VOLATILE PROFILE OF CASHEWS (ANACARDIUM OCCIDENTALE L.),
ALMONDS, AND HONEYS FROM DIFFERENT ORIGINS BY SELECTED ION
FLOW TUBE MASS SPECTROMETRY
Dissertation
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of
Philosophy in the Graduate School of the Ohio State University
By
Amal Agila, M.S.
Graduate Program in Food Science and Technology
The Ohio State University
2012
Dissertation Committee
Professor Dr. Sheryl A. Barringer, Advisor
Professor Dr. W. James Harper
Adjunct Professor Dr. John H. Litchfield
Professor Dr. V.M. Balasubramaniam
© Copyright by
Amal Agila
2012
ABSTRACT
Volatile compounds were quantified in the headspace of Indian, Vietnamese and
Brazilian cashews, both raw and during roasting by Selected Ion Flow Tube-Mass
Spectrometry (SIFT-MS). The optimum roasting times based on color measurements
were also determined. Raw cashews were oil roasted for 3 to 9 min at 143 ºC and color
and volatiles measured. An excellent correlation, following a pseudo-first order reaction,
was found between L value and roasting time; darkness increases as roasting time
increases. The optimum roasting time was 6, 8 and 9 min for Vietnamese, Indian and
Brazilian cashews, respectively. Raw cashews had lower concentrations of volatiles than
roasted cashews. Most volatiles significantly increased in concentration during roasting
of Brazilian, Indian and Vietnamese cashews. Only a few volatiles significantly
decreased during roasting. Ethanol and 1-heptene significantly decreased during roasting
in Brazilian cashews and toluene decreased in Vietnamese cashews. Brazilian cashews
had significantly higher levels (p = 0.05) of most volatiles than Indian and Vietnamese
cashews. Most volatile levels in Indian and Vietnamese cashews were not significantly
different. Of the volatiles, Strecker aldehydes including methylbutanal, 2-methylpropanal
and acetaldehyde were at the highest concentration in roasted cashews. The Maillard
reaction contributed to formation of most of volatiles in cashews from the three countries.
ii
There was also degradation of sugars to form furan type compounds and oxidation of
lipids to form alkanals such as hexanal.
Microwave, oven and oil roasting of almonds were used to promote almond flavor
and color formation. Raw pasteurized almonds were roasted in a microwave for 1 to 3
min, in an oven at 177 ºC for 5, 10, 15, and 20 min; and at 135 and 163 ºC for 20 min,
and in oil at 135, 163, and 177 ºC for 5 min and 177 ºC for 10 min. Volatile compounds
were quantified in the headspace of ground almonds, both raw and roasted, by Selected
Ion Flow Tube-Mass Spectrometry (SIFT-MS). Strong correlations were found between
L value, chroma, and 5-hydroxymethyl furfural (HMF); and were independent of roasting
method. Raw almonds had lower concentrations of most volatiles than roasted almonds.
Conditions that produced color equivalent to commercial samples were 2 min in the
microwave, 5 min at 177 ºC in the oven, and 5 min at 135 ºC in oil. Microwave heating
produced higher levels of most volatiles than oven and oil roasting at commercial color.
Sensory evaluation indicated microwave-roasted almonds had the strongest aroma and
were the most preferred. Oil-roasted almonds showed significantly lower levels of
volatiles than other methods, likely due to loss of these volatiles into the oil. Alcohols
such as benzyl alcohols and Strecker aldehydes including benzaldehyde and methional
were at higher concentrations than other volatiles in roasted almonds. The oxidation of
lipids to form alkanals such as nonanal and degradation of sugars to form furan type
compounds was also observed. The Maillard reaction contributed to the formation of
more of the total volatiles in almonds than the lipid oxidation reaction.
iii
Ten Ohio and Indiana honey samples from star thistle (Centaurea Americana),
blueberry (Vaccinium spp.), clover (Trifolium spp.), cranberry (Vaccinium spp.),
wildflower, and an unknown source were collected. The headspace of these honeys was
analyzed by selected ion flow tube mass spectrometry (SIFT-MS) and soft independent
modeling of class analogy (SIMCA). SIMCA was utilized to statistically differntiate
between honeys based on their composition. Ohio honeys from star thistle, blueberry, and
clover were similar to each other in volatile composition, while Ohio wildflower honey
was different. Indiana honeys from star thistle, blueberry, and wildflower were different
from each other in volatile composition while clover and cranberry honeys were similar.
Honeys from Ohio and Indiana with the same floral origins were different in volatile
composition. Furfural, 1-octen-3-ol, butanoic and pentanoic acids were the volatiles with
the highest discriminating power between types of floral honey. Methanol and ethanol
followed by acetic acid were at the highest levels in most honeys, through furfural was at
the highest concentration in Indiana blueberry honey while 1-octen-3-ol was at the
highest concentration in Indiana wildflower honey. The highest concentration of volatile
compounds was in Indiana wildflower honey followed by Ohio wildflower honey while
the lowest concentration of volatile compounds was observed in Ohio clover honey
followed by Indiana clover honey.
iv
Bismillah Al-rahman Al-raheem
In the Name of Allah, the Infinitely Compassionate, the Most Merciful
v
AKNOWLEDGMENTS
First, I would like to express my gratitude to my advisor, Professor, Dr. Sheryl Ann
Barringer. She taught me how to write academic papers, had confidence in me when I
doubted myself, and brought out the good ideas in me. She was always there to listen and
to give advice. One of the luckiest things in my life was having her as my advisor.
Second, I want to thank my committee members Dr.Harper, Dr.Litchfield, and Dr. Bala
for their insightful comments, invaluable support and encouragement.
I must thank my great mother, Nagia Agila, the most special one in my life. She
sacrificed so much that I still have not found the words that describe or express how I feel
for this woman and what her presence in my life has meant. I must also thank my
husband, Youssef Binkhayal, he supported me like no one else and changed me for the
better and encouraged me to pursue my interests. Thank you with all my heart and soul. I
thank my father, Rajab Agila, for his encouragement and my daughter Hanin, for giving
me life in the first place. Thank you and I love you much.
I thank Dr. Luis E. Rodriguez-Saona, Dr Monica Giusti and Dr Ahmed Yousef for
allowing me to use some equipment from their labs.
I thank all my current and previous labmates, especially Carolina, Elijandra, Offy, Yang,
Peren, Ruslan and Neda for their friendship, support, help and happiness brought to me.
Last, but not least, I would like to thank Kraft Foods and Planters Company, who
provided the cashew samples.
vi
VITA
August 1996 …………………………….. …………… B.S., Laboratory Medicine
The Omar Almokhtar University, Derna, Libya
August 2004…………………M.S., Analytical Chemistry & Instrumental Analysis
The Malaya University, Kuala Lumpur, Malaysia
October 2004…………………………………...... Instructor, Analytical Chemistry
The Omar Almokhtar University, Derna, Libya
January 2009 – present………………… ………..Ph.D Candidate in Food Science
The Ohio State University, Columbus, OH
PUBLICATIONS
Research Publications
1. Agila A, Barringer SA. 2011. Volatile profile of cashews (Anacardium
occidental L.) from different geographical origins during roasting. J Food Sci,
76(5): C768 – C774.
2. Agila A, Barringer SA. 2012. Effect of roasting conditions on color and
volatile profile including HMF in sweet almonds (Prunus dulcis). J Food Sci,
77(4): C461 – C467.
FIELD OF STUDY
Major Field: Food Science and Technology
vii
Table of Contents
ABSTRACT ............................................................................................................................................ ii
AKNOWLEDGMENTS ........................................................................................................................ vi
VITA...................................................................................................................................................... vii
CHAPTER 1 ............................................................................................................................................ 1
LITERATURE REVIEW ........................................................................................................................ 1
1.1 Volatile Compounds in Cashew Nuts ................................................................................... 1
1.1.1 Introducing the Cashew Tree to the World .................................................................... 1
1.1.2 The Components of Cashew Trees ................................................................................. 2
1.1.3 Importance of Cashew .................................................................................................... 3
1.1.4 Diseases and Pests in Cashew ........................................................................................ 4
1.1.5 Harvest and Post-Harvest Handling ............................................................................... 5
1.1.6 The Main Steps in Cashew Processing .......................................................................... 6
1.1.7 Flavor .............................................................................................................................. 8
1.1.8 Roasting ........................................................................................................................ 13
1.1.9 Maillard Reaction in Food ............................................................................................ 14
viii
1.1.10 Lipid Oxidation ............................................................................................................ 16
1.1.11 The Color of the Cashew Nuts ..................................................................................... 17
1.1.12 Aroma Volatiles in Cashew Nuts ................................................................................. 18
1.2 Volatile Compounds in Almond Nuts .................................................................................. 19
1.2.1 Introducing Almond Nuts to California ......................................................................... 19
1.2.2 Importance of Almonds .................................................................................................. 19
1.2.3 Uses of Almonds ............................................................................................................ 20
1.2.4 Diseases and Pests in Almond Nuts ............................................................................... 21
1.2.5 Almond Harvest and Post-Harvest Handling ................................................................ 22
1.2.6 Maillard Reaction in Almond Nuts ................................................................................ 22
1.2.7 Lipid Oxidation in Almond Nuts ................................................................................... 23
1.2.8 Almond Aroma ............................................................................................................... 24
1.2.9 Measring the Color in Foods .......................................................................................... 28
1.3 Volatile Compounds in Unifloral American Honeys ........................................................... 29
1.3.1 Honey History ................................................................................................................ 29
1.3.2 Honey Composition ........................................................................................................ 30
1.3.3 Importance of Honey ...................................................................................................... 31
1.3.4 Heating of Honey ........................................................................................................... 31
1.3.5 Honey Aroma ................................................................................................................ 32
1.3.6 Honey Quality ................................................................................................................ 35
viii
1.3.7 Honey Adulteration ........................................................................................................ 36
1.3.8 Honey Storage ................................................................................................................ 37
1.3.9 Chemometrics to Discriminate Volatile Compounds in Honeys ................................... 39
1.4 Techniques for Analyzing the Volatile Compounds ............................................................ 40
1.4.1 Analysis of Volatile Compounds ................................................................................... 40
1.4.2 Isolation and Separation Techniques.............................................................................. 40
1.4.3 Identification Techniques .............................................................................................. 42
CHAPTER 2 .......................................................................................................................................... 52
VOLATILE PROFILE OF CASHEWS (ANACARDIUM OCCIDENTALE L.) FROM
DIFFERENT GEOGRAPHICAL ORIGINS DURING ROASTING .................................................. 52
2.1 Abstract ........................................................................................................................... 52
2.2 Practical Application ....................................................................................................... 53
2.3 Introduction ..................................................................................................................... 53
2.4 Materials and Methods .................................................................................................... 55
2.5 Results and Discussion .................................................................................................... 60
2.6 Conclusion ....................................................................................................................... 71
CHAPTER 3 .......................................................................................................................................... 72
EFFECT OF ROASTING CONDITION ON COLOR AND VOLATILE PROFILE
INCLUDING HMF LEVEL IN SWEET ALMONDS (PRUNUS DULCIS) ....................................... 72
3.1 Abstract ............................................................................................................................ 72
3.2 Practical Application ........................................................................................................ 73
ix
3.3 Introduction ...................................................................................................................... 73
3.4 Materials and Methods ..................................................................................................... 75
3.5 Results and Discussion ..................................................................................................... 82
3.6 Conclusion ........................................................................................................................ 96
CHAPTER 4 .......................................................................................................................................... 97
APPLICATION OF SELECTED FLOW TUBE MASS SPECTROMETRY COUPLED WITH
CHEMOMETRICS TO STUDY THE EFFECT OF LOCATION AND BOTANICAL ORIGIN
ON VOLATILE PROFILE OF UNIFLORAL AMERICAN HONEYS .............................................. 97
4.1 Abstract ........................................................................................................................... 97
4.2 Practical Application ....................................................................................................... 98
4.3 Introduction ..................................................................................................................... 98
4.4 Materials and Methods .................................................................................................. 100
4.5 Results and Discussion .................................................................................................. 105
4.6 Conclusion ..................................................................................................................... 115
REFERENCES .................................................................................................................................... 116
x
List of Tables
Table 1.1. Some Compounds Found in Roasted Cashew Nuts Flavor ................................................ 11
Table 1.2. Some Compounds Found in Roasted Almond Nuts Flavor ................................................ 26
Table 1.3. Some Compounds Found in Honey Flavor ......................................................................... 34
Table 2.1. Kinetics Parameters for SIFT-MS Analysis of Selected Volatile Compounds .................. 58
Table 2.2 Concentration (µg/L) of Selected Organic Volatiles in Raw and Roasted Cashews .......... 63
Table 2.3. Concentration (µg/L) of Selected Organic Volatiles in Brazilian Cashew during
Roasting ................................................................................................................................................. 66
Table 3.1. Kinetic Parameters for SIFT-MS Analysis of Selected Volatile Compounds in
Almonds ............................................................................................................................................... 79
Table 3.2. L Value, Chroma, Headspace HMF Concentration (µg/L) of Almonds at Different
Roasting Conditions .............................................................................................................................. 82
Table 3.3. Concentration (µg/L) of Organic Volatiles in Raw Almonds and Almonds Roasted to
L Value of 45 (Light Color) .................................................................................................................. 86
Table 3.4. Concentration (µg/L) of Organic Volatile in Almonds Roasted to L Values 44
(Medium) and 43 (Dark) ....................................................................................................................... 89
Table 3.5. The Volatiles in Oil Samples Heated with and without Almond Present ........................... 93
xiii
Table 4.1. Kinetics Parameters for SIFT-MS Analysis of Selected Volatile Compounds in Honey 102
Table 4.2. Interclass Distances between Honey Samples from Different Plant Origins and
Location ............................................................................................................................................. 106
Table 4.3. SIMCA Discriminating Power (DP) of Honey Samples Based on Volatile
Concentrations ..................................................................................................................................... 108
Table 4.4. Concentration (µg/L) of Honeys from Different Origins and Locations .......................... 113
xiii
List of Figures
Figure 1.1. Major Pathway for Pyrazines Formation via Strecker Degradation (Figure Adapted
from Mabrouk 1979) ............................................................................................................................. 10
Figure 1.2. Biological Pathway of Ester Formation (Figure Adapted from Olias and others 1995) ... 12
Figure 1.3. The Formation Pathway of Maillard Reaction Molecules (Figure Adapted from
Hodge and others 1953) ........................................................................................................................ 16
Figure 1.4. Principle of Selected Ion Flow Tube Mass Spectrometry .............................................. 45
Figure 2.1. L Value for Different Cashews versus Roasting Time and Equations for Pseudo-1st
Order Reactions ..................................................................................................................................... 61
Figure 2.2. Organic Volatiles in Raw Brazilian Cashew Decrease in Concentration during
Roasting ................................................................................................................................................ 65
Figure 2.3. Maillard Reaction Volatiles in Brazilian Cashew during Roasting .................................. 68
Figure 2.4. Lipid Degradation Volatiles in Brazilian Cashew ............................................................. 70
Figure 3.1. Headspace Concentration (µg/L) of HMF versus L Value and Chroma of Almonds
after Different Roasting Conditions ..................................................................................................... 85
Figure 3.2. Concentration (µg/L) of the Sum of Maillard Reaction and Lipid Oxidation Volatiles
in Almonds Roasted at Different Roasting Conditions ......................................................................... 95
xiii
CHAPTER 1
LITERATURE REVIEW
1.1 Volatile Compounds in Cashew Nuts
1.1.1 Introducing the Cashew Tree to the World
The cashew tree Anacardium occidentale is a native of Brazil (Akinwale 2000).
This tree belongs to the Anacardiaceae family of plants, which also includes many trees
such as the mango and the pistachio. The tree was introduced to India, West Africa, East
Africa, Mexico, South and Central America by the Portuguese in the fifteen and sixteen
centuries to control coastal soil erosion (Akinhanmi 2008; Azam-Ali and others 2004).
This because the tree has strong roots which are as long as the tree and for this reason, it
is typically used for erosion control purpose (Aremu and others 2007). In the nineteenth
century, the plantations were developed and then the tree spread to a number of other
countries in Africa, Asia and Latin America. In the first half of the twentieth century,
manual cashew processing was begun in India and then was exported to the United States.
In 1960, many countries started to process the nuts rather than sending them to India.
This enables them to benefit from the sale of processed cashew nuts. At the presen
1
time, cashew trees are cultivated for its nuts in many countries in Africa, Asia and
America (Azam-Ali and others 2004).
1.1.2 The Components of Cashew Trees
Food products from the cashew tree are divided into two groups, one is the
cashew nut and another is the fruit (Damasceno and others 2008). The cashew nut is
about 2.5 - 4.0 cm long and has a kidney like shape. Its shell is about 5 mm thick, with a
soft outer skin and a thin hard inner skin (Azam-Ali and others 2004). The cashew nut
contains valuable amount of lipids (47%), proteins (21%), carbohydrates (22%) and
vitamins, especially thiamine (Dwomoh and others 2008). It is also considered as a good
source of some minerals such as calcium, phosphorus and iron (Garruti and others 2006).
Generally, cashew nuts are rich in oleic acid and have a good level of linoleic acid. They
produce about half its weight in edible oil but no evidence of it being commercially used
(Akinhanmi 2008).
The cashew apple (the pseudo-fruit) which is attached to the nut (Garruti and
others 2006) and has a sour and astringent taste unless it is fully ripe, when it becomes
edible. Cashew apple juice contains high amount of vitamin C (203.5 mg/100 ml)
(Akinhanmi 2008), which is five times more than in an orange. The apple is also a good
source of calcium and iron (Azam-Ali and others 2004). Cashew apple juice can be
extracted. It has a pleasant flavor and is rich in vitamin C, but has little acceptance
because of its astringency. However, the clarified cashew apple juice has greater
acceptance because of its low astringency (Damasceno and others 2008).
2
The cashew nut shell contains a viscous liquid, which is extremely caustic. The
liquid inside the shell or cashew nut shell liquid (CNSL) represents 20 to 25% of the
content of the raw nut (Azam-Ali and others 2004). It is basically phenolic in nature
(Jayalekshmy and Narayanan 1989).
1.1.3 Importance of Cashew
In cashew-producing countries, the nuts are considered one of the products
preferred by a large number of the people. 60% of cashew nuts are used in the form of
snacks while the remaining 40% are consumed in confectionery .They are often
consumed in three ways: directly by the consumer; as roasted and salted nuts and in
confectionery and bakery products such as in the production of sweets, ice creams, cakes
and chocolates, and as paste to spread on bread (Azam-Ali and others 2004). Recently,
they have been used as edible nuts by consumers interested in quality and health aspects
of food because of the presence of valuable levels of lipids, proteins and carbohydrates
(Akinhanmi 2008).
The cashew apple is very rich in vitamin C and a glass of cashew apple juice
meets the requirements of the adult daily intake of vitamin C (30 mg). In addition, the
fruit is claimed to have medicinal properties such as in treating diarrhea (Azam-Ali and
others 2004). Cashew fruit can be suitable for use by removing the undesirable tannins
that are responsible for the astringent taste and processing the apples into useable
products such as juices, pickles, jams, dried and canned fruits.
3
The juice can be fermented into cashew wine which is a very popular drink in
West Africa. In Brazil, the apple is used to manufacture jams, and soft and alcoholic
drinks (Azam-Ali and others 2004). In the United States, the cashew apple juice is used
in ices and jelly (Akinwale 2000). Cashew apple juice is considered a very good raw
material for alcoholic fermentation because it is rich in sugar and mineral contents
(Garruti and others 2006). The recommended methods for removing the astringent
properties of the cashew apple include washing it in cold water and then boiling the fruit
in salted water for five minutes.
In addition, the leaf and shell of cashew are claimed to have many medical
applications such as improving heart disease, decreasing toothache and treating calcium
deficiency (Azam-Ali and others 2004). The cashew nut shell liquid (CNSL) is used for
making resins and paints (Akinhanmi 2008).
1.1.4 Diseases and Pests in Cashew
Cashew can be affected by a number of different plant pathogens such as fungi
including Colletotrichum glocosporioides. This fungus is one of the most common
pathogens in cashew. It leads to decay in the nuts and apples, the leaves become
crumpled, and the flowers turn black and fall off. The trees can be sprayed with various
fungicides including Bordeaux mixture to control the fungus (Azam-Ali and others
2004). Also, the molds such as Aspergillus flavus and Aspergillus parasiticus produce the
toxin “aflatoxin” which may lead to the cashew nuts becoming unsuitable for consumer
consumption (Mexis and Kontominas 2009). The fungal infections cause many
4
symptoms include withering, yellowing of the lower leaves and darkened tissue around
the stems.
The other major source of crop loss is insect pests such as sap-sucking bugs, leafchewing caterpillars and spider mites. They can lead to damage the tree and the crop by
early abortion of immature nuts and loss of yield. Fungi can be also controlled by a
copper containing fungicide and insects can be controlled by chemical spray containing
of Carbaryl and also spray containing "Folidol E.605". Rodents such as rats and squirrels
may cause serious damage to cashew seedlings especially when they appear above the
ground. Also, cashew apples may be attacked by monkeys, bats and parrots which lead to
damage to the fruit. The most important protection against infestation of any type of
insects or rodents is cleanliness of the rooms used for drying, peeling, grading,
conditioning, and packaging (Azam-Ali and others 2004).
