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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
harvesting time (abc represents the statistical significant difference between different oils).
........................................................................................................................................... 41
Figure 5: Comparison of stigmasterol of cranberry seed oil by different extraction methods and
harvesting time (abc represents the statistical significant difference between different oils).
........................................................................................................................................... 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
Data were expressed as mean (n = 3). Different letters within each row represent significance difference (P < 0.05).
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)
Parameter COM CP1 CP2 ASE
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
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. 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
harvesting time (abc represents the statistical significant difference between different oils).
Figure 4. Comparison of γ-tocotrienol of cranberry seed oil by different extraction methods and
harvesting time (abc represents the statistical significant difference between different oils).
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
harvesting time (abc represents the statistical significant difference between different oils).
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.
Parameter COM CP1 CP2 ASE
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
Data were expressed as mean (n = 3). Different letters within each row represent significance difference (P < 0.05).
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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