total phenolic content was

determined to be 5.7 Gallic Acid equivalent (GAE) mg/g fresh weights and total anthocyanin’s

were determined to be 90 mg/100 g. MCF-7 proliferation was significantly reduced in a dose

dependent manner from 5 to 30 mg/mL cranberry extracts. Apoptosis was observed to be 25 %

higher than control at 50 mg/mL, and cell cycle was arrested in a dose dependently in the G1 and

G2/M phases.

Potential Value-Adding Components Detected in Cranberry Seeds

As mentioned, cranberry seed oil is rich in nutrients such as essential fatty acids,

phytosterols, antioxidants (vitamin E), carotenoids, and phospholipids. Together, these nutrients

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make cranberry seed oil beneficial to the human diet, as many of them may aid in disease

prevention and contribute to overall health and well-being.

Fatty acids. Fatty acids can be divided into saturated and unsaturated fatty acids on the

basis of the absence or presence of a double bond in their alkyl chains. Fatty acids derived from

plant sources contain higher concentrations of unsaturated fatty acids; whereas saturated fatty

acids are more dominant in animal sources. Essential fatty acids (EFAs) are fatty acids that must

be consumed in order to maintain proper health. They are called essential because the human

body is unable to synthesize them on its own. Alpha-linolenic acid (ALA) is an essential fatty

acid and the main omega-3 fatty acid 20 found in food. Linoleic acid (LA) is also an essential

fatty acid and is the major omega-6 fatty acid found in food. Pre-agricultural humans ate diets

that were rich in fish, lean meat, nuts, berries, fruits, green leafy vegetables, and honey

(Simopoulos, 2002). Cranberry seed oil contained 35-40% linoleic acid and 30-35% α linolenic

acid, along with 20-25% oleic acid, a trace amount of arachidonic acid (20:4n-6), and possibly

environmental protection agency (EPA) (Heeg et al., 2002). In another study, Kequan et al.,

reported 44.3% linoleic acid and 22.3% α-linolenic acid, along with 22.7% oleic acid; however,

arachidonic acid and EPA were not detected in the cold-pressed cranberry seed oil (Adams, et

al., 2003). Significant levels of phytochemicals including β-sitosterol (1.3 g/kg oil), α-

tocopherol (341 mg/kg oil) and γ-tocopherol at 110 mg/kg oil were detected (Heeg et al., 2002),

and significant antioxidant activities were also seen in cold-pressed cranberry seed oil extract

(Parry, 2006). Cranberry seed oil components have also shown potential to reduce the oxidation

of human LDL that may help reduce the risk of heart disease. Additionally, cold pressed

cranberry seed oil demonstrated similar oxidative stability to commercial soybean and corn oils.

In a recent study, Carmire et al., 2007 examined whole dehydrated fruit powder from 4 fruits

including cranberry, blueberry, Concord grape, and raspberry for anthocyanin content. Cranberry

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seed may be an excellent dietary source of α-linolenic and linoleic acid, may be used to improve

the dietary ratio of n-6/n-3 fatty acids, may provide a significant level of natural antioxidants

including phenolic compounds and tocopherols, and may contain antproliferative compounds

(Parry, 2006). Cranberry seed oil, with a balanced ratio of 1-3:1 omega-6 fatty acids to omega-3

fatty acids could play a positive role in altering the current unbalanced ratio in the Western diet.

Depending on the source (Adams et al., 2003; Luke, 2006), cranberry seed oil is shown to

contain approximately 22-35% omega-3 fatty acids. In addition, it is a great source of

antioxidants.

Phytosterols. Sterols are found in animals, whereas phytosterols are plant compounds

with a structure similar to cholesterol. Their role in plants is similar to that of cholesterol in

animals or humans. They serve a structural purpose in cell membrane walls. Phytosterols have

cholesterol reducing properties by lowering the amount of cholesterol absorbed in the gut (Ohr,

2003). In 2000, companies were allowed to use labeling health claims regarding plant sterols and

their role in reducing coronary heart disease (CHD) due to authorization by the food and drug

administration (FDA). In order for plant sterols to have a significant effect in lowering

cholesterol, 1.3 grams must be consumed daily. Phytosterols are naturally found in vegetable

oils, but only in small quantities (Ohr, 2003).

Cranberry seed oil contains approximately 145.3 mg/100g. Corn oil and soybean oil

contain larger amounts at 968 mg/100 g and 327 mg/100 g, respectively. Consuming cranberry

seed oil alone would not be a sufficient way to meet the serving recommended to affectively

lower cholesterol, but it could be combined with other phytosterol-containing foods to help

reach that goal (Ohr, 2003).

Cranberry seed oil is rich in the phytosterols; stigmasterol, campesterol, and

betasitosterol. Studies in humans on unrestrictive diets have shown that supplementing

23

phytosterols may inhibit the absorption of cholesterol and lower serum cholesterol levels by

competing for intestinal absorption (De Quattro, 2000).

Plant sterols have been shown to reduce LDL cholesterol as much as 20% when

supplemented at 2 grams per day. Plant sterols have also been shown to affect non-lipid factors

for atherosclerosis (Naruszewicz & Kozlowska-Wojciechowska, 2007).

Tocopherols. Tocopherols (vitamin E) are lipid soluble antioxidants. Tocopherols play

an important role in preventing the oxidation of biological material and lipoproteins caused by

free radicals. Clinical trials have shown that mixed tocopherols inhibit platelet aggregation in

humans. Oxidation of lipoproteins is one of the causes of atherosclerosis. There also appears to

be a strong correlation between damage to DNA and cancer. Normal physiological processes

such as aerobic respiration and metabolism produce free radicals. Some of the free radicals

formed by the body include superoxide anion and hydroxyl radical. These free radicals are

extremely reactive and attack damaged body cells. Antioxidants react with these free radicals and

get oxidized, thereby protecting the body cells from oxidation. In this way, tocopherols help in

the inhibition of cancer and atherosclerosis.