1.1.5 Harvest and Post-Harvest Handling
The harvesting and processing of cashew are labor intensive. The true cashew fruit
should be picked after the clusters of the flowers appear, the apple becomes mature and
the nut is covered by a heavy shell. Generally, when the fruit is fully ripened, it falls to
the ground. Harvesting basically includes collecting the nuts once they have dropped to
the ground after maturing and the surface under the tree should be free from weeds and
dry leaves. The nuts are often collected in baskets or sacks. The cashew fruit are left to
fall to the ground before being collected as an indication the kernel is mature. The type of
climate effects harvest of the nuts. In dry climates, the nuts can be left under the trees for
5
several weeks without their quality being affected. However, in humid climate, the nuts
should be collected at least twice a week.
Extraction of the nut from the shell is the main step during post-harvest of cashew
nuts. The whole shell can be either soaked in water or roasted. It also can be softened by
air-drying to a maximum moisture content of 9%. The nuts should be stored for export in
a clean and dry location. They must be inspected and tested prior to export and in the
importing countries (Azam-Ali and others 2004) for the toxin “aflatoxin” which is
produced by some dangerous molds such as Aspergillus flavus and Aspergillus
parasiticus (Mexis and Kontominas 2009).
1.1.6 The Main Steps in Cashew Processing
There are generally five steps involving a number of operations that should be
used during the processing of cashew nuts and are essential to improve the desired quality
of cashews. The main steps in processing of cashew nuts are:
1.1.6.1 Shelling
Shelling or removal of the outer shell and CNSL is done to produce clean, whole
kernels without cracks (Azam-Ali and others 2004). Shelling is presently the greatest
processing problem of cashew nuts. The difficulties in shelling cashew nuts are due to the
irregular shape of the nut, presence of CNSL in the outer shell, and the CNSL within the
shell must not be allowed to wet the kernel during its removal from the shell (Ogunsina
and Bamgboye 2007). Shelling can be performed by roasting or soaking.
6
Roasting can cause brittleness of the shell, loosening of the kernel within the
shell and release of the CNSL from the shell. Soaking increases the moisture content of
the kernel as well as decreasing the risk of it being burned during roasting and increasing
its flexibility to make it less likely to crack (Azam-Ali and others 2004).
1.1.6.2 Drying
Drying is a step prior to peeling of the shell and leads to a decrease of the nut’s
size. It allows the testa to be easily removed. The moisture content of the kernel should
be reduced about 6 to 3% by drying (Azam-Ali and others 2004).
1.1.6.3 Peeling
Manual peeling or removal of the testa can be carefully performed by gently
rubbing with the fingers or using a knife without any cutting or damage during the
process. The use of knives increases the numbers of the nuts becoming damaged, but it
removes all of the testa (Azam-Ali and others 2004).
1.1.6.4 Grading of Different Sizes and Color According to Standard Grading
Cashews are categorized for export based on size, color and condition of the nuts.
The grading system is known as the American Standard. The grading is divided into three
groups: white whole such as super large (120 and 180 nuts per lb), white pieces such as
baby bits (Very small pieces of kernel which are white in color) and scorched grades such
as Butts (Butts that have been scorched) (Azam-Ali and others 2004).
7
1.1.6.5 Packaging
The general packaging for the export of nuts is air-tight cans of 25lbs (11.34 kg)
weight capacity. The package should be low cost and impermeable to protect the cashew
nuts from rancidity. The air must be removed from the can and substituted with carbon
dioxide (CO2) because it will not support the growth of any infestation (Azam-Ali and
others 2004). The use of suitable packaging materials, selection of good quality raw
materials and good processing conditions of temperature and time of heating will prevent
contaminating materials such as dirt, metal and stones from mixing with the nuts and
protect the nuts after processing (Azam-Ali and others 2004).
1.1.7 Flavor
The developed aroma and flavor in foods are determined by the type and amount
of generated organic volatiles in the food item (Coleman and others 1994). Flavor is
defined as a complex sensation determined mainly by the senses of taste in the mouth and
smell in the nose. Aroma compounds are volatile compounds vaporizing at room
temperature to travel through the air and reach the receptors in the upper part of the
human nose. They produce a chemical response which is expressed as an odor or smell.
They can be found in food, perfume, wine, spices and essential oils (Bosland and Votava
2000). The taste happens when the components of the food mix with the saliva in the
mouth; and the odor comes afterwards from the transferred air to the nose either by
chewing or swallowing (Land 1996) or by sniffing food via the nose (Diaz 2004). During
eating, the flavor of food depends on the amount of volatile compounds in the mouth and
their interactions with the nose and mouth (Dattetreya and others 2002). Flavor release of
8
food is affected by temperature alteration of the eaten food, saliva penetration into the
food, and enzyme activity of the food (Taylor 1996).
1.1.7.1 Cashew Nuts Aroma
Among dry foods, cashew nuts are consumed for their characteristic aroma
(Mexis and Kontominans 2009). The aroma characteristics of raw cashew nuts are
enhanced as a result of roasting such as hot oil-roasting. Twenty six compounds are
identified in cashew nuts as previously reported by Jayalekshmy and Narayanan (1989).
They detected that raw cashew nuts had a strong pungent aroma.
Additionally, acids strongly contribute to pungent aroma in cashew apple to
which the cashew nut is attached (Maarse 1991). Mexis and Kontominans (2009) also
previously reported that acetaldehyde and acetic acid contribute to pungent aroma in
cashew nuts. Roasted cashews had mild and nutty aromas. Pyrazines are heterocyclic
nitrogen-containing compounds (Huang and Barringer 2010) which mostly contribute to
roasted and nutty aromas in oil-roasted cashews (Jayalekshmy and Narayanan 1989). The
major pathway for pyrazine formation via Strecker degradation is shown in Figure 1.1.
9
H2N-CH2COOH + H-CO-CO-H
CHO-H2C-NH2
CHO-CH=N-CH2COOH -CO2
+ HCHO
CHO-H2C-NH2 + CHO-H2C-NH2
-2H2O
Figure 1.1: Major Pathway for Pyrazine Formation via Strecker Degradation
(Figure Adapted from Mabrouk 1979).
In addition, flavor in aqueous extracts of cashew nuts can be developed from non
enzymatic reaction such as the Maillard reaction between amino acids and reducing
sugars (Coleman and others 1994). Pyrazines, esters, ketones, acids, lactones, furan
deravatives compounds are identified in cashew nuts (Jayalekshmy and Narayanan 1989);
and aliphatic aldehydes, alcohols and aliphatic hydrocarbons are also detected in cashew
nuts (Mexis and Kontominans 2009). A list of some compounds found in roasted cashew
nut flavor is shown in Table 1.1.
10
Table 1.1: Some Compounds Found in Roasted Cashew Nuts Flavor (Jayalekshmy
and Narayanan 1989).
Compound
Acids (analyzed as methyl ester)
Hexanoic acid
Octanoic acid
Decanoic acid
Dodecanoic acid
Tetradecanoic acid
Alcohols
Phenyl alcohol
2-Butylbenzothiazole
5-Ethyl-2-methyl oxazole
Aldehydes
Benzaldehyde
2-Thiophene carbowaxaldehyde
2.4-Undecadienal
Aromatic Compounds
Butoxy methylbenzene
Myrcene
Naphthalene
Esters
Butyl acetate
1,3-Propane diol diacetate
Ethyl octanoate
Methyl tetradecanoate
Methyl hexadecanoate
Furan Containing Compounds
Furfural
2-Pentyl furan
5-Methyl-2-furanylmethyl-2-furanone
Ketones
5-Heptene-2-one
Butyrolactone
Hexalactone
Pyrazines
Methylpyrazine
2,6-Dimethylpyrazine
2-Ethyl-6-methylpyrazine
2-Ethyl-3-methylpyrazine
2-Ethyl-5-methylpyrazine
2,6-Diethylpyrazine
Sensory Properties
Rancid oil-like
Unpleased rancid
Stored oil-like
Slightly pungent
Rancid oil-like, slightly fruity
Flowery, ester-like
Slightly pungent
Mild, roasted
Smell of bitter almond
Raw green smell
Raw, green
Mild hydrocarbone-like
Green, mango-like
Aromatic
Ester-like, fruity
Fresh, green, cashew nut-like
Oily, nutty
Mildy, nutty, oily
Mildy, nutty, oily
Fermented
Green, raw
Raw green smell
Intense, fresh, green
Pleased nutty
Nutty smell
Raw, green, mildy, nutty
Green, nutty
Mild roasted
Green, raw, nut-like
Raw green smell
Steam cooked smell
11
Short chain esters such as ethyl butanoate are responsible for a sweet, fruity and
cashew-like aroma (Franco and Janzantti 2005). Alcohols in food item may contribute to
ester formation (Ozcan and Barringer 2010). The biological pathway of volatile ester
formation is shown in Figure 1.2. 3-Methylbutanal, 2-methylbutanal and 2-methyl
propanal are formed from reaction of free amino acids such as isoleucine, leucine and
valine with reducing sugars; and they are responsible for a pleasant odor in many roasted
foods (Coleman and others 1994).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Fatty Acids
Amino Acids
Aldehydes
Oxidation
Reduction
Acids
Alcohols
Alcohols Acyltransferase
Esters
Figure 1.2: Biological Pathway of Ester formation (Figure Adapted from Olias
and others 1995).
12
1.1.8 Roasting
Roasting is a thermal process preceded by drying (Mayer 1985) to increase the
chemical reactions in the foods such as the Maillard reaction (DeRovira 2006) and
degradation of lipids (Mexis and Kontominans 2009) to provide a pleasant color and
aroma (Mayer 1985). The optimum roasting time used in industry is mainly based on the
characteristics of the heat transfer of the roaster.
A roaster with the higher heat transfer efficiency normally requires a shorter
roasting time (Nebesny and Rutkowski 1998). The choice of roasting conditions for
different cashew nuts depends on the geographical origin and uniformity of heat during
isolation of the nuts from the shell (Franco and Janzantti 2005). Also, the maturity at
harvest because the mature nut has the maximum level of flavor precursor (Mayer 1985).
1.1.8.1 Hot Oil Roasting: A Major Process to Develop Cashew’s Aroma
Hot oil roasting at a high temperature of 200 °C was developed in the mid 1930s
and used for processing large amounts of nuts (Azam-Ali and others 2004). The roasted
cashew nuts are usually fried in vegetable oil to a light brown color (Jayalekshmy and
Narayanan 1989). The application of heat should therefore be uniform and constant to
maintain the product quality (Azam-Ali and others 2004). Heating is also applied to the
cashew nuts to facilitate extraction of the nut from the shell, release the CNSL and make
the shell brittle (Azam-Ali and others 2004; Sabarez and Noomhorm 1993). The degree
of chemical changes depends on the temperature and time applied during the roasting
because Maillard reaction occurs at high roasting temperature to develop the volatile
13
compounds. For example, high temperatures favor Strecker degradation, which is an
important step for converting dicarbonyl compounds formed in the Maillard reaction to
flavor compounds such as aldehydes and pyrazines (Davies and Labuza 1997). Also, the
increase in the temperature leads to an increase of the reactivity between the sugar and
the amino group in heated cashew nuts (Coleman and others 1994).
In many foods, a high temperature favors the open chain configuration of the
reducing sugar which is more reactive than the closed ring configuration (Van Boekel
2001). However, extensive browning reaction or over-roasting changes the nutrients
profile, flavor, color and consistency during processing and storage of food products
(Franco and Janzantti 2005; Mayer 1985; Mexis and Kontominas 2009).
1.1.9 Maillard Reaction in Food
Application of heat is used to preserve food products in the industry. However,
the negative effect of exposure to the heat is non enzymatic browning associated with the
loss of the nutrient and formation of undesirable products resulting in very dark color
such as 5-hydroxymethylfurfural (Damasceno and others 2008). The Maillard reaction or
non-enzymatic browning in nuts such as cashews and peanuts is preceded by a set of
reactions between amino acids and carbonyl compounds such as reducing sugars
(Coleman and others 1994). It can happen in heated, dried and roasted foods.
In food, the Maillard reaction contributes to the changes in color, flavor, and
nutritive value. It is initiated by the condensation of the amino compound with the
carbonyl group of the reducing sugar to produce a N-glycosylamine compound.
14
Subsequently, this intermediate rearranges and dehydrates by deoxyosones to low
molecular weight yielding products such as furfural. Then, these molecules react with
other reactive compounds such as amino acids and dicarbonyl compounds and undergo
an important reaction which is the Strecker degradation. The amino acid is then
decarboxylated and deaminated to form an aldehyde, whereas the dicarbonyl is converted
to an aminoketone or aminoalcohol.
These reactions result in the formation of many active molecules characterizing
the heated food aromas such as furans, pyrazines, pyrroles, oxazoles, thiazole and other
heterocyclic compounds. The last stage includes the condensation of Maillard reaction
products such as pyrrole derivatives leading to the formation of brown high molecular
weight melanoidin polymers (Fay and Brevard 2005). The formation pathway of many
Maillard reaction molecules is shown in Figure 1.3.
15
Figure 1.3: The formation Pathway of Maillard Reaction Molecules (Figure
adapted from Hodge 1953).
1.1.10 Lipid Oxidation
The cashew nut is one of the lipid foods. It is called a “good fat” which means
that the cashew nut is completely natural and best for humans. It contains a valuable
amount of fats (47%) (Akinhanmi 2008). Of the fats, 61% is oleic acid and 17% is
linoleic acid (Mexis and Kontominas 2009). Cashew has the right ratio of saturated to
mono unsaturated and polyunsaturated (1:2:1) fats, which is suitable for the consumption
by humans (Akinhanmi 2008).
16
The presence of fat in lipid foods significantly increases the sensory properties of
foods and is responsible for many desirable qualities in foods, including texture,
structure, mouth feel, flavor and color. However, unsaturated lipids are chemically
unstable in food and may rapidly undergo oxidation in free-radical chain reactions.
Generally, lipid oxidation and rancidity are the main factors determining the shelf life of
lipid-containing foods (Brich and others 2010).
The free-radical chain reactions may cause deterioration of the lipids and rancid
off flavor (German 1999) such as a card board flavor that is generated from the increase
of the concentration of short chain aldehydes during long storage such as pentanal,
hexanal, heptanal, octanal and nonanal. In cashew nuts, hexanal should be in low
concentration to minimize the development of the rancid flavor (Mexis and Kontominas
2009). One of the reactions in foods contributing to aroma formation is lipid degradation
(thermal and oxidative). Lipid degradation products can produce a variety of oxygen
containing compounds such as aliphatic aldehydes, esters and alcohols (Coleman and
others 1994; Mexis and Kontominas 2009).
1.1.11 The Color of the Cashew Nuts
Different cashew nuts from various origins and different locations have
differences in color. This may be related to the geographical origins of cashews, improper
post harvest treatment of the nuts during storage, an extensive use of pesticides in the
crop and unavailability of even heat during shelling (Franco and Janzantti 2005; Sabarez
and Noomhorm 1993; Mexis and Kontominas 2009; Mayer 1985).
17
1.1.12 Aroma Volatiles in Cashew Nuts
The aroma compounds in cashew nuts are isolated by simultaneous distillation
extraction and by steam distillation as previously reported by Jayalekshmy and
Narayanan (1989). The flavor constituents in cashew nuts have also been identified by
gas chromatography (GC) and mass spectrometry coupled with gas chromatography (GCMS) as previously documented by Jayalekshmy and Narayanan (1989).
In cashew nuts, acetic acid and acetaldehyde contribute to a pungent aroma, and
phenylethanol is responsible for sweet and floral aromas. The main aromas come from
pyrazines and aldehydes.
Pyrazines are responsible for a nutty aroma and aldehydes such as methylbutanal
and 2-methyl propanal are responsible for a pleased aroma in many roasted foods. Lipid
oxidation products such as hexanal and nonane are also contributed to green and
herbaceous odors, while ethyl butanoate is responsible for sweet, fruity and cashew-like
aromas. In lipid foods, 1-Heptene, toluene, and nonnal are responsible for a fatty aroma
(Brewer 2009; Clark and Nursten 1976; Coleman and others 1994; Franco and Janzantti
2005; Garruti and others 2006; Jayalekshmy and Narayanan 1989; Mexis and
Kontominans 2009).
18
1.2 Volatile Compounds in Almond Nuts
1.2.1 Introducing Almond Nuts to California
Almond is a fruit similar to the cherry, the plum, and the peach (Nutfarm 2005).
Almond agriculture takes place in Spain because of the mild climate that is essential for
almond growth (Vazquez-Araujo and others 2008). In the mid 1700’s, the almond tree
was brought to California by the Spanish Franciscan Padres (Nutfarm 2005).
Recently, the Spanish production of almonds has reduced, while the production of
almonds has increased rapidly in the United States of America because of the large
investment of money for developing new agricultural almond crops (Vazquez-Araujo and
others 2008). California is the only source in North America (Nutfarm 2005), and
represents the most producers of almonds, supplying about 80% of the world’s needs and
100% of those of the United States. The California almond industry is valued
approximately $2 billion per year (Beck and others 2009). This “leads to a competition
between American and Spanish almonds” (Vazquez-Araujo and others 2008).
1.2.2 Importance of Almonds
The almond contains approximately 72% fats, 15% carbohydrates, and 13%
proteins (Nutrition Facts 2011). The composition of almonds may be effected by various
factors such as almond cultivar, moisture, and mixing almonds from different seasons
(Vazquez-Araujo and others 2009). They are good sources for vitamins and minerals and
are known as dietary super foods.
19
Almonds can decrease cholesterol level, improve heart function, and prevent
cancer because they have low levels of saturated fats and contain a valuable amount of
protective nutrients such as vitamin E, mono-saturated fat, magnesium, and antioxidants
that assist in preventing heart related diseases. They also protect from high blood pressure
because they contain a good amount of potassium that regulates high blood pressure in
humans. Almonds also promote tooth and bone strengthening because they contain a
good level of calcium (Pollick 2011). Almonds contain folic acid that is claimed to
decrease the risk of cervical cancers (Nutfarm 2005). The addition of other foods
containing amino acid profiles complimentary to almonds mostly provides a complete
protein source (Nutrition Facts 2011).
1.2.3 Uses of Almonds
Almonds are the most popular nuts in the world and are widely used raw, or
processed in candies, baked products and confectioneries (Wirthensohn and others 2008).
They are fruits found in Asia and North Africa and have two types, sweet and bitter.
Sweet almonds are used in desserts, puddings, ice cream, and many Asian and European
foods. It can be also processed to form almond oils or extracts that can be substituted for
vanilla extract, and are mostly roasted for consumer perception.
The bitter almonds are required to be processed before using it in cooking because
bitter almonds may have a toxic level of hydrogen cyanide that may lead to death.
Therefore, hydrogen cyanide must be removed before the bitter almonds are used as food
(Pollick 2011). In Jamaica and India, roasted and boiled almonds are used as snacks
20
during tea time or can be used over cereal or yogurt for breakfast in Malaysia (Lasekan
and Abbas 2010).
1.2.4 Diseases and Pests in Almond Nuts
In California, the navel orange worm (NOW) Amyelois transitella is the main
insect that affects the tree crops of almonds (Beck and others 2011a; Beck and others
2009). The adult worm has “silver gray and black forewings and legs, and a snout like
projection at the front of the head”. The females start egg laying approximately two
nights after emergence. They lay the eggs on nut crop trees and stay inside nuts or
between hulls and shells (UC IMP 2009). The infection by this insect leads to decrease in
nut kernel quality because of its feeding and leads to economic decline in the almond
industry. This feeding damage by the navel orange worm may contribute to invasion by
Aspergillus flavus. This fungus can damage the tree crops of almond nuts and produce
aflatoxins which are carcinogenic for humans (Beck and others 2011a; Beck and others
2009). In addition, whole raw almond crops may be infected by Salmonella enterica,
which is an organism causing food-borne illness. Infection by Salmonella enterica in raw
almonds can be controlled by using chlorine dioxide (ClO2), chemical solutions such as
acetic acid or citric acid, and the exposure to high hydrostatic pressure.Steam
pasteurization can also control the growth of Salmonella enterica. Steam is approved by
the Federal and Drug Administration (FDA), but it may result in increase in moisture
content and in decrease in the quality of almonds including flavor and structure integrity
(Bingol and others 2011).
21
1.2.5 Almond Harvesting and Post-Harvest Handling
The almonds can be harvested manually or by knocking the nuts from the tree
limbs, and letting them stay on the ground for a week to ten days to facilitate drying. The
fallen almonds are then swept into rows and gathered to the almond huller or sheller for
transportation. Then, harvested almonds undergo two processes, one is post-harvest
processing and another is finishing processing. The almond post-harvest processing
facilities include pre-cleaning, hulling and shelling, and the final products are known as
hullers and shellers. The shelled almonds are shipped to large production facilities to
undergo further processing into several final products. “The hulled, in-shell almonds are
separated from any remaining hull pieces in a series of air legs (counter-flow forced air
gravity separators) and are then graded, collected, and sold as finished product”(Food
And Agricultural Industry 1995).
1.2.6 Maillard Reaction in Almond Nuts
Non-enzymatic browning (NEB) is a chemical reaction that generates brown color
in foods without the need for enzyme catalysis (Laroque and others 2008). It normally
occurs in two forms: caramelization and Maillard reaction. Although they can both
involve application of heat, caramelization refers to the degradation of sugars (Ajandouz
and others 2001); whereas, the Maillard reaction entails chemical interactions between
amines and carbonyl compounds, usually reducing sugars, to generate various aroma
compounds such as Strecker aldehydes and pyrazines (Coleman and others 1994).
