- Faculty of Biosciences and Medical Engineering

Transcription

1
CHAPTER 1
INTRODUCTION
1.1
Introduction
Cockroaches (order: Blatteria) are among the most persistent pests that thrive
in protected locations all over the world. American, brown-banded, German and
Oriental cockroaches are the four most common species in human habitats (Hahn and
Ascerno, 2005). They can be found in boiler rooms, heated steam tunnels, floor
drains, water heaters, bath tubs (Eggleston and Arruda, 2001), kitchen sink, wall
cracks, underneath or inside cupboards, behind drawers, around pipes or conduits,
behind window or door frames, in radio and TV cabinets (Koehler et al., 2007).
Cockroaches are objectionable because they compete with humans for food,
carry allergens in saliva, fecal material, secretions, cast skins, debris and dead bodies
that can lead to asthma in humans (Eggleston and Arruda, 2001), harbor pathogenic
bacteria, contaminate paper and fabrics, and impart repulsive stains and odours
(Koehler et al., 2007) .
The life cycle of cockroach consists of egg, nymph and adult and they can
live for one year. Nymphs are more than adults in natural populations (Hahn and
Ascerno, 2005). Cockroaches are nocturnal and omnivorous and will eat anything
though they can stay for about one month without food and for about two weeks
without water. Cockroach population in an enclosure is governed by presence of food
and water, height of location (for some species), humidity, temperature darkness
(Eggleston and Arruda, 2001) and history of control measures (Wang and Bennett,
2006).
2
Trapping, mating distruptions, attracticides are used in cockroach control.
Organophosphates, carbamates, pyrethrins, chlorinated hydrocarbons and inorganics
are some of the pesticides used to control cockroaches (Eggleston and Arruda, 2001).
Traps are relatively more effective in controlling cockroach (Rust et al., 1999).
Attractiveness of methylcyclohexyl n-alkanoates to the German cockroach was
reported by Iida and co-workers (Iida et al., 1981). Many chemical attractants used in
commercial cockroach traps are injurious to humans and the environment (Quesada
et al., 2004). Pest resistance, toxicity and non-sustainable raw materials of pesticides
are some of the factors behind the trend to replace chemical feedstock, processes and
end products with bio-based alternatives.
Cockroaches emit pheromones for communication and other activities. Three
semiochemicals from stale beer and peanut butter are capable of being used as
cockroach bioattractants were identified by Karimifar and co-workers (Karimifar et
al., 2011). Foods such as bread, peanut butter, molasses and beer can act as
cockroach attractants. Food baits are not used in commercial pest control strategies
as they often turn out to be breeding grounds for pests (Cox and Collins, 2002).
Many insects respond to chemicals released by same species or chemicals released
by other organisms. Many plants release volatile chemicals that are able to affect
other
organisms
such
as
insects.
Semiochemicals
are
pheromones
and
allelochemicals, many of which are responsible for insect behaviors such as feeding
and reproduction. They are mostly volatile metabolites that are involved in signaling
and other functions (Maffei, 2010).
Metabolites are involved in metabolism either as intermediates or as the final
products. Metabolism can be divided into anabolism in which simple molecules are
used to build large, complex molecules and catabolism in which complex, large
molecules are broken into simple molecules. Most of the time, each metabolite can
serve more than one purpose (Kooijman and Segel, 2005). Most metabolites are
within cells where they are utilized by enzymes in various biochemical reactions.
Some of the metabolites have control functions. Metabolites include lysine,
riboflavin (Damain, 1980), ethanol, acetic acid (Styger et al., 2011). Two subgroups
of metabolites are primary and secondary metabolites. Primary metabolites have
physiological functions (Gika et al, 2012). Secondary metabolites serve ecological
3
functions (Demain, 1998). Primary pathways normally yield few products but are
present in many organisms. Secondary metabolic pathways yield many products but
are present in few organisms. Antibiotics make up most of the 50 most important
secondary metabolites (Demain, 1999).
Volatile metabolites are intermediates and final products of metabolism. A
major feature of volatile metabolites is the ease of evaporation at normal temperature
and pressure. They act over a wide range of distances, concentration and
environment (Humphris et al., 2002). Yeasts produce volatile metabolites which can
be grouped into alcohols, acids, esters, ketones and phenols. Such volatiles include
ethanol, 3- methyl-1-butanol, 3- hydroxyl-2-butanone, ethyl propanoate, 2-phenylethanol (Nout and Bartelt, 1998). Volatile metabolites are useful to both the
organism producing them and other organisms. Volatiles are useful as flavors,
fragrances (Berger, 2009), inhibitors (Ting et al., 2010), and attractants (Rowan,
2011), as well as for communication (Maffei, 2010), identification and profiling
(Frisvad et al., 2007), and detection of contaminants (Berge et al., 2011). Volatile
metabolites are produced by plants, animals, microorganisms and through chemical
synthesis. Yields from plants and animals are low compared to microbial sources
(Vandamme, 2003). Plant cell cultures are not economically viable for producing
volatile metabolites (Verpoorte et al., 2002). Chemical (synthetic) volatile
metabolites are relatively cheap with large market share but disadvantaged by
perceived environmental toxicity and possible formation of recemic mixtures (Longo
and Sanroman, 2006). Major advantages of microbial based production of volatile
metabolites include low energy input, reduced emission of pollutants, easy
purification and renewable raw materials (Chelmer et al., 2006). Some volatile
metabolites are produced through microbial fermentation of various substrates.
Microbial volatile production depends on medium, species (Borjesson et al., 1992),
temperature, pH, initial aeration (Reddy and Reddy, 2011), culture a ge and volatiles
from other microorganisms (Tirranen and Gitelson, 2006).
Yeasts are the most suitable source for microbial volatile production as they
tolerate high sugar concentration, anaerobic growth, high salt concentration,
resistance to inhibitors in the biomass (Nevoigt, 2008). An alcoholic fermentation
takes place in the absence of oxygen and presence of yeasts, nutrient medium,
4
suitable pH and temperature. Alcohols, vitamins, hormones, antibiotics, enzymes,
acids are made through fermentation. Yeast utilization of glucose can be through the
fermentation of glucose, oxidation of glucose or oxidation of ethanol.
Saccharomyces cerevisiae can completely ferment glucose, fructose, galactose,
sucrose and maltose (Yoon et al., 2003). Volatile metabolites are produced during
bacterial and yeast fermentations but in an experiment, only volatile metabolites
from yeast fermentation were able to attract insects (Nout and Bartelt, 1998).
The Saccharomycetaceae family has about 20 genera, which include
Saccharomyces, Candida and Pichia. Members of the family reproduce by budding
and live in environments rich in carbohydrates. Saccharomyces cerevisiae is the most
important yeast as a model eukaryotic organism. Saccharomyces cerevisiae is used in
baking, brewing and wine making. Saccharomyces cerevisiae and closely related
yeasts dominate alcoholic fermentations as they are tolerant of high alcohol content
and they also use the produced ethanol as feedstock (Piskur et al., 2006).
Candida ethanolica was first isolated from industrial fodder yeast and can
grow on ethanol as the only carbon source. C. ethanolica is one of the yeasts
involved in shubat (a fermented camel milk) production (Shori, 2012), Ghanaian
heaped cocoa bean fermentation (Daniel et al., 2009), mescal (Mexican distilled
beverage) (Valdez, 2011), sour cassava starch fermentation (Lacerda et al., 2005).
Pichia kudriavzevii is synonymous with Issatchenkia orientalis based on
molecular, biochemical and phenotypic characterization (Bhadra et al., 2007). Pichia
kudriavzevii was shown to degrade phytate in ‘togwa’, a cereal food in Tanzania
(Hellstrom et al., 2012), produce ethanol from alkali- treated rice straw (Oberoi et al.,
2012), ferment cotton stalks in a combined saccharification and fermentation process
for ethanol production (Kaur et al., 2012), part of fen-daqu, starter culture for
Chinese liquor production (Zheng et al., 2012), among the yeasts involved in
‘tchapalo’, a local beer in Cote d’ Ivoire (N’guessan et al., 2011) and among the
yeasts involved in Ghanaian heap cocoa bean fermentation (Daniel et al., 2009).
Media composition is important in yeast fermentation performance (HahnHagerdal et al., 2005). Agar plates and nutrient broth are used in most yeast
fermentations. Among the common media for yeast fermentation are wort (Lodolo et
5
al., 2008), molasses (Alegre et al., 2003) and potato dextrose broth (Kalyani et al.,
2000). Yeast stock cultures are normally kept at 4 o C on potato dextrose agar
(Lakshmanan and Radha, 2012). Most yeast utilize hexose sugars (monosaccharides
and disaccharides). The media used can improve yield, increase biomass formation
and decrease fermentation period (Chunkeng et al., 2011). The ecology and profile of
microorganisms during fermentation could be affected by the media used (Hsieh et
al., 2012). Potato dextrose broth is commonly used for the cultivation of yeasts and
molds. Its low pH discourages bacterial growth (Condolab, 2012).
Chromatography is used to separate complex mixtures but it cannot readily
identify the constituents of separated unknown mixtures. It is always desirable to
couple separation processes to identification steps. Liquid chromatography (LC) and
gas chromatography (GC) are two of the leading chromatographic methods. LC is
favoured over GC for samples susceptible to degradation as LC does not involve
derivation step. In order to identify, LC is normally coupled to mass spectrometry
(MS) that is in tandem. Liquid chromatography – tandem mass spectrometry (LCMS/MS) was found to have lower limit of quantification, less costly to run, reduced
preparation time, does not involve derivation of sample, and reduced run time (Coles
et al., 2007). LC-MS/MS is best suited for the analysis of chemically and thermally
unstable compounds (Johnson, 2005).
Downstream processing involves the recovery of the desired product from the
fermentation broth. Removal of insolubles, product isolation, product purification
and product polishing are the major groupings of the unit operations of fermentation
products recovery. Low product concentration makes product recovery expensive
(Schugerl, 2000).
Freeze drying (lyophilization) is a dehydration process in which solvent is
removed through sublimation. It is used for the recovery of heat sensitive products
such as volatile metabolites. Compared to air drying, the retention of volatile
materials was more in the freeze dried sample (Krokida and Philippoules, 2006).
6
1.2
Statement of Problem
Cockroach control using insecticides and other chemicals are not desirable
because they are toxic to organisms and the environment (Quesada et al., 2004).
Pests can develop resistance to the chemicals. In addition, chemicals are
unsustainable as the raw materials of the chemicals are from petroleum. Hence, there
is consumer demand for natural product based compounds.
On the other hand, foods as attractants favour all organisms thereby providing
breeding grounds (Cox and Collins, 2002). Foods have limited range of effectiveness
and are non-specific.
Biological based attractants from plants and animals are very low in yields
(Vandamme, 2003). Extraction from plants is very difficult, depends on the season
and some of the exotic plants have lost the ability to produce desired volatile
metabolites (Dudareva and Pichersky, 2000).
Microorganisms produce volatile metabolites that can potentially act as insect
attractants. Volatile metabolites from yeasts have been found to be better at attracting
insects than volatile metabolites from bacteria (Nout and Bartelt, 1998).
Therefore, there is the need to test if volatile metabolites from yeast
fermentation can attract cockroaches and the leading volatile metabolites responsible
for the attraction. In this study, two locally isolated yeasts, namely Candida
ethanolica M2 and Pichia kudriavzevii M12 were tested for their potential in
bioattraction of domestic cockroaches and production of exometabolites.
7
1.3
Objectives of the Research
The specific objectives of this study were:
1. To determine the potential of yeast strains C. ethanolica M2 and P.
kudriavzevii M12 in producing metabolites for bioattraction of domestic
cockroaches
2. To compare the fresh and freeze-dried yeast culture supernatants in its
efficiency in bioattraction of domestic cockroaches
3. To study the exometabolites of P. kudriavzevii M12
1.4
Scope of Research
This research work focused on the production of volatile metabolites from
yeast strains C. ethanolica M2 and P. kudriavzevii M12 which were cultured in
potato dextrose broth (PDB) up to a period of a week. The culture supernatant
containing volatile metabolites were tested as bait of commercial trap for the
attraction of domestic cockroaches at six different locations at the student residential
halls in Universiti Teknologi Malaysia. Freeze-drying of the culture supernatant was
attempted as a preservation technique and the efficiency of the freeze-dried
metabolites in bioattraction of cockroach was determined. As the fermentation of P.
kudriavzevii M12 was found to be significant, LC-MS/MS profiling was performed
to determine the leading metabolites in the supernatant from the four-days
fermentation broth.
1.5
Significance of Research
This research is significant as it has the potential to discover non-toxic,
natural bait from yeasts which may replace the harmful chemical b aits in domestic
cockroach traps. In the future, the identified metabolites could be optimized for the
production of cheap bait for domestic cockroach attraction.
8
CHAPTER 2
LITERATURE REVIEW
2.1
Metabolites
Metabolites are molecular intermediates and final products of metabolism.