Others. Carotenoids are plant pigments and are what give cranberry seed oil its rich

color. Carotenoids are precursors to vitamin A and are often referred to as provitamin A because

they can be converted into vitamin A if needed. Some major functions of vitamin A are to

promote vision and growth, prevent drying of the skin and eyes, and to promote resistance to

bacterial infection (Wardlaw, 2000). Deficiencies of vitamin A can lead to growth retardation,

dryness of skin, night blindness, and xerophthalmia. Xerophathalmia is the drying of the eyes

due to lack of mucus production. This is a cause of blindness from deficiency of vitamin A.

Phospholipids are a natural part of fat and are built similar to triglycerides but have a compound

containing phosphorous that is connected to the glyceride (Wardlaw, 2000). Phospholipids act as

24

surfactants that can stabilize emulsions. Lecithin is an example of common phospholipids and is

found in egg yolk and soybeans. Lecithin is found in cells throughout the body, but also helps aid

in fat digestion in the intestinal tract. Different types of phospholipids exist in the body and are

commonly found in the brain. They help form the membrane of cells. Normally vegetable oils

contain 0.1 -3% phospholipids and these are removed during the refining process (Stauffer,

1996). Since cranberry seed oil undergoes a unique processing method, the phospholipids are

retained in the oil. The human body produces its own phospholipids, so it is not essential to get it

from dietary intake.

Extraction of Oil

Lipids, for a long time, have been removed from lipid-containing materials by primarily

two methods: mechanical crushing and solvent extraction (Williams, 1997). In mechanical

crushing, the oilseed is cleaned, cracked, flaked, sometimes cooked, and conditioned to optimum

moisture and temperature and then entered into a mechanical screw press, which generates

enough pressure to cause oil to flow out of the oilseed. In solvent extraction, the prepared oilseed

passes through a percolation vessel on a moving screen (or perforated plate or some similar

device) that transports the material through the vessel. Hot solvent, sprayed on top, percolates

through the material, extracting the oil. Finally, the oil is desolventized by a general method.

Farsaie, (1985) reported maximum oil recovery was obtained when sunflower seeds were

expressed at 6% moisture content and increasing the moisture content to 14% decreased oil

recovery by 16%. R. Croteau and J. Fogerson extracted lipids from cranberry seeds with boiling

isopropanol, followed by CHCl3-isopropanol, and finally CHCl3. The three extracts were

combined and concentrated under vacuum and were purified by elution through Sephadex with

CHCl3-methanol (1:1) which was saturated with water. The lipids were found to constitute

23.3% (w/w) of the total isopropanol/CHCl3 extract (Bhagdeo, 2004). The yield of cranberry

25

seed oil via Soxhlet extraction was 23.1% and via SFE was 21.4%. This result is similar to the

lipid composition (23.3%) of cranberry seed oil obtained by R.Croteau and I. Fagerson

(Bhagdeo, 2004).

The two methods described above have many disadvantages. Mechanical pressing has a

very poor yield and it also damages the nutrient profile of the oil due to the heat generated during

pressing. Solvent extraction, on the other hand, requires a long extraction time and large solvent

usage. Solvent residue in oil also raised questions on associated human exposure and health risks

(Luthria et al., 2004). Due to these reasons, numerous other efficient methods of extraction have

been studied and used that include cold pressing, microwave, centrifugal, high pressure, osmosis,

preheating, filtration fractionation, accelerated solvent extraction, filtration fractionation,

supercritical fluid, etc. The primary objectives of extraction should be lower cost, less time and

solvent usage, more nutrient retention, and environmentally and human health friendly.

Cold press extraction. One of the earliest methods of obtaining oil from oilseeds was the

use of mechanical press to squeeze out oil from oil containing matrix. However, when this

method is followed, there is friction between the molecules which generates heat (Parry et al.,

2005). Heat is not desirable in phytonutrient extraction, as it destroys some of the valuable

nutrients. To overcome this disadvantage, Bernard Lager, in 1992, developed the cold processing

method to recover oil from the cranberry seeds, without compromising the natural nutrients

contained in the oil (cited in Fruit Essentials, 2006, n.d.). The processing of oil in the cold press

extraction is performed below 100°F so that the original phytonutrient profile of the seed is

maintained and there is little or no nutritional value lost. The oil is then filtered to remove any

oil or debris present, and no chemical processes are used to do this. After the filtering process,

the oil is immediately packed in nitrogen to reduce exposure to oxidation, thus allowing the oil to

retain the majority of all of its natural nutrients, colors, odors, and flavors.

26

The key benefit of using the cold expeller pressing process is that the processed product

is not limited to cranberry seed oil (Fruit Essential, 2006). In addition to cranberry seed oil, the

expeller produces a cranberry seed flour (or cranberry seed meal). This result is surprising in that

a single process for cranberry waste products yields two beneficial products (flour and oil), each

having many uses.

Accelerated solvent extraction. Traditional solvent extraction techniques use large

volumes of solvent and long extraction time to remove oil from the seeds. Accelerated solvent

extraction (ASE) uses high temperature and pressure to reduce the solvent use and extraction

time for solvent extraction.