22
The complexity of the Maillard reaction is such that it is affected by various
parameters including type and percentage of amino acid and reducing sugars; also water
activity, heating time, temperature and pH (Laroque and others 2008). The almond
industry indicates that the Maillard reaction is responsible for browning in almonds
(Pearson 1999), and the roasting process can enhance chemical reactions such as the
Maillard browning reaction (Franco and Janzantti 2005). The roasting process can also
decrease the moisture, reduce the water activity, increase the amount of carbon dioxide,
and produce brittle roasted almonds (Severini and others 2000).
1.2.7 Lipid Oxidation in Almond Nuts
Lipids are also considered important compounds for the formation of flavor
(Brich and others 2010). The almond nuts that are exposed to a humid environment for a
long period of the time may contain oxidized fats (Pearson 1999). Oleic acid is the main
fatty acid present in almonds, followed by linoleic acid (Beck and others 2011b). Linoleic
and linolenic acids represent the precursors of most aldehydes, acids, alcohols and esters
(Perez and others 1999). There are also many volatiles formed from lipid oxidation such
as hexanal, hexanoic acid, and nonanal. Hexanal is an off-flavor volatile generated from
auto-oxidation of linoleic acid and the thermal oxidation of linoleates. Hexanoic acid is
formed from auto-oxidation of almond oil. Another volatile is nonanal, which comes
from decomposition of oleic acid (Beck and others 2011b). Some volatile compounds
such as ethanol and hexanal can delay the growth of microorganisms in foods (Severini
and others 2000).
23
During the exposure of the food to heat, the moisture is reduced down to the BET
monolayer value; therefore, heat can generate food products stable from lipid oxidation.
Moreover, Maillard reaction volatiles generated from the roasting process of almonds
have antioxidant ability to extend the shelf life and prevent lipid oxidation, but may
decrease the aroma of the product. This antioxidant capability is related to the presence of
the active volatile compounds in the headspace of the roasted almonds package rather
than the melanoidins related to the Maillard reaction (Severini and others 2000).
1.2.8 Almond Aroma
Approximately seventy four organic volatile compounds are identified and
quantified in roasted almonds (Lasekan and Abbas 2010). Takei and Yamanishi (1974)
previously identified fifty five compounds responsible for 90 % of the aromas in roasted
almonds. They found that pyrazines and pyrroles contributed to aromas in roasted
almonds. Pyrazines in almonds include 2,5-dimethylpyrazines, 2-ethyl-3-methylpyrazine,
2-methylpyrazine, and 2,4-dimethylpyrazine (Vazquez-Araujo and others 2009).
Pyrroles contribute to burnt aroma and are formed in most of heated foods (VazquezAraujo and others 2008). They are important for all kinds of fragrance compositions, and
provide naturalness and long lasting effect (Indiamart 2011). Pyrazines and pyrroles are
generated during the Maillard reaction between amino acids and reducing sugars under
thermal conditions (Vazquez-Araujo and others 2008). Furans and their derivatives are
also formed from a reaction between amino acids and reducing sugars through the
Maillard reaction (Vazquez-Araujo and others 2008). They are mostly formed in foods
from the thermal degradation of glucose and fructose. Fructose generates the highest
24
level of furan because it is more reactive than glucose due to the rise in the rate of
mutarotation resulting in more of the reactive open chain furanose. Furan can also be
formed from the oxidation of poly-unsaturated fatty acids (Fan 2005; Kim and Lee 2009;
Vranova and Ciesarova 2009). They are responsible for a caramel-like aroma in heated
foods containing carbohydrate, and the major furans are furfuryl alcohol and furfural.
These furan type compounds are known as “cooked sugar, sweet, woody, almond,
fragrant, and baked bread”. Other furans such as 2-methyl furan, 2-acetyl furan, and 5methyl furfural increase in concentration with temperature and time. However, furans
have high threshold levels making them have no significant role in the almond aromas.
The other volatile compounds in almonds are alcohols. They may be formed from
decomposition of fatty acids or by reduction of the aldehydes during heating (VazquezAraujo and others 2008), and are not responsible for aroma in foods (Vitova and others
2007). Moreover, (Z)-3-Hexen-1-ol is one of the major flavor compound found in all
types of fruit and vegetable compositions. It contributes to green grass and green leaves
aromas (Alchemist 2010). Also, methional is found in various types of foods and is
responsible for various aromas such as vegetable and creamy aromas (Indiamart 2011).
Almond aromas are shown in Table 1.2.
25
Table 1.2: Some Compounds Found in Roasted Almond Nuts Flavor.
Compound
Alcohols
benzyl alcohol
(Z)-3-hexen-1-ol
1-octanol
1-octen-3-ol
Aldehydes
(E)-2-heptenal
nonanal
octanal
decanal
(E,E)-2,4-decadienal
(E)-2-octenal
(E)-2-nonenal
heptanal
methional
Aromatic compounds
toluene
vanilin
carvone
benzaldehyde
Furans
2-pentylfuran
Furans
5-methylfurfural
furfural
Ketones
2-heptanone
Pyrazines
dimethylpyrazine
2-methylpyrazine
Pyrroles
N-furfuryl pyrrole
Sensory Properties
Ref.
Sharp burning taste, faint aromatic
green grass and green leaves
Sharp, fatty, waxy, citrus fresh, ogange-rose
Herbaceous, earthy
4
1
4
4
Pungent, green
Tallow fruity
Citrus-like
Green soapy
Powerful, fatty, citrus
Fresh cucumber
Fatty tallow
Oily, fatty, heavy, woody
vegetable and creamy
4
3
3
3
4
3
3
4
2
Paint
Vanilla-like
Warm, herbaceous
Bitter almond, fragrant, aromatic, sweet
4
3
4
4
Beany-like
3
Sweet, spicy, caramel, bitter almonds
Sweet, woody, almond, baked bread
5
5
Fruity, spicy, cinnamon
4
Cocoa, roasted nuts, woody
Nutty, cocoa, green, roasted
5
5
Vegetable, earthy
4
References: [1]: Alchemist 2010; [2]: Indiamart 2011; [3]: Lesekan and Abbas 2010; [4]:
Vazquez-Araujo and others 2008; [5]: Vazquez-Araujo and others 2009.
26
1.2.8.1 Type of Aromas in Almonds
Wirthensohn and others (2008) reported three types of aroma in almonds
including non-bitter, semi-bitter, and bitter aromas. The sweet almonds contain a slightly
nutty flavor and the semi-bitter almonds contain marzipan-like flavor. They also
documented that the major volatile components of semi-bitter and bitter almonds are
aromatic hydrocarbons such as benzaldehyde and benzyl alcohol. The concentration of
benzaldehyde is higher in bitter almonds than in semi-bitter and non-bitter almonds,
while the concentration of benzyl alcohol is higher in bitter and semi-bitter almonds than
in non-bitter almonds (Wirthensohn and others 2008).
Benzaldehyde represents the major compound in bitter almond oil (Beck and
others 2011a). The bitter flavor is formed in roasted and cooked almonds through
concealed damage. Concealed damage of almonds is “a browning of the kernel interior
after moderate to high heat processing”. This browning does not occur in the exterior of
heated almonds, therefore this browning “does not fall into any of USDA defect
categories for almonds” (Pearson 1999).
In addition, Wirthensohn and others (2008) implied that benzyl alcohol may be
formed from benzaldehyde during reversible enzymatic reactions. Also, benzaldehyde
and benzyl alcohol represent the major compounds in almond oil (Picuric-Jovanovic and
Milovanovic 1993).
27
1.2.8.2 Analysis of Aroma Volatiles in Almonds
Previous analysis of almond volatile compounds was mainly performed by gas
chromatography-mass spectrometric (GC-MS) technology (Vazquez-Araujo and others
2008; Takei and Yamanishi 1974; Wirthensohn and others 2008; Vazquez-Araujo and
others 2009; Lasekan and Abbas 2010; Picuric-Jovanovic and Milovanovic 1993). Also,
some conventional sample extraction methods, such as simultaneous steam-distillation
extraction have been used in gas chromatography mass spectrometric analysis of organic
volatiles in almonds (Vazquez-Araujo and others 2009). Solid-phase micro-extraction
(SPME) is also used as an alternative extraction method for almond volatile compound
analysis (Wirthensohn and others 2008), because it is an economic and rapid technique
(Beltran and others 2006).
1.2.9 Measuring the Color in Foods
The color in various foods can be economically measured by a colorimeter
instrument. A colorimeter contains a sensor, simple data processor and a set of
illuminants at a view angle usually of 10º. This instrument is used in routine analysis for
providing measurements that correlate with the perception of human eye-brain. The data
provided by a colorimeter is represented as L, a and b values (Hunter Lab 2008a). L value
indicates lightness while a and b represent red-green and yellow-blue chromatic
coordinates, respectively (Vazquez-Araujo and others 2009). There are many
colorimeters such as Color Quest XE Colorimeter that can be used for evaluating the
color in raw materials and finished products. It can also measure the color in solid and
28
liquid materials of food products and the color calculations are performed from 400 nm to
700 nm (Hunter Lab 2008b).
1.3 Volatile Compounds in Unifloral American Honeys
1.3.1 Honey History
Honey has been known for a long period of time as the “nectar of gods” (Odeh
and others 2007) and is mostly produced from Apis mellifera bees (Cuevas-Glory and
others 2006). It is a sweet liquid which provides medicinal and sensory benefits (Moreira
and others 2010) without the need for any additive or preservative substance (CuevasGlory and others 2006).
Honey may be produced from mixed floral types or one botanical source. The
term “unifloral” honey means that honey is formed from one plant species (Soria and
others 2004), while the term “multifloral” honey means the honey nectar is from different
flowers (Ceballos and others 2010).
Adult bees include a queen, female workers and male drones. The queen mates
with several drones and then starts to protect the eggs lain, while worker bees serve as
labors and pollen baskets (Horn 2008). Honey bees have been found in North America
ever since human-assisted migration in the 17th century. English colonists took Apis
mellifera bees to New Zealand, Australia and Tasmania, and then the human-assisted
migration of Apis mellifera bees was distributed around the world.
29
The U.S. Congress issued a Honey Bee Restriction Act in 1922, for protecting
Apis mellifera bees until a species of mites expanded in 1980 which caused 50 to 80 %
loss in honey hives. At the present time, China, Argentina, USA, Turkey, and Mexico are
the largest honey producers in the world (Viuda-Martos and others 2010).
1.3.2 Honey Composition
The composition of honey may be affected by many external features such as the
plant source, region, season, and storage time (Kukurova and others 2008). Honey
consists of at least 181 components such as sugars including fructose (38%) and glucose
(31%); and 0.18% of ash. Honey also consists of vitamins and minerals, the vitamins
include niacin, pyridoxine, and ascorbic acid (Viuda-Martos and others 2010), while
potassium is the most abundant mineral and is mainly found in dark honeys. The amount
of minerals enhances the color and the taste of honey (Kukurova and others 2008). Honey
also contains enzymes such as invertase, phosphatase, glucose oxidase, and catalase. It
contains organic acids such as gluconic acid, pyruvic acid, malic acid, and citric acid. The
main proteins in honey are amino acids which are essential to develop Maillard reaction
products. Honey does not represent a complete food based on human nutritional
standards, but it can be used as a dietary supplement (Viuda-Martos and others 2010).
Honey has a large quantity of flavonoids and phenolic compounds that work as
antioxidants and may be serving as markers of floral origin of honeys (Kukurova and
others 2008).
30
1.3.3 Importance of Honey
Honey is used around the world as a sweet and natural food because of its unique
characteristics. Small amounts of honey can be utilized as a good source for energy and
sugar replacer. Honey is also used for healing complicated wounds and ulcers, especially
diabetic ulcers because it can reduce the infection by microorganisms. Honey contains a
lot of important nutrients such as vitamins and minerals that provide people the necessary
nutrients needed. Moreover, it contains antioxidants which serve to decrease the free
radicals and keep a healthy body (Kumar 2009). Antioxidants in honeys can be enzymatic
antioxidants such as catalase, glucose oxidase and peroxidase and non-enzymatic
antioxidants such as organic acids, amino acids, ascorbic acid, and carotenoids.
There are also phenolic compounds that mostly act as active antioxidants in
honey including caffeic acid, ferulic acid, and coumaric acid (Santos and others 2011).
Honey also has antimicrobial properties due to the presence of hydrogen peroxide that
produced throughout enzymatic glucose oxidase reactions. Hydrogen pyroxide is
generated in honey when glucose forms gluconic acid and hydrogen pyroxide in the
presence of water molecules and oxygen (Waikato Honey Research Unit 2006).
1.3.4 Heating of Honey
Exposing honey to heat treatment may lead to enhancing the formation of volatile
compounds due to complex reactions such as caramelization and Maillard reaction
(Moreira and others 2010). Heating may affect some volatile compounds in honey that
are not stable and are rapidly destroyed, or may lead to the formation of others different
31
volatile compounds because of non enzymatic browning reactions. For example, furanic
compound such as hydroxymethylfurfural or HMF is used as a marker of heating of
honey (Castro-Vázquez and others 2008). It is formed from degradation of fructose
(Bogdanov 2009), also it can be formed by cyclic glucose through protonation of the
second hydroxyl group in the ring (Dee and Bell 2011). Other examples of furanic
compounds including furfural, 1-(2-furanyl)-ethanone, furfuryl alcohol (Soria and others
2003), and furanmethanol are formed by heating (Baroni and others 2006). Furanic
compounds come from Maillard reactions or dehydration of sugars in an acid medium;
these reactions may be increased if honey is exposed to high temperatures during
processing (Castro-Vázquez and others 2009). Additionally, the increase in concentration
of some volatiles generated during heating such as pyrazines, pyrroles, and furanones are
used as an indicator of honey heating (Castro-Vázquez and others 2008).
1.3.5 Honey Aroma
The organic volatile compounds represent the fingerprint of honey used to detect
the honey origin and are the main factors contributing to aroma, taste, and flavor (Baroni
and others 2006). Honey has a distinctive aroma with organoleptic properties (Shimoda
and others 1996) and contains low concentration of aroma components (Cuevas-Glory
and others 2007). More than 700 volatiles compounds are detected in honeys from
various floral sources and locations (Ampuero and others 2004). These compounds are
found in honey at low levels as mixtures of volatile compounds with relatively low
molecular weight (Cuevas-Glory and others 2006), and may be proposed as markers for
distinction of unifloral honeys (Cuevas-Glory and others 2007). For example, honey-like
32
aroma is produced by many benzene derivatives components such as benzaldehyde,
phenylethanol, benzylalcohol, and phenylacetaldehyde (Castro Vázquez and others
2006), while a honey-haze aroma is formed by linalool (Shimoda and others 1996).
Linalool is generated from oxidative degradation of carotenoids (Moreira and others
2010). Linalool derivatives ((E)-2,6-dimethyl-6-hydroxy-2,7-octadienoic acid, (E)-2,6dimethyl-3,7-octadiene-2,6-diol, (Z)-2,6-dimethyl-2,7-octadiene-1,6-diol, (Z)-2,6dimethyl-6- hydroxy-2,7-octadienal, lilac aldehydes and lilac alcohols) were been
detected as markers for nodding thistle honey (Soria and others 2003). Acids also can
produce a positive aroma in honey such as benzoic acid; however, isovaleric acid may
contribute to a negative aroma (off flavor) in honey (Moreira and others 2010).
In addition, ethanol, furfural, benzene acetaldehyde, acetone, and dimethyl
sulfide have been documented as common components of several unifloral honeys at
different concentrations (Pérez and others 2002). Methyl anthranilate and lilac aldehyde
were previously detected as characteristic compounds of citrus honey. While, nonanol,
nonanal, nonanoic acid and acetoin were determined as marker compounds of eucalyptus
honey. Other distinct marker compounds including acetophenone, 1-phenylethanol and 2acetophenone were found in chestnut honey; and isophorone (3,5,5-trimethylcyclohexen2-enone) was detected at high level in heather (Cuevas-Glory and others 2007) and
rosemary honey (Soria and others 2003). Also, polyoxygenated terpenes including (Z)2,6-dimethyl-2,7-octadien-1,6-diol and 2,6-dimethyl-3,7-octadien-2,6-diol had been
proposed as marker compounds in orange honey (Castro Vázquez and others 2006).
33
Table 1.3: Some Compounds Found in Honey Flavor
Compound
Sensory Properties
Ref.
pleasant, sweet odor
floral odor
[4]
[2]
green, grass, and paraguay tea
smokey odor
grass, lemon, and mint
honey, rose, spicy, flowery
[4]
[2]
[4]
[1]
citrus honey aroma
bitter almond and burnt sugar
bread. Almond, sweet, woody, fragrant
fruity odor
fruity odor
sweet, musty, aldehydic
[3]
[1]
[3]
[3]
[3]
[3]
oily, flowery, and green
honey, rose, and green
sweet odor like vanilla
[1]
[1]
[5]
estery and fruity odors
minty and cool odor
[3]
[2]
ripe fruit and resin odor
buttery and cabbage-like
burnt sugar and toasted caramel
[7]
[4]
[6]
cabbage, sulfur, gasoline
vegetable, cabbage, putrid
powerful, fish, fresh onion
[3]
[3]
[3]
Acids
benzoic acid
acetic acid
Alcohols
benzyl alcohol
guaiacol
menthol
2-phenylethanol
Aldehydes
acetaldehyde
benzaldehyde
furfural
heptanal
hexanal
3-methylbutanal
Aldehydes
nonanal
phenylacetaldehyde
vanillin
Esters
ethyl acetate
methyl salicylate
Ketones
2-aminoacetophenone
2,3-butanedione
maltol
Sulfur Compounds
dimethyl sulfide
dimethyl disulfide
dimethyl trisulfide
References: [1]: Mannas and Altug 2007; [2]: Shimoda and others 1996; [3]:
Alissandrakis and others 2005; [4]: Moreira and others 2002; [5]: Moreira and others
2010; [6]: Castro-Vázquez and others 2008; [7]: Castro-Vázquez and others 2009.
34
1.3.6 Honey Quality
The quality of unifloral honey may be determined by its aroma-related
components which are affected by its floral origin (Pérez and others 2002). The floral
origin of honey is economically important because consumers sometimes prefer honey
types from some botanic sources over others (de la Fuente and others 2005). Honey is
important for the characterization of honey (Soria and others 2004) since honey
composition, flavor, color, and texture are influenced by the type of plant where the bees
take the nectar (Ampuero and others 2004).
The diversity of honey floral types makes no honey exactly the same as any
another (Viuda-Martos and others 2010) and leads honey to be a complex natural food
that is difficult to control (Soria and others 2004). A previous study by Shimoda and
others (1996) reported that the quality of honey is correlated to the HMF compound. This
compound is formed naturally over time in honey and may be produced rapidly when
honey is exposed to heat. HMF is not toxic, but its level in honey from non tropical
climates is regulated to be no more than 40 mg/kg HMF. Also honey from tropical
climates and blends may contain no more than 80 mg/kg HMF as a maximum
concentration (Allen and others 2011). In addition, the loss of freshness in honey can be
identified by many other organic volatiles other than HMF. These compounds were
produced from the Maillard reaction and may negatively affect the sensory properties of
eucalyptus extracts such as maltol (3-hydroxy-2-methyl-4H-pyran-4-one), 2-methoxy-6methyl pyrazine, and pantolactone (Castro Vázquez and others 2006).
35
1.3.6.1 Factors Affecting Honey Quality
Some factors affect the properties of honey which affect its quality such as
weather conditions including temperature, humidity and bees foraging habits. For
example, the moisture content of honey should be about 17.7% because it affects honey
stability. The higher moisture content may cause undesirable honey fermentation by
osmotolerant yeasts especially during storage leading to production of carbon dioxide and
ethyl alcohol that may be converted to acetic acid and water producing honey with a sour,
off taste, or surface foaming (Viuda-Martos and others 2010). Also, bees sometimes
forage any type of flower they reach to produce mixed honey aromas, or the bees reach
one type of flower to produce honey with specific characteristic and organoleptic
properties (Ampuero and others 2004).
1.3.7 Honey Adulteration
For centuries, foods have been adulterated by using cheaper and lower quality
materials to cheat consumers. These materials rarely cause health hazards but they add to
defrauding the consumer out of hundreds of thousands of dollars each year and may
undercut the competition (Kurtzweil 1999). In the case of honey, it can be adulterated
with cheaper products such as high fructose corn syrup (HFCS) and invert syrup (IS)
(Ruiz-Matute and others 2007), or addition of sugars into honey by feeding of bees. Also,
addition of water to adulterate honey, but this is rarely used because of the risk of
undesirable fermentation (Kukurova and others 2008).
36
Adulteration of honey may be hard to detect because the composition of honeys is
dependant on its plant or geographical origins, and the chemical composition of the added
syrups may be similar to that in natural honey (Ruiz-Matute and others 2007). Sometimes
the adulteration of honey can be detected by observing how the poured honey is
distributed in a glass of water because pure honey does not rapidly dissolve in water and
requires some efforts to stir it. However, different honeys have different viscosities; some
are denser or thicker than others, even when they are adulterated with other substances
(Tan 2008).