Each molecule normally has a functional group that determines much of its
characteristics. Metabolites can be building blocks of anabolism or one of the
degradative products of catabolism (Kooijman and Segel, 2005). As they are mostly
found inside cells, they are used as substrates by enzymes (Saghatelian et al., 2004).
An organism can contain many metabolites. Saccharomyces cerevisiae has been
determined to have 584 metabolites (Forster et al., 2003). Products of metabolites
serve as substrates in subsequent metabolic reactions. Metabolites do not accumulate
in cells and some are involved in metabolic controls. Each metabolite serves a
specific useful biological function. Lysine, glutamic acid (amino acids), riboflavin,
cyanocobalamin (vitamins) are examples of metabolites (Damain, 1980). Other
examples of metabolites include ethanol, acetic acid, acetaldehyde, esters, and fatty
acids (Styger et al., 2011).
Metabolites can be subdivided into primary and secondary metabolites. While
primary metabolic pathways lead to few products, secondary metabolic pathways
lead to many products. Ethanol, glycerol, and acetic acid are primary metabolites;
esters, higher alcohols are secondary metabolites (Styger et al., 2011). Also grouped
as primary metabolites are vitamins (B2 ), amino acids (glutamic acid), antioxidants
(isoascorbic acid), and nucleotides (5’guanylic acid). Primary metabolites have
physiological functions in development, reproduction and growth (Gika et al., 2012).
Primary metabolites are present in many organisms. Secondary metabolites have
ecological functions (Izhaki, 2002). Even though secondary metabolites are mainly
9
for defense and communications, the same secondary metabolite can act in different
modes depending on the organism being acted upon. Each secondary metabolite is
present in limited number of organisms. Secondary metabolites aid producing
organisms in competition and defense (Vaishnav and Demain, 2010). Many
secondary metabolites have been identified, most of which are from plants and
microorganisms. Most of the 50 most important microbial secondary metabolites are
antibiotics (Demain, 1999). More secondary metabolites are still being discovered.
Some secondary metabolites of plant origin (isoprene, limone ne, naringin, coumarin
and carvone) are useful in bioremediation (Luo et al., 2007). Pigments, antibiotics,
toxins, pheromones, enzyme inhibitors, immunomodulatory agents, receptor
antagonists, growth promoters and pesticides are some of the microbial seco ndary
metabolites (Demain, 1998). Secondary metabolites, especially those from
myxobacteria are important in drug discovery processes (Diez et al., 2012).
Saccharomyces cerevisiae produces not just alcohol during alcoholic fermentation
but also an array of secondary metabolites such as isoprenoids, long chain
polyunsaturated fatty acids and flavonoids (Chemler et al., 2006).
2.2
Volatile Metabolites
Volatile metabolites are distinguished by their ease of vaporization (Seto,
1994). Volatility enables effectiveness over a wide range of distance, concentration
and environment (Humphris et al., 2002).
Volatile metabolites (mostly secondary metabolites) are intermediates and
products of metabolism that evaporate readily at normal temperature and pressure.
Volatile metabolites are mostly organic compounds such as carboxylic acids,
alcohols and esters that are produced by plants and microorganisms. An analysis of
volatile metabolites from the leaves and flowers of Acacia cyclops revealed six
classes of compounds – benzenoids, aliphatic compounds, nitrogen containing
compounds, monoterpenoids, sesquiterpenoids and irregular terpenes (Kotze et al.,
2010). Yeast fermentations produce variety of volatile metabolities, including
(alcohols, acids, ketones, aldehydes, esters and phenolic compounds). Examples of
these metabolites are ethanol, acetaldehyde, 3- methyl-1-butanol, 2- methyl-1-butanol,
acetone, ethyl acetate, isopropyl acetate, ethyl butyrate, 1-propanol, 2- methyl-1-
10
propanol, 1-butanol, 2- methyl-1-butanol, 3-methyl-1-butanol, hexyl acetate and 1hexanol (Nout and Bartelt, 1998; Ragaert et al., 2006). Some volatile metabolites
can be detected by humans at the level of parts per trillion (Rowan, 2011). Twenty
strains of basidiomycete cultured on glucose/asparagine/yeast extract medium
produced volatile metabolites, 123 of them identified as alcohols, aldehydes, esters,
ketones and phenols (Abraham and Berger, 1994). Molecular weight varies from 50
– 200 Daltons (Rowan, 2011). In plants, they are used for protection, such as against
pathogens and for reproduction, such as attracting pollination agents (Figueiredo et
al., 2008). Microorganisms, especially yeasts produce volatile metabolites by direct
medium transformation, secreting enzymes that carry out the transformation of
medium constituents and the de novo synthesis of primary and secondary volatile
metabolites (Styger et al., 2011). Many of the volatile metabolites are isomers. An
analysis of 196 different volatile metabolites produced by 47 Penicillium taxa
showed that 70 were sesquiterpene hydrocarbon (C 15 H24 ) isomers (Larsen and
Frisvad, 1995). While pure cultures are used in yeast volatile metabolites production,
mixed cultures are also used as some mixed culture fermentations are capable of
producing more complex volatile metabolites (Lee et al, 2010). Not all volatiles from
yeasts fermentations are desirable. Some thiol compounds give rise to unpleasant
aromas while others, especially volatile thiol compounds with additional functional
groups that impact positively with their fruity flavours (Coetzee and du Toit, 2012).
Sensory qualities of many products are due to the volatile metabolites generated
during yeast fermentations (Nova et al., 2009).
2.3
Importance of Volatile Metabolites
Volatile metabolites, though not required for physiological functions of
organisms, are useful to organisms producing the volatile metabolites; to organisms
not produce them or both. Flavours, fragrances (Berger, 2009), communications
(Maffei, 2010), profiling (chemotaxonomic studies) (Frisvad et al., 2007), and
inhibitory functions (Ting et al., 2010) are some of the uses of volatile metabolites.
11
2.3.1
Flavors and Fragrances
Flavor is a term used for volatile metabolites that are ingested while fragrance
is normally reserved for volatile metabolites that are not ingested. Flavors and
fragrances are important to the food, pharmaceutical, feed, cosmetic and chemical
industries (Vandamme, 2003). Volatile metabolites as flavors and fragrances are the
basis of sensory attributes for consumer acceptance of foods and cosmetics (Berger,
2009). Interactions of volatile metabolites enhance the sensory perception of each
interacting metabolite (Styger et al., 2011). Beyond the sensory attributes, volatiles
have antimicrobial, antifungal, antioxidant, fat reducing, blood pressure regulating
and anti- inflamatory properties (Berger, 2009).
2.3.2
Identification, Profiling and Che motaxonomic Studies
In addition to DNA sequencing, morphological and biochemical features,
secondary metabolites profile is increasingly being used for identification of
microorganisms (Frisvad et al., 2007). In a study by Pontes and co-workers, a total of
68 volatile metabolites were used to identify the different cultivars of banana (Pontes
et al., 2012). Volatile metabolites have been used in metabolic footprinting in
functional genomics studies in which even single gene deletion mutants could be
distinguished from wild type yeasts (Mas et al., 2006). A combination of species
identification and detection of contamination by microorganisms was achieved by
Magan and Evans using ‘electronic nose’ technology involving volatile metabolites
(Magan and Evans, 2000).
2.3.3
Communication
Many volatile metabolites in plants are used for signaling and other functions
(Maffei, 2010). Semiochemicals, also known as chemical signals, comprised of
pheromones and allelochemicals and are responsible for some insect behavior such
as feeding and reproduction (Norin, 2007).
12
2.3.4
Inhibitory Function
Volatile metabolites are used in biocontrol such as the inhibition of growth of
Fusarium oxysporum F. sp. cubense, the organism responsible for Fusarium wilt
disease (Ting et al., 2010). Some volatile metabolites are used to ward off pests and
pathogens (Dudareva and Pichersky, 2008).
2.3.5
Detection of Contamination
Berge and co-workers used volatile metabolites as metabolic signatures to
detect exposure of poultry to xenobiotics (Berge et al., 2011). In indoor
environments, visual and bioaerosol monitoring of mold growth behind walls and
barriers are not as effective as the use of volatile metabolites to detect mold growth
(LeBouf et al., 2010). Volatile metabolite profiling was used by Bianchi and coworkers for the early detection of orange juice spoilage by microbial contaminations
(Bianchi et al., 2010). In milk quality control and shelf life evaluation studies, some
spoilage yeasts volatile metabolites were detected by electronic nose and the profiles
were used to separate regular milk from the ones contaminated by yeas ts (Magan et
al., 2001).
2.3.6
Attraction
Chemical signals can make organism elicit responses such as attraction,
repellance or aggregation. Volatile metabolites in plants are used for attracting
pollinating agents (Maffei, 2010), attracting pollinating agents and seed dispersers
(Rowan, 2011). A large variety of plant volatile metabolites are attractants for
pollinators and enemies of herbivores while other volatile metabolites ward-off
herbivores (Pichersky and Gershenzon, 2002). Currently, a lot of chemicals are used
as pesticides. Major disadvantages of pesticides are pests resistance, environmental
pollution and health hazards (Heuskin et al., 2011).
13
2.4
Sources of Volatile Metabolites
Volatile metabolites can be sourced from plants, animals, microorganisms, as
well as from chemical synthesis. Yields from plants and animals are low compared to
microorganisms (Vandamme, 2003; Gounaris, 2010). While transgenic plants
increased yields by 50%, 100- fold increase was observed using microorganisms
(Gounaris, 2010). Very few plants are cultivated for their volatile metabolite
constituents and many have lost the ability to produce volatile metabolites due to
breeding for other traits such as colour, shape, and shelf- life (Dudareva and
Pichersky, 2000). Production of volatile metabolites from plants by using plant cell
cultures though technically feasible, are not economical (Verpoorte et al., 2002).
Volatile metabolites can also be produced through chemical means. Chemical
manufacturing of volatiles are cheaper, with a large market share (Gounaris, 2010).
However, pollution and consumer perceptions of leftover impurities from
manufacturing make them unpopular (Longo and Sanroman, 2006). High value
chemicals sourced from microbial sources are being developed to replace the current
petrochemical sources (Scalcinati et al., 2012). There are increasing demands for the
replacement of the petrochemical industry with bio- based industry and processes
(Botella et al., 2009). Increasingly, flavours and fragrances are being produced
through biotechnological processes using microorganisms (Krings and Berger, 1998;
Vandamme, 2003). Preference for natural flavors by consumers has made microbial
fermentation the better choice compared to plant and chemical sources (Jiang, 1995).
Microbial production of volatiles requires low energy input, reduced carbon dioxide
emissions, less generation of toxic wastes from processing, biological, renewable
sources of raw materials input, and easier to purify (Chemler et al., 2006).
A major drawback of volatile metabolites production is the costly purification
process which end- users may not be willing to pay for (Perrut, 2000). It is much
easier to produce volatile metabolites from microorganisms than from plants
(Gounaris, 2010).
14
2.5
Volatile Metabolites from Microorganis ms
Fermentation of various substrates, especially carbohydrates, is the method of
producing volatile metabolites by microorganisms. In microorganisms, productions
of volatile metabolites through many pathways (Figure 2.1), depend on species
(Romano et al., 2003), medium and species (Sunesson et al., 1995), medium, species
and duration (Borjesson et al., 1992). In volatile metabolites production through
yeast fermentations, temperature, pH, SO 2 and aeration all have effects on yeast
growth, duration of growth, fermentation rate and volatile composition (Reddy and
Reddy, 2011). Production of volatile metabolites is also influenced by concentration
of medium components, culture age as well as the volatile metabolites p roduced by
other microorganisms (Tirranen and Gitelson, 2006). Flavour and aroma (due to the
presence of volatile metabolites), are some of the attributes responsible for
differences in beverages (Swiegers et al., 2005). Yeasts such as Saccharomyces
cerevisiae are efficient converters of sugars to ethanol, in addition to tolerating low
pH, high sugar concentration, high ethanol concentration, anaerobic growth and
resistance to inhibitors in biomass (Nevoigt, 2008).
Tolerance of low pH, low aw, and high salt concentrations by yeasts (Fleet,
1990) make them indispensible in the production of dairy products such as koumis,
kefir, viili, longfil and some varieties of cheese (Florez et al., 2010). Compared to
yeasts, Escherichia coli is one of the bacteria being considered for ethanolic
fermentation but narrow pH band in the neutral region, public perception of the
pathogenicity of some of its strains as well as the production of mixtures of organic
acids and ethanol from sugar fermentation are serious disadvanta ges (Dien et al.,
2003). In an experiment, both bacteria and yeasts produced volatile metabolites
during fermentation but only the volatile metabolites from yeasts were able to attract
insects (Nout and Bartelt, 1998). Mixed bacterial and yeast fermentatio n can lead to
competition between microorganisms, reduced fermentation speed and altered
secondary (volatile) metabolites (Rossouw et al., 2012). Some volatile metabolites
showing variations in some physical, chemical characteristics as well as source and
bioactivity diversity are shown in Table 2.1.
15
Aromatic secondary
metabolites
Ethanol
Glucose
Amino acids
(e. g.