ASE makes use of enhanced solubility that occur as the temperature of a liquid solvent is

increased (Luthria et al., 2004). Increasing temperature of the solvent results in a decrease in

viscosity and, allowing better penetration of the sample matrix. In addition, analyte diffusion

from the sample matrix into the solvent and overall solvent capacity is also increased. Applied

pressure maintains the solvent in its liquid state beyond its atmospheric boiling point. This

provides continuous high temperature for the enhanced extraction process.

The ASE technique consists, briefly, in enclosing a solid sample in a cell and sealing it

(Dionex, 2004). A commonly used solvent is then pumped into the extraction cell under elevated

temperature and pressure. ASE has been used for the extraction of various commodities in many

industries, Environmental: pesticides and herbicides, explosives, dioxins and furans, active

ingredients in pharmaceutical and natural products; Foods: vitamins and antibiotics, fats and

lipids, natural products, phenols, pesticides residues, contaminants, herbal and dietary

supplements; Consumer products: detergents, biofuels, paper and pulp, textiles and fibers, etc.

(Accelerated Solvent Extraction, n.d.).

27

Chapter III: Methodology

This chapter includes sample collection and preparation, instrumentation, bioactivity

tests, and data analysis. At the end, the chapter will cover the limitations of the methodology.

Sample Collection and Preparation

Cranberry seeds were obtained from local cranberry seed oil processors (Simply

Incredible Foods Inc. Port Edwards, WI). Seeds were naturally dried to moisture content of 5-6%

and then stored in high density polyethylene bags under room storage until used. All the solvents

to be used were analytical grade.

Oil Extraction

Cold pressing method. The cranberry seed oil was extracted using cold press extractor

with small scale ‘Komet’ expeller (IBG Monforts and Reiners, German) in a pilot scale. Five

kilograms of cranberry seeds (approx. 99.9% pure seed) were pressed at temperatures between

40°C and 60̊C and cold-filtered using muslin cloth (20 micron size) at a temperature below 100C.

Raw oils were drawn through tubes immersed in ice-water. (Hoed et al., 2009).

Accelerated solvent extraction (ASE) method. The cranberry seed oil was extracted

using solvent extractor (ASE 200, Dionex Corporation, Sunnyvale, CA). The cranberry seeds

were ground to particle diameter less than 3 mm, and were mixed with diatomaceous earth and

then the extraction was carried out using 100% hexane. The oil was collected in ASE collecting

vial (22ml). (Peterson et al., 2009). The operating conditions of ASE were oven temperature: 105

°C, pressure: 1500 psi., oven heat up time: 5 min, static time: 10 min, flush volume: 100%, purge

time: 60 s, solvent: hexane, and static cycles: 3 times.

Percentage yield. Recovery of the oil was defined as the ratio of the quantity of oil

extracted to the quantity originally contained in the seed sample. The recovery factors depend on

28

the properties of the oil extraction conditions and the natural oil contained in the seed.

Percentage yield of the extraction was determined by using equation:

Percent yield= actual yield (g)*100/theoretical yield (g)

Actual yield was measured by below equation and theoretical yield was calculated following by

Croteau and Fogerson method (1969)

Actual yield of oil = oil obtained (g)*100/seed taken (g)

Moisture Content Measurement

Moisture content was expressed as the amount of water present in the cranberry seed

sample. The mass of the test sample was determined and recorded as the “wet basis” then the

cranberry seed sample was dried to constant mass in oven at 110±5⁰C for 4 hrs. The drying time

was required until to achieve constant mass less than 0.1% of the sample’s wet mass during an

additional exposure to the drying process. Moisture content percentage was calculated on dry

basis and represented by the equation:

% Moisture Content= (W1-W2)*100/ W1

W1 = weight of the sample before drying

W2 = weight of the sample after drying

Antioxidant Activity Measurements

Sample extraction. To measure antioxidant activity of cranberry seed oils, the phenol

compounds in oil samples were extracted by modified method of methanolic extraction method

(Parry et al., 2005). For the extraction of oils, 15mL of 90% methanol was added to 5.0 g oil,

vortexed for 3 min and centrifuged for 10 min at 3,500 rpm. The supernatant was collected for

the measurement of total phenolic content (TPC) and 1,1-diphenyl-2-picrylhydrazyl (DPPH)

radical scavenging activity.

29

Total phenolic content. The total phenolic content (TPC) was determined based on the

Folin-Ciocalteu’s colourimetric method (Yu & Zhou, 2004). The reaction mixture contained 50

μL of extracted sample, 250 μL of the Folin-Ciocalteu reagent and 0.75 mL of 20 % sodium

carbonate and 3 mL of pure water. Reactions were carried out for 2 h. at ambient temperature,

and the absorbance was measured at 765 nm using Varian Cary 50 Bio UV/Visible

spectrophotometer (Agilent Co., Santa Clara, CA). The TPC of the oils was expressed in gallic

acid equivalents (GAE) as described by Parry et al. (2006). The gallic acid calibration line has

the equation of y = 0.6662x + 0.6044 (R2 = 0.9747), where y is the absorbance at 765 nm and x

is the concentration of phenolic compounds in mg/g of the sample (the graph is not shown).

DPPH scavenging activity. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical

scavenging activity of each extracted samples was determined using Varian Cary 50 Bio

UV/Visible spectrophotometer (Agilent Co., Santa Clara, CA), based on the decrease absorbance

of ethanolic DPPH solution at 517 nm (Parry et al. (2005). One mL of fresh 0.2 mM DPPH

solution was added to 1 mL extracted sample and the samples were vortexed to thoroughly mix

it. The samples were then left to stand in the dark for 10 min for the radical-antioxidant reaction.