1.3.8 Honey Storage
The chemical composition of honey may undergo modifications during storage
which affects honey properties and quality (Cuevas-Glory and others 2006). The storage
conditions can lead to change the percentage of volatile compounds of honeys
(Kaškonienė and others 2008) and may lead to reduce the aroma-related compounds,
especially at 35 to 40 ºC over three to six months (Moreira and others 2010). In the case
of the storage at room temperature for three months, the volatile compounds of white
clover honey decreased in concentration approximately 70%; however, the volatile
compounds of spring rape honey were increased 30% compared to fresh honey. Some
compounds may decrease or not found during storage such as hexane, 3-methylbutanal,
2-methyl propanoic acid, 2-methyl butanoic acid, 2-nonanone, undecane and decanoic
acid (Kaškonienė and others 2008). In comparison, some compounds may be found only
during storage at room temperature in white clover and spring rape honey such as
dimethyl sulfide, 2-methylbutanenitrile, dimethyl disulfide, hexanal, nonane, dimethyl
37
trisulfide, octanal, heptanoic acid (Kaškonienė and others 2008). Also, other compounds
were increased in the concentration during prolonged storage at 35 to 40 ºC such as
octadecanol, benzeneethanol, and benzenemethanol (Moreira and others 2010). These
volatiles are produced from the aldehyde reduction processes; however, the presence of
antioxidants and antimicrobials may minimize the production of these volatiles.
Benzenemethanol and benzenethanol have a pleasant impact on the aroma of fresh honey.
Benzenemethanol contributes to green, grass, and Paraguay tea aromas, while
benzenethanol is responsible for floral, herb-like, and spicy aromas (Moreira and others
2010). Moreover, volatile compounds such as pyrroles, furanones, and pyrazines mostly
increase in the concentration during storage especially at high temperature (Castro
Vázquez and others 2006).
Some furanic compounds in fresh citrus honey including furfural, 2-acetylfuran,
5-methylfurfural, and furfuryl alcohol were had a little increase with storage at 10 and 20
°C, but they greatly increased when honey was stored at 40 °C (Castro-Vázquez and
others 2008). The freshness and sensory properties of honeys stored at 40 °C were
changed and being not accepted by the assessors and honeys had an increase in the
acidity due to the increase in the concentration of organic acids, especially acetic acid.
Therefore, to prevent the damage of some sensitive compounds by heating or developing
any other unneeded compounds, honey should not be heated to more than 40 to 50 °C
during storage and honey should be stored at 20 ºC or lower to keep its aroma compounds
(Castro- Vázquez and others 2008).
38
1.3.9 Chemometrics to Discriminate Volatile Compounds in Honeys
The discrimination between diverse honey types is essential to know honey
features such as aroma, taste, and nutritional characters which affect consumer perception
(Aliferis and others 2010). Chemometrics (pattern recognition techniques) represent
multivariate analysis to utilize mathematical and statistical methods for discriminating the
chemical data. This technique can apply accurate, fast, and easy methods for
classification of sample components in complex food matrices. These methods were used
to discriminate the volatile compounds in various floral honeys to choose a small number
of compounds and provide good discrimination among various volatile compounds
(Baroni and others 2006).
One powerful method of multivariate data analysis is soft independent modeling
of class analogy (SIMCA). SIMCA is a principal component analysis (PCA) method that
can examine the variations in the entirely data and in one particular data (Christy and
others 2003). With this technique, principle component analysis (PCA) is utilized for
each class separately, resulting in a principal component (PC) model for each class
(Gurdeniz and Ozen 2009). A class space is built around the data with a 95% confidence
interval. The SIMCA discriminating power algorithm is able to identify values or
numbers of interest and also remove that do not assist in separating classes. Interclass
distance represents the distance between classes based on factor loadings and can detect
whether it is possible to completely separate the classes. Distances larger than or equal to
three are considered significantly different (Dunn and Wold 1995).
39
SIMCA were previously used to discriminate and classify the volatile compounds
in honeys such as thyme and citrus according to their plant sources and geographical
locations (Aliferis and others 2010). These volatile compounds include different chemical
classes such as phenolics, terpenoids, and aliphatics compounds such as acids, esters,
alcohols, and aldehydes. By SIMCA discrimination, the volatile compounds such as
lilacaldehyde, limonene and methyl anthranilate were reported as distinct compounds of
citrus honey. Volatile compounds such as phenylacetaldehyde, 1-phenyl-2,3-butanedione,
3-hydroxy-4-phenyl-2-butanone, 3-hydroxy-1-phenyl-2-butanone, and 3-hydroxy-4phenyl-3-buten- 2-one were documented as discriminating compounds for thyme honey
(Aliferis and others 2010).
1.4 Techniques for Analyzing the Volatile Compounds
1.4.1 Analysis of Volatile Compounds
Analysis of volatile compounds that are generated by cooking, baking or roasting
are responsible for a pleasant aroma in food products was started in the 19th century.
Then the attempts continued for identifying the major volatile compounds in foods in the
mid 20th century (Maarse 1991). Then later, the analysis for the isolation, separation,
and identification of volatile compounds in foods has been made to enhance knowledge
in the field of flavor chemistry (Maarse 1991; Teranishi and others 1999).
1.4.2 Isolation and Separation Techniques
The traditional analytical methods for the flavor profile mainly need an isolation
step before identifying the compounds. The popular techniques for the isolation and
40
separation of compounds involve distillation, extraction, and liquid and gas
chromatography. But, some decomposition of generated compounds may occur due to the
application of heat (Maarse 1991; Teranishi and others 1999). The isolation technique
should be simple, fast and compatible with a range of analytical instrumentation as well
as used to remove the volatile compounds from the original food with minimum artifacts
(Sousa and others 2006).
In 1960 up to the present, gas chromatography technique was widely used to
separate the complex mixture of volatile compounds present in foods and has contributed
to understanding flavor chemistry (Teranishi and others 1999). Recently, headspace
isolation techniques have been developed such as dynamic headspace analysis and
headspace solid phase microextraction (SPME) to isolate compounds directly from the
vapor phase of the sample according to equilibrium partitioning coefficients. These
techniques are simple, fast, efficient, and less expensive artifacts compared to traditional
methods (Maarse 1991). This change in the analysis method means the sensory
perception of aroma can be determined by the concentration of volatiles in the air phase
rather than in the food product itself (Teranishi and others 1999). However, the relative
concentration of volatiles in the headspace of the food sample is effected by the
differences in the affinity of each volatile compound to the polymeric fiber (Maarse
1991).
41
1.4.3 Identification Techniques
There are several spectrometric methods used for the characterization and
identification of flavor compounds such as mass spectrometry. Gas chromatography
coupled with mass spectrometry (GC-MS) has been mainly used for the identification of
compounds in various foods and beverages (Maarse 1991; Teranishi and others 1999).
GC-Olfactometry, based on sniffing the effluents from GC columns, has also been used
to identify the individual compounds in foods, as well as to assign sensorial odor
descriptors to chemical compounds (Teranishi and others 1999).
In addition, “time-of-flight” (TOF) based GC-MS has high sensitivity and
selectivity to determine a large number of volatiles in single analysis (Waters Corporation
2008). Solid phase micro extraction (SPME) gas chromatography with a flame ionization
detector is also a highly sensitive and helpful method to detect all organic compounds
containing carbon, but it destroys the sample (SHU 2010). In addition, matrix assisted
laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is used
to indentify the components of the sample at low quantities of detection limit 10-15 to 1018
mole with an accuracy of 0.1 - 0.01 %. Most of these techniques were used to identify
and quantify the organic volatiles in cashews, almonds and honeys (Jayalekshmy and
Narayanan 1989, Garruti and others 2006, Lasekan and Abbas 2010, Cuevas-Glory and
others 2006). However, chromatographic and spectrometric methods such as GC-MS
have difficulty in monitoring the real-time release of the volatiles in foods because of the
need for sample preparation (Smith and Spanel 2005).
42
Another advanced technique such as proton transfer reaction mass spectrometry
(PTR-MS) is a recently developed technique which provides rapid detection of aroma
compounds in alcoholic beverages (Aprea and others 2007). Also, atmospheric pressure
ionization (API-MS) allows real-time quantifying of volatiles in the mixture in the
headspace directly without sample preparation (Boukobza and others 2001). Both these
technique lack the ability to distinguish the isomers because they do not have the
separation ability provided by gas chromatography although they can monitor the realtime analysis of volatile release (Xu and Barringer 2009).
1.4.3.1 Selected Ion Flow Tube Mass Spectrometry (SIFT-MS)
SIFT-MS is an analytical technique that allows selected precursor positive ions
for chemical ionization reactions coupled with mass spectrometric detection to rapidly
identify and quantify volatile organic compounds (VOCs). This technique monitors the
real-time analysis of the complex mixtures of the volatile compounds without preconcentration (Spanel and Smith 1999a).
1.4.3.1.1 Principle of SIFT-MS
The principle of SIFT-MS is based on soft chemical ionization (Spanel and Smith
1996) and is explained in Figure 1.9. The selected mass precursor ions generated by
microwave gas enter into a flow tube with an inert carrier gas at a controlled velocity via
quadrupole mass filter. The sample gas mixture is introduced into the flow tube to react
with precursor ions to generate the characteristic product ions. The precursor and product
ions count rates are then determined by a quadrupole mass spectrometer system (Spanel
43
and Smith 1996; Spanel and Smith 1999a). Three precursor ions such as H3O+, NO+, and
O2+ are used during the analysis to ionize volatile compounds (Spanel and Smith 1996)
because they do not interact with major components of air such as N2, O2, H2O and CO2
but can react rapidly with the volatile compounds in the headspace above the sample
(Spanel and Smith 1999a). The concentration of a volatile compound A can be calculated
using the following reaction:
[A]=Ip/Ikt,
where, Ip is a product count rate, I is a precursor ions count rate, k is reaction rate
coefficient and t is a reaction time.
The reaction rate coefficient, k, was obtained from a previous study of the ion
chemistry of the precursor ions, H3O+, NO+, and O2+ with a wide variety of volatile
compounds, and was stored in the kinetic database of the SIFT-MS. The reaction time t is
precisely detected by a known flow velocity of the helium carrier gas and the length of
the flow tube (Spanel and Smith 1999a). In addition, SIFT-MS allows fast identification
and quantification of volatile compounds in a large range of sample formats, including
whole air and headspace. Also, SIFT-MS technique uses multiple reagent ions (H3O+, O2+
and NO+) to chemically ionize volatile compounds. Therefore, the identification of some
isomers without the need for chromatographic separation can be possible (Smith and
Spanel 2005).
44
Figure 1.4: Principles of Selected Ion Flow Tube Mass Spectrometry (Syft
Technologies Inc. 2011).
1.4.3.1.1.1 The Precursor Ion H3O+
The H3O+ precursor ion can react with a large number of organic compounds via
proton transfer with unit efficiency to generate one or two product ions (Smith and
Spanel 2005). When ions are introduced into humid air, clustering H3O+ (H2O)n may
occur (Smith and Spanel 1996) and be present in the carrier gas of the SIFT-MS; thus,
they must be taken into account in the analysis. They can be used as an additional
analytical tool because they can rapidly react with many gas molecules, especially with
polar molecules (Spanel and Smith 1995). They may produce a secondary reaction, which
effect the distribution and intensities of product signal in SIFT-MS analyses (Wang and
others 2004c). In addition, the product ion of the proton transfer reaction of H3O+ with
molecule A is AH+, whose mass represents the mass of the reactant molecule plus 1.
Thus, precursor ion H3O+ is not suitable for separate isomers because they can generate
the same mass spectra (Smith and Spanel 1996). If A represents an alcohol, aldehyde or a
carboxylic acid, the dihydrate ions (AH+ (H2O)2) readily form. The increase in the
number of the final product ions generates more conflicts with other masses. Therefore,
45
H3O+ reaction masses should be avoided for these compounds. But if A is a ketone or
ester, only the monohydrate ions (AH+ H2O) readily form. Protonated phenols mainly
form monohydrates and very small fractions of dihydrates (Wang and others 2004c).
1.4.3.1.1.2 The Precursor Ion NO+
The precursor ion NO+ reacts with organic molecules more than the precursor ion
H3O+, but they often produce one or two product ions. The precursor ion NO+ reacts
with organic volatile A to generate charge transfer producing A+ ions, hydride ion
transfer producing AH+, and ion-molecule association producing NO+A ions. Charge
transfer can only occur if the ionization energy (IE) of the organic volatile A is lower
than the ionization energy of NO+. Also, hydride ion transfer is produced by the reactions
of NO+ with aldehydes and ethers (Spanel and others 1997, Spanel and Smith 1998c).
Ion-molecule association is generated from the reactions of NO+ with some types of polar
organic molecules at internal thermal energy occurred due to the random movements of
atoms and molecules, especially the carboxylic acids and esters (Spanel and Smith
1998c) and ketones (Spanel and others 1997).
1.4.3.1.1.3 The Precursor Ion O2+
The precursor ion O2+ reacts rapidly with most organic molecules, and produces
one or two or more product ions. It also has higher energy than H3O+ or NO+, and can
react with some aromatic organic compounds to produce a single product ion, which is
useful for detection and quantification of these compounds. Also, it can be used for the
detection and quantification of small molecules such as NO, NO2 and CS2 that do not
46
react with either H3O+ or NO+ ions (McIntosh and others 1988, Hunter and Lias 1998).
On the other hand, the reaction of O2+ with aliphatic hydrocarbons generates many
product ions increasing the complexity of the mass spectra. Therefore, the use of the
precursor ion O2+ is limited in SIFT-MS analyses.
1.4.3.1.2 Application of SIFT-MS in Food Science
The SIFT-MS method has been successfully used in various applications such as
monitoring air pollution in environmental science (Smith and Spanel 1996), analysis of
breath volatiles for medical diagnosis and therapeutic monitoring in human physiology
(Senthilmohan and others 2000; Smith and others 2003a), and detection of bacterial
metabolites in microbiology (Scotter and others 2005; Allardyce and others 2006).The
SIFT-MS technique can be also used for the analysis of oxidation in dry fermented
sausage (Olivares and others 2010) and it is used to analyze trace gas analysis to monitor
freshness in foods and food preparation (Smith and Spanel 2005).
In recent times, this technology has been used in the analysis of volatile
compounds in several foods such as the detection of important components in milk for
the deodorization of malodorous odor in breath after ingestion of raw garlic
(Hansanugrum and Barringer 2010), the effect of the alkalization of cocoa beans before
or after roasting on the formation of alkylpyrazines (Huang and Barringer 2010), the
analysis of volatiles in tomatillo during chewing (Xu and Barringer 2010) and the effect
of enzyme activity and frozen storage on jalapeno pepper volatiles (Azcarate and
Barringer 2010).
47
CHAPTER 2
VOLATILE PROFILE OF CASHEWS (ANACARDIUM OCCIDENTALE L.)
FROM DIFFERENT GEOGRAPHICAL ORIGINS DURING ROASTING
2.1 Abstract
Volatile compounds were quantified in the headspace of Indian, Vietnamese and
Brazilian cashews, both raw and during roasting by Selected Ion Flow Tube-Mass
Spectrometry (SIFT-MS). The optimum roasting times based on color measurements
were also determined. Raw cashews were oil roasted for 3 to 9 min at 143 ºC and color
and volatiles measured. An excellent correlation, following a pseudo-first order reaction,
was found between L value and roasting time; darkness increases as roasting time
increases. The optimum roasting time was 6, 8 and 9 min for Vietnamese, Indian and
Brazilian cashews, respectively. Raw cashews had lower concentrations of volatiles than
roasted cashews. Most volatiles significantly increased in concentration during roasting
of Brazilian, Indian and Vietnamese cashews. Only a few volatiles significantly
decreased during roasting. Ethanol and 1-heptene significantly decreased during roasting
in Brazilian cashews and toluene decreased in Vietnamese cashews. Brazilian cashews
had significantly higher levels (p = 0.05) of most volatiles than Indian and Vietnamese
cashews. Most volatile levels in Indian and Vietnamese cashews were not significantly
52
different. Of the volatiles, Strecker aldehydes including methylbutanal, 2-methylpropanal
and acetaldehyde were at the highest concentration in roasted cashews. The Maillard
reaction contributed to formation of most of volatiles in cashews from the three countries.
There was also degradation of sugars to form furan type compounds and oxidation of
lipids to form alkanals such as hexanal.
Key Words: Volatile compounds, selected ion flow tube mass spectrometry, optimum
roasting time, Maillard reaction
2.2 Practical Applications
The volatile profile during roasting of cashews can be used to determine the best
roasting time for each type of cashew. The rate of color development and the production
of volatiles differ for the cashews from the three geographical locations.
2.3 Introduction
Cashew nuts are widely eaten for their desirable sensory characteristics as well as
health benefits. They are heated by boiling or roasting to facilitate shelling, which is the
biggest limitation in processing because of the uneven shape of cashew nuts and the
presence of caustic liquid within the shell. Roasting is the most important step in
developing cashew aroma (Mexis and Kontominans 2009). Roasting accelerates chemical
reactions such as the Maillard reaction and degradation of lipids to generate a pleasant
color and aroma. Various reactions are responsible for roasted aroma: the Maillard
reaction between amino acids and sugars, lipid degradation (thermal and oxidative),
thermal degradation of sugars and ascorbic acid and non-enzymatic lipid-protein
53
browning. The products of the Maillard reaction include aldehydes, furan derivatives and
nitrogen heterocyclic compounds such as pyrazines. Lipid degradation products can
produce either deterioration or improvement by producing a variety of oxygen containing
compounds such as alkanals, esters and alcohol. Thermal degradation of sugars yields
heterocyclic oxygen-containing compounds such as furan type compounds (Coleman and
others 1994). Ascorbic acid degradation yields maltol (Franco and Janzantti 2005) and 5hydroxymethyl-2-furfural (Damasceno and others 2008). In non-enzymatic lipid-protein
browning, proteins such as amino acids can react with lipid related hydroperoxides and
their degradation products causing browning (Mexis and Kontominas 2009).
Selected ion flow tube mass spectrometry (SIFT-MS) allows real-time analysis of
complex mixtures of volatile compounds without trapping or pre-concentration (Spanel
and Smith 1999a) by chemically ionizing the volatile compounds with H3O+, NO+, or
O2+. SIFT-MS has been used for many applications. It has been applied in the analysis of
volatiles generated from the lipoxygenase pathway in tomato (Xu and Barringer 2009),
detection of important components in milk for the deodorization of garlic breath
(Hansanugrum and Barringer 2010), the effect of alkalization of cocoa beans before or
after roasting on the formation of alkylpyrazines (Huang and Barringer 2010), effect of
enzyme activity and frozen storage on jalapeno pepper volatiles (Azcarate and Barringer
2010), effect of enzymes on strawberry volatiles during storage (Ozcan and Barringer
2010), the oxidation of olive oil (Davis and McEwan 2007) and the analysis of aroma
volatiles in dry fermented sausages (Olivares and others 2010).
54
The objectives of this study are to quantify the level of volatiles generated from
cashews of different geographical origins, determine how roasting time affects volatile
generation, and determine the correlation between the optimum roasting time and color
formation.
2.4 Materials and Methods
Raw cashews from India, Vietnam and Brazil (Kraft Foods and Planters
Company, Fort Smith, Arkansas, U.S.A) were stored in a sealed container under
refrigeration in the dark to minimize lipid oxidation.
2.4.1 Optimum Roasting Time
Samples (50 g) were roasted in 1L cottonseed cooking oil (Kraft Food and
Planter Company, East Hanover, New Jersey, U.S.A) using an electric fryer (National
Presto Industries Inc., Eau Claire, Wisconsin, U.S.A), then put on a sieve to drain
overnight. All roasting was carried out at 143 ºC on whole nut. The cashews were roasted
from 3 to 9 min. This test was repeated in triplicate. The optimum roasting time was
selected based on industry standards for color development.
2.4.2 Color Measurements
A Color Quest XE colorimeter (Hunter Associate Laboratory, Inc., Virginia,
U.S.A) with D65 light source and 10º viewing angle was used to measure the reflectance
spectral included (RSI) of the external surface of the side of raw and oil-roasted cashews.
The nuts were filled to the top of a 30 ml clear glass container (Corning Inc., Corning,
55
New York, U.S.A), and the external surface was measured.
2.4.3 Sample Preparation
Samples (50 g) of cashews were blended in an electric blender (Sunbeam Oskar
Jr., Niles, Illinois, U.S.A) for 30 s. The ground cashews were transferred into a 500 ml
Pyrex bottle and capped with open top caps coupled to polytetrafluoroethylene (PTFE)
faced silicone septa. The samples were held in a temperature controlled water bath
(Precision, Jouan Inc., Winchester, Virginia, U.S.A) at 50 ˚C for 60 min before volatiles
were measured.
2.4.4 Analysis of Volatiles in Ground Cashew
A selected ion flow tube mass spectrometer (SIFT-MS) (SYFT Voice 100, Syft
Ltd, Christchurch, New Zealand) was used to detect and quantify the volatile compounds
released from ground cashews. The headspace volatile compounds were sampled directly
by coupling the inlet port of the instrument with an 18 gauge 3.8-cm-long stainless steel
piercing the septa. The septa were also pierced with a 14 gauge 15-cm-long stainless steel
needle to maintain the pressure in the bottle at atmospheric pressure. The tips of the short
and long needle were located 12 cm and 1 cm from the surface of the sample,
respectively. Measurement of volatile compounds in the headspace above the cashew was
started immediately and continued for 2 min. A blank air sample for each bottle was run
and the order of analysis was randomized in order to account for any interference. The
flow tube pressure during SIFT-MS run was 0.038 ± 0.003 Torr. The temperature of the
capillary and arm was automatically maintained at 120 °C.