Leucine,
Valine)
Pyruvate
Acetate
Aromatic amino
acids (Phenylalanine)
Malonate
(Acetyl –CoA)
Nitrogen
compounds
Alcohols
Threonine
Ochrato xin s
Polyketides
Mevalonate
Geranyl
pyrophosphate
Famesyl
pyrophosphat
e
Fatty
acids
Lactones
Methyl
ketones
Secondary
alcohols
Alcohols
Sesquiterpenes
Geosmin
Aldehydes
Trichothecene
s
TCA-cycle:
Oxalo-acetate
Monoterpenes
Aspartate
2-methylisoborneol
Esters
Methionine
Sulphur
compounds
Figure 2.1: Pathways for volatile metabolites (Magan and Evans, 2000).
16
Table 2.1: Some volatile metabolites showing variations in some
physical, chemical characteristics as well as source and bioactivity diversity
(Rowan, 2011).
Chemical cl ass/
bi osynthesis
Source/
bi oacti vity
28
bpo C
(760)
mm Hg
-102
Hydrocarbon
fro m
methionine
CH3 SH
48
6
sulfide
Isoprene
C5 H8
68
34
terpenoid
Ethanol
C2 H5 OH
46
78
Alcohol
Dimethyl
disulfide
CH3 SSCH3
94
109
Disulfide
Hexanal
C6 H12 O
100
131
Aldehyde via
lipo xygenase
Ethy 2methybutanoate
C7 H14 O2
130
133
Ester fro m
isoleucine
3-Z-hexenol
C6 H12 O
100
156
Alcohol via
lipo xygenase
Methional
C4 H8 S1 O1
104
165
Thioether
aldehyde
Plant
hormone
with ro les in
fruit
ripening,
senescence
and stress
response
Biogenic,
odor of
rotten egg
Oxidative
stress
response of
plants
Anaerobic
respiration
Onion,
garlic, coffee
flavoring
Green leaf
volatile,
green, grassy
odor
Key flavor
compound
of fru it
Green leaf
volatile,
herbaceous
odor
Flavour
compound
Linalool
C10 H18 O
154
198
Monoterpene
alcohol
22, 6Enonadienal
C9 H14 O1
138
203
Aldehyde via
lipo xygenase
Vol atile
Molecul ar
formulas
Molecul ar
weight
ethylene
C2 H4
methanethiol
Co mmon
plant volatile
with floral
odor
Cucu mber
flavour
17
Table 2.1: (continued)
Molecul ar
weight
bpo C (760)
mm Hg
furaneol
Molecul ar
formulas
C2 H8 O3
128
216
∂-octalactone
C8 H14 O2
142
238
Fatty acid
oxidation
Hexy l hexanoate
C12 H24 O2
200
245
skatole
C9 H9 N1
131
265
Ester fro m fatty
acid degradation
Aromatic
heterocycle
α-farnesene
C15 H24
204
280
Sesquiterpene
androstenone
C18 H28 O1
272
372
Steroid
Vol atile
2.6
Chemical cl ass/
bi osynthesis
Sugar derived
Source/
bi oacti vity
Strawberry
furanone,
meaty
flavouring
agent
Microbial,
dairy foods –
coconut,
creamy,
fruity
Fruity ester
Tryptophan,
faecal, fru ity
odor at low
levels
Flower
volatile and
flavour
precursor
Mammalian
pheromone
Yeast Ferme ntation
Fermentation is the oxidation of carbon compounds by microorganisms in the
absence of oxygen to produce energy and other compounds such as ethanol and lactic
acid. Microorganisms, carbon (organic compounds) and absence of oxygen are
necessary for fermentation to take place. Fermentation also depends on
environmental factors such as temperature and pH. Many chemicals (alcohols,
vitamins, hormones, antibiotics, enzymes, acids) are made through fermentation,
most being antibiotics and other drugs. Fermentation takes place in yeasts, bacteria
and muscle cells of animals. Yeasts are much more commonly used for fermentation
among microorganisms because they can tolerate low pH, higher substrate
concentration, higher product concentration, anaerobic growth, and resistance to
inhibitors (Nevoigt, 2008). Yeast metabolism such as the utilization of glucose can
be through the fermentation of glucose, oxidation of glucose or oxidation of ethanol.
In metabolic reactions, yeasts such as Saccharomyces cerevisiae can completely
18
ferment the monosaccharides glucose, fructose, galactose and the disaccharides
sucrose and maltose (Yoon et al., 2003).
2.7
Attractants and Traps
Attractants are volatiles (chemical signals) that can move great distances and
are easily detected by organisms such as insects. Some of the terms used for
attractants are shown in Figure 2.2. Chemical signaling enable organisms to identify
pathogens, kin, mating partner, toxins, prey, food and predators. Since the chemistry
of pheromones was known to be small organic compounds, many semiochemicals
have been identified. Many semiochemicals have functional groups such as esters,
ketones, alcohols, amines, aldehydes, carboxylic acids, phenols, diols and quinines.
Semiochemicals are communication molecules used in insect- insect and plant-insect
interactions (Heuskin et al., 2011). Pheromones or their analogues are also used by
predators to attract potential preys or in the case of plants, to attract potential
pollinators. Semiochemicals are used to control insects thro ugh trapping, attraction
and killing of insects (Norin, 2007).
19
SEMIOCHEMICALS
Gr. Semeon, signal
Acting between individuals
Acting between individuals of different
within a species
species
Pheromones
Allelochemicals
Gr. Phereum, carry
Gr. Allo, different
Alarm
Trail
Sexual
Aggregation
Allomones
advantage for sender
Kairomones advantage for receiver
Synomones
advantage for both
parties
Apneumones from non- living
sources
Figure 2.2: Terms used for semiochemicals (Norin, 2007).
Semiochemicals are natural, safe for humans, animals and the environment,
sustainable, specific and do not involve insect resistance (Heuskin et al., 2011).
Within semiochemicals, volatiles with the same characteristics as with the volatiles
from foods can act as good attractants. While semiochemicals effects are similar to
smell in humans, they are more sensitive and specific. Receptors of organisms such
as insects are able to filter out irrelevant chemical messages. In addition, the
receptors are able to detect chemical messengers at extremely low concentrations. In
order to produce attractants, specific active components are identified and isolated
20
and sometimes produced synthetically. The attractants can be for feeding, alarm, sex
or aggregating. Attractants are used in four ways: lure to traps for population studies,
lure to trap for destruction, signal to discourage mating, and lure to traps containing
insecticides. Commonly used attractants include bread, peanut butter, molasses, food
scent, roach pheromone, and beer (Wang and Bennett, 2006). Many attractants are
chemicals such as carbamates, organophosphates, pyrethroids that are injurious to
animals, humans and the environment (Quesada-Moraga et al., 2004). Pest resistance
and toxicity to humans is driving the pressure to replace synthetic pesticides with
biocontrol measures (Jiang et al., 2008). Food baits are not common in commercial
pest control strategies despite their effectiveness as they often turn out to be breeding
grounds for pests (Cox and Collins, 2002). Pheromones, apart from being target
specific, do not pose any harm to the environment and can trap pests at low density
populations (Schwalbe and Mastro, 1988). Food and synthetic volatiles have been
used as attractants in experiments but results have been inconsistent (Nalyanya and
Schal, 2001). Sticky traps are the most commonly used traps for cockroach compared
to jar traps as these are safe, non-toxic, more convenient and easy to use (Wang and
Bennett, 2006). Traps can be differentiated on the basis of size, shape, glue,
attractants and placement methods. Factors affecting the location of cockroaches
include height of location, humidity, temperature, feed, darkness (Eggleston and
Arruda, 2001).
2.8
The Saccharomycetaceae Family
Yeast classification based on phenotypic characteristics has been replaced by
molecular identification. Doubts on the accuracy of using morphology and metabolic
characteristics such as sexual state, cell division type, sugar fermentation, growth
pattern on several carbon and nitrogen sources prompted the transition to molecular
identification of yeasts (Suh et al., 2006). Members of the Saccharomycetaceae
family reproduce by budding, are found in most habitats especially those rich in
carbohydrates (Nevoigt, 2008, Yoon et al., 2003). The Saccharomycetaceae family is
divided into several genera among which are Saccharomyces, Pichia and Candida
(Jung et al., 2010). They are found in a wide variety of habitats especially those
habitats rich in carbohydrates. The family contains the species Saccharomyces
21
cerevisiae, the most important yeast, first eukaryote to be sequenced, with 16 haploid
chromosomes, about 6000 protein encoding genes in a genome of about 12 million
base pairs (Goffeau et al., 1996). Ethanol is toxic to most microorganisms but
Saccharomyces cerevisiae and its closest relatives are able to dominate fermentation
by producing ethanol and later using it as feedstock (Piskur et al., 2006).
2.8.1
Candida ethanolica
Isolated from industrial fodder yeast, it can grow on ethanol as the only
carbon source (Rybarova et al., 1980). Fe3+ and not Fe2+ is the growth limiting iron
form for Candida ethanolica (Emelyanova, 2000). C. ethanolica is part of the
dominant microflora in shubat, a fermented camel milk product (Shori, 2012),
among the 91 yeast isolates of microbial community of Ghanaian heap cocoa bean
fermentation (Daniel et al., 2009), part of the yeast communities of mescal, (a
Mexican distilled beverage) fermentation (Valdez, 2011) and likely involved in sour
cassava starch fermentation (Lacerda et al., 2005). C. ethanolica can be used to
control bacterial wilt disease in tomato and improvement of yield of the tomato
(Nguyen and Ranamukhaarachchi, 2010; Nguyen et al., 2011). C. ethanolica is one
of the yeast strains isolated from fermentation of grape marc (pomace) in grappa
production (Iacumin et al., 2012), a reference strain for chemiluminescent DNA
optical fiber sensor for Brettanomyces bruxellensis detection (Cecchini et al., 2012),
C. ethanolica is part of the yeast genera present during some stages in wine
fermentation (Cocolin et al., 2000).
2.8.2
Pichia kudriavzevii
P. kudriavzevii, and Issatchenkia orientalis are synonymous organisms
(Kurtzman et al., 1980; Casaregola et al., 2011; Isono et al., 2012). Using
phenotypic, biochemical and molecular techniques, P. kudriavzevii and I. orientalis
were shown to be synonymous yeasts (Bhadra et al., 2007). Phytate, an inhibitor of
minerals availability in ‘togwa’ a cereal food in Tanzania, was degraded by P.
kudriavzevii TY13 and other yeasts (Hellstrom et al., 2012). Oberoi and co-workers
22
used a thermotolerant P. kudriavzevii to produce ethanol from alkali- treated rice
straw (Oberoi et al., 2012). A galactose medium adapted P. kudriavzevii was able to
produce 30% more ethanol from sugarcane juice than a non-adapted cell (Dhaliwal et
al., 2011). In a study using simultaneous saccharification and fermentation, P.
kudriavzevii was used to ferment hydrolyzed cotton stalks (Kaur et al., 2012). P.
kudriavzevii is a potential high phytase active yeast for improved minerals
availability in sourdoughs (Nuobariene et al., 2012), part of fen-daqu, starter culture
for Chinese liquor production (Zheng et al., 2012), part of the yeast species involved
in cocoa bean fermentations (Papalexandratou and De Vuyst, 2011), one of the
yeasts involved in alcoholic fermentation of tchapalo, a local beer in Cote d’Ivoire
(N’guessan et al., 2011), able to decolourize textile wastewater (Pajot et al., 2011),
among 91 yeast isolates of the microbial community of Ghanaia n heap cocoa bean
fermentation (Daniel et al, 2009), can produce ethanol under various stress
conditions (Isono et al., 2012).
2.9
Media for Volatile Metabolites Production by Yeasts
Medium composition is one of the determinants of the fermentation
performance of microorganisms such as yeasts (Hahn-Hagerdal et al., 2005). While
cell culture is used for animal and plant cells, microbiological cultures are used for
the growth of microorganisms such as yeasts and bacteria. Agar plates and nutrient
broth are used extensively in yeast fermentations. Wort (Lodolo et al., 2008),
molasses (Alegre et al., 2003) and potato dextrose broth (Kalyani et al., 2000) are
common in yeast fermentation. Yeast stock cultures are usually maintained at 4 o C in
potato dextrose agar (Lakshmanan and Radha, 2012). PDB is used in many yeast
fermentations such as in the production of ethanol from water hyacinth (Eichhornia
crassipes) (Kasthuri et al., 2012), as subcultures that can be used as inocula for
fermentations (Lakshmanan and Radha, 2012). When molasses is used, the pH is
sometimes adjusted to 4-5. Polysaccharides including dextrins, pectins and gums are
not utilized by yeasts; most yeasts utilize hexose sugars (- mono and disaccharides).