After 10 minutes of reaction, the absorbance was measured at 517 nm against a blank. Synthetic

antioxidants, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and ascorbic

acid (AA), were used as a positive control for this assay. The sample absorbance was used to

estimate the remaining (or disappearing) radical percentages compared to control. Triplicate

measurements were taken. The antioxidant activity was expressed as:

% disappearance = [(A control – A sample)/ A control] × 100

A control = Absorbance reading of the control

A sample = Absorbance reading of the sample

30

Lipid Oxidation Measurement (Peroxide Value)

Peroxide Value (PV) was determined by Titration Method of AOAC Official Method

965.33. Fats and oils, weigh 5.00 ±0.05 g test portion into 250 mL glass-stoppered Erlenmeyer.

Add 30 mL acetic acid and chloroform mixture (CH3COOH-CHCI3) (a) and swirl to dissolve.

Add 0.5 mL saturated KI solution, (b) from Mohr pipet, let stand with occasional shaking 1 min,

and add 30 mL water (H2O). Slowly titrate with 0.l M sodium thiosulphate (Na2S2O3) with

vigorous shaking until yellow is almost gone. Add 0.5 mL 1% starch solution, and continue

titration, shaking vigorously to release all iodine (I) from chloroform (CHCl3) layer, until blue

just disappears. Conduct blank determination daily (must be ~0.1 mL 0.1 M Na2S2O3) and

subtract from test portion titration.

Peroxide value (milliequivalent peroxide/kg oil or fat) = S x M x1000/g test portion

S = mL of sodium thiosulphate (Na2S2O3) solution (blank corrected)

M= molarity of sodium thiosulphate (Na2S2O3) solution

Fatty Acid Analysis

Fatty acids were converted to fatty acid methyl esters before analysis by heating1ml of

borontrifluoride methanol and 400µl of oil in 90⁰C water bath for 1 hour. After cooling to room

temperature, 3ml of hexane and 5ml of water were added, mixed thoroughly, and left at room

temperature overnight for phase separation. The top hexane layer containing methylated fatty

acids (1.5ml) was collected in a GC vial and analyzed with a gas chromatography (GC, Hewlett-

Packard HP 4890D) equipped with a capillary column (Supelcowax; 30 m × 0.25 mm; 0.25 μm),

a split-splitless injector, and a flame ionization detector (FID). The carrier gas was helium and

used at a flow rate of 2.0 mL/min. The temperatures of the injector, detector, and oven were held

at 250, 260, and 200 °C for 40min, respectively.

31

Phytosterol Analysis

0.5 ml of oil was weighed into a test tube with cap and then 10 ml of freshly prepared

saponification reagent (ethanol and 33% KOH at the ratio of 94:6), 0.5 ml of 20% ascorbic acid

solution and 50 µl of 5α-cholestane solution (1µg/µl in hexane) were added immediately (Fenton

and Sim 1991). The sample was homogenized with a polytron for 5 s at full speed, capped and

then incubated for 1 hr at 50⁰C. After cooling in ice water for 10min, 5 ml deionized distilled

water and 3 ml of hexane were added. Tubes were capped tightly and then the contents were

mixed thoroughly by shaking After 15 hour for phase separation, the hexane layer containing

unsaponifiables was carefully transferred to a scintillation vial and dried under airflow. To the

dried sample 200ul of pyridine and 100ul of Sylon BFT (99% BSFTA +1% TMCS) were added.

The sample were derivatized either at 50⁰C in a water bath for 1 hour. Analysis of tocopherols

was performed with a Hewlett- packard (HP) 6890 gas chromatography (GC) equipped with an

on-column capillary injector and a FID detector (Hewlett- Packard Co., Wilmington, Del.,

U.S.A.). A 0.25-mm (i.d.)*30-m Supelco Equity-5 (bonded 5% diphenyl and 95%

dimethylsiloxane phase) column was used. A splitless inlet was used to inject samples (1ml) into

the capillary column: and ramped oven temperature was used. A splitless inlet was used to inject

samples (1ml) into the capillary column: and ramped oven temperature was used (from 260⁰C

increased to 283⁰C @1⁰C/min). Inlet temperature was 300⁰C, and the detector temperature was

320⁰C. Helium was the carrier gas at constant flow of 0.9ml/min. Detector air, H2 and make up

gas He flows were 450ml/min, 40ml/min and 39ml/min, respectively. The area of each peak

(pA*sec) was integrated using the chemstation software (Hewlett- Packard Co., Wilmington,

Del., U.S.A.) and the amounts of tocopherols and sterols were calculated using an internal

standard, 5α-cholestane (Du and Ahn 2002).

32

Data Analysis

The experimental design was a randomized complete block using a mixed effects model.

Statistical analysis was performed for all measurements using Statistical Package for Social

Science (SPSS) statistical software (SPSS-21, IBM). All least significant differences were found

using the Tukeys’ HSD (honest significant difference) tests. The model included replication

(n=3) and significance levels were determined at P < 0.05.

Limitations

The condition of the seed obtained from the juice processing plant was greatly influence

the parameters of the final extracted oil. Temperature control was a major problem in cold press

extraction method. If the temperature control was not achieved properly, then there was a

significant loss of phytonutrient profile of the oil.

33

Chapter IV: Results and Discussion

Effect of Different Extraction Method

Moisture content of cranberry seed and yield of cranberry seed oil with different

extraction methods were shown in Table 1. Moisture contents of cranberry seed were ranged

from 5.94% to 5.97% and it was not significantly different by extraction method and harvesting

period. Moisture content of seeds was important factor to extract high yield of oil. Farsaie,

(1985) reported that maximum oil recovery yield of sunflower seeds was obtained when seed

moisture content was low (6%) and 16% oil yield was decreased when seed moisture content

were increased up to 14%.