56
A selected ion mode (SIM) method with H3O+, NO+, or O2+ as precursors was
developed based on compounds known to be present in cashews (Jayalekshmy and
Narayanan 1989; Franco and Janzantti 2005; Maia and others 2000). SIFT-MS analyses
do not identify the compounds unambiguously, however on the assumption of their
presence, they provide robust values of their concentrations that can be used to compare
between samples, which is the focus of this study.
The measured m/z produced by reaction with the reagent ions must be carefully
chosen because many m/z are produced by several different volatiles, which create an
irresolvable interference that must be removed or the results must be reported as a
mixture (Spanel and Smith 1997). Initial full mass scans were performed and for each
compound the m/z that produced the lowest concentration was chosen, to minimize
interference from unknown compounds. If that m/z was produced by multiple compounds
in cashews, it was either not reported, or the compound was reported as a mixture.
Methylbutanal represents a mixture of 2-methylbutanal and 3-methylbutanal;
dimethylpyrazine is 2,3- 2,5 and 2,6-dimethylpyrazine and 2-ethylpyrazine; mono and
cyclic terpenes include terpenes such as limonene, terpienene, ocimene, myrcene and
pinene.
The known rate coefficients were used by the software for the calculations and
are provided in Table 2.1:
57
Table 2.1: Kinetics Parameters for SIFT-MS Analysis of Selected Volatile
Compounds.
Compound
Acids
acetic acid
Aldehydes
acetaldehyde
benzaldehyde
(E)-2-hexenal
heptanal
hexanal
methylbutanal
2-methylpropanal
nonanal
propanal
Alcohols
Precursor
Ion
Product Ion
K(10-9 cm3
s-1)
m/z
Ref.
NO+
NO+.CH3COOH.H2O
0.9
108
7
O2+
NO+
NO+
NO+
NO+
H3O+
O2+
O2+
NO+
C2H4O+
C7H5O+
C6H9O+
C7H13O+
C6H11O+
C5H10O.H+
C4H8O+
C10H18+
C3H5O+
2.3
2.8
3.8
3.3
2.5
3.7
3
3.2
2.5
44
105
97
113
99
87
72
138
57
6
6
6
8
6
5
8
9
6
ethanol
1-phenylethanol
NO+
NO+
methanol
2-methyl-1butanol
1-pentanol
Alkanes
decane
heptane
hexane
nonane
pentane
Alkenes
1-heptene
Aromatic
Hydrocarbons
styrene
toluene
Esters
ethyl buanoate
methyl benzoate
H3O+
C2H5O+, C2H5O+.H2O,
C2H5O+.2H2O
C8H10+
CH5O+, CH3OH2+.H2O,
CH3OH.H+.(H2O)2
O2+
NO+
2.7
45,
63, 81
106
33,
51, 69
C5H8+
C5H11O+
2.3
2.5
68
87
10
6
NO+
H3O+
O2+
H3O+
O2+
C10H21+
C7H16+
C6H14+
C9H20.H3O+
C3H6+
1.5
0.26
1.76
1.3
1.6
141
119
86
147
42
7
1
7
4
7
NO+
CH4.NO+
2
46
2
O2+
NO+
C8H8+
C7H8+
1.8
1.7
104
92
9
7
O2+
NO+
C6H12O2+
NO .C6H5COOCH3
2.5
1.5
116
166
3
7
+
58
1.2
2.2
7
10
9
Table 2.1 Continued
Furans
furans
furfural
5-hydroxymethyl2-furfural
Ketones
acetone
butanone
6-methyl-5heptene-2-one
Pyrazines
dimethylpyrazines
2-methylpyrazine
trimethylpyrazines
Terpenes
mono and cyclic
NO+
NO+
C4H4O+
C5H4O2+
1.7
3.2
68
96
10
10
NO+
C6H6O3+
2.5
126
9
NO+
NO+
C3H6O+
C4H8O+
1.2
2.8
88
102
6
6
O 2+
C8H14O+
2.5
126
9
O 2+
NO+
O 2+
C6H8N2+
C5H6N2+
C7H10N2+
2.7
2.8
2.5
108
94
122
9
9
9
NO+
C10H16+
2
136
11
References: [1] Arnold and others 1998, [2] Diskin and others 2002, [3] Francis and
others (2007a), [4] Francis and others (2007b), [5] Michel and others (2005),[6] Spanel
and Smith (1997), [7] Spanel and Smith (1998c), [8] Spanel and others (2002a), [9] Syft
Technologies (2011), [10] Wang and others (2004b), [11] Wang and others (2003).
2.4.5 Statistical Analysis
The mean, standard deviation and % relative standard deviation (RSD) values of
three replicates of each ground cashew were calculated by 2007 Microsoft Excel. Data
were analyzed by one-way analysis of variance (ANOVA) using Tukey’s procedure.
Significance was defined as p ≤ 0.05 acceptance of null hypothesis by using SPSS
(version 18, 2009, SPSS Inc., Chicago, Illinois, U.S.A).
59
2.5 Results and Discussion
The darkness of the color after roasting was measured by the L value, which
measures the black to white scale (Damasceno and others 2008). The darkness
(browning reaction) increased as roasting time increased (Figure 2.1). The industry
standard of color for the roasted nut is an L value of 54 for Indian and Vietnamese
cashews and 52 for Brazilian cashew. Each cashew required different roasting times to
reach the commercial standard for L value, which was 6, 8 and 9 min for Vietnamese,
Indian and Brazilian cashews, respectively. The difference in the time to reach the
desired level of browning is affected by differences in geographical origin and
availability of uniform heat during shelling (Franco and Janzantti 2005).
The browning in the heated cashew nuts is due to the Maillard reaction, a nonenzymatic reaction between amino acids and the carbonyl group of reducing sugars
which produces a wide array of strecker aldehydes, pyrazines and other compounds
(Coleman and others 1994). While it is a complicated reaction, many authors have shown
that both the Maillard reaction and the L value used to monitor the color generated by the
Maillard reaction, follow a pseudo first order reaction. The change in the L value with
roasting time fits a first order reaction equation with an R2 > 0.92 for all of the cashews
(Figure 2.1). The equations for Indian and Vietnamese cashews were very similar while
the equation for the Brazilian cashews was different. Brazilian cashews are generally
considered to be the best quality.
60
67
Indian Cashew
y = 0.1196x2 - 2.4786x + 66.514
65
Brazilian Cashew
y = -0.0356x2 - 0.7233x + 61.631
63
Vietnamese Cashew
y = 0.0847x2 - 2.0613x + 63.543
Poly. (Vietnamese
Cashew)
Poly. (Vietnamese
Cashew)
Poly. (Vietnamese
Cashew)
L Value
61
59
57
55
*
53
*
*
51
0
2
4
6
8
10
Roasting Time (min)
Figure 2.1: L Value for Different Cashews versus Roasting Time and Equations for
Pseudo-1st Order Reactions.
*: L Value for Indian, Vietnamese and Brazilian cashews at Optimum Roasting
Times.
2.5.1 Raw Cashews
In raw Brazilian cashews, methanol and ethanol were at the highest
concentrations among the organic volatiles (Table 2.2). They do not contribute to aroma;
however, alcohols contribute to the formation of esters which usually have fruity odors
(Ozcan and Barringer 2010; Vitova and others 2007). Acetic acid, acetone, 1phenylethanol and 1-heptene were at the next highest concentration. Acetic acid
contributes to a pungent aroma (Mexis and Kontominans 2009) and was in lower
concentration in Indian cashews than Brazilian cashews. Raw cashew has a strong
pungent aroma (Jayalekshmy and Narayanan 1989). Additionally, acids strongly
61
contribute to pungent aroma in cashew apple to which the cashew nut is attached (Maarse
1991). Phenylethanol is responsible for a sweet and floral aroma (Garruti and others
2006), while heptane does not contribute to aroma (Vitova and others 2006). Most
volatiles in raw Brazilian cashew were significantly higher than in Indian and Vietnamese
cashews. Most volatile levels in raw Indian and Vietnamese cashews were not different.
2.5.2 Roasted Cashews
Most volatiles significantly increased in concentration during roasting of
Brazilian, Indian and Vietnamese cashews (Table 2.2). Only a few volatiles significantly
decreased during roasting. Ethanol and 1-heptene significantly decreased during roasting
in Brazilian cashews (Figure 2.2) and toluene decreased in Vietnamese cashews (Table
2.2). 1-Heptene is responsible for a fatty aroma in many lipid foods (Brewer 2009) and
toluene is responsible for a green, fatty and lard odor (Clark and Nursten 1976) while
ethanol does not have a major contribution to aroma (Vitova and others 2007).
Roasted cashews have a mild and nutty aroma. This is due to pyrazines which are
generated from the Maillard reaction and are mostly responsible for a roasted and nutty
aroma in oil roasted cashews (Jayalekshmy and Narayanan 1989). There was a significant
increase in the concentration of Maillard reaction volatiles during roasting (Figure 2.3).
62
Table 2.2: Concentration (µg/L) of Selected Organic Volatiles in Raw and Roasted
Cashews.
Compound
Acids
acetic acid
Aldehydes
acetaldehyde
2-methylpropanal
benzaldehyde
hexanal
methylbutanal
propanal
heptanal
nonanal
(E)-2-hexenal
Alcohols
methanol
ethanol
1-phenylethanol
2-methyl-1-butanol
1-pentanol
Alkanes
heptane
pentane
hexane
decane
nonane
Alkenes
1-heptene
Aromatic
Hydrocarbons
toluene
styrene
Esters
ethyl buanoate
Furans
furan
HMF
furfural
Indian Cashew
Raw 8 min*
Vietnamese Cashew
Raw 6 min* 8 min
Brazilian Cashew
Raw
8 min 9 min*
707e
1430ab
821de
908de
850de
1040cd
1310bc
1630a
36e
33c
22e
22c
19d
11d
6c
5b
4d
368cd
148b
63cd
226a
495b
73c
13b
24b
15c
64e
70c
20e
40c
43c
20d
20a
15b
13c
351cd
129b
47d
182ab
713ab
92bc
19a
41b
15c
494bc
178b
55cd
187ab
937a
131ab
20a
43b
11c
174de
74c
70c
76bc
154c
32cd
6c
17b
17bc
903a
457a
109b
201ab
914a
192a
14b
114a
21b
912a
492a
133a
203ab
941a
194a
17ab
120a
30a
5000c
435c
153c
21e
6d
29600b
260c
139c
114cd
14cd
5960c 25500b
224c
318c
292b
177c
36de
102cd
d
7
20bc
27300b
322c
126c
122c
26b
28200b 86600a
7530a 3700b
413a
370a
73cde 326b
16c
28ab
93600a
3160b
377a
419a
35a
49b
34d
32c
15cb
5b
1160b
398b
196bc
30abc
93b
93b
56d
57c
35ab
7b
15c
13c
11c
19c
41d
13b
47cd
47b
133a
11b
62bcd
39b
3b
54b
21b
82b
3b
3b
2c
19b
11b
6b
4b
2b
2c
11b
8b
7b
63
1050b 1390b
154cd
203c
280b
315b
46a
43a
87b
98b
553b
109d
100c
11c
51b
5410a
1110a
496a
27abc
341a
6000a
1160a
541a
19cb
408a
20c
181a
105b
104b
47cd
52b
73bc
71b
51bcd
184a
76b
233a
118b
25b
355a
421a
14b
9b
9b
13b
3b
6b
47a
20a
17a
50a
31a
20a
Table 2.2 Continued
Ketones
acetone
butanone
6-methyl-5-heptene2-one
Pyrazines
dimethylpyrazines
2-methylpyrazine
trimethylpyrazines
Terpenes
mono and cyclic
70e
16c
332cd
26c
195de
35bc
404cd
38bc
431c
53b
1080b
48b
2130a
100a
2340a
111a
5c
10c
12c
15c
15c
8c
46b
86a
4b
2c
0b
16b
7bc
5b
4b
3c
2b
19b
8bc
6b
29b
11b
8b
18b
10bc
4b
82a
22a
25a
87a
25a
35a
25b
98a
20b
98a
99a
14b
95a
100a
*: Optimum roasting time for each three cashew type.
abcde
: Different letters in the same row indicate significant differences between concentrations.
HMF is 5-hydroxymethyl-2-furfural.
64
ethanol
9000
8000
200
7000
6000
150
5000
4000
100
3000
2000
50
Concentration (µg/L)
Concentration (µg/L)
250
1-heptene
1000
0
0
0
2
4
6
8
10
Roasting Time (min)
Figure 2.2: Organic Volatiles in Raw Brazilian Cashew Decrease in Concentration
during Roasting. 1-heptene is plotted on the right hand axis.
In Brazilian cashew, methylbutanal, 2-methypropanal, acetaldehyde, and
dimethylpyrazine increased in concentration by more than a factor of 6, 6, 5 and 4,
respectively compared to raw cashew (Table 2.3). The increase in concentration as
roasting progresses is directly correlated to the Strecker aldehyde formation pathway
during the Maillard reaction (Maarse 1991). Methylbutanal (a mixture of 3 and 2methylbutanal) is formed from free amino acids such as isoleucine and leucine; and 2methylpropanal is formed from valine. They are responsible for a pleasing odor in many
roasted foods (Coleman and others 1994). Among the pyrazines, dimethylpyrazines are in
the highest concentration (Table 2.3). Dimethylpyrazines are formed from the reaction of
many different amino acids and glucose during Strecker degradation (Hwang and others
1993).
65
Also, acetaldehyde is formed from the reaction of alanine with reducing sugar
(Salem and others 1967), and is responsible for a pungent aroma in cashew nuts (Mexis
and Kontominans 2009).
Table 2.3: Concentration (µg/L) of Selected Organic Volatile in Brazilian Cashew
during Roasting.
Compound
Raw
3 min
Acids
acetic acid
1040b 1150b
Aldehydes
acetaldehyde
174d
297cd
methylbutanal
154d
343cd
hexanal
76c
113bc
d
2-methylpropanal
74
116cd
benzaldehyde
70c
86bc
propanal
32e
59de
nonanal
17c
23bc
(E)-2-hexenal
17cd
18cd
heptanal
6d
7d
Alcohols
methanol
28200c 30900c
ethanol
7530a
5400b
1-phenylethanol
413a
361ab
2-methyl-1-butanol
73c
138c
1-pentanol
16c
18c
Alkanes
heptane
553c
848bc
pentane
109e
209e
hexane
100b
141b
c
decane
51
72c
nonane
11b
14ab
Alkenes
1-heptene
181a
153ab
Aromatic
Hydrocarbons
toluene
73a
58ab
Brazilian Cashew Roasted at Different Times
4 min 5 min
6 min
7 min
8 min 9 min
1050b
377cd
389bcd
124bc
158cd
94b
66de
24bc
16cd
8cd
1010b
467bc
474bc
142abc
216bc
96b
86cde
32bc
18cd
9bcd
895b
1360ab
1310ab
1630a
485bc
485bc
146abc
268b
100ab
96cd
39bc
12d
12bc
671b
675ab
155ab
395a
106ab
135bc
90ab
24ab
13bc
903a
914a
202a
457a
109ab
192ab
114a
21bc
14ab
912a
941a
203a
492a
133a
194a
120a
30a
17a
37300c 45500c
4950bc 4930bc
344ab
314b
150c
160c
19bc
21bc
47200c 70800b
4000bcd 3870bcd
291b
302b
161c
278b
23bc
27ab
86600ab 93600a
3700cd 3160d
370ab 377ab
326ab 419a
28ab
35a
1150bc 1480bc
358ed 663cd
165b
191b
bc
100
101bc
17ab
19ab
1620bc
767bc
223b
122bc
14ab
3000b
1080ab
399a
201b
19ab
5410a
1110ab
496a
341a
27a
6000a
1160a
541a
408a
19ab
151ab
137abc
115bc
110bc
105c
104c
56ab
45b
49b
64ab
51b
76a
66
Table 2.3 Continued
Aromatic
Hydrocarbons
styrene
Esters
ethyl buanoate
methyl benzoate
Furans
furans
furfural
HMF
Ketones
acetone
butanone
6-methyl-5heptene-2-one
Pyrazines
dimethylpyrazines
2-methylpyrazine
trimethylpyrazines
Terpenes
mono and cyclic
71c
72c
77c
84c
90c
148bc
184ab
233a
25c
0b
50c
43ab
73c
55ab
101c
55ab
143bc
26ab
270abc
50ab
355ab
45ab
421a
105a
13c
6d
3d
14c
7cd
6d
15c
8cd
7cd
20c
9cd
8cd
23bc
11cd
10cd
36abc
13bc
17bc
47ab
17ab
20b
50a
20a
31a
1080f
48c
1370ef
50c
1470ed 1560ed
51c
53c
1730cd
56c
1940bc
80b
2130ab
100a
2340a
111a
8d
11c
17c
19bc
20bc
46b
46b
86a
18c
10c
4c
19c
11c
7c
21c
13bc
8c
25bc
14bc
9c
27bc
15bc
12bc
49bc
16bc
15bc
82a
22ab
25ab
87a
25a
35a
20c
50bc
63abc
79ab
81ab
96a
98a
99a
*: Optimum roasting time for Brazilian Cashew.
Abcdef
: Different letters in the same row indicate significant difference in concentrations.
67
methylbutanal
dimethylpyrazine
1000
Concentration (µg/L)
120
2-methylpropanal
100
2-methylpyrazine
trimethylpyrazine
800
80
600
60
400
40
200
20
0
Concentration (µg/L)
1200
0
0
2
4
6
8
10
Roasting Time (min)
Figure 2.3: Maillard Reaction Volatiles in Brazilian Cashew during Roasting. Di,
2- and trimetheylpyrazines are plotted in the right.
The concentration of Maillard volatiles formed in aqueous extracts of cashew
increases as the amount of free amino acids increase (Coleman and others 1994), thus
amino acid concentration is the limiting factor in the reaction. Maillard reaction volatiles
such as Strecker aldehydes and dimethylpyrazines are at a higher concentration in
Brazilian cashews than in Indian and Vietnamese cashews, which were not significantly
different from each other (Table 2.2). Differences in the amino acid profile may be
responsible for the higher concentration of Maillard products in the Brazilian cashews,
which are known to be preferred over Indian and Vietnamese cashews.
The volatiles measured in the three cashews were mostly alcohols, alkanes,
aldehydes, acids and ketones with a small number of pyrazines, esters, alkenes and
68
oxygen and nitrogen containing heterocyclic compounds. During roasting, most volatiles
in the three varieties of cashews increased by roughly the same percentage. Excluding the
volatiles that decreased in concentration, acetic acid, butanone, acetone, methanol, 1phenylethanol and 1-pentanol increased noticeably less than the other volatiles during
roasting. When roasted, Brazilian cashews had significantly higher levels of most
volatiles than Indian and Vietnamese cashews (Table 2.2). Most volatile levels in Indian
and Vietnamese cashews were not significantly different.
Acetone and acetic acid were in high concentration in the three roasted cashews
especially in Brazilian cashew (Table 2.2). The maximum concentration of acetic acid in
the headspace of the Brazilian cashew was significantly higher than in the Indian and
Vietnamese cashews. Acetic acid is formed in the early stages of the Maillard reaction,
between glycine and D-xylose (Davidek and others 2008), and acetone is a result of the
reaction of D-xylose with valine. Additionally, acetic acid is formed from decomposition
of oleic acid and ketones are formed from decomposition of linoleic acid (Mexis and
Kontominans 2009).
Cashew nut is a good source of lipids (47.8 g/100 g cashew). Of the lipids, 25.9 g
is oleic acid and 8.5 g is linoleic acid (USDA 2010). Lipid decomposition volatiles in
roasted cashews were significantly higher in concentration than in raw cashews (Figure
2.4).
69
Many aliphatic aldehydes such as hexanal and nonanal and aliphatic hydrocarbons
such as nonane are formed from decomposition of linoleic acid and oleic acid, and
increased in concentration during roasting. Hexanal and nonane are responsible for green
and herbaceous odors in cashew nuts while nonanal is responsible for a fatty aroma.
Hexanal should be present only at low concentrations to improve the aroma and avoid a
rancid flavor (Mexis and Kontominans 2009). Short chain esters such as ethyl butanoate
are responsible for a sweet, fruity and cashew like aroma (Franco and Janzantti 2005).
600
ethylbutanoate
nonane
Concentration (µg/L)
500
hexanal
nonanal
400
300
200
100
0
0
2
4
6
8
10
Roasting Time (min)
Figure 2.4: Lipid Degradation Volatiles in Brazilian Cashew.
70
There was also degradation of sugars to form furan type compounds such as
furan, furfural and 5-hydroxymethyl-2-furfural (Table 2.2). Furan and its derivatives are
formed in many foods from the thermal degradation of fructose and glucose. Fructose
produces the highest level of furan (Fan 2005). Theoretically, fructose is more reactive
than glucose due to the increase in the rate of mutarotation resulting in more of the
reactive open chain furanose (Kim and Lee 2009). Furan type compounds were at a low
concentration in the three roasted cashews because the levels of fructose and glucose in
the raw and oil-roasted cashews are only 0.05 g and 0.08 g, respectively (USDA 2010).
Most Maillard reaction volatiles were at higher concentrations than the lipid
decomposition and sugar degradation volatiles. Based on level and type of characterized
volatiles, the Maillard reaction contributed the most to volatile formation in the three
varieties of cashew nuts.