Spiecies, substrate composition and growth duration all influence the volatile
metabolites produced by microorganisms (Borjesson et al., 1992). Media can
improve yield, decrease fermentation period, or increase biomass formation
23
(Chunkeng et al., 2011). Medium can also affect the profile and ecology of
microorganisms during fermentation (Hsieh et al., 2012). In an experiment in which
Penicillium aurantiogriseum was grown on six different substrates, production of
terpenes was more on artificial substrate (Czapek agar and Norkrans agar) but
alcohol production was more on cereal based substra tes (barley meal agar, oat meal
agar, wheat meal agar, malt extract agar)
(Borjesson et al., 1990). Common
fermentation media include potato dextrose broth (Kalyani et al., 2000), PMP (2.4%
PDB, 1% malt extract, 0.1% peptone) (Park et al., 2000), and Sabouraud dextrose
broth (Maghsoodi et al., 2009). Potato dextrose broth is a general purpose medium
for the cultivation of yeasts and molds. Its low pH (5.6 ± 0.2 at 25 o C) discourages
bacterial growth (Condolab, 2012).
2.10
Cockroach as Pest
Pests are plants and animals detrimental to humans directly or indirectly.
They carry human disease vectors or compete with humans in agriculture by feeding
on crops or becoming parasites to livestock. Allergens produced by cockroach
infestation are responsible for asthma, a significant public health disease (Arruda et
al., 2001). Many isolated bacteria from cockroaches obtained from health care
facilities were found to have resistance to common antibiotics (Pai, 2013). Only five
out of the 3,500 known cockroach species inhabit homes and cause indoor allergens
(Eggleston et al., 2001). Ecologically, cockroaches are in every part of the world but
American (Periplaneta americana), German (Blattella germanica) and brownbanded (Supella longipalpa) cockroaches are the most common in tropical
environments. Cockroach allergens, mainly Bla g 1 and Bla g 2, can be found in gut,
saliva and malphighian tubules (De Lucca et al., 1999). They are everywhere
wherever there are structures such as homes, restaurants, schools, hospitals, offices
and industries. They contaminate foods, destroy paper products, fabrics and impact
repulsive stains and odours. They are omnivorous. They are capable of transmitting
Salmonella and Shigella food poisoning, Staphylococcus and Streptococcus bacteria,
and have been implicated in typhoid, dysentery, hepatitis virus and coliforms.
24
2.11
Cockroach Control
Cockroaches are found in dark, sheltered, humid, warm places such as around
kitchen sinks, cracks around or underneath cupboards, cabinet, behind drawers,
around pipes or conduits, behind window or door frames, underside of tables and
chairs, in bathroom, radio and TV cabinets, garages, sewers, attics, basements and
storerooms. Cockroach
population in a location depends on height, humidity,
temperature, and darkness. Natural population also depends on the history of the site
due to insecticide treatment date and intensity and environmental factors make
comparison of data difficult (Rivault, 1989). Trapping, mating distruptions,
attracticides are used to control many pests including cockroach. Traps are relatively
more effective in cockroach control (Rust et al., 1999). Cockroaches share the same
three phase development cycles of egg, nymph and adult during a life span of 100200 days (Wu et al., 2007). In natural populations, nymphs are usually more than
adult cockroaches (Hahn and Ascerno, 2005). In a survey of 69 health care facilities,
45 were infested with cockroaches comprising 250 adults and 308 nymphs (Pai,
2013). Jiang and co-workers reported a biocontrol method for cockroach that
employed virus in rat chow as bait (Jiang et al., 2008). Fungi spores were used as
entomopathogenic agent in an experimental biological pest control carried out by
Quesada-Moraga and co-workers (Quesada-Moraga et al., 2004). However, fungal
biocontrol has limitation of reduced efficacy due to many factors such as temperature
and moisture (Ekese et al., 2003). In another experiment, mice vaccinated with Bla g
2 DNA vaccine (Bla g 2 is one of the most potent cockroach alle rgens) helped
minimize the effect of the cockroach allergen (Zhou et al., 2012). With sticky catch
(no release), the daily catch decreases with time (Wang and Bennett, 2006).
Cockroaches do not require large amounts of food to survive especially in the
presence of water. However, baits normally compete with food sources in the
surroundings (Lucas and Invest, 1993; Koehler et al., 2007). Cockroaches (especially
the German cockroach), consume a wide range of macro- and micro-nutrients
including carbohydrates, proteins, and fats (Kells, 2005).
25
2.12
Yeast Volatile Metabolites as Attractants
While yeast fermentation produced volatile metabolites that can attract sap
beetles, volatile metabolites from bacteria fermentation could not attract the sap
beetles (Nout and Bartelt, 1998). Yeasts produce many volatile metabolites during
fermentation. In an experiment, volatile metabolites were able to attract Rhodnius
prolixus, a disease vector in South America (Lorenzo et al., 1999). Active volatile
metabolites (alcohols, aldehydes, acetates, esters, ketones) are usually in a complex
mix as attractants for insects (Nout and Bartelt, 1998). Volatile metabolites as
attractants are natural, safe for humans, animals and the environment, sustainable,
specific and does not involve insect resistance (Heuskin et al., 2011).
2.13
Identification of Volatile Metabolites from Yeasts Fermentation
Chromatography is used in the separation of complex mixtures where
separation is determined by affinity of the compound partitioned between the two
immiscible solvents of the stationary and mobile phases. Chromatography cannot
readily identify the constituents of the separated mixtures and mass spectroscopy is
used to identify compounds but not from complex mixtures. Therefore, it is always
desirable to couple separation processes to identification steps. Part of the output
from the detector is normally channeled to a mass spectrometer where its
fragmentation pattern can be used to identify the compound (Matuszewski et al.,
1998). Two most important chromatographic techniques in use are liquid
chromatography (LC) and gas chromatography (GC). In mass spectroscopy, sample
components are ionized in one of the several ways leading to formation of ions. Ions
are separated according to their mass-charge ratio, detected quantitatively and signal
is processed into mass spectra. LC is favored over GC due to less demanding and
simple handling of instrument and preparing sample, faster analysis, better at
handling volatiles and higher masses, need no derivation, uses tandem mass
analyzers, and best for researching unknown metabolite. In the analysis of body
fluids for various drugs, LC-MS/MS was found to be better in reduced lowest limit
of quantification, reduced cost, reduced preparation time, no need for sample
derivation and reduced run time (Coles et al., 2007). The introduction of electrospray
26
ionization (ESI) and matrix-assisted laser desorption - ionization (MALDI) has made
LC-MS/MS the favorite tool of biomolecular analysis. LC - MS is especially suited
to the analysis of chemically and thermally unstable compounds such as fatty acids
(Johnson, 2005).
2.14
LC-MS/MS
There is no direct link between LC-MS/MS signals and biochemical
knowledge of the investigated organism. Unlike proteins, metabolites structures are
not repeated with limited alphabets but a combination of elements (C, H, O, S, N, P)
with diverse structures. Their physical and chemical diversity make it difficult to
derive a general rule for fragmentation patterns. It is difficult to link mass (from m/z
values) to a specific metabolite because (i) metabolites with different elemental
formulas may still share highly similar masses (ii) metabolites can have same
elemental formulas but different structures (Breitling et al., 2006). Only LC-MS
analysis cannot identify the different structures having same mass even though there
are separated by the retention time scale. Therefore, for metabolite identification that
ensures less ambiguity and more confidence, tandem mass spectroscopy is used. LCMS/MS combines the resolving power of chromatography and sensitivity and
specificity of mass spectrometry. Steps in LC-MS/MS include sample preparation,
sample introduction, sample separation, vapourization (desorption, electron injection,
electron capture), ionization (protonation, deprotonation, cationization), analyte
sorting (separate by space or time), detection, and conversion. LC-MS/MS offers
speed and increased sensitivity over conventional methods (Dear et al., 1998). Most
LC-MS/MS systems have triple quadruple mass analyzer, atmospheric pressure
ionization source, electro spray source, +/- mass spectrometer, curved collision cell,
10-1500 amu mass range and 105 linear dynamic range. Metabolites with different
elemental formulas can share highly similar masses and metabolites with different
structures can have the same elemental formulas. This LC-MS/MS is able to
differentiate the metabolites as retention time in LC is not enough to unambiguously
identify the different metabolites. The comb ination is used to separate, identify and
quantify components in a compound. LC-MS/MS identification is based on
molecular weight, retention and fragmentation properties (Bajad et al., 2006). It can
27
analyze compounds across different classes such as polar, semi volatiles, pesticides,
mycotoxins, thermally labile compounds, and steroids. There are more positive ions
produced than negative ions. Formation of parent ion is through ionization without
cleavage. Compounds can be identified through MS at 1 part in 1012 . Liquid
chromatography – tandem mass spectroscopy is the preferred technique used for the
analysis of drugs, metabolites and other biomolecules due to its sensitivity, speed and
specificity (Jemal, 2000). LC-MS/MS is not only a superior analytical tool to
immunoassays, HPLC, faster than GC-MS, but is spreading to smaller laboratories
(Grebe and Singh, 2011). Introduction of automated 96-well sample preparation, and
-piercable caps have increased throughput such that analysis that usually take 4-6
hours for instrument and analyst’s time now take 20-120 minutes and 10 minutes for
the analysis and the analyst respectively (Jemal, 2000). LC-MS/MS is used to
identify unknown compounds, determine the isotopic composition of elements in a
molecule, determine chemical structure by examining its fragmentation, quantifying
the amount of a compound. LC-MS/MS was used to identify and quantify nonvolatile metabolites from the biodegradation of environmentally persistent
compounds (Dinglasan et al., 2004). LC-MS/MS with electrospray ionization was
used by Bajad and co-workers to quantify 141 metabolites from Escherichia coli
(Bajad et al., 2006). Direct analysis of a wide variety of drugs in samples using LCMS/MS is of benefit to several applications (Fitzgerald et al, 1999). Along with GC
– based tests, LC-MS/MS was used to detect 50 environmental contaminants in baby
foods (Vukovic et al., 2012). Some varieties of grape were fermented by yeast and
LC-MS/MS was used to confirm that melatonin, a neurohormone, is not present in
grapes or musts but is produced during the alcoholic fermentation of grapes (must) to
produce wines under strictly controlled conditions (Rodriguez-Naranjo et al., 2011).
In profiling the volatile metabolites in wines obtained from pure starter culture
fermentation by Saccharomyces cerevisiae and mixed starter culture involving
Saccharomyces cerevisiae and other yeasts, LC-MS/MS was used to show that
metabolic pathways and hence, metabolites can be altered by the interaction between
yeasts in a mixed culture of Saccharomyces cerevisiae and other yeasts (Sadoudi et
al., 2012).
28
2.15
Downstream Processing of Ferme ntation Broth
Downstream processing is the recovery of biosynthetic products from plants
and animal tissue cultures as well as from fermentation broth. It also includes
recycling and waste treatment and disposal. In the case of fermentation, it is the
processes after fermentation to get useful products. Products from downstream
processing include hormones, antibiotics, antibodies, vacc ines, enzymes, fragrance
and flavors. The unit operations involved in downstream processing are divided into
four categories; the removal of insolubles (filtration, sedimentation, centrifugation,
precipitation, flocculation), product isolation (solvent extraction, adsorption,
ultrafiltration, precipitation), product purification (chromatography, cyrstallization),
and product polishing (crystallization, dessication, freeze drying, lyophilization,
spray drying). Fermentations occur mostly at close to physiolo gical conditions and
low substrate and product concentrations thus making products recovery expensive
(Schugerl, 2000). However, advances have been recorded in processing of
fermentation products in many unit operations such as, adsorption, extraction,
centrifugation, flocculation and precipitation (Cramer and Holstein, 2011).
Centrifugation is the most common way of separating cells from medium. Some
product recovery operations may contain more than one unit operation per step such
as affinity chromatography which involves isolation and purification. Pervaporation
has been used to extract dairy aroma compounds (Baudot and Marin, 1996), extract
aroma compounds from fruit juices using membranes (Sampranpiboon et al., 2000).
Centrifugation at 10,000 rpm for 15 minutes was used to remove cells from
fermentation broth in the process of pullulan production (Wu et al., 2009).
Separation of two metabolites from fermentation broth was reported to constitute
over 50% of the total cost of microbial production (Xiu and Zeng, 2008). In the
purification of surfactin from fermentation broth, a combination of adsorption, ion
exchange and ultrafiltration was used (Chen et al., 2008). Solvent sublation was used
by Bi and co-workers to purify Penicillin G from fermentation broth (Bi et al., 2008).
In the case of highly viscous and heterogeneous broth, ultrafiltration was used for the
purification of demethylchlrotetracycline (DMCT) (Morao et al., 2001).
29
2.16
Freeze Drying
Freeze drying (lyophilization) is a dehydration process in which low pressure
is applied to a frozen material in such a way that the solvent (water) sublimes to
vapor. It is essentially the removal of solvent such as water by sublimation. It is
normally employed to separate water from volatile materials with minimal loss or
degradation of the volatile components. In a comparison of air and freeze drying, the
retention of volatile materials was more in freeze dried samples than in air dried
samples of the same product (Krokida and Philippoulos, 2006). Even though freeze
drying is expensive, the cost may be less than that of preservation, storage and
transportation of the bulk liquid product. Products for freeze drying must be in frozen
form. Most samples for freeze drying are eutectics, however, the presence of
suspended materials can lead to glass formation with the attendant high viscosity.