Cranberry seed oil was obtained 18.41±0.18 g per 100 g seeds (CP1, July harvested seed

and cold pressed seed oil), 18.36±0.21 g per 100g seeds (CP2, September harvested seed and

cold pressed seed oil) and 20.79±0.20 g per 100g seeds (ASE, July harvested seed and

accelerated solvent extracted seed oil). This corresponds to the percentage yield of 79.01%

(CP1), 78.80% (CP2) and 89.23% (ASE) based on the initial fat content (23.3%) of cranberry

seed following by Croteau and Fogerson method (1969) (Bhagdeo, 2004).

The yield of oil samples extracted by accelerated solvent extraction (ASE) process was

significantly higher than those of the cold pressed (CP) process which uses mechanical press to

squeeze out oil without use of any chemical. This dramatic increase could be due to use of

organic solvent (hexane) with high temperature and pressure, which ultimately decrease the use

of solvent and time of extraction (Figure 1). ASE is same as traditional solvent extraction process

and uses high temperature and pressure to reduce the solvent use and extraction time. High

temperature of the solvent in ASE results in a decrease in viscosity, allowing better penetration

of the sample matrix and also applied pressure maintains the solvent in its liquid state beyond its

34

atmospheric boiling point thus providing the continuous high temperature for the enhanced

extraction in accelerated extraction process.

Table 1

Moisture Contents of Cranberry Seeds and Yields of Cranberry Seed Oil by Different Extraction

Methods and Harvesting Time.

Parameter CP1 CP2 ASE

Moisture content (%) 5.97a 5.94a 5.94a

Yield (%) 79.01b 78.80b 89.23a

Data were expressed as mean (n = 3). Different letters within each row represent significance difference (P < 0.05).

CP1: July harvested seed, cold pressed cranberry seed oil, CP2: September harvested seed, cold pressed cranberry

seed oil, ASE: July harvested seed, accelerated solvent extracted cranberry seed oil.

Figure 1. Percentage yield of cranberry seed oil by different extraction methods and harvesting

time (abc represents the statistical significant difference between different oils).

Fatty Acid Profile

The primary fatty acids detected in the cranberry seed oils were palmitic (16:0), stearic

(18:0), oleic (18:1), linoleic (18:2), and linolenic (18:3) acids (Table 2). All the cranberry seed

b b a

-

20

40

60

80

100

CP1 CP2 ASE

% Y

ield

35

oil samples contained high amounts of polyunsaturated fatty acids (PUFAs) ranging from

69.33% (ASE) to 73.17% (Commercial cranberry seed oil, COM) (Table 2). ASE contained the

highest amount of saturated fatty acids (SFA, 7.56%) and monounsaturated fatty acids (MUFAs,

23.13%) compared with the other cranberry seed oils (Table 2). COM has highest

polyunsaturated (PUFA): saturated (SFA) ratio of 10:1 similarly CP1 has 9.6:1 while CP2 and

ASE has lower ratio of 9:1. According to Heeg et al. (2012), cranberry seed oil has a high

PUFA:SFA ratio of 10:1. This ratio is regarded as having in reducing serum cholesterol,

atherosclerosis and in preventing heart disease (Heeg et al, 2012). Sadeghi and Talaii (2000)

explained that SFA and MUFA of seed oils were influenced by environmental conditions such as

temperature, rainfall and genotypes.

The results of fatty acid composition in cranberry seed oils showed that cold-pressed

samples were contained significant amount of omega-3 fatty acid (alpha-linolenic acid, ALA).

The ALA content of the cranberry seed oils were 30.40±0.35%, 32.37±0.49%, 33.77±0.21% and

34.80±0.01% observed in ASE, CP1 and CP2 and COM samples, respectively. ALA contents

were significantly different by extraction methods and harvesting times. Hoed and other (2009),

Nawar (2010) and Heeg and other (2012) reported that the amounts of linolenic acid in cranberry

seed oil were ranged from 30 % to 35 %. Linoleic acid, omega-6 fatty acid, was the most

prevalent fatty acid in all seed oils and ASE samples contained the highest amounts of linoleic

acid (38.93±0.15%) compared to COM (38.37±0.06%), CP1 (38.57±0.25) and CP2

(37.83±1.00%). Linoleic acid contents of cranberry seed oils were significantly different by

extraction method and harvesting times (Figure 2).

The ratio of omega-6 to omega-3 in the typical Western diet is usually as high as 15:1 to

17:1 and this high n-6/n-3 ratio promotes the pathogenesis of many diseases including

cardiovascular disease, inflammatory diseases and cancer. A lower n-6/n-3 ratio improves tissue

36

function and controls many disease states. Ratio of 2-3:1 n-6/n-3 ratio suppressed inflammation

in patients with rheumatoid arthritis and ratio as low as 1.4:1 has been recommended for infants.

Therefore the ratio of n-6/n-3 in oil products is important factor for nutrition values. The ratios of

n-6/n-3 in COM, CP1 and CP2 were 1.1:1 while ASE on the contrary, exhibited a high ratio of n-

6/n-3 (1.3:1) (Table 2). However, all cranberry seed oils had a very favorable n-6/n-3 ratio,

compared with other vegetable oils. Simpoulous (1999) reported that the ratio of n-6/n-3 in the

cold-pressed edible seed oil was 1:1, which was ideal balanced ratio of n-6/n-3.