2.6 Conclusion
The Maillard reaction is a major mechanism in the formation of most volatiles in
cashew nuts. Raw cashews have a lower concentration of volatiles than roasted cashews.
During roasting, the concentration of most volatiles in Brazilian, Indian and Vietnamese
cashews increased as a result of oil roasting with excellent correlations between roasting
time and L value. Only a few volatiles significantly decreased during roasting. Roasting
reduced the levels of ethanol and 1-heptene in Brazilian cashews and the level of toluene
in Vietnamese cashews. The cashews from different origin generate different
concentrations of volatile compounds.
71
CHAPTER 3
EFFECT OF ROASTING CONDITIONS ON COLOR AND VOLATILE
PROFILE INCLUDING HMF LEVEL IN SWEET ALMONDS (PRUNUS
DULCIS)
3.1 Abstract
Microwave, oven and oil roasting of almonds were used to promote almond flavor
and color formation. Raw pasteurized almonds were roasted in a microwave for 1 to 3
min, in an oven at 177 ºC for 5, 10, 15, and 20 min; and at 135 and 163 ºC for 20 min,
and in oil at 135, 163, and 177 ºC for 5 min and 177 ºC for 10 min. Volatile compounds
were quantified in the headspace of ground almonds, both raw and roasted, by Selected
Ion Flow Tube-Mass Spectrometry (SIFT-MS). Strong correlations were found between
L value, chroma, and 5-hydroxymethyl furfural (HMF); and were independent of roasting
method. Raw almonds had lower concentrations of most volatiles than roasted almonds.
Conditions that produced color equivalent to commercial samples were 2 min in the
microwave, 5 min at 177 ºC in the oven, and 5 min at 135 ºC in oil. Microwave heating
produced higher levels of most volatiles than oven and oil roasting at commercial color.
Sensory evaluation indicated microwave-roasted almonds had the strongest aroma and
were the most preferred. Oil-roasted almonds showed significantly lower levels of
volatiles than other methods, likely due to loss of these volatiles into the oil. Alcohols
72
such as benzyl alcohols and Strecker aldehydes including benzaldehyde and methional
were at higher concentrations than other volatiles in roasted almonds.The oxidation of
lipids to form alkanals such as nonanal and degradation of sugars to form furan type
compounds was also observed. The Maillard reaction contributed to the formation of
more of the total volatiles in almonds than the lipid oxidation reaction.
Key Words: Roasting time, temperature, HMF, Maillard reaction.
3.2 Practical Application
The level of 5-(hydroxy methyl)-2- furfural (HMF), color, volatile profile, and
sensory perception can be used to develop the best roasting method, time, and
temperature for almonds. The rate of color development and the production of volatiles
differ under different roasting conditions. Based on the color, volatile, and sensory
assessments of the three almonds, the use of microwave technology as a process for
roasting almonds reduces processing time and leads to an almond product with better
flavor than oven or oil roasting.
3.3 Introduction
The almond is a widely used nut and its composition is influenced by many
factors such as cultivar, moisture content, and season (Vazquez-Araujo and others 2009).
Sweet almonds are often roasted for consumer consumption. Application of heat by
roasting can enhance the Maillard reaction and degradation of lipids to produce a pleasant
color and aroma.
73
Also, roasting decreases the level of moisture, reduces the water activity,
increases the amount of carbon dioxide, and produces brittle roasted almonds (Severini
and others 2000). Legally, almonds must be pasteurized to kill the microorganisms in raw
almonds. Almonds can be roasted in vegetable oil, by microwave oven, and by hot air.
Roasting temperature and time affects the production of volatile compounds through the
Maillard reaction between amino acids and reducing sugars. Volatiles travel through the
olfactory epithelium and are expressed as a flavor in foods. Sweet almonds have a faint
and slightly nutty aroma (Vazquez-Araujo and others 2008, Wirthensohn and others
2008). Benzyl alcohol and benzaldehyde, which are well known to be important to the
aroma of bitter almonds, are potentially important aroma compounds in sweet almonds as
well (Wirthensohn and others 2008). Pyrazines such as dimethylpyrazine and 2methylpyrazine are generated during the Maillard reaction under thermal conditions
(Vazquez-Araujo and others 2008), and contribute to roasted and nutty aromas in roasted
almonds (Vazquez-Araujo and others 2009).
Heating of foods containing reducing sugars and amino acids also leads to
generation of 5-(hydroxy methyl)-2- furfural (HMF) which is an intermediate compound
of the Maillard reaction and is an indicator of the severity of the temperature. HMF
comes from enolization of reducing sugars that react with amino acids after Amadori
rearrangement and then condenses with nitrogenous compounds and polymerizes to
produce brown pigment in foods during heating. The quality of food is an important
issue; therefore, the browning in food should be controlled.
74
Several analytical techniques can be used to quantify the volatile organic
compounds in food applications such as selected ion flow tube mass spectrometry (SIFTMS). This method allows chemical ionization reactions to monitor the real-time analysis
of complex mixtures of the volatile compounds without pre-concentration (Spanel and
Smith 1999a). It utilizes the three positive precursor ions H3O+, NO+, and O2+ to ionize
volatile compounds (Spanel and Smith 1996) because they do not interact with major
components of air such as N2, O2, H2O and CO2, but can react rapidly with the volatile
compounds in the headspace above the sample (Spanel and Smith 1999a).The objectives
of this study are to identify the effect of method, time, and temperature on the generation
of color, HMF and other volatiles in almonds by roasting in a microwave, oven and
vegetable oil, using selected ion flow tube mass spectrometry (SIFT-MS). It is also to
determine the roasting conditions equivalent to commercial almonds.
3.4 Materials and Methods
Commercially steam pasteurized raw sweet almonds of the Prunus dulcis cultivar
(Paramount Farms, California, U.S.A) were used for the roasting experiments. The same
almonds dry roasted by the company were labeled Commercial #1. Another
commercially roasted almond (Nutcracker Brands Inc, Dothan, Alabama, U.S.A) was
labeled Commercial #2.
75
3.4.1 Roasting Conditions
Three roasting processes were used. The roasting was performed on 50 g samples
roasted in a 120 voltage microwave oven (Daewoo Electronics Inc., Lyndhurst, New
Jersey, U.S.A), in a regular oven (Oster, Boca Ration, Florida, U.S.A), and in 1L
cottonseed cooking oil (Kraft Food and Planter Company, East Hanover, New Jersey,
U.S.A) using an electric fryer (National Presto Industries Inc., Eau Claire, Wisconsin,
U.S.A).
The samples roasted in cottonseed oil were put on a sieve to drain overnight.
All roasting was carried out on the whole nut. The almonds were roasted in a microwave
from 1 to 3 min. The approximate internal temperature of the microwave-roasted almond
kernels meausered by a thermocouple meter (HH21, Type K thermocouple, Omega
Engineering, Inc. Stamford, Connecticut, U.S.A) and was 67, 108, and 152 ºC after 1, 2,
and 3 min, respectively. The oven-roasting was performed at 177 ºC for 5, 10, 15, and 20
min; and at 135 and 163 ºC for 20 min. The oven temperature was measured by a
thermocouple (Omega Inc., Stamford, Connecticut, U.S.A). The oil-roasting was done at
135, 163, and 177 ºC for 5 min and 177 ºC for 10 min. The temperatures and times for
the three roasting treatment were selected based on commercial processes and designed to
produce samples with acceptable flavor and color, but ranging from slightly under to
slightly over roasted. The tests were repeated in five replicates.
76
3.4.2 Color Measurements
Color determinations were made, at 25 ± 1 ºC, using a Color Quest XE
colorimeter (Hunter Associate Laboratory, Inc., Reston, Virginia, U.S.A). This
spectrophotometer utilizes an illuminant D65 and a 10º observer as references to measure
the reflectance spectral included (RSI) of the external surface of the raw and roasted
almonds. The nuts were filled to the top of a 30 ml clear glass container (Corning Inc.,
Corning, New York, U.S.A), and the external surface was measured. Color data are
Hunter CIELab coordinates, which represent the color in a three-dimensional space. L
indicates the lightness from 0 – 100. The chroma (coordinate C), is 0 at the center of a
color sphere and rises according to the distance from the center. It is calculated as C =
(a) 2+ (b) 2 . Color analysis was performed on 5 replicates for each heating process at each
time and temperature, and the value obtained was averaged.
3.4.3 Sample Preparation
Almond samples (50 g) were blended in an electric blender (Sunbeam, Oskar Jr.
Niles, Illinois, USA) for 30 sec. The ground almonds were transferred into a 500 ml
Pyrex bottle and capped with open top caps coupled to polytetrafluoroethylene (PTFE)
faced silicone septa. The samples were held in a temperature controlled water bath
(Precision, Jouan Inc., Winchester, Virginia, U.S.A) at 50˚C for 60 min before volatiles
were measured to allow equilibration of the volatiles released from the ground almonds.
77
Cottonseed oil samples (50 ml) that had been heated in an electric fryer (National
PrestoIndustries Inc., Eau Claire, Wisconsin, U.S.A) at 135 ºC for 5 min with or without
almonds present were tested for volatiles by the same method.
3.4.4 Measurement of Volatile Concentrations
A selected ion flow tube mass spectrometer (SIFT-MS) (SYFT Voice 100, Syft
Ltd, Christchurch, New Zealand) was used to detect and quantify the volatile compounds
released from ground almonds, using the method described by Agila and Barringer
(2011). The analysis was performed using selected ion mode (SIM) with H3O+, NO+, or
O2+ as precursors developed based on compounds known to be present in almonds
(Lesekan and Abbas 2010; Vazquez-Araujo and others 2008; Vazquez-Araujo and others
2009). SIFT-MS does not identify the compounds unambiguously; however, it can
provide robust values of their concentrations that can be used to compare between
samples. The absolute concentration was calculated by using the branching ratios and
pre-determined reaction rate constant for both volatiles and precursor ions (Smith and
Spanel 1996). The concentration [M] of selected volatiles was calculated using the
product count rate (Ip), reaction rate constant (k), precursor ions count rate (I) and
reaction time (t) as follows: [M] = Ip/Ikt (Spanel and Smith 1999a). Concentrations are
reported in µg/L in the headspace. Some compounds generate the same mass for a given
precursor ion, in which case the interfering compounds have to be reported as a mixture.
In this study menthone is a mixture of eucalyptol and menthone, acetophenone is a
mixture of phenylacetaldehyde and acetophenone, dimethylpyrazine represents
2, 3- 2, 5 and 2, 6-dimethylpyrazine and 2-ethylpyrazine; and terpenes includes limonene,
78
terpienene, ocimene, myrcene and pinene. The concentration of volatile compounds was
calculated using known kinetic parameters (Table 3.1). Compounds with irresolvable
conflicts or concentrations near the detection limit are not reported. Other conflicts were
removed by selecting different precursor ions in the method.
Table 3.1: Kinetics Parameters for SIFT-MS Analysis of Selected Volatile
Compounds in Almonds.
Precursor
Ion
Product Ion
k (10-9
cm3 s-1)
m/z
Ref.
NO+
C7H7O+
2.3
107
8
1-butanol
NO+
C4H9O+
2.2
73
4
ethanol
NO+
C2H5O+, C2H5O+.H2O,
C2H5O+.2H2O
1.2
45.63,81
4
2-heptanol
NO+
C7H14O.NO+
3.4
144
8
1-hexanol
NO
+
C6H13O
2.4
101
4
(Z)-3-hexen-1-ol
NO+
C6H10+
2.5
82
1
1-octanol
NO+
C8H17O+
2.3
129
4
methanol
H3O+
2.7
33,51,69
4
1-octen-3-ol
H3O+
CH5O+, CH3OH2+.H2O,
CH3OH.H+.(H2O)2
C8H15+
3.1
111
1
benzaldehyde
NO+
C7H5O+
2.8
105
4
heptanal
NO+
C7H13O+
3.3
113
5
(E)-2-heptenal
NO+
C7H11O+
3.9
111
5
(E)-2-hexenal
NO+
C6H9O+
3.8
97
4
methional
NO+
C4H8OS+
2.5
104
1
Compound
Alcohols
benzyl alcohol
+
Aldehydes
79
Table 3.1 Continued
Aldehydes
nonanal
O2+
C10H18+
3.2
138
1
(E)-2-nonenal
NO+
C9H15O+
3.8
139
5
(E)-2-octenal
+
C8H13O
+
4.1
125
5
Alkanes
dodecane
NO+
C12H25+
1.5
169
2
undecane
H3O+
C11H24.H3O+
2.4
175
1
NO+
C10H16+
2.2
136
6
NO+
C 7H 8+
1.7
92
2
furfural
NO+
C5H4O2+
3.2
96
8
5-(hydroxy
methyl)-2-furfural
Ketones
O2+
C6H6O3+, C6H6O3.H+
2.5
126, 127
1
acetoin
NO+
C4H8O2.NO+
2.5
118
1
1,4-butyralactone
O2+
C2H3O+ or C3H6+
3.4
42
8
Aromatic
Hydrocarbons
terpenes
toluene
NO
Furans
2-undecanone
+
+
H 3O
C11H22OH ,
C11H22OH+.H2O
4.3
171,
189
7
dimethyl pyrazine
NO+
C6N2H8+
2.8
108
1
2-methyl pyrazine
NO+
C5H6N2+
2.8
94
1
trimethylpyrazine
NO+
C7H10N2+
2.5
122
1
NO+
C4H5N+
2.5
67
3
Pyrazines
Pyrroles
pyrrole
[1] Syft Technologies (2011), [2] Spanel and Smith (1998a), [3] Spanel and Smith
(1998b), [4] Spanel and Smith (1997), [5] Spanel and others (2002a), [6] Wang and
others (2003), [7] Smith and others 2003b, [8] Wang and others (2004c).
80
3.4.5 Sensory Evaluation
Sensory analysis of almonds roasted by the three methods was performed by a 90
member untrained panel. Panelists were aged 22 to 80 and had no known food allergies.
Samples were prepared 1 hr prior to evaluation by roasting in a microwave for 2 min, an
oven at 177 ºC for 5 min, or oil at 135 ºC for 5 min. Panelists were asked to taste each
sample (5 almonds per treatment) and determine which sample had the most almond
flavor and aroma, and which sample was preferred most. The total Maillard reaction
volatiles were the sum of methional, benzyl alcohol, benzaldehyde, dimethylpyrazine,
trimethylpyrazine, HMF, pyrrole, toluene, 5-methylfurfural, and terpenes. The total lipid
oxidation volatiles were the sum of (E)-2-heptenal, nonanal, 1-octen-3-ol, (Z)-3-hexen-1ol, 1,4-butyrolactone, and 1-butanol, (E)-2-octenal, (E)-2-nonenal, undecane, 1-hexanol,
(E)-2-hexenal, heptanal, 2-heptanol, 1-octanol, octanal, decanal, 2-heptanone, hexyl
acetate, 2-undecanone, and dodecane.
3.4.6 Statistical Analysis
All analytical testing was done in 5 replicates. Data were subjected to
independent samples t-test and analysis of variance (ANOVA) using Tukey’s procedure
to determine significant difference among almonds processed at different times and
temperatures. Significance was defined as p ≤ 0.05 by using IBM SPSS Statistics
software (version 19, 2010, SPSS Inc., Chicago, Illinois, U.S.A).
81
3.5 Results and Discussion
3.5.1 Color
The value of almond lightness (L) significantly decreased during roasting, as the
samples became darker because of browning reactions (Table 3.2). Simultaneously, the
chroma (C) increased during roasting as the color intensity increased. The L and chroma
values were highly correlated (R2 = 0.81), with a small group of outliers. When all of the
lightly roasted samples (L = 45.1 to 45.9, Chroma = 11.5 to 11.9) were removed, the
correlation increased to R2 = 0.97. For the lightly roasted sample, there was a smaller
increase in chroma than was expected from the decrease in L, in other words, less red and
more black was formed than for the other samples. This may indicate a different pathway
during initial roasting compared to later roasting. The roasting conditions which produced
an L value equivalent to the two commercially roasted almonds were 2 min in a
microwave, 5 min at 177 ºC in an oven, and 5 min at 135 ºC in oil.
Table 3.2: L Value, Chroma, and Headspace HMF Concentration (µg/L) of
Almonds at Different Roasting Conditions. (n=5)
Roasting Process
L Value
Chroma
Raw
Commercial #1
Commercial #2
Microwave 1 min (67 ºC)
Microwave 2 min (108 ºC)
Microwave 3 min (152 ºC)
Oil 135 ºC 5 min
Oil 163 ºC 5 min
Oil 177 ºC 5 min
Oil 177 ºC 10 min
51.8a
45.9c
45.6cd
48.7b
45.1cdef
43.7hij
45.4cde
44.8defg
44.7efg
43.3ij
9.1f
11.5e
11.6e
10.9e
11.9e
15.5bcd
11.7e
14.3d
14.7cd
16.0bc
82
HMF (µg/L)
6f
41e
42e
18e
47e
302bc
43e
54e
64e
389b
Table 3.2 Continued
Roasting Process
L Value
Chroma
Oven 177 ºC 5 min
Oven 135 ºC 20 min
Oven 177 ºC 10 min
Oven 163 ºC 20 min
Oven 177 ºC 15 min
Oven 177 ºC 20 min
45.3cde
44.4fgh
44.1ghi
43.4ij
43.1j
40.2k
11.8e
14.8bcd
14.9bcd
15.7bc
16.1b
19.9a
HMF (µg/L)
45e
83de
119cd
341b
392b
905a
a-k
Different letters in the same column indicate significant differences in L value,
calculated chroma, or HMF concentration.
3.5.2 HMF
Browning can be used to estimate the progress of the Maillard reaction (Ledi and
Nursten1990). The rate of chemical reaction during roasting increases with increasing
temperature and time which accelerates the reaction of amino acids and reducing sugars
during heating and affects the production of volatile compounds including 5(hydroxymethyl)-2-furfural (HMF) (Surh and Tannenbaum 1994). HMF is produced in
foods exposed to heat and considered a significant indicator of excess heat treatment
(Burdulu and Karadeniz 2003); therefore, heating should be controlled. The headspace
concentration of HMF in commercial almonds was not significantly different from the
concentration of HMF in samples roasted in the microwave, oven, or oil to equivalent L
value (Table 3.2). In the headspace of raw almonds, HMF was at low concentration,
approximately 6 µg/L, while the concentration of HMF significantly increased with
increasing roasting time and temperature. HMF concentration was 41 µg/L for
83
commercial #1 and 42 µg/L for commercial #2. The highest level of HMF was in the
headspace of almonds roasted in an oven at 177 ºC for 20 min, at 905 µg/L. This
indicates exposure to severe temperatures.
HMF had a good correlation to L value and chroma, and was independent of the
roasting method (Figure 3.1); the lower the L value and higher the chroma, the higher the
HMF levels. The plot of L value and chroma versus HMF concentration shows two linear
relationships in a broken curve. There is an initial steep change in color with little HMF
accumulation, followed by a more gradual change in color with greater HMF
accumulation. Since HMF is an intermediate in the Maillard reaction, which produces a
variety of brown colored compounds as an endproduct, the change in slope likely
indicates a change in pathway. During roasting, moisture is lost, concentrating the acids
and possibly lowering the pH. As pH decreases, HMF formation increases as the pathway
shifts so that reducing sugars and amino acids produce more N-substituted glycosylamine
condensation products that rearrange to an Amadori product that forms hydroxymethyl
furfural (HMF), and at the same time color formation decreases (Martins and others
2001). Therefore this may indicate a shift in pH, but it is also possible that the decrease in
water activity or increase in temperature caused the shift in pathway. The HMF values of
both commercial almonds are located on the initial curve where large changes in color
occurred with slower HMF formation.
84
L Value
52
17
Chroma
51
16
50
15
14
48
47
13
46
12
*
45
Chroma
L Value
49
11
44
10
43
42
0
50
100
150
200
250
300
350
9
400
HMF Concentration
Figure 3.1: Headspace Concentration (µg/L) of HMF versus L Value and Chroma
(C) of Almonds after Different Roasting Conditions. Chroma is plotted on the right
hand axis. * Commercially roasted samples.
3.5.3 Raw Almonds
A few of the volatiles in raw almonds (Prunus dulcis) were at significantly lower
concentrations than in lightly roasted almonds, while most were not significantly
different, especially from oven and oil roasted almonds (Table 3.3). Methanol and
ethanol were at the highest concentrations among the volatiles. They may be generated
from decomposition of fatty acids or aldehyde reduction (Vazquez-Araujo and others
2008), but they do not contribute to aroma (Vitova and others 2007).
85
Toluene and benzaldehyde were at the next highest concentration. Toluene is
responsible for a paint aroma and benzaldehyde contributes to bitter and sweet almond
aromas (Vazquez-Araujo and others 2008, Wirthensohn and others 2008). Mexis and
Kontominas (2009) also found that benzaldehyde, but not toluene, was at higher levels
than any other volatiles in raw almonds.