Such materials freeze at glass transition temperatures. Freeze drying takes place
below the triple point of water for aqueous solutions. Below the triple point, water is
present either in ice or vapour phase. Any vapour from the product must be
condensed immediately to maintain vacuum. It is easier for water to migrate from the
interior of a product when in vapour form and hence easier to dry the product. Loss
of volatile metabolites is minimized in freeze drying than in other forms of drying. In
comparing oven drying and freeze drying methods, loss of volatiles from grape skins
was less in freeze drying method than in oven drying method (de Torres et al., 2010).
Temperature and vacuum are the two most important parameters in freeze drying but
they depend on product, shape, water content, structure and composition of product.
Freeze drying has a wide range of applications in preparation of active
pharmaceutical ingredients, vaccines, diagnostic kits, reagents, proteins, hormones,
viruses, bacteria, tissue, bone, blood plasma, enzymes, pathological samples,
nutraceuticals, vitamins, herbal extracts and the recovery and concentration of
reaction products. Freeze drying retained most of the basic volatiles of saffron (the
dried stigma of a flower, used as spice, drug component) compared to toasting, sun
drying and 35o C dryer (Kanakis et al., 2004). There are three phases of freeze drying;
prefreezing, primary and secondary drying. During freeze drying, end point,
contamination, backfilling and product stability will have to be monitored. In a
freeze dryer, vapour molecules migrate from the higher pressure sample to the lower
pressure area of the ice collector. Some products do have collapse temperature, such
30
as sucrose that has a collapse temperature of -32o C. A vacuum pump ensures that
water flows from the product being freeze dried (Christ, 2012).
31
CHAPTER 3
MATERIALS AND METHODS
3.1
Experime ntal Design
Several factors, many of them confounding, affect bioattraction of
cockroaches. They include history of routine pest control measures in place, location
(proximity to food water), humidity, temperature, darkness, altitude, initial
population, bait (1-day, 4-day fermented broth, freeze dried fermented broth,
control), trap and time. Bait type was the independent variable, and number of
cockroach trapped as dependent variable. The experiments (Figure 3.1) were
conducted with either eliminating or minimizing the effect of the remaining
(confounding) variables such as the use of the same locations and conducting
experiments at regular intervals.
3.2
Materials and Equipme nt
The materials used were potato dextrose agar (PDA), potato dextrose broth
(PDB), petri dishes, centrifuge tubes, Schott bottles, wire loops, measuring cylinders,
distilled water, cockroach trap, cotton wool, parafilm, beaker, spatula, rack, cuvettes,
glycerol, beads tubes, pipette tips, marker, hand gloves, clean wipes, weighing boat,
70% (v/v) ethanol solution, sealer, autoclave plastic, rubber bands, and scissors. The
equipments used include autoclave, laminar flow chamber, incubator, shaking
incubator, freeze dryer, liquid chromatography tandem mass spectrometer (LCMS/MS), UV-visible spectrophotometer, pipettes, centrifuges, weighing balance,
refrigerators, freezers, and digital camera.
32
15.6 g PDA + 400 ml d istilled
H2 O, autoclaved
PDA on plates
13.25 g PDB + 500 ml
distilled H2 O, autoclaved
Yeast colonies on agar
plate
Streaked agar p late with
yeast, incubated overnight
at 30o C
Single yeast colony on agar
plate
10 ml starter culture 30o C,
150 rp m
500 ml
PDB
10 ml PDB
Starter culture OD = 1.00 at
600 n m
1 ml starter culture
21 ml fermentation culture,
30o C, 150 rp m
Fermented broth OD at 600
nm
1 day (or 4 days)
fermentation culture
17.5 ml
Freeze dried 5.5 ml
Trap
Bioattraction
(control) 5.3 ml
Centrifuged
4000 rp m, 10
minutes
10 ml (LCMS/MS)
control
16 ml supernatant
LC-M S/MS (10
ml)
ml
0.13 g solids
20 ml PDB
5.3 ml supernatant
Bioattraction
Bioattraction
Trap
Trap
Freeze dried 5.3 ml
0.15 g solids
control
Trap
Bioattraction
Figure 3.1: Flowchart of the experimental procedure for the bioattraction of
cockroaches.
33
3.3
Yeasts Source
Candida ethanolica M2 and Pichia kudriavzevii M12 yeasts were obtained
from the culture collection of the Microbiology Laboratory of the Faculty of
Biosciences and Bioengineering, UTM.
3.4
Media Preparation
Potato dextrose agar (PDA) and potato dextrose broth (PDB) were used as
media to culture the yeasts. In preparing the PDA, 15.6 g of PDA powder (OXOID)
were dissolved in 400 ml of distilled water and autoclaved for 20 minutes at 121 o C.
In a sterile laminar flow hood, the PDA solution was poured into 10 agar plates to
solidify and cooled to ambient temperature. The plates were sealed with parafilm and
kept in a refrigerator at 4o C. In preparing the PDB, 13.25 g of PDB powder (CONDA
PRONADISA) were dissolved in 500 ml of distilled water and autoclaved for 20
minutes at 121o C. The PDB solution was kept in a refrigerator at 4 o C. A 30% (v/v)
glycerol (10 ml) was prepared and placed in a 50 ml Schott bottle prior to
autoclaving for 20 minutes at 121o C and this was kept at 4o C in a refrigerator.
3.5
Yeast Maintenance and Subculturing
For long-term preservation of pure cultures of C. ethanolica and P.
kudriavzevii, glycerol stocks were prepared. A single yeast colony was inoculated in
10 ml of PDB in a 50 ml Schott bottle under a sterile laminar flow hood. The
inoculated broth was placed in a shaking incubator (Hotech Instruments Corp., New
Taipei City, Taiwan) set to 30o C and 150 r.p.m. When the optical density at 600 nm
of the fermenting culture was approximately 1, 0.5 ml of the culture was transferred
aseptically to the autoclaved 30% (v/v) glycerol solution (0.5 ml) in a 1ml centrifuge
tube and mixed thoroughly and the glycerol stocks were kept at -80o C. In preparing
the stock culture utilizing preservative beads, 0.5 ml of the solution in the bead tube
was removed and 0.5 ml of the yeast culture was added to the tube containing beads.
Two of such culture stocks were separately prepared for each of C. ethanolica M2
and P. kudriavzevii M12 and kept at -80o C.
34
C. ethanolica M2 and P. kudriavzevii M12 yeasts were subcultured
approximately every biweekly. During subculturing, for each of C. ethanolica M2
and P. kudriavzevii M12, two labeled streaked plates were produced using yeast from
previous plate culture. A flame-sterilized and cooled wire loop was used to remove a
single colony from the culture and inoculate the sterile PDA plate. The newly
streaked plates were sealed with parafilm and kept in an incubator overnight at 30 o C.
When there was good growth on a plate, the plate was stored in the refrigerator at
4o C.
3.6
Production of Volatile Metabolites
Starter culture preparation was carried out under aseptic conditions. A single
colony of C. ethanolica M2 or P. kudriavzevii M12 was used to inoculate the 10- ml
PDB in each 50- ml centrifuge tube. The centrifuge tubes were transferred to a
shaking incubator set at 30o C and 150 r.p.m. When the culture was reasonably
cloudy, the optical density (OD), was measured (Jenway 6300, Baroworld Scientific
Ltd, Essex, United Kingdom). Approximately 3.5 ml of PDB was transferred into a
cuvette using a pipette. The spectrophotometer was blanked using the PDB. Then 3.5
ml of the starter culture was transferred into the cuvette and the OD reading was
taken. Culture with OD600 of about 1 was used for fermentation. Under aseptic
condition in the laminar flow hood, 20 ml of the sterile PDB and 1ml of the starter
culture were transferred into each of 6 labeled 50- ml centrifuge tubes. They were
placed in shaking incubator set at 30o C and 150 r.p.m. After 1-, and 4 days, the OD
was determined for each tube.
3.7
Identification of Volatile Metabolites: using LC-MS/MS
Two of the tubes containing 1-day and 4-day fermented cultures were
centrifuged at 4000 rpm for 10 minutes. Under aseptic conditions, supernatant was
obtained by decanting approximately 16 ml into a sterile 50 ml centrifuge tube.
Supernatant (10 ml) from each of the two tubes as well as 10 ml of PDB (control)
35
were transferred into separate sterile, labeled 50 ml centrifuge tubes and kept at 4 o C
in the refrigerator prior to submission for the LC-MS/MS analyses at Institute of
Bioproduct Development (UTM).
3.8
Cockroach Attraction Using Yeast Metabolites
The supernatant of 1-day and 4-day cultured broth were centrifuged
(Universal 32, Hettich Zentrifugen, Tuttlingen, Germany) at 4000 r p m for 10
minutes. Under aseptic conditions in the laminar flow hood, the supernatant was
obtained by decanting approximately 16 ml into a sterile, 50- ml centrifuge tube.
Approximately 5.3 ml of the supernatant was transferred into each of 6 labeled sterile
50 ml centrifuge tube and kept at 4 o C in the refrigerator. Additionally, control was
prepared from 5.3 ml of PDB which was transferred into each of 6 sterile, labe led 50
ml centrifuge tube. Samples and controls were kept at 4 o C in the refrigerator. Six
locations at the student residential hostels (Kolej Tun Ghafar Baba and Kolej Dato’
Onn Jaafar) were used for most of the bioattraction tests. Trap-a-roach hoy hoy
(Earth Chemical Co., Ltd., Tokyo, Japan) commercial cockroach trap was set up as
trap according to manufacturer’s instructions. Instead of the bait for roaches supplied
in a sachet, two layers of white facial cotton (Guardian Health and Beauty, Malaysia)
was placed firmly on top of the glued surface of the roach trap. Each sample or
control was sprinkled on the surface of the cotton wool in such a way as to avoid
flooding the glued surface. Traps were placed in the enclosures that were damp, dark,
warm and mostly on the 8th floor of the buildings. Traps placed by about 8 p.m. were
checked for trapped cockroaches every morning between 6 a.m. and 7a.m. for 3
consecutive days in order to observe the efficacy of the volatile metabolites as a
function of time in an ambient environment.
3.9
Cockroach Attraction Using Freeze-Dried Metabolites
Three tubes containing fermented broth were used to produce 9 freeze-dried
samples. Under aseptic conditions in the laminar flow hood, each tube was agitated
to ensure yeast dispersion. Approximately 5.5 ml of the fermented broth was
36
transferred into a sterile, labeled and weighed 50 ml centrifuge tube. The weight of
each of the centrifuge tubes were further determined after transferring the 5.5 ml of
fermentation broth into each of them. The samples were kept overnight at -80o C in a
freezer. Each frozen sample was attached to the freeze dryer (Martin Christ Dryers,
Osterode, Germany) set to a vacuum of 0.37 mbar. Dried samples were weighed,
kept in a cool dry place and used almost immediately. Approximately 0.15 g from
freeze dried 5.3 ml PDB served as control. Six locations at the student residential
hostels (Kolej Tun Ghafar Baba and Kolej Dato’ Onn Jaafar) were used for most of
the bioattraction tests. Trap-a-roach hoy hoy (Earth Chemical Co., Ltd., Tokyo,
Japan) commercial trap was set up as trap according to manufacturer’s instructions.
Instead of the bait for roaches supplied in a sachet, two layers of white facial cotton
(Guardian Health and Beauty, Malaysia) were placed firmly on top of the glued
surface of the roach trap. Each freeze dried sample was sprinkled on the surface of
the cotton wool. Traps were placed in enclosures that were damp, dark, warm and
mostly on the 8th floor of the buildings. Traps placed by about 8 p.m. were checked
for trapped cockroaches every morning between 6 a.m. and 7 a.m. for 3 consecutive
days in order to observe the efficacy of the volatile metabolites as a function of time
in an ambient environment.
37
CHAPTER 4
RESULTS AND DISCUSSION
4.1
Bioattraction
Results of the bioattraction tests were grouped according to the yeast utilized
for the fermentation, length of fermentation and form in which the bioattraction bait
was used. The average number of trapped cockroaches using co ntrol (PDB), 1-day
fermented broth by P. kudriavzevii M12, 4-day fermented broth by P. kudriavzevii
M12, 1-day fermented broth by C. ethanolica M2, 4-day fermented broth by C.
ethanolica M2 along with their freeze-dried forms are shown in Figure 4.1.
As shown in Figure 4.1, the highest average number of trapped cockroach
was recorded for the 4-day fermented broth by P. kudriavzevii M12. On the other
hand, the freeze-dried samples of the 1-day fermented broth by P. kudriavzevii M12
and 4-day fermented broth by C. ethanolica M2 trapped the least number of
cockroaches. This could be due to reduced population of cockroaches on the sites
due to previous bioattraction experiments, pest control programs or reduced
availability of volatiles in the freeze dried prod uct due to the effects of the formation
of dry microstructures (Flink and Karel, 1970) or the effects of glass transition
temperature in which the viscous, sticky, rubbery state slows down the rate water is
removed from the material (Abbas and Lasekan, 2010; Bhandari and Howes, 1999).