Cranberry seed oil contained the significant amount of palmitic (5-8%), stearic (1-2%),

and oleic (20-25%) acids (Table 2). ASE, CP1 and COM have the more palmitic acid than CP2.

ASE has highest oleic acid content than other tested sample. CP2 has more oleic acid content

than CP1. Commercial oil has lowest oleic acid among all. Heeg and others (2012) reported that

cold pressed cranberry seed oil has 20 to 25% oleic acid, 5 to 6% of the palmitic acid, and 1% of

the stearic acids in the total fatty acid. The amount of oleic acid was high in the warm weather

but the other fatty acids amount was not significant. Baccouri and others (2006) explained that

the amount of oleic acid decrease when the amount of linoleic acid increases.

From the fatty acid profile it was clear that berry seed oils are very interesting from a

nutritional point of view. These results were in the line with those reported in the literatures

(Bushman et al., 2004; Nawar 2004; Parry et al., 2005; Parry et al., 2006; Oh et. al., 2007,

Nawar, 2010; Heeg et al., 2012). This indicated that the entire cranberry seed oils were an

excellent dietary source for linoleic (n-6) and linolenic (n-3) essential fatty acids. Linoleic and

linolenic acid were essential fatty acids that cannot be synthesized by humans and must be

obtained through the diet (Heeg et al., 2012). This study demonstrated that the all cold-pressed

cranberry seed oils (CP1, CP2 & COM) were rich dietary sources for essential fatty acids than

ASE. Fatty acid contents, especially linolenic acids, of cranberry seed oils may depend on

37

different growing, oil processing and storage conditions. Recently, several industries have tried

to use cranberry seed oil as edible oils, cosmetics, fragrance, soap, skin and hair care products

(Heeg et al., 2012)

Table 2

Fatty Acid Composition of Cranberry Seed Oils by Different Extraction Methods and Harvesting

Time.

Parameter COM CP1 CP2 ASE

Palmitic acid (16:0) 6.13ab 6.20ab 6.00b 6.43a

Stearic acid (18:0) 1.03a 1.33a 1.73a 1.13a

Oleic acid (18:1) 19.67c 20.13c 22.03b 23.13a

Linoleic acid (18:2 n−6) 38.37ab 38.57ab 37.93b 38.93a

Linolenic acid (18:3 n−3) 34.80a 33.77b 32.37c 30.40d

Saturated (SFA) 7.16d 7.53c 7.73a 7.56b

Polyunsaturated (PUFA) 73.17a 72.34b 70.30c 69.33d

Monounsaturated (MUFA) 19.67d 20.13c 22.03b 23.13a


The results are expressed in percentage. CP1: July harvested seed, cold pressed cranberry seed oil, CP2:

September harvested seed, cold pressed cranberry seed oil, ASE: July harvested seed, accelerated solvent extracted

cranberry seed oil. COM: Commercially available cold press cranberry seed oil (Botanical Oil Innovation)

38

Figure 2. Fatty acid profile of cranberry seed oil by different extraction methods and harvesting

time (abc represents the statistical significant difference between different oils).

Phytosterol Profiles

Phytosterol profiles included α-tocopherol, γ-tocotrienol, stigmasterol and β-sitosterol

were shown in Table 3. Alpha-tocopherol contents of cranberry seed oil samples were

significantly different following by extraction method. ASE has lower α-tocopherol (84.70 ppm)

compared with cold pressed oils (COM: 197.50 ppm, CP1: 261.75 ppm, and CP2: 273.55 ppm).

Nawar (2010) reported 130 ppm of α-tocopherol contents from cranberry seed oil while Heeg

and others (2012) reported 341 ppm of α-tocopherol contents from cranberry seed oil. Therefore,

α-tocopherol contents of cranberry seed oil may depend on various factors including extraction

methods and harvesting time.

Gamma-tocotrienol contents of cranberry seed oil samples were also significantly

different between commercial product (COM: 1378.85 ppm) and lab-scale extraction oils (CP1:

1096.80 ppm, CP2: 1056.95 ppm and ASE: 1149.10 ppm) (Table 3). This result indicated that γ-

tocotrienol was more stable in the cranberry seed oil after refining oil processing. Nawar (2010)

ab

a

c

ab a

ab a

c

ab b

b a

b

b

c

a

a

a

a

d

-

5

10

15

20

25

30

35

40

45

Palmitic Acid Stearic Acid Oleic Acid Linoleic Acid Linolenic Acid

% F

atty

Aci

d COM.CP1CP2ASE

39

reported that γ-tocotrienol content of cranberry seed oil was 1500 ppm and it also indicated that

γ-tocotrienol content of cranberry seed oil might depend on various factors including extraction

methods and harvesting time.

The total tocopherol and tocotrienol contents of cranberry seed oil are varied depending

on different growing, processing and storage conditions, which can cause certain oil class

consequently (Boskou, 2006). Nakagawa and others (2007) reported that tocotrienols exhibit a

higher bio-potency than tocopherols against different diseases. This difference may be reflective

of the influence of growing conditions and variation among raspberry genotype on the

phytochemical production. Alterations in phytochemical compositions of berries have been noted

among genotype, growing condition, and the interaction between genotype and environmental

conditions (Yu et al., 2005).

The sterol contents of cranberry seed oil were significantly different by extraction

methods (Table 3). Stigmasterol content of ASE sample was significantly higher (34.15 ppm)

than cold-pressed samples (CP1: 14.35 ppm and CP2: 18.70 ppm). Similarly, β-sitosterol content

of ASE sample was also significantly higher (1001.60 ppm) than cold-pressed samples (CP1:

660.20 ppm and CP2: 642.90 ppm). This indicated that solvent extraction (ASE) was more

effective to extract phytosterols from berry seeds compared with traditional cold-pressed (CP)

extraction method. Detail oil processing steps of commercial cranberry seed oil (COM) were not

available because company did not allow to release. However, using sterol contents, it might be

assuming to use both extraction methods, cold-pressed and solvent extraction methods.