Table 3.3: Concentration (µg/L) of Organic Volatiles in Raw Almonds and Almonds
Roasted to L Value of 45 (Light roasting). (n=5)
Compound
Raw
Microwave
2min
Oil
135 ºC
5min
Oven
177 ºC
5 min
Commercial Commercial
#1
#2
acetoin
24c
272a
26c
38c
184b
39c
benzaldehyde
32e
173a
68d
112b
124b
83c
benzyl alcohol
10d
501a
110c
126c
325b
107c
1,4-butyrolactone
10c
56b
14c
18c
51b
105a
1-butanol
27d
97a
54c
49c
68b
55bc
dimethylpyrazine
2c
112a
40b
53b
33b
30b
dodecane
3c
17a
3c
6bc
8b
4c
2940ab
3280a
ethanol
3350a
2810ab
2150bc
1810c
(E)-2-heptenal
3c
16b
4c
5c
12b
24a
(E)-2-hexenal
3b
28a
3b
8b
22a
7b
(E)-2-nonenal
1d
13b
1d
2d
17a
7c
(E)-2-octenal
1c
17a
3c
1c
10b
11b
furfural
3c
19a
3c
8bc
18a
11b
heptanal
9d
15c
9d
9d
18b
24a
86
Table 3.3 Continued
2-heptanol
2c
58a
2c
3c
30b
6c
1-hexanol
3b
14a
4b
6b
15a
14a
HMF
6b
43a
45a
47a
41a
42a
71300a
37800b
methanol
19400c
6500d
26500c
methional
10e
317b
98d
99d
402a
162c
nonanal
7b
85a
4b
15b
90a
38b
2-methylpyrazine
4c
52a
14b
17b
18b
14b
1-octen-3-ol
17c
55b
44b
54b
144a
57b
1-octanol
1c
24a
2c
2c
19a
9b
pyrrole
1c
24a
2c
2c
11b
3c
terpenes
6b
18a
6b
7b
16a
10b
toluene
33c
53b
56b
58b
128a
11d
trimethylpyrazine
5b
40a
5b
5b
36a
11b
undecane
1b
60a
4b
5b
70a
16b
13c
35a
13c
27ab
35a
21bc
(Z)-3-hexen-1-ol
a-d
31700bc
Different letters in the same row indicate significant differences between almonds.
3.5.4 Roasted Almonds
The concentration of volatile compounds increased with increasing roasting time
and temperature in the microwave, oven, and oil heating. The volatiles generated from
87
roasting almonds were mostly alcohols and aldehydes with a small number of pyrazines,
alkanes, ketones, and oxygen and nitrogen containing heterocyclic compounds. All
volatiles except ethanol significantly increased in concentration during roasting in the
microwave, oven, and oil (Tables 3.3, 3.4). There are few references in the literature on
the volatiles in roasted almonds, but the pyrazines, benzyl alcohol and benzaldehyde have
been reported to strongly contribute to the aroma of roasted almonds (Vazquez-Araujo
and others 2008; Vazquez-Araujo and others 2009, Wirthensohn and others 2008). In this
study, pyrazines such as dimethylpyrazine, trimethylpyrazine, and 2-methylpyrazine were
found in low concentrations, increasing in concentration during roasting, but they may
play an important role in the sensory aroma profile of almonds. The pyrazines, which
contribute to nutty and roasted aromas in roasted almonds, are formed during the
Maillard reaction between amino acids and reducing sugars under thermal conditions
(Vazquez-Araujo and others 2008). Benzaldehyde and benzyl alcohol, which were found
at higher levels than the pyrazines, have also been reported by others as major
compounds in almonds and almond oil (Beck and others 2011a; Wirthensohn and others
2008). Benzaldehyde is produced from phenylalanine under heat (Chu and Yaylayan
2008), and benzyl alcohol comes from the Maillard reaction (Mancilla-Margalli and
Lopez 2002), and is responsible for a sharp, faint, and burning taste (Vazquez-Araujo and
others 2008). Methional was also at higher concentrations in roasted almonds, similar to
the results of Vazquez-Araujo and others (2008). Methional contributes to vegetable and
creamy aromas (Indiamart 2011).
88
Table 3.4: Concentration (µg/L) of Volatiles in Almonds Roasted to L Values 44
(Medium) and 43 (Dark). (n=5)
Medium Color, L Value 44
Dark Color, L Value 43
Compound
Oil 163
ºC 5
min
Oil 177
ºC 5
min
Oil 177
ºC 10
min
Oil 177
ºC 10
min
acetoin
54e
84e
233d
169d
310c
384ab
331bc
409a
benzaldehyde
72d
109d
309bc
226c
398b
516a
382b
560a
benzyl alcohol
1,4butyrolactone
68c
120c
388b
495ab
559ab
599ab
431ab
617a
34c
56c
100b
74bc
160a
193a
162a
203a
dimethylpyrazine
4e
41de
174d
147de
582ab
491b
331c
695a
dodecane
5d
8d
8d
16cd
65ab
54abc
33bcd
94a
2270b
1370cd
1670cd
2950a
734e
1660cd
1250de
1830bc
(E)-2-hexenal
12e
13e
35de
35de
160a
100bc
63cd
117b
(E)-2-heptenal
8d
10d
27cd
21cd
84ab
70ab
56bc
95a
(E)-2-nonenal
3d
3d
15bc
14c
25b
25b
15bc
36a
(E)-2-octenal
5d
8d
18d
22cd
62a
51ab
40bc
59a
furfural
13d
20d
52cd
37cd
310a
165b
93bc
208a
heptanal
11e
11e
24de
20de
58ab
48bc
38cd
70a
2-heptanol
5e
41de
309cd
149de
570bc
824b
1170a
1-hexanol
8d
13d
38c
24cd
75ab
68b
98a
HMF
54c
64c
83bc
119b
302a
389a
392a
ethanol
methanol
Oven
135 ºC
20 min
39800d 45600d 58500abc
Oven
177 ºC
10 min
1150a
92ab
341a
57100bc 43800d 63400ab 47800cd
methional
115f
385ef
1020cd
nonanal
14c
43c
255c
182c
2-methylpyrazine
5d
19d
92d
72d
89
MW
3 min
707de 2530a
Oven177
ºC 15
min
69300a
1990b
1250c
2650a
656ab
586b
255c
945a
553a
325b
192c
370b
Table 3.4 Continued
1-octen-3-ol
38c
48c
245bc
142c
510a
394ab
204bc
557a
1-octanol
4d
17d
175c
73cd
397b
597a
406b
589a
pyrrole
3c
8c
87bc
46c
595a
594a
213b
537a
terpenes
8e
9e
27de
24de
68ab
54bc
41cd
85a
toluene
56d
55d
76cd
74cd
118ab
124a
95bc
128a
trimethylpyrazine
4d
11d
56c
45c
165a
144a
89b
156a
2-undecanone
6c
11c
62c
35c
366b
446b
239c
573a
undecane
7d
26d
239bc
97cd
330b
640a
389b
697a
24d
21d
88cd
57cd
574a
252bc
131cd
(Z)-3-hexen-1-ol
a-f
Different letters in the same row indicate significant differences between almonds.
MW: microwave.
Almonds are a high lipid food containing oleic acid as the major fatty acid,
followed by linoleic acid (Beck and others 2011b). Linoleic acid is a precursor to many
aldehydes and alcohols in foods (Perez and others 1999). The lipid oxidation volatiles
were aliphatic aldehydes such as (E)-2-heptenal and nonanal which formed from
decomposition of linoleic acid and oleic acid (Min and Smouse 1985). (E)-2-Heptenal is
responsible for pungent and green aromas (Vazquez-Araujo and others 2008), and
nonanal is responsible for tallow and fruity aromas (Lesekan and Abbas 2010).
90
269ab
Alcohols also were generated through lipid oxidation such as 1-octen-3-ol which
comes from thermal decomposition of methyl linoleate hydroperoxide (Min and Smouse
1985), and contributes to an herbaceous aroma (Vazquez-Araujo and others 2008).
Nonanal and 1-octen-3-ol were also found in high concentration in toasted almonds by
Vazquez-Araujo and others (2008). Also, (Z)-3-hexen-1-ol is produced from oxidation of
linolenic acid (Stone and others 1975) and is responsible for green grass and green leaf
aromas (Alchemist 2010). 1-Butanol is formed from decomposition of linolenic acid
(Min and Smouse 1985), and is responsible for an unripe apple aroma (Nykanen 1983).
Other lipid oxidation volatiles such as lactones including butyrolactone contribute to
milky and creamy aromas (Burdock 2004).
There was also thermal degradation of sugars such as fructose and glucose to
produce furan containing compounds such as furfural (Table 3.3). Furfural was also
found in toasted almonds by Vazquez-Araujo and others (2008). Almonds contain a low
level of sugars with levels of fructose and glucose in almonds at 0.09 and 0.12 g/100 g,
respectively (USDA 2011). This explains the low level of furfural in the light and
medium almonds.
3. 5. 5 Comparisons of Roasted Almonds
Microwave roasting produced the highest level of volatiles of the three roasting
methods in the commercially equivalent light colored almonds, L value 45. The higher
level of volatiles in microwave-roasted almonds may be due to the internal as well as the
external heating that occurs in the microwave.
91
If the entire almond were heating at once, volatile formation would occur
throughout, rather than just on the surface. The greater amount of reactants would create
a greater total amount of volatiles. After roasting, all of the microwave-roasted almonds
were browner on the inside than any of the oven or oil-roasted almonds, as another
indication that internal heating was occurring. Microwave roasting was also the closest in
volatile level to the commercially roasted samples. During sensory evaluation of the
almonds roasted by the three methods to produce the commercial L value, 83% of
panelists said that microwave-roasted almonds had the highest flavor and aroma,
followed by oil (10%) and oven (7%). The most preferred almonds were microwaveroasted (60%) followed by oven-roasted (28%) and oil-roasted (12%). Several of the
panelists commented that oven roasted almonds had a soft texture, oil roasted had an oily
texture, and microwave roasted had a crunchy texture which produced a cracking sound
In the medium and dark samples, oil-roasted almonds showed significantly lower levels
of volatiles than other methods at the same L value (Table 3.4). The low volatile
concentration in oil-roasted almonds compared to oven and microwave-roasted almonds
may be due to the transfer of some volatiles from the almonds into the oil during oil
roasting. The oil was heated with and without almonds present, and there was a
significant increase in many volatiles in the oil roasted with almonds present (Table 3.5).
Many of the volatiles that were lower in the oil roasted almonds were the same volatiles
that were higher in the oil heated with almonds. This may indicate the oil roasted
almonds were lower in volatiles because the volatiles were solubilized into the oil.
92
For seven of the volatiles, concentrations were higher in the oil roasted without
almonds than the oil roasted with almonds (Table 3.5). This implies that these volatiles
were absorbed from the oil into the oil-roasted almonds. The levels for these volatiles in
the almonds are inconclusive, in that they are not clearly higher in the oil roasted almonds
than in the other almonds.
Table 3.5: The Volatiles in Oil Samples Heated with and without Almonds Present.
(n=3)
Compound
Oil without Almonds
Oil with Almonds
acetoin
4a
5a
benzaldehyde
14b
22a
benzyl alcohol
5a
2b
438b
712a
1-butanol
6a
6a
dimethylpyrazine
3a
1b
dodecane
14a
13b
ethanol
41a
54a
(E)-2-hexenal
5b
23a
(E)-2-heptenal
48b
175a
(E)-2-octenal
6b
16a
furfural
5a
5a
heptanal
39a
25b
2-heptanol
1a
2a
1-hexanol
3b
8a
HMF
6a
10a
methanol
142b
375a
methional
5a
5a
1,4-butyrolactone
93
Table 3.5 Continued
Compound
Oil without Almonds
Oil with Almonds
b
12a
2-methylpyrazine
3a
1b
octanal
8b
23a
1-octen-3-ol
16b
43a
pyrrole
2a
1a
terpenes
0a
0a
toluene
3a
2b
trimethylpyrazine
5a
5a
2-undecanone
2a
2a
undecane
0a
0a
(Z)-3-hexen-1-ol
18a
15b
nonanal
10
a, b
Different letters in the same row indicate significant differences between oil with and
without almonds present.
The total concentration of Maillard-reaction-generated volatiles is greater than the
total concentration of lipid-oxidation-generated volatiles in all roasted almonds (Figure
3.2). Maillard reaction volatiles were also at higher levels than lipid oxidation volatiles
when comparing the same volatiles measured in three varieties of almonds by VazquezAraujo and others (2008). Maillard-reaction-generated volatiles have been shown to
reduce lipid oxidation in stored almonds (Severini and others 2000). The total Maillardreaction-produced volatiles were 33, 37, and 13 % greater than the total lipid-oxidation
volatiles at light, medium and dark degrees of color, respectively. In the dark samples, the
severe heat treatment significantly enhanced the production of both Maillard-reaction and
94
lipid-oxidation volatiles, and the percentage difference between Maillard-reaction and
lipid-oxidation generated volatiles decreased. This implies that the Maillard reaction
contributed the most to volatile formation in the roasted almonds at all temperatures and
times. However, lipid oxidation also significantly contributed to the generation of aroma
volatiles in roasted almonds. Thermal degradation of sugars also took place during
roasting but contributed a low percentage of the total volatiles.
Maillard Reaction/ MW
6200
Lipid Oxidation/ MW
Maillard Reaction/ Oven
Concentration (ppb)
5200
Lipid Oxidation/ Oven
Maillard Reaction/ Oil
4200
Lipid Oxidation/ Oil
3200
2200
1200
200
43
43.5
44
44.5
45
45.5
L Value
Figure 3.2: Concentrations (µg/L) of the Sum of Maillard Reaction and Lipid
Oxidation Volatiles in Almonds Roasted at Different Roasting Conditions.
MW: Microwave.
Unlike roasted almonds, in raw almonds, the lipid-oxidation-generated volatiles
were 21 % greater than Maillard-reaction-generated volatiles. Almonds contain 31 and 12
g/100 g of monounsaturated and polyunsaturated fatty acids (USDA 2011). This
95
composition makes almonds susceptible to oxidation during storage. The Maillard
reaction requires much higher temperature or longer times to produce significant
volatiles. This may explain why the lipid-oxidation-generated volatiles are higher in raw
almonds than Maillard-reaction-generated volatiles.
3.6 Conclusion
During roasting, the concentration of most volatiles in almonds increased as a
result of microwave, oven, and oil heating with excellent correlations between L values,
chroma, HMF concentrations, roasting times and temperatures. The roasting conditions
which produce color equivalent to commercial almonds were microwave heating for 2
min, oven heating at 177 ºC for 5 min and oil heating at 135 ºC for 5 min, with HMF
concentrations of 47, 45 and 43 µg/L, respectively. Microwave heating enhanced the
production of volatiles compared to oil and oven heating, and the flavor was the most
preferred by panelists. Oil heating showed significantly lower levels of volatiles than
other methods, due to loss of these volatiles into the oil. The percentage of Maillard
reaction generated volatiles was higher than the lipid decomposition and sugar
degradation volatiles. This implies that the Maillard reaction contributed the most to
volatile formation in the roasted almonds at all temperatures and times. However, lipid
oxidation also significantly contributes to the generation of aroma volatiles in roasted
almonds. Based on the color, volatile, and sensory assessments of the three almond
roasting methods, the use of microwave technology as a process for roasting almonds
reduced processing time and led to almonds with better flavor than oven and oil heating.
96
CHAPTER 4
APPLICATION OF SELECTED ION FLOW TUBE MASS SPECTROMETRY
COUPLED WITH CHEMOMETRICS TO STUDY THE EFFECT OF
LOCATION AND BOTANICAL ORIGIN ON VOLATILE PROFILE OF
UNIFLORAL AMERICAN HONEYS
4.1 Abstract
Ten Ohio and Indiana honey samples from star thistle (Centaurea Americana),
blueberry (Vaccinium spp.), clover (Trifolium spp.), cranberry (Vaccinium spp.),
wildflower, and an unknown source were collected. The headspace of these honeys was
analyzed by selected ion flow tube mass spectrometry (SIFT-MS) and soft independent
modeling of class analogy (SIMCA). SIMCA was utilized to statistically differentiate
between honeys based on their composition. Ohio honeys from star thistle, blueberry, and
clover were similar to each other in volatile composition, while Ohio wildflower honey
was different. Indiana honeys from star thistle, blueberry, and wildflower were different
from each other in volatile composition while clover and cranberry honeys were similar.
Honeys from Ohio and Indiana with the same floral origins were different in volatile
composition. Furfural, 1-octen-3-ol, butanoic and pentanoic acids were the volatiles with
the highest discriminating power between types of floral honey. Methanol and ethanol
followed by acetic acid were at the highest levels in most honeys, through furfural was at
97
the highest concentration in Indiana Wildflower honey. The highest concentration of
volatile compounds was in Indiana wildflower honey followed by Ohio wildflower honey
while the lowest concentration of volatile compounds was observed in Ohio clover honey
followed by Indiana clover honey.
Key Words: Selected ion flow tube mass spectrometry, floral origins, geographical
locations.
4.2 Practical Application
Using chemometrics, concentrations of volatile compounds in different honeys
can be used to determine the influence of botanical and geographical origins on aroma,
which is important for the quality of honey. Characterization of volatile compounds can
also be a useful tool for assessing honey quality.
4.3 Introduction
Honey is one of the oldest foods used by man and is known as a sweet, flavorful
and neutral food identified by its characteristic taste and aroma (Kaškonienė and others
2008). It is popular around the world and in every state in the US; however, some honey
types can be produced only in a few locations because of their floral sources. Aromarelated volatile compounds may be useful to determine honey origin and location. The
US produces more than 300 types of honey. These honeys can be unifloral and come
from a single plant such blueberry (Vaccinium spp.), clover (Trifolium spp.), star thistle
(Centaurea Americana) and cranberry honey (Vaccinium spp.).
98
Other honeys are polyfloral such as wildflower (mixed seasonal wildflowers)
which comes from various unknown flower sources collected in the summer. Some
volatile compounds are found at high levels in different honeys such as furfural and
benzaldehyde in clover, blueberry, wildflower (Overton and Manura 1994) and thistle
honeys (Bianchi and others 2011). Other volatiles compounds such as acetic acid (de la
Fuente and others 2005) and acetone (Pérez and others 2002) are also detected at high
levels in different honeys. Some volatile compounds are considered to be characteristic of
different unifloral honeys including benzene derivatives such as benzaldehyde in acacia
honey and terpenes including linalool in marmeleiro honey (Cuevas-Glory and others
2007).
The industry is looking for a fast way to characterize unifloral honeys, and
selected ion flow tube mass spectrometric (SIFT-MS) technology may be ideal for rapid
identification of volatile compounds in foods with minimal sample preparation. This
technique does real-time analysis of complex mixtures of volatile compounds (Spanel
and Smith 1999a) by utilizing three positive precursor ions H3O+, NO+, and O2+ to ionize
volatile compounds. These precursor ions are ideal because they do not interact with
major components of air such as N2, O2, H2O and CO2, but can interact quickly with the
volatile compounds in the sample headspace (Spanel and Smith 1999a).
99
SIFT-MS has been used to analyze the volatile compounds in various foods such
as the volatiles produced from the lipoxygenase pathway in tomato (Xu and Barringer
2009), the volatiles generated from the oxidation of dry fermented sausage (Olivares and
others 2010), the volatiles in strawberry during storage (Ozcan and Barringer 2010) and
the volatile compounds in roasted pumpkin seeds (Bowman and Barringer 2012).
Chemometrics involve multivariate analysis to provide accurate, robust, and
simple methods for classification of sample components in complex food matrices
(Christy and others 2003). It involves soft independent modeling of class analogy
(SIMCA) based on principal component analysis (PCA) to examine variations of samples
in their entirety, rather than only one single sample. Thus SIMCA can be used to classify
samples and determine which volatiles are most important to the differentiation. The aim
of this work is to identify the major volatiles in star thistle, blueberry, clover, cranberry
and wildflower honeys using SIFT-MS combined with chemometrics to classify honeys
by their volatile compounds and evaluate their capability as indicators of botanical and
geographical origin.
4.4 Materials and Methods
A total of 10 honey samples were analyzed. Honey samples from star thistle,
blueberry, clover, and wildflower were acquired from beekeepers at the Ohio State
University Bee Laboratory, Columbus, Ohio. Honey samples from star thistle, blueberry,
clover, wildflower, and cranberry were also acquired from Laney Honey Co, North
Liberty, Indiana. An unknown honey was acquired from Wal-Mart, Columbus, Ohio
100
(distributed by Tut’s Int’l Expert and Import Co., Dearborn, Michigan).
Triplicate (10 g) samples of honey were transferred into a 500 ml Pyrex bottle and
capped with open top caps coupled to polytetrafluoroethylene (PTFE) faced silicone
septa. The samples were held in a temperature controlled water bath (Precision, Jouan
Inc. Winchester, Virginia, USA) at 50˚C for 60 min before volatiles were measured to
allow equilibration of the volatiles released from the honey samples into the headspace.
4.4.1 Measurement of Volatile Concentrations
A selected ion flow tube mass spectrometer (SIFT-MS) (SYFT Voice 100, Syft
Ltd, Christchurch, New Zealand) measured and quantified the volatile compounds
released from 3 replicates of each honey sample, using the method described by Agila
and Barringer (2011). The analysis was done using selected ion mode (SIM) and the
concentrations of volatile compounds were detected from their reactions with H3O+, NO+,
or O2+ as precursors generated based on compounds previously present in honeys with
known kinetic parameters (Alissandrakis and others 2007; Castro- Vázquez and others
2009; Escriche and others 2009; Jerković and others 2009; Soria and others 2008). The
concentration of the volatile was calculated by applying the predetermined reaction rate
constant for the volatile with a selected precursor ion and accounting the dilution of the
sample gas into the carrier gas in the flow tube (Smith and Spanel 1996). Concentrations
were measured in µg/L in the headspace above the honey sample.