38
Figure 4.1: Bioattraction of domestic cockroaches to P. kudriavzevii M12 and C.
ethanolica M2. Values are means ± standard deviation of three replicates.
As the 4-day fermented broth of P. kudriavzevii M12 was found to be a better
attractant, successive experiments of cockroach bioattraction at the previously
selected location were carried out. Changes in the average number of cockroach
trapped at a location with time is shown in Figure 4.2
39
Figure 4.2: Successive tests of cockroach attraction to the 4-day fermented broth of
P. kudriavzevii M12 at the same location (i.e. phone cable riser room). Values are
means ± standard deviation of three replicates.
The 4-day fermented broth attracted more cockroaches than the 1-day
fermented broth from both P. kudriavzevii M12 and C. ethanolica M2. An
organoleptic test indicated the 1-day fermented broth to have a ‘sharp’ smell (Garcia
et al., 2013), much like unripened fruit. However, this flavor note was not observed
on the 4-day fermented broth. Primary metabolites such as ethanol and organic acids
are likely to be more abundant in the 1-day fermented broth. Primary metabolites
productions depend on cell growth (Bai et al., 2008) which is at the early phases (lag
and growth phases) of fermentation. Secondary metabolites are produced after
growth phase (Bunch and Harris, 1986). Secondary (volatile) metabolites cover great
distances (Humphris, 2002). As chemical signals for bioattraction (Maffei, 2010), the
more volatile metabolites with likely higher concentrations in the 4-day fermented
broth will attract more cockroaches than the 1-day fermented broth.
Mostly American cockroach (Periplaneta americana) was attracted by each
bait type from P. kudriavzevii M12 fermentation and this is shown in Figure 4.3.
Some control traps (those at the same location where the cockroach population was
40
relatively high) did trap some cockroaches. This is in agreement with the previous
study on attractants evaluation in which control also trapped a minimal number of
cockroaches (Nalyanya and Schal, 2001). As shown in Figure 4.3(a), the nymph
population is more than the adult population. The first set of bioattraction tests were
on natural populations. There are more nymphs than adult cockroac hes in natural
populations (Hahn and Ascerno, 2005). However, the average number of
cockroaches attracted decreased as the same locations were used because the
population was depleted much faster than the rate of population increase (Figure
4.2). The egg of an American cockroach hatch in about 45 days, the nymph mature
in about 215 to 400 days while the adult lifespan of the female cockroach is about
440 days (Hahn and Ascerno, 2005).
(a)
(b)
2.8 cm
2.8 cm
Figure 4.3: Bioattraction of domestic cockroaches to P. kudriavzevii M12 (a) 4-day
fermented broth, and (b) freeze-dried 4-day fermented broth.
4.2
Profiling of Exometabolites from Pichia kudriavzevii M12
In this study, the potential of locally isolated yeasts, namely Pichia
kudriavzevii M12 and Candida ethanolica M2 in cockroach attraction was
elucidated. P. kudriavzevii M12 and C. ethanolica M2 were respectively isolated
from a local preparation of Effective Microorganism (Chan et al., 2012), and
fermented rotten pineapple. As the 4-day fermented potato dextrose broth of P.
kudriavzevii M12 was found to be a better cockroach attractant, the metabolites were
profiled using liquid chromatography-mass spectrometry (LC-MS/MS), thus
revealing the presence of secondary metabolites from the yeast strain for cockroach
41
attraction. P. kudriavzevii M12 and C. ethanolica M2 were observed to be bottom
fermenting yeasts. Optical density at the end of one and four days are shown in
Figure 4.4. This is in agreement with the optical density of the broth of a bottom
fermenting yeast at the end of fermentation when secondary metabolites have been
produced (Virtual Labs, University of Leeds). Distance covered by communication
molecules depends on volatility, its stability in air, diffusion rate, efficiency of
receptor, and wind currents and lipids have the best combination of these properties
(Regnier and Law, 1968). Appendices A, B, D and E show the various mass spectra
of metabolites from PDB P. kudriavzevii M12 fermented supernatant detected using
LC-MS/MS. On the other hand, Appendices C and F show the mass spectra of
metabolites originated from PDB. Figure 4.5 shows the distribution, based on subgroups, of metabolites from 4-day P. kudriavzevii M12 fermented PDB supernatant
from analysis of the mass spectra of LC-MS/MS, while Table 4.1 shows mass
spectral profiling of each metabolite.
Figure 4.4: Optical density of P. kudriavzevii M12 and C. ethanolica M2 at the end
of fermentation in PDB.
42
Figure 4.5: Ratio of the exometabolites (n = 44) identified from LC-MS/MS
profiling of the 4-day fermented PDB of P. kudriavzevii M12.
43
Table 4.1: Mass spectral profiling of exometabolites found in the 4-day fermented PDB of P. kudriavzevii M12.
Mass
Molecul ar
weight
Formul a
m/z
Major
frag ment
m/z
129.062
85.0, 101.0
130.1849
188.119
64.8, 90.8,
114.9
195.181
197.182
Structure
IUPAC name
Synonym
Classification
C7 H14 O2
3-methylbutyl acetate
isoamyl acetate;
isopentyl acetate
ester
187.1931
C8 H13 NO4
6-acetamido-2oxohexanoate
2-o xo -6acetamido
caproate
carboxy lic acid
69.8, 97.8
195.1705
C6 H13 NO6
2-amino-2-deo xy-Dgluconate
D-glucosaminate;
glucosaminate
monosaccharide
67.8, 69.8
196.1634
C7 H8 N4 O3
3,7-dimethyluric acid
(NA)
purine
44
Table 4.1 (continued)
Mass
Molecul ar
weight
Formul a
m/z
Major
frag ment
m/z
203.094
185.0
203.1941
211.206
69.4, 69.8,
153.0, 183.0
227.232
243.137
Structure
IUPAC name
Synonym
Classification
C11 H9 NO3
3-(1H-indol-3-y l)-2oxopropanoic acid
indolepyruvate
carboxy lic acid
210.3126
C13 H22 O2
4-(3-hydroxybutyl)3,5,5-trimethylcyclohex2-en-1-one
3-o xo -7,8dihydro-alphaionol
cycloalkene
69.8, 83.8,
99.9, 181.1,
209.0
226.2325
C9 H14 N4 O3
(2S)-2-(3aminopropanamido)-3(1H-imidazo l-5yl)propanoic acid
carnosine
carboxy lic acid
110.0, 199.0
244.2014
C9 H12 N2 O6
5-[(2S,3R,4S,5R)-3,4dihydroxy-5(hydroxy methyl)o xo lan2-yl]-1,2,3,4tetrahydropyrimid ine2,4-dione
betapseudouridine
nucleoside
45
Mass
Major
frag ment
m/z
Molecul ar
weight
Formul a
315.112
198.9,
216.9, 296.9
316.2623
340.728
99.9, 182.1,
210.1
339.1959
m/z
Structure
IUPAC name
Synonym
Classification
C16 H12 O7
3,5,7-trihydro xy-2-(4hydroxy-3metho xyphenyl)-4Hchromen-4-one
isorhamnetin
polyketide
C9 H14 N3 O9 P
5-amino-1[(2R,3R,4S,5R)-3,4dihydroxy-5[(phosphonooxy)methyl]
oxo lan-2-yl]-1Himidazo le-4-carbo xy lic
acid
5-amino-1-(5phospho-Dribosyl)imidazole 4-carbo xy late
carboxy lic acid
46
Mass
Molecul ar
weight
Formul a
m/z
Major
frag ment
m/z
365.245
185.0, 203.0
364.2054
381.190
69.9, 227.0
385.467
69.8, 215.1,
272.1
Structure
IUPAC name
Synonym
Classification
C10 H13 N4 O9 P
{[(2R,3S,4R,5R)-5-(2,6dio xo-2,3,6,9-tetrahydro1H-purin-9-y l)-3,4dihydroxyo xo lan-2yl]metho xy}phosphonic
acid
xanthosine 5'phosphate;
xanthylic acid
nucleoside
379.386
C13 H21 N3 O8 S
(2S)-2-amino-4-{[(1R)1[(carbo xy methyl)carbam
oyl]-2-{[(2R)-2hydroxypropanoyl]sulfan
yl}ethyl]carbamoyl}buta
noic acid
(R)-S-lactoyl
glutathione;
S-D-Lactoyl
glutathione
carboxy lic acid
384.6377
C27 H44 O
(2S,5S,7S,11R,14R,15R)
-2,15-d imethyl-14-[(2R)6-methylhept-5-en-2yl]tetracyclo[8.7.0.0^{2,
7}.0^{11,15}]heptadec1(10)-en-5-o l
zy mosterol;
delta8,24cholestadien-3betaol;
5alpha-cholesta8,24-dien-3beta-ol
steroid
47
Mass
Major
frag ment
m/z
Molecul ar
weight
Formul a
387.995
99.9, 182.1,
211.1, 338.2
387.1834
394.659
88.8, 251.2
401.626
82.8, 99.9,
182.1, 210.1
m/z
Structure
IUPAC name
Synonym
Classification
C25 H25 NO3
4'-[[(1oxopentyl)phenylamino]
methyl]-[1,1'-biphenyl]2-carbo xy lic acid
(NA)
carboxy lic acid
394.6325
C28 H42 O
(2S,5S,11R,14R,15R)14-[(2R,3E,5R)-5,6dimethylhept-3-en-2-yl]2,15dimethyltetracyclo[8.7.0.