Heeg (2012) reported that significant levels of phytochemicals including β-sitosterol

(1319 ppm) and stigmasterol (68 ppm) were detected in cranberry seed oil. The phytosterol

profile seems to be relatively similar within same species of berry seeds, even when produced

40

under different growing conditions. Thus, this could be a useful parameter in authentication

studies (Hoed et al., 2009).

Table 3

Phytosterol Profiles of Cranberry Seed Oils by Different Extraction Methods and Harvesting

Time. (Unit: ppm or mg/kg Oil)


α-tocopherol 197.50a 261.75a 273.55a 84.70b

γ-tocotrienol 1378.85a 1096.80ab 1056.95b 1149.10ab

Stigmasterol 31.65a 14.35b 18.70b 34.15a

β-sitosterol 828.40ab 660.20b 642.90b 1001.60a


CP1: July harvested seed, cold pressed cranberry seed oil, CP2: September harvested seed, cold pressed cranberry

seed oil, ASE: July harvested seed, accelerated solvent extracted cranberry seed oil. COM: Commercially available

cold press cranberry seed oil (Botanical Oil Innovation)

41

Figure 3. Comparison of α-tocopherol of cranberry seed oil by different extraction methods and


Figure 4. Comparison of γ-tocotrienol of cranberry seed oil by different extraction methods and


a

a a

b

0

50

100

150

200

250

300

350

COM. CP1 CP2 ASE

ppm

α-tocopherol

a

ab b ab

-

500

1,000

1,500

COM. CP1 CP2 ASE

ppm

γ-tocotrienol

42

Figure 5. Comparison of stigmasterol of cranberry seed oil by different extraction methods and


Figure 6. Comparison of β-sitosterol of cranberry seed oil by different extraction methods and

harvesting time (abc represents the statistical significant difference between different oils)

Antioxidant Property

Total phenol content. Total phenol contents (TPC) of cranberry seed oils were shown in

Table 4. The TPC were ranged from 1.23 to 1.32 mg of the gallic acid equivalents (GAE) in each

a

b

b

a

0

5

10

15

20

25

30

35

40

COM. CP1 CP2 ASE

ppm

Stigmasterol

ab

b b

a

0

200

400

600

800

1000

1200

COM. CP1 CP2 ASE

ppm

Beta-sitosterol

43

gram of the cranberry seed oils and ASE sample was the lowest TPC content compared with CP

samples (Table 4). Even though this amounts were slightly lower than the results (1.61 ± 0.14

mg GAE/g) of Yu and Parry (2004), it indicated that cranberry seed oil can be served as a dietary

source of phenolic substances, which may act as antioxidants for disease prevention and/or

general health promotion. This difference of TPC in the sample may be due to non-specific

nature of spectrophotometric method compared to HPLC (Hoed et al., 2011).

Since the cold pressing procedure has been not involved any heat and chemicals during

oil extraction processing, the cold pressed oil may contain more phytochemicals including

natural antioxidants. Harvesting time does not affect the phenolic compounds of the cranberry

seed oil as shown in Figure 7.

Table 4

Antioxidant Properties and Lipid Oxidation of Cranberry Seed Oils by Different Extraction

Methods and Harvesting Time.


TPC (mg GAE/g) 1.32a 1.27ab 1.32a 1.23b

DPPH Inhibition (%) 55.29a 54.35a 55.62a 55.02a

PV (meq/kg) 3.48d 7.09c 8.88b 10.89a


TPC: Total phenolic content expressed by gallic acid equivalents (GAE) in each gram of the cranberry seed oils, %

DPPH: 2,2-diphenyl-1-picrylhydrazyl Inhibition activity, PV: Peroxide Value expressed in milli equivalents of

peroxide per kg oil. CP1: July harvested seed, cold pressed cranberry seed oil, CP2: September harvested seed,

cold pressed cranberry seed oil, ASE: July harvested seed, accelerated solvent extracted cranberry seed oil.. COM:

Commercially available cold press cranberry seed oil (Botanical Oil Innovation)

44

Figure 7. Comparison of total phenol content (TPC) of cranberry seed oil by different extraction

methods and harvesting time (abc represents the statistical significant difference between different

oils).

Scavenging activity. The DPPH radical-scavenging activity of antioxidants is influenced

by the radicle system and testing conditions. Two or more radicle system is required to better

study selected antioxidants for its radicals scavenging properties. DPPH is a stable radical, and

has been used to estimate the radical-scavenging capacities of antioxidants and to evaluate the

kinetics and thermodynamic properties of radical- antioxidants reactions (Yu, Zhou and Parry,

2005). In this study, all the extracted and commercial cranberry seed oils directly reacted with

and quenched DPPH radicals. The DPPH scavenging activity of the cranberry seed oil was not

significantly different by different extraction methods and harvesting time (Figure 8).

a ab a b

-

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

COM. CP1 CP2 ASE

mg

GA

E/g

Total Phenol Content

45

Figure 8. Comparison of scavenging activities of cranberry seed oil by different extraction

methods and harvesting time (abc represents the statistical significant difference between different

oils).