101
During the analysis, some compounds produce the same mass for a given
precursor ion, so the interfering compounds have to be reported as a mixture. In this
study, benzoic acid is a mixture of benzoic acid and 2-phenylethanol; methyl-1-butanol is
a mixture of pentanol and methyl-1-butanol; acids represent a mixture of butanoic acid
and pentanoic acid; and terpenes include limonene, terpienene, ocimene, myrcene and
pinene. The concentration of volatile compounds was calculated using known kinetic
parameters (Table 4.1). Compounds with concentrations close to the detection limit were
not included.
Table 4.1: Kinetics Parameters for SIFT-MS Analysis of Selected Volatile
Compounds in Honey.
Compound
(E)-2-hexenal
(E)-2-methyl-2-butenal
1,3-butanediol
1-octen-3-ol
2,3-butanedione
2-butanol
2-heptanol
2-methyl-2-butanol
2-methylfuran
3-methylbutanal
4-ethyltoluene
5-(hydroxy methyl)-2furfural
5-methylfurfural
acetic acid
acetoin
acetone
acids
benzaldehyde
Precursor
Ion
NO+
NO+
O 2+
H3O+
NO+
O 2+
NO+
H3O+
NO+
NO+
O 2+
O 2+
NO+
NO+
NO+
NO+
O 2+
NO+
m/z
Ref.
C6H9O+
C5H7O+
C4H8O+
C8H15+
C4H6O2+
C3H6+
C7H14O.NO+
C5H11+
C5H6O+
C5H9O+
C9H12+
K(10-9 cm3
s-1)
3.8
4
3.3
3.1
1.3
2.1
3.4
2.8
1.7
3
1.9
97
83
72
111
86
42
144
71
82
85
120
[4]
[9]
[10]
[14]
[3]
[4]
[13]
[14]
[14]
[9]
[14]
C6H6O3+,C6H6O3.H+
C6H6O2+
NO+.CH3COOH.H2O
C4H8O2.NO+
C3H6O+
CH3COOH+
C7H5O+
2.5
3.1
0.9
2.5
1.2
2.1
2.8
126, 127
110
108
118
88
60
105
[14]
[13]
[6]
[14]
[3]
[6]
[4]
Product Ion
102
Table 4.1 Continued
benzoic acid
benzyl alcohol
chloroform
dimethyl disulfide
dimethyl sulfide
dimethyl trisulfide
dodecane
NO+
NO+
O 2+
H3O+
O 2+
O 2+
NO+
ethanol
ethyl acetate
NO+
O 2+
ethyl benzoate
furfural
heptanal
heptane
hexanal
hexane
hexanoic acid
isopropyl benzene
lemonol
maltol
menthol
H3O+
NO+
NO+
H3O+
NO+
O 2+
H3O+
NO+
NO+
NO+
NO+
methanol
methylbutanoic acid
nonanal
nonane
octanal
octane
phenol
phenyl methanol
phenylacetaldehyde
p-isopropenyl toluene
propanoic acid
terpenes
H3O+
NO+
O 2+
H3O+
NO+
O 2+
NO+
NO+
NO+
O 2+
O 2+
NO+
C7H6O2+
C7H7O+
CH(Cl35)(Cl37)+
(CH3)2S2.H+
(CH3)2S+
C 2H 6 S 3+
C12H25+
C2H5O+, C2H5O+.H2O,
C2H5O+.2H2O
C2H5O2+
C6H5COOC2H5.H+,
C6H5COOC2H5.H+.H2O
C5H4O2+
C7H13O+
C7H16+
C6H11O+
C6H14+
C6H12O2.H+
C9H12+
C10H17+
C6H6O3.NO+
C10H19+, C10H19+.2H2O
CH5O+, CH3OH2+.H2O,
CH3OH.H+.(H2O)2
C5H10O2.NO+
C10H18+
C9H20.H3O+
C8H15O+
C8H18+
C6H6O+
C7H7O+
C8H8O.NO+
C10H12+
C2H5COOH+
C10H16+
103
2.5
2.3
1.8
2.6
2.2
2.2
1.5
1.2
2.4
3.1
3.2
3.3
0.26
2.5
1.76
3
1.2
2.5
2.5
2.6
2.7
2.5
3.2
1.3
3
1.9
2
2.3
2.5
1.8
2.2
2.2
122
107
85
95
62
126
169
45, 63,
81
61
[14]
[13]
[8]
[7]
[7]
[11]
[5]
151, 169
96
113
119
99
86
117
120
137
156
139, 175
33, 51,
69
132
138
147
127
114
94
107
150
132
74
136
[14]
[13]
[9]
[1]
[3]
[6]
[14]
[14]
[14]
[14]
[4]
[4]
[6]
[4]
[14]
[14]
[2]
[9]
[5]
[4]
[12]
[14]
[14]
[6]
[11]
Table 4.1 Continued
toluene
Z-3-hexen-1-ol
NO+
NO+
C7H8+
C6H10+
1.7
2.5
92
82
References: [1] Arnold and others (1998), [2] Francis and others (2007b), [3] Spanel and
others (1997), [4] Spanel and Smith (1997),[5] Spanel and Smith (1998a), [6] Spanel and
Smith (1998c), [7] Spanel and Smith (1998d), [8] Spanel and Smith (1999b), [9] Spanel
and others (2002a), [10] Spanel and others (2002b), [11] Wang and others (2003), [12]
Wang and others (2004a), [13] Wang and others (2004c), [14] Syft Technologies (2011).
4.4.2 Data Analysis
4.4.2.1 One-Way Data Analysis
The concentrations of volatile compounds in each honey sample were
analyzed in triplicate. One-way analysis of variance (ANOVA) using Tukey’s procedure
with a 95% confidence interval was carried out to determine statistical differences among
honey samples; significance was defined as p ≤ 0.05 by using IBM SPSS Statistics
software (version 19, 2010, SPSS Inc., Chicago, Illinois, U.S.A).
4.4.2.2 Multivariate Data Analysis
The concentrations (µg/L) of volatile compounds of three replicates for each
honey sample were imported into Pirouette software for Windows Comprehensive
Chemometrics Modeling, version 4.0 (Infometrix Inc, Bothell, Washington, U.S.A).
104
[6]
[14]
The concentrations were analyzed by soft independent modeling of class analogy
(SIMCA) which applied principal component analysis (PCA) to the whole data set and to
each of the classes to discriminate honeys based on their volatile compound compositions
according to their botanical origins and locations. Data was not normalized. The identities
of honey samples were predicted by where they fell in relation to the classes.
4.5 Results and Discussion
Aroma related volatiles of 9 honey samples of known floral source and
geographical location were analyzed by SIMCA to discriminate between honey samples
(Table 4.2). Interclass distances (ICD) of >3 indicate honeys are significantly different.
The greater the interclass distances between two honeys the better separation of the
honeys. Most of the samples were significantly different, indicating separation of the
honey types based on the volatile composition.
4.5.1 Effect of Geographical Location
Comparing between different locations but the same floral source, honeys from
Ohio were always classified as different from those from Indiana even though it seems
like they should not be different (Table 4.2). Environmental conditions including sunlight
and moisture, and soil properties may cause differences in volatile composition between
honeys from different geographical locations even when they are from the same botanical
origin (Kaškonienė and Venskutonis 2010).
105
Table 4.2: Interclass Distances between Honey Samples from Different Plant
Origins and Locations.
OST
IST
OBB
IBB
OWF
IWF
OCL
ICL
ICB
OST
0
4.4
1.3
5.6
6.2
6.7
1.8
4.4
6.9
IST
OBB
IBB
OWF
IWF
OCL
ICL
ICB
0
3.7
8.1
10.1
7.9
3.6
4.3
7.2
0
4.8
4.5
6.5
1.5
3.7
5.2
0
6.7
6.2
5.4
6.1
6.8
0
7.6
5.5
9.7
12.2
0
6.1
6.4
7.4
0
3.5
5.6
0
1.4
0
O: Ohio; I: Indiana; ST: Star Thistle; BB: Blueberry; WF: Wildflower; CL: Clover; CB:
Cranberry. Numbers in bold indicate no separation.
4.5.2 Effect of Botanical Origin
Comparing between floral sources within one location, Ohio star thistle,
blueberry, and clover honeys did not classify as being different (Table 4.2). Only Ohio
wildflower was different from the other Ohio honeys. Honey samples collected from
Indiana were all significantly different from each other, except clover and cranberry
honeys. It is difficult to identify the floral source accurately (Cuevas-Glory and others
2007). Unifloral honeys are never actually unifloral because the bees rarely collect nectar
from a specific botanical source (Kaškonienė and Venskutonis 2010). Thus the Ohio
honeys may be from overlapping floral sources while the Indiana honeys were not.
Wildflower honeys from Ohio and Indiana were different from each other and the
honeys from other sources (Table 4.2). The aroma of wildflower honeys can differ
106
greatly from each other depending on the region and location because they are mixtures
of unknown flowers and different wildflowers generate honeys with different flavors.
4.5.3 Volatiles Important For Differentiating Honeys
SIMCA determined the most significant volatile compounds for differentiating
honeys from different geographical and botanical sources. Between the Ohio and Indiana
honey samples, furfural, 1-octen-3-ol, butanoic, and pentanoic acids had the most
significant discriminating power (Table 4.3). Furfural contributes to sweet, fragrant
(Alissandrakis and others 2005), fruit, cherry, and soft almond aromas (Manyi-Loh and
others 2011); while 1-octen-3-ol imparts an herbaceous aroma (Vázquez-Araújo and
others 2008). Butanoic acid imparts a pungent aroma (Pérez and others 2002). Other
volatiles with high discriminating power included acetonitrile, 3-methylbenzaldehyde,
(E)-2-hexenal, terpenes, and benzaldehyde. 3-Methyl benzaldehyde contributes to a
pungent odor (Shimoda and others 1996) and E-2-hexenal can impart green, grassy, and
fresh aromas in foods (Teranishi and others 1999). Terpenes are responsible for imparting
floral, rose-like, green, and herbaceous aromas (Marais 1983).
107
Table 4.3: SIMCA Discriminating Power (DP) of Honey Samples Based on Volatile
Concentrations.
Compound
Discriminating Power
furfural
1-octen-3-ol
acids
acetonitrile
3-methylbenzaldehyde
(E)-2-hexenal
terpenes
benzaldehyde
4-ethyltoluene
(Z)-3-hexen-1-ol
2-methyl-2-butanol
phenyl methanol
5-methylfurfural
acetic acid
acetoin
anise alcohol
methyl salicylate
2,3-butanedione
2-heptanol
acetone
ethyl benzoate
3-methylbutanal
octane
xylene
dimethyl sulfide
menthol
p-isopropenyl toluene
methylbutanoic acid
heptanal
p-hydroxybenzoic acid
terpinolene
octanal
p-hydroxybenzaldehyde
hexane
(E)-2-octenal
2-butanol
2-butoxyethanol
166
149
121
89
75
66
62
61
51
49
42
38
34
32
30
26
25
22
19
17
17
17
16
15
14
12
12
12
11
10
9
9
7
7
6
6
6
108
Table 4.3 Contineud
Compound
Discriminating Power
ethyl acetate
2-methylbutanal
decanal
hexanoic acid
5
5
4
4
4.5.4 Volatile Composition of Honeys
By ANOVA, methanol and ethanol were at the highest concentrations among the
volatiles, but do not produce an aroma due to their high threshold of detection values
(Table 4.4) (Vitova and others 2007). Ethanol is commonly found at high concentrations
in honey, especially in thyme and Spanish honeys (Pérez and others 2002). Alcohols may
be formed from growth of yeasts in honey (Kaškonienė and others 2008), or from
reduction of aldehydes (Vázquez-Araújo and others 2008) that are catalyzed by
reductases produced from bees (Moreira and others 2010). Acetic acid was at the next
highest concentrations in the honeys used in this study, except for Indiana blueberry and
wildflower honeys. Acetic acid is produced from break down of alcohols (Viuda-Martos
and others 2010) and imparts a floral odor in honeys (Shimoda and others 1996). It has
previously been reported at high concentrations in honey (de la Fuente and others 2005).
Furfural was at the next highest concentration in Indiana blueberry honey while 1-octen3-ol was at the next highest concentration in Indiana wildflower honey (Table 4.4).
109
Furfural was previously detected at high concentration in wildflower, clover,
blueberry (Overton and Manura 1994), and thistle honeys (Bianchi and others 2011).
Hexanoic acid and heptane were at high concentration in the honey from an unknown
source (1640 µg/L) (Table 4.4); however, they were at low concentration in the other
honeys. Hexanoic acid is a green odor compound and comes from hexanal during
oxidation (De Pooter and Schamp 1989).
Hexanoic acid has been reported as a characteristic volatile compound in
eucalyptus honey (Castro Vázquez and others 2006) and is responsible for a pungent odor
(Shimoda and others 1996).
Benzaldehyde was at high concentrations in all Indiana honeys used in this study
(Table 4.4). It was previously identified at high concentrations in wildflower, clover,
blueberry (Overton and Manura 1994), and thistle honeys (Bianchi and others 2011).
Benzaldehyde is responsible for sweet, almond, and marzipan aromas (Manyi-Loh and
others 2011) and also contributes to a honey-like aroma (Castro Vázquez and others
2006). Benzene derivatives are frequently considered to be major markers of floral origin
(Pérez and others 2002; Viuda-Martos and others 2010). Comparing to honeys from
different plant sources such as thyme, benzaldehyde was detected as a characteristic
compound in honeys from Thymus capitatus and Thymelaea hirsute (Odeh and others
2007).
110
Indiana clover and cranberry honeys also had high concentrations of acetone
while Indiana star thistle and wildflower honeys had high concentrations of isopropyl
benzene. Other volatiles at high concentrations were ethyl acetate and acetone in Ohio
blueberry, clover, and wildflower honeys. Acetone contributes to a pungent or fruity odor
and ethyl acetate is responsible for a fruity aroma in honey (Alissandrakis and others
2005). (E)-2-methyl-2-butenal was also found at high concentrations in Indiana
wildflower honey. (E)-2-methyl-2-butenal contributes to coffee aroma (Chin and others
2011).
The unknown honey had the highest concentration of most volatiles. Among the
Ohio and Indiana honeys, the highest concentration of volatile compounds was in Indiana
wildflower honey followed by Ohio wildflower honey (Table 4.4). The lowest
concentration of volatile compounds was observed in Ohio clover honey followed by
Indiana clover honey. Volatiles were previously found to be at higher concentration in
wildflower honey than clover honey in Lithuania (Kaškonienė and others 2008). Clover
honeys also had lower concentrations of compounds than other honeys in New Zealand
(Tan and others 1988).
The most important volatiles were typically acetic acid, furfural, 1-octen-3-ol,
ethyl acetate, benzaldehyde, and acetone, which were usually at high levels in the honey
(Table 4.4). These volatiles are commonly detected in different types of honeys including
furfural, acetone, (Pérez and others 2002), and acetic acid (de la Fuente and others 2005).
111
When the absolute concentration of each volatile in the honey samples was
converted to percentage and analyzed by SIMCA, very similar separation and interclass
distances were produced. This indicates that the separation is based on the volatile
distribution and not dominated by some samples having higher concentrations of total
volatiles. For each honey type, the volatiles that contributed most to the SIMCA loading
were similar.
112
Table 4.4: Concentration (µg/L) of Honeys from Different Origins and Locations
113
Compound
(E)-2-hexenal
(E)-2-methyl-2-butenal
1,3-butanediol
1-octen-3-ol
2,3-butanedione
2-butanol
2-methyl-2-butanol
2-methylfuran
3-methylbenzaldehyde
3-methylbutanal
4-ethyltoluene
5-methylfurfural
acetic acid
acetoin
acetone
acetonitrile
acids
benzaldehyde
dimethyl disulfide
dimethyl sulfide
dimethyl trisulfide
ethanol
ethyl acetate
OST
IST
OBB
IBB
OWF
IWF
c
c
c
b
a
8
7
9
26
38
21b
7c
24c
6c
8c
60b
126a
46bc
71ab
42bc
74ab
65abc
66abc
c
c
c
c
b
36
44
27
42
198
393a
49b
29b
26b
29b
39b
30b
44bc
41bc
43bc
54ab
53ab
34bc
16bc
15bc
13bc
13bc
17bc
28bc
7ef
16bc
4f
10cde
18b
27a
3d
7cd
5cd
9bc
7cd
13b
32bcd
28cd
19d
36bcd
54b
46bc
8c
29b
5c
12c
27b
55a
bc
cd
bc
cd
a
16
13
17
14
38
21b
414bc
562ab
440bc
347c
701a
311c
31c
32c
16c
24c
30c
23c
63e
95bcde 94bcde
105bcde 137ab
92cde
28b
10de
46a
48a
26b
16cd
28c
32c
36c
39c
26c
30c
95cde 176ab
87de
129b
71de
127bcd
10c
7c
6c
8c
20b
27a
57cd
60cd
37d
61cd
91bc
120b
b
b
b
b
b
35
35
28
32
26
22b
1760de 5950c
1110e 2900de
12600b 21400a
104c
90c
96c
102c
161b
98c
OC
5c
4c
32c
18c
53b
22c
4c
4f
3d
16d
6c
11cd
326c
16c
72de
8e
17c
43e
6c
38d
26b
1520de
73c
IC
8c
11c
53abc
15c
42b
25bc
31b
9def
13b
17d
12c
7d
334c
36c
114bcd
10de
95b
149ab
4c
48cd
25b
3440d
78c
ICB
UH
c
8
13c
9c
14c
52abc
83a
c
22
46c
28b
166a
41bc
77a
36b
48a
6ef
15bcd
20a
19a
14d
102a
11c
36b
cd
11
17bc
394bc
628a
66b
186a
175a
134abc
22bc
20bc
132a
20c
189a
48e
5c
14bc
49cd
270a
b
22
128a
2590de 2750de
103c
242a
Table 4.4 Continued
114
ethyl benzoate
furfural
heptanal
heptane
hexanal
hexane
hexanoic acid
HMF
isopropyl benzene
lemonol
methanol
methylbutanoic acid
nonanal
nonane
octanal
octane
phenol
phenyl methanol
phenylacetaldehyde
p-isopropenyl toluene
terpenes
terpinolene
toluene
xylene
a-f
13bc
102cd
19b
62b
30b
49b
13b
27ab
45cd
6c
826ef
15cd
18
40b
36b
40b
8
13cd
16cd
12cd
20d
31abc
7c
14bc
18bc
44e
13bc
51b
34b
39b
13b
22bc
153b
23b
621f
32b
24
34b
34bc
15d
12
31b
62abc
23abc
41b
33ab
9c
19bc
18bc
124c
17b
30b
16b
54b
13b
23b
38cd
6c
604f
21bcd
28
23b
29bcd
34bc
6
10cd
40bcd
13cd
13de
20bcd
6c
12bc
44a
383a
13bc
49b
12b
35b
17b
21bc
56cd
6c
713f
32b
22
10b
39b
16d
12
11cd
84ab
35a
20d
16cd
8c
16bc
21b
272b
17b
87b
35b
44b
20b
35a
146b
8c
1450ab
22bc
18
32b
41b
23cd
31
29b
46abcd
11cd
11ef
24abcd
25b
8c
18bc
119c
20b
61b
42ab
54b
13b
25ab
337a
21ab
1210bc
12cd
19
41b
31bc
16d
33
34b
91a
15bcd
25cd
19bcd
66a
18bc
3c
56e
7c
40b
25b
30b
9b
19bc
23d
2c
962de
8d
16
21b
25bcd
15d
5
9d
2d
7d
5f
13d
6c
8c
9bc
37e
8c
23b
7b
43b
5b
22bc
46cd
11bc
1190cd
16cd
16
18b
12d
9d
8
28b
26cd
11cd
38bc
18bcd
7c
20ab
12bc
46a
72de
111cd
8c
97a
25b 1690a
15b
120a
35b
143a
13b
1640a
c
11
24b
72c
136b
10bc
30a
775ef 1570a
15cd
90a
22
28
b
18
126a
16cd
91a
d
14
99a
3
16
a
48
23bc
30cd
32cd
11cd
28ab
28cd
66a
22abcd
62a
5c
24b
ab
21
31a
Different letters in the same row indicate significant differences between honeys O: Ohio; I: Indiana; ST: Star Thistle; BB:
Blueberry; WF: Wildflower: CL: Clover; CB: Cranberry; UH: Unknown Honey.
4.6 Conclusion
The interclass distances between most honey varieties indicated they were
significantly different. Honey from different locations but the same floral origin showed
differences in volatile composition. Among the honey samples, furfural, 1-octen-3-ol,
butanoic, and pentanoic acids had the most significant discriminating power. Methanol
and ethanol, followed by acetic acid were at the highest levels in most honeys. Furfural
was at the highest concentration in Indiana blueberry honey while 1-octen-3-ol was at the
highest concentration in Indiana wildflower honey. The unknown honey had the highest
concentration of most volatiles. Among the Ohio and Indiana honeys, the highest
concentration of volatile compounds was observed in Indiana wildflower honey followed
by Ohio wildflower honey. The lowest concentration of volatile compounds was
observed in Ohio clover honey followed by Indiana clover honey. The aroma of honey is
determined by volatile composition, which is affected by factors such as plant source and
environmental location. These factors cause no two honeys to have the exact same
composition and makes honey a complex food that is hard to control.
115
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