0^{2,7}.0^{11,15}]hepta
deca-1(17),7,9-trien-5-o l
dehydroergosterol
steroid
400.6801
C28 H48 O
(1R,2S,5S,6S,7S,11R,15
R)-2,6,15-trimethyl-14[(2R)-6-methylheptan-2yl]tetracyclo[8.7.0.0^{2,
7}.0^{11,15}]heptadec9-en-5-o l
4-alpha-methyl-5alpha-cholest-7en-3-beta-ol
steroid
48
Mass
Major
frag ment
m/z
Molecul ar
weight
Formul a
413.122
260.9,
278.9,
296.9, 394.9
414.6636
435.437
273.3
435.637
69.8, 83.7
m/z
Structure
IUPAC name
Synonym
Classification
C28 H46 O2
(2S,5S,7S,11R,14R,15R)
-6-(hydro xy methyl)2,15-dimethyl-14-[(2R)6-methylhept-5-en-2yl]tetracyclo[8.7.0.0^{2,
7}.0^{11,15}]heptadec1(10)-en-5-o l
zy mosterol
intermediate 1a
steroid
436.5198
C21 H41 O7 P
[(2R)-2-hydro xy-3[(9Z)-octadec-9enoyloxy]propo xy]
phosphonic acid
lysophosphatidic
acid
(18:1(9Z)/0:0)
glycerolip id
435.2807
C25 H41 NO3 S
N-acetyl-S-(3,7,11,15tetramethyl2E,6E,10E,14hexadecatetraenyl)-Lcysteine
N-acetyl-Sgeranylgeranyl-Lcysteine
carboxy lic acid
(amino acid
derivative)
49
Mass
m/z
453.609
Major
frag ment
m/z
Molecul ar
weight
Formul a
99.9, 113.9,
182.1,
209.2, 435.2
453.5503
C21 H44 NO7 P
Structure
IUPAC name
Synonym
(2-aminoethoxy)[(2R)-3(hexadecanoyloxy)-2hydroxypropoxy]phosphi
nic acid
1-palmitoyl-2hydroxy-snglycero-3phosphoethanola
mine;
Classification
glycerophospholipid
lysophosphatidyle
thanolamine(16:0)
455.052
99.8, 437.0
454.2833
C13 H19 N4 O12 P
(2S)-2-({5-amino-1[(2R,3R,4S,5R)-3,4dihydroxy-5[(phosphonooxy)methyl]
oxo lan-2-yl]-1Himidazo l-4yl}formamido)butanedio
ic acid
(S)-2-[5-amino-1(5-phospho-Dribosyl)imidazole 4carboxamido]succ
inate; SAICAR
carboxy lic acid
50
Mass
Major
frag ment
m/z
Molecul ar
weight
Formul a
455.382
69.8, 83.8,
215.1
454.4707
457.587
457.1
456.432
m/z
Structure
IUPAC name
Synonym
Classification
C28 H22 O6
(1R,8R,9R,16R)-8,16bis(4hydroxyphenyl)tetracycl
o[7.7.0.0^{2,7}.0^{10,1
5}]hexadeca2,4,6,10,12,14-hexaene4,6,12,14-tetro l
pallidol
indane
C20 H22 N7 O6
3-amino-8-(4-{[(1S)-1,3dicarbo xypropyl]carbam
oyl}phenyl)-1-o xo1H,4H,5H,6H,6aH,7H,8
H2,4,5,8,10000000$l^{5}imidazo[1,5-f]pteridin10-yliu m
5,10methenyltetrahydrofolate
carboxy lic acid
51
Mass
Major
frag ment
m/z
Molecul ar
weight
Formul a
501.371
69.8, 83.8,
129.9, 241.0
500.0755
525.548
67.9, 69.8,
244.1
566.743
95.9, 113.9,
209.2,
322.2, 548.4
m/z
Structure
IUPAC name
Synonym
Classification
C6 H16 O18 P4
{[(1S,2S,3S,4S,5R,6S)-2,4dihydroxy-3,5,6tris(phosphonooxy)cyclohexyl]o x
y}phosphonic acid
1D-myo-inositol
1,3,4,5tetrakisphosphate
monosaccharide
525.613
C24 H48 NO9 P
(2S)-2-amino-3-({hydro xy[(2R)2-hydro xy-3(octadecanoyloxy)prop
oxy]phosphoryl}oxy) propanoic
acid
lysophosphatidyl
serine acid
(18:0/ 0:0)
glycerophospholipid
566.3018
C15 H24 N2 O17 P2
{[(2R,3S,4R,5S)-5-(2,6-d io xo1,2,3,6-tetrahydropyrimidin-4-yl)3,4-dihydro xyo xo lan-2yl]metho xy}({[hydro xy({[(2S,3R
,4S,5R,6R)-3,4,5-trihydro xy-6(hydroxy methyl)o xan-2yl]o xy})phosphoryl]oxy })phosphi
nic acid
UDP-galactose;
UDP-Dgalactopyranose
nucleoside
52
Mass
Major
frag ment
m/z
Molecul ar
weight
Formul a
571.608
69.8, 215.1,
230.1
570.6072
C25 H47 O12 P
617.713
88.8, 132.9,
177.1,
459.1, 600.5
616.4873
634.132
589.6
635.8528
m/z
Structure
IUPAC name
Synonym
Classification
[(2R)-3-[(9Z)-hexadec9-enoylo xy]-2hydroxypropoxy]({[(1s,3
R)-2,3,4,5,6pentahydroxycyclohexy l
]o xy})phosphinic acid
Lysophosphatidyl
inositol acid
(16:1(9Z)/0:0)
glycerophospholipid
C34 H32 FeN4 O4
(NA)
heme; protoheme,
heme B;
protoheme IX
tetrapyrrole
C33 H66 NO8 P
(2-aminoethoxy)[(2R)-3(dodecanoyloxy)-2(hexadecanoyloxy)propo
xy ]phosphinic acid
phosphatidylethanolamine
(12:0/ 16:0)
glycerophospholipid
53
Mass
Major
frag ment
m/z
Molecul ar
weight
Formul a
674.064
69.9, 403.2,
573.3
672.9127
C37 H69 O8 P
675.082
69.8, 404.2,
547.3, 630.5
674.9286
679.885
99.9, 182.1,
209.1, 661.6
679.8623
m/z
Structure
IUPAC name
Synonym
Classification
[(2R)-3-[(9Z)-hexadec9-enoylo xy]-2-[(9Z)octadec-9enoyloxy]propo xy]
phosphonic acid
phosphatidic acid
(16:1(9Z)/
18:1(9Z))
glycerophospholipid
C37 H71 O8 P
[(2R)-3(hexadecanoyloxy)-2[(9Z)-octadec-9enoyloxy]propo xy]
phosphonic acid
phosphatidic acid
(16:0/ 18:1(9Z))
glycerophospholipid
C34 H66 NO10 P
(2S)-2-amino-3-({[(2R)3-(dodecanoyloxy)-2(hexadecanoyloxy)propo
xy ](hydro xy)phosphoryl
}o xy)propanoic acid
phosphatidyl
serine (12:0/ 16:0)
glycerophospholipid
54
Mass
Major
frag ment
m/z
Molecul ar
weight
Formul a
691.111
563.3,
646.5, 674.5
691.1635
C48 H82 O2
696.706
69.8, 425.2,
568.3, 651.4
696.1818
712.716
69.8, 308.0,
584.3
712.1812
m/z
Structure
IUPAC name
Synonym
Classification
(2S,5S,7R,11R,14R,15R
)-2,6,6,11,15pentamethyl-14-[(2R)-6methylhept-5-en-2yl]tetracyclo
[8.7.0.0^{2,7}.0^{11,15
}]heptadec-1(10)-en-5-y l
(9Z)-octadec-9-enoate
lanosteryl oleate
steroid
C44 H89 NO4
N-[(2S,3R)-1,3dihydroxyoctadecan-2yl]-26hydroxyhexacosanamide
N-(26hydroxyhexacosa
noyl)sphinganine
sphingolipid
C44 H89 NO5
26-hydro xy-N[(2S,3S,4R)-1,3,4trihydro xyoctadecan-2yl]hexacosanamide
N-(26hydroxyhexacosa
nyl)phyto
sphingosine
sphingolipid
55
Mass
Major
frag ment
m/z
Molecul ar
weight
Formul a
741.053
94.9, 467.3,
495.4, 523.5
740.045
C42 H78 NO7 P
749.136
69.8, 167.0,
244.2,
408.1, 650.4
748.1085
762.060
283.0
762.092
m/z
Structure
IUPAC name
Synonym
Classification
(2-{[2-(hexadec-1-en-1ylo xy)-3-(octadeca6,9,12trienoylo xy)propyl
phosphonato]oxy}ethyl)
trimethylazaniu m
Phosphatidylcholi
ne (18:3(6Z,9Z,
12Z)/P-16:0)
glycerophospholipid
C42 H86 NO7 P
(2-{[3-(hexadecylo xy)2(octadecanoyloxy)propyl
phosphonato]oxy}ethyl)t
rimethylazaniu m
phosphatidylcholi
ne (o-16:0/18:0)
glycerophospholipid
C42 H84 NO8 P
(2-{[(2R)-3(hexadecanoyloxy)-2(octadecanoyloxy)propyl
phosphonato]oxy}ethyl)t
rimethylazaniu m
phosphatidylcholi
ne (16:0/ 18:0)
glycerophospholipid
56
Mass
Major
frag ment
m/z
Molecul ar
weight
Formul a
790.030
227.1,
284.3, 537.6
790.1037
C46 H80 NO7 P
790.051
227.2,
296.2, 537.7
790.1452
813.960
69.8, 183.1,
217.1, 244.1
814.0359
m/z
Structure
IUPAC name
Synonym
Classification
(2-{[3-(icosa-5,8,11,14,17pentaenoyloxy)-2-(octadeca1,11-dien-1-y lo xy)propyl
phosphonato]oxy}ethyl)trime
thylazaniu m
phosphatidylcholi
ne (20:5(5Z,8Z,
11Z,14Z,17Z)/P18:1(11Z))
glycerophospholipid
C44 H88 NO8 P
(2-{[(2R)-2,3bis(octadecanoyloxy)propyl
phosphonato]oxy}
ethyl)trimethylazaniu m
phosphatidylcholi
ne (18:0/18:0)
glycerophospholipid
C40 H80 NO13 P
{[(2S,3S,4R)-3,4-dihydro xy2-[(9R)-9hydroxyhexadecanamido]oct
adecyl]o xy}({[(1s,3R)2,3,4,5,6-pentahydroxy
cyclohexy l]o xy}) phosphinic
acid
Inositolphosphoceramide
18:0;3/16:0;1
sphingolipid
57
Mass
Major
frag ment
m/z
Molecul ar
weight
Formul a
874.807
69.8, 199.1,
241.0, 645.4
873.6561
C28 H42 N7 O17 P3 S
890.445
69.8, 217.1,
229.1, 383.2
889.6555
972.253
311.4
971.7129
m/z
(NA) Not available
Structure
IUPAC name
Synonym
Classification
(NA)
cyclohex-2,5diene-1-carbo xylCoA
coenzyme
C28 H42 N7 O18 P3 S
(NA)
6-keto xycyclo
hex-1-ene-1carboxy l-CoA
coenzyme
C32 H44 N7 O20 P3 S
(NA)
2-succinyl
benzoyl-CoA;
o-succinyl
benzoyl-CoA;
succinylbenzoylCoA
coenzyme
58
Many of the exometabolites found in the 4-day PDB fermented broth of P.
kudriavzevii M12 have characteristics that enable them to be used in many
applications apart from their potential use as bioattractants. Some of the
exometabolites serve as bioattractants to some organisms while some closely related
compounds of the exometabolites have potential as bioattractants to some other
organisms such as isoamyl acetate which could be an attractant to insects and
larvaes. Characteristics of the exometabolites and their close analogues are shown in
Table 4.2
59
Table 4.2: Characteristics of exometabolites found in the 4-day PDB fermented of P. kudriavzevii M12.
IUPAC name
Synonym
Characteristics
3-methylbutyl
Isoamyl
Isoamyl acetate confers banana or pear flavor when used in food flavoring, solvents, cigarette additive,
acetate
acetate
shoe polish, water proof varnishes, and metallic paints (Teo and Saha, 2004).It is produced by the
esterification of acetyl Co A and isoamyl alcohol (Singh et al., 2008; Fukuda et al., 1998).It is a honey
bee pheromone used to initiate stinging operations (Batra, 1985; Boch et al., 1962), attracts insects
lavae (Oppliger et al., 2000), one of the main contributors to the flavor of sake, a Japanese wine
fermented from rice (Fukuda et al., 2000), used to formulate a micro-emulsion of banana aroma in an
experimental nano delivery system (Edris and Malone, 2011), among the volatiles released to attract
insects in some tropical plants (Kite and Hetterschieid, 1997), and one of the flavor compounds in wine
made by a new process (Tsakiris et al., 2010). An increase in isoamyl acetate level in sake fermented by
S. cerevisiae has been shown to depend on the ratio of an alcohol acetyltransferase and an acetate
hydrolyzing esterase (Fukuda et al., 1998). Erten and Tanguler found isoamyl acetate to be in higher
concentration in mixed culture than in regular fermentation culture (Erten and Tanguler, 2010).
60
IUPAC name
Synonym
Characteristics
6-acetamido-2-
2-oxo-6-acetamido
The exometabolite 6-acetamido-2-oxohexanoate is a conjugate base of caproic acid involved in
oxohexanoate
caproate
lysine degradation to glutarate. Lysine degradation varies in different organisms as there are nine
pathways (Zemriskie and Jackson, 2000). In S. cerevisiae, L- lysine is degraded through N6Acetyl- L- lysine,
2-keto-6-acetoamidocaproate,
5-acetoamidovalerate,
5-aminopentanoate,
glutaratesemialdehyde, and glutarate which enters the citric acid cycle through glutaryl CoA
(KEGG pathway, lysine degradation – S. cerevisiae)
2-amino-2-deoxy-D-
D-glucosaminate;
D-glucosaminate is involved in amino sugar metabolism which leads to glycolysis (pentose
gluconate
glucosaminate
phosphate pathway) ,purine, pyrimidine and histidine metabolism (KEGG, pentose phosphate
pathway – reference pathway).
61
IUPAC name
Synonym
Characteristics
3-(1H-indol-3-yl)-2-
Indolepyruvate
The exometabolite 3-(1H- indol-3-yl)-2-oxopropanoic acid is an aromatic heteropolycyclic
oxopropanoic acid
intermediate involved in tryptophan metabolism in S. cerevisiae. Tryptophan deamination is
followed by the formation of 3- indolepyruvate that is subsequently decaboxylated to trytophol.
D-amino acid oxidase, aromatic lactate dehydrogenase and aromatic L- amino aminotransferase
and the principal enzymes required for the catabolism of aromatic amino acids (Bode and
Birnbaum, 1987).
4-(3-hydroxybutyl)-3,5,5-
3-oxo-7,8-dihydro- The exometabolite 3-oxo-7,8-dihydro-alpha-ionol is an aromatic volatile released by S.
trimethylcyclohex-2-en-1-
alpha-ionol
one
cerevisiae during alcoholic fermentation of grapes (Loscos et al., 2007). Male fruitflies
(Bactrocera latifrons) were attracted to cut eggplant and one of the attractants was 3-oxo-7,8dihydro-alphaionol (Nishida et al., 2009).
62
IUPAC name
Synonym
Characteristics
(2S)-2-(3-
Carnosine
Carnosine is a dipeptide of beta-alanine and histidine, an antioxidant, metal chelator and
aminopropanamido)-3(1H-
antiglycator that is involved in beta-alanine and histidine metabolism (Boldyrev et al., 2010).
imidazol-5-yl)propanoic
Through histidine metabolism, it is involved in pentose phosphate pathway, thiourocanic acid
acid
and imidazole lactate production, purine, alanine, aspartate, and glutamate metabolism (KEGG:
Histidine metabolism). In beta-alanine metabolism, carnosine is an intermediate for pyrimidine,
alanine, aspartate, glutamate and propanoate metabolism (KEGG: beta-alanine metabolism).
3,5,7-trihydroxy-2-(4-
Isorhamnetin
Isorhamnetin is a flavonol, along with alkaloids, terpenoids, fatty acid related compounds and
hydroxy-3-methoxyphenyl)-
amino acids related compounds are grouped under phytochemicals. Isorhamnetin was found to
4H-chromen-4-one
be an oviposition stimulant (Tsukasa, 2003).