The ASE cranberry seed oil, at a concentration of 4.50 mg oil equivalents per ml of

reaction mixture was shown the lowest DPPH scavenging activity (50.95%) compared with

COM (4.03 mg oil equivalent/ml, 55.29%), CP1 (3.99 mg oil equivalent/ml, 54.30%), and CP2

(4.13 mg oil equivalent/ml, 55.62%), respectively. The free radical scavenging potentials of the

synthetic antioxidant BHT, BHA and AA were determined as controls and scavenging activities

of BHT, BHA and AA were 74.18%, 73.09% and 79.51%, respectively and these results were

consistent with the results of Nigella sativa seeds (Burits & Bucar, 2000).

Lipid oxidation (peroxide value). Oxidative status of oils were the most important

factor to estimate the quality of oil because the oxidative processes in lipids are the main

chemical reaction related the deterioration of the principal organoleptic and nutritional

characteristics of food stuffs. Peroxidation values (PV) of cranberry seed oils were shown in

100.00a

73.09b 79.51c

74.18c

54.3d 55.62d 50.95d

55.29d

-

20

40

60

80

100

Control BHA AA BHT CP1 CP2 ASE COM

% In

hibi

tion

% Inhibition Activity of DPPH

46

Table 4. Lab-scale extracted oils (CP1: 7.09±0.75, CP2: 8.88±0.46 and ASE: 10.89±0.50

meq/kg) were detected high peroxide values while commercial seed oil (COM) was low

(3.48±0.61 meq/kg) (Figure 9).

The main differences between commercial oil processing and lab-scale processing were

that COM had refining processing, but lab extraction had not. Therefore lab-scale extracted oils

(CP & ASE) had high turbidity and impurity, which can accelerate lipid oxidation of oil,

consequently. Generally, filtering process can reduce PV in oil. Hoed and other (2011) explained

that slower conversion of primary to secondary oxidation products in filtered cranberry seed oil

sample were observed during storage and consequently, resulting in lower PV.

In addition, because cranberry seed oils contained high PUFA and MUFA contents, it

was highly susceptible to oxidation. Among lab-scale extracted oils, solvent extracted oil (ASE)

was higher PV than traditional cold-pressed oils (CP1 and CP2). ASE method was needed an

additional processing step to evaporate organic solvent (hexane) from oil after the extraction

step, compared with cold-pressed method. During this evaporation step, oils have more chance to

exposure in the air and consequently, it can cause to accelerate lipid oxidation (Parry et al., 2005;

Hoed et al., 2009). This PV results agreed with previous results of alpha-tocopherol content

(Figure 3) and antioxidant activities (Table 4). Kim and others (2007) reported that oxidized

alpha-tocopherol, which contain polar and nonpolar groups in the same molecule, may reduce

the surface tension of oil and increase the transfer of headspace oxygen to oil, which can

accelerate oxidation.

All PV in this study were exceeded the reference value (5 meq peroxide (O2)/kg oil) of

good oxidative stability oils, while the Codex Standard 19-1981 of PV for virgin oils and cold

pressed fats and oils were 15 meq O2/ kg (Hoed et al., 2011). COM oil was stored in

manufactured bottle (brown glass bottle) and all other oil samples (CP1, CP2 and ASE) were

47

stored in transparent glass bottle. Peroxide value can be increased in the glass transparent bottle

for storage time due to the lack of protection from light. Light was the predominant factor that

can accelerate oxidation by catalysis of radical initiation or, in the presence of photo-sensitizers,

and by formation of singlet oxygen (Velasco & Dobarganes, 2002).

Figure 9. Comparison of peroxide value of cranberry seed oil by different extraction methods

and harvesting time (abc represents the statistical significant difference between different oils).

d

c

b

a

-

2

4

6

8

10

12

14

COM. CP1 CP2 ASE

meq

/kg

Peroxide Value

48

Chapter V: Conclusions

In conclusion, solvent extraction method (ASE) applied with high temperatures and

pressures to produce cranberry seed oils were very effective method to achieve high yield of oil

production and to extract high amounts of phytosterols such like stigmasterol and β-sitosterol

from cranberry seeds. However, ASE method needed an additional oil processing step for

removing solvent from samples, which can exposure oil to the air and then accelerate lipid

oxidation. This additional step was not acceptable especially to cranberry seed oil because of

high PUFA and MUFA contents of oils, which can be highly susceptible to oxidation. ASE

samples were also detected low alpha-tocopherol content and antioxidant activities.

Traditional cold-pressed method is excellent extraction method for cranberry seed oils,

but extraction temperature was important factor for oil quality and refining processing to remove

impurities of crude oil was also very important. For storage, the protection of light using ambient

bottle was the main factor to prevent lipid oxidation. Fatty acid content may depend on different

growing, processing and storage conditions.

There is growing consumer awareness for healthy food products. In the fat and oils

industry, this is reflected in a trend toward low trans-fat and more (poly) unsaturated fatty acids.

Specialty oils such like berry seed oils have a unique fatty acid profile and phytochemicals.

Because cranberry seed oil is produced from by-products of the juice extraction process, it is

high sustainable products as economical point of view. The use of these fruit seed oils in food

and cosmetic products may enhance the profitability of fruit production and processing

industries.

49

Recommendations for Further Study

1. Comprehensive and detailed profile of the different components of cranberry seed oil

extracted by supercritical CO2 extraction in comparison with accelerated solvent

extraction and cold press extraction methods.

2. Determination of phytosterol and triacylglycerol composition using GC-MS and MALDI-

TOF/MS to ensure the authenticity of these specialties, high-value oils.

3. Further studies are needed to examine the oxidative stability and to characterize the oils

polyphenol content and radical scavenging capacities with respect to time and difference

in methanol content (80 and 100%).

50

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