63
IUPAC name
Synonym
Characteristics
5-amino-1-[(2R,3R,4S,5R)-
5-amino-1-(5-
The exometabolite 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxylate is an intermediate
3,4-dihydroxy-5-
phospho-D-
of purine, alanine, aspartate, glutamate, thiamine, histidine, riboflavin, glycine, serine, threonine
[(phosphonooxy)methyl]oxo
ribosyl)imidazole-
metabolism and the biosynthesis of folate and secondary metabolites in S. cerevisiae (KEGG,
lan-2-yl]-1H- imidazole-4-
4-carboxylate
purine metabolism).
(2S)-2-amino-4-{[(1R)-1-
(R)-S-lactoyl
S-D-Lactoyl glutathione is a peptide that is involved in pyruvate, glycine, serine, threonine,
[(carboxymethyl)carbamoyl
glutathione;
nicotine and nicotinamide metabolism; an intermediate in the production of lactic acid and
]-2-{[(2R)-2-
S-D-Lactoyl
glutathione from methylglyoxal detoxification; as well as valine, leucine, isoleucine, ketone
hydroxypropanoyl]sulfanyl}
glutathione
bodies, lysine, and fatty acid biosynthesis (KEGG: Pyruvate metabolism).
carboxylic acid
ethyl]carbamoyl}butanoic
acid
64
IUPAC name
Synonym
Characteristics
(2S,5S,7S,11R,14R,15R)-
zymosterol;
Zymosterol is an intermediate in steroids biosynthesis as well as the biosynthesis of primary bile
2,15-dimethyl-14-[(2R)-6-
delta8,24-
acid and Vitamin D2 (KEGG: Steroids biosynthesis). C-mavelonic acid was converted to
methylhept-5-en-2-
cholestadien-
zymosterol as an intermediate in the synthesis of ergosterol (Katsuki and Bloch, 1967).
yl]tetracyclo[8.7.0.0^{2,7}.
3beta-ol;
0^{11,15}]heptadec-1(10)-
5alpha-Cholesta-
en-5-ol
8,24-dien-3beta-ol
4'-[[(1-
(NA)
oxopentyl)phenylamino]met
hyl]-[1,1'-biphenyl]-2carboxylic acid
65
IUPAC name
Synonym
Characteristics
(1R,2S,5S,6S,7S,11R,15R)-
4-alpha- methyl-5-
-
2,6,15-trimethyl-14-[(2R)-6- alpha-cholest-7methylheptan-2-
en-3-beta-ol
yl]tetracyclo[8.7.0.0^{2,7}.
0^{11,15}]heptadec-9-en-5ol
[(2R)-2-hydroxy-3-[(9Z)-
PA (18:1(9Z)/0:0)
PA (18:1(9Z)/0:0) is a lysophosphatidic acid and a precursor of phosphotidic acid. PA
octadec-9-
(18:1(9Z)/0:0) is an acid involved in glycerolipid metabolism and lipid enzymes such as lipid
enoyloxy]propoxy]phospho
phosphate phosphatises use glycerolipids and sphingolipids to regulate their signalling functions
nic acid
(Brindley et al., 2002).
N-acetyl-S-(3,7,11,15-
N-acetyl-S-
N-acetyl-S-geranylgeranyl- L-cysteine is an isoprenoid-derived inhibitor of one of the
tetramethyl-2E,6E,10E,14-
geranylgeranyl-L-
phosphatases needed for the conversion of farnesyl pyrophosphate to juvenile hormone in
hexadecatetraenyl)-L-
cysteine
insects, thus having the possibility of disrupting the normal metamorphosis of the insects (Cao et
cysteine
al., 2009).
66
IUPAC name
Synonym
Characteristics
(2-aminoethoxy)[(2R)-3-
1-palmitoyl-2-
Lysophosphatidylethanolamine, LPE(16:0), is an acid and a lysophospholipid (LPL). LPE is
(hexadecanoyloxy)-2-
hydroxy-sn-
formed from phosphatidylethanolamine (Park et al., 2007). Lipids, apart from structural and
hydroxypropoxy]phosphinic
glycero-3-
energy functions, are involved in cell signalling which enable the cells to respond to its
acid
phosphoethanolam
environment (Wang, 2004). Many LPLs have signalling functions (Heringdorf et al., 2007). LPE
ine;
has signalling activities such as calcium signalling through the activation of some lipases (Park
LPE(16:0)
et al., 2007). Cell signalling is regulated by phosphotidylinositol 3-kinase (PI3K) in yeasts
(Gillooly et al., 2000).
(2S)-2-({5-amino-1-
(S)-2-[5-amino-1-
SAICAR (21 synonyms) is involved in purine and ADE2 (p rotein) metabolism as intermediate
[(2R,3R,4S,5R)-3,4-
(5-phospho-D-
and substrate respectively. SAICAR is connected to the metabolism of alanine, aspartate,
dihydroxy-5-
ribosyl)imidazole-
glutamate, thiamine, histidine, riboflavin, glycine, serine, threonine as well as the folate
[(phosphonooxy)methyl]oxo
4-
biosynthesis (KEGG: Purine metabolism). SAICAR has regulatory function in purine
lan-2-yl]-1H- imidazol-4-
carboxamido]succi
metabolism by stimulating interaction of transcription factors (Pinson et al, 2009).
yl}formamido)butanedioic
nate; SAICAR
acid
67
IUPAC name
Synonym
Characteristics
(1R,8R,9R,16R)-8,16-bis(4-
Pallidol
Pallidol is an oligomer in the stilbene group of compounds that are known to aid in preventing
hydroxyphenyl)tetracyclo[7.
cancer and reducing the effects of oxidative damage of lipoproteins (Landrault et al., 2002).
7.0.0^{2,7}.0^{10,15}]hexa
Pallidol in red wine is a potent natural antioxidant and also with antifungal activities (He et al.,
deca-2,4,6,10,12,14-
2008). Also, pallidol was among the most potent antioxidants out of the 10 isolates from
hexaene-4,6,12,14-tetrol
Caragana sinica that were tested for antioxidant activities (Landrault et al., 2002).
3-amino-8-(4-{[(1S)-1,3-
5,10-
The metabolite, 5,10-methenyltetrahydrofolate is a type of tetrahydrofoliate that is involved in
dicarboxypropyl]carbamoyl
methenyltetrahydr
biosynthesis of purine, pyrimidine and methionine as well as histidine catabolism and 5,10-
}phenyl)-1-oxo-
ofolate
Methenyltetrahydrofolate is a substrate in the tetrahydrofolate salvage pathway. Folates (vitamin
1H,4H,5H,6H,6aH,7H,8H2,4,5,8,10000000$l^{5}imidazo[1,5- f]pteridin-10ylium
B9) are responsible for single-carbon unit transfer in biochemical reactios (Appling, 1991).
68
IUPAC name
Synonym
Characteristics
{[(1S,2S,3S,4S,5R,6S)-2,4-dihydroxy-3,5,6-
1D-myo- inositol
The metabolite 1D- myo- inositol 1,3,4,5-tetrakisphosphate (14 synonyms) is
tris(phosphonooxy)cyclohexyl]oxy}phosphonic
1,3,4,5-
an intermediate in inositol pyrophosphate biosynthesis with links to
acid
tetrakisphosphate
glycolysis,
gluconeogenesis,
TCA
cycle,
and
glycerophospholipid
metabolism (KEGG: Inositol phosphate metabolism). Messaging that is
involved in Ca2+ regulation is another area in which 1D-myo- inositol
1,3,4,5-tetrakisphosphate is useful in metabolism (Xia and Yang, 2005).
{[(2R,3S,4R,5S)-5-(2,6-dioxo-1,2,3,6-
UDP-galactose;
UDP-galactose (40 synonyms) is important in starch, sucrose, fructose,
tetrahydropyrimidin-4- yl)-3,4-dihydroxyoxolan- UDP-D-
mannose, galactose metabolism (KEGG: Amino sugar, nucleotide sugar,
2-yl]methoxy}({[hydroxy({[(2S,3R,4S,5R,6R)-
galactose metabolism). UDP-galactose is an important transporter in the
3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2yl]oxy})phosphoryl]oxy})phosphinic acid
galactopyranose
Golgi apparatus (Olczak and Guillen, 2006).
69
IUPAC name
Synonym
Characteristics
[(2R)-3-[(9Z)-hexadec-9-enoyloxy]-2-
PI (16:1(9Z)/0:0)
PI (16:1(9Z)/0:0) is a lysophosphatidylinositol acid, an important lipid in
hydroxypropoxy]({[(1s,3R)-2,3,4,5,6-
membrane component and metabolic processes.
pentahydroxycyclohexyl]oxy})phosphinic acid
(NA)
(2-aminoethoxy)[(2R)-3-(dodecanoyloxy)-2(hexadecanoyloxy)propoxy]phosphinic acid
heme; protoheme,
Heme is a component of most essential proteins, involved in oxidative
heme B; protoheme
phosphorylation and in many regulatory functions (Tsiftsoglou et al., 2006).
IX
Heme is a combination of cytochrome, haemoglobin and myoglobin
PE (12:0/16:0)
PE
(12:0/16:0)
is
a
zwitterion,
and
glycerophospholipid.
Glycerophospholipids as a group form part of the structural components of
cell membranes. Each molecule consists of a polar head and two
hydrophobic tails. Glycerophospholipids
emulsifying agents (Tao, 2007).
are amphiphilic and serve as
70
IUPAC name
Synonym
Characteristics
[(2R)-3-[(9Z)-hexadec-9-enoyloxy]-2-[(9Z)-
PA (16:1(9Z)/
PA (16:1(9Z)/ 18:1(9Z)) is a phosphatidic acid and intermediate in
octadec-9-enoyloxy]propoxy]phosphonic acid
18:1(9Z))
synthesis of triacylglycerols and phospholipids, signalling molecule,
important in cell physiology and biochemistry, reduce or elevate the
activities of some proteins (Chritie, 2012).
[(2R)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9enoyloxy]propoxy]phosphonic acid
PA (16:0/18:1(9Z))
PA (16:0/18:1(9Z)) is a phosphatidic acid and intermediate in synthesis of
triacylglycerols and phospholipids, signalling molecule, important in cell
physiology and biochemistry, reduce or elevate the activities of some
proteins (Chritie, 2012).
71
IUPAC name
Synonym
Characteristics
(2S)-2-amino-3-({[(2R)-3-(dodecanoyloxy)-2-
PS (12:0/16:0)
PS (12:0/16:0) is a phosphatidylserine, a chelating agent whose biosynthesis
(hexadecanoyloxy)propoxy](hydroxy)phosphor
involves
exchange
reaction of
serine
for
ethanolamine.
As
a
yl}oxy)propanoic acid
glycerophospholipid, form part of the structural components of cell
membranes. Each molecule consists of a polar head and two hydrophobic
tails. Glycerophospholipids
are amphiphilic and serve as emulsifying
agents (Tao, 2007).
(2S,5S,7R,11R,14R,15R)-2,6,6,11,15-
lanosteryl oleate
Lanosteryl oleate is an acid ester of lanosterol, a compound required for all
pentamethyl-14-[(2R)-6- methylhept-5-en-2-
steroids synthesis. Sterols are components of cell membrane while their
yl]tetracyclo
esters are the storage form of sterols (Billheimer et al., 1983).
[8.7.0.0^{2,7}.0^{11,15}]heptadec-1(10)-en-5yl (9Z)-octadec-9-enoate
72
IUPAC name
Synonym
Characteristics
26-hydroxy-N-[(2S,3S,4R)-1,3,4-
N-(26-
N-(26-hydroxyhexacosanyl) phyto sphingosine is a phytoceramide, an
trihydroxyoctadecan-2-yl]hexacosanamide
hydroxyhexacosany
intermediate in the production of sphingolipids. Sphingolipids act as
l)phyto sphingosine
signalling molecules that affect cancer, diabetes, heart disease, microbial
infection, neurological disorder and immune dysfunction as well as
structural role in actin cytoskeleton (Dickson, 2008).
(2-{[2-(hexadec-1-en-1-yloxy)-3-(octadeca-
PC
PC (18:3(6Z,9Z,12Z)/P-16:0), (14 synonyms), is a phosphatidylcholine, a
6,9,12-trienoyloxy)propyl
(18:3(6Z,9Z,12Z)/P
glycerolphospholipid and phospholipid subgroup involved structurally in
phosphonato]oxy}ethyl)trimethylazanium
-16:0)
lipid bilayer, cell metabolism and cell signalling (Ohvo-Rekila et al., 2002;
Geiger et al., 2012). It is involved as lecithin in glycerophospholipid,
arachidonic, linoleic, alpha- linolenic metabolism as well as retrograde
endocannabnoid signalling (KEGG: glycerophospholipid, arachidonic,
linoleic,
signalling).
alpha- linolenic
metabolism,
retrograde
endocannabnoid
73
IUPAC name
Synonym
Characteristics
(2-{[3-(hexadecyloxy)-2-
PC (o-16:0/18:0)
PC (o-16:0/18:0) is a phosphatidylcholine, a glycerolphospholipid and
(octadecanoyloxy)propyl
phospholipid subgroup involved structurally in lipid bilayer, cell
metabolism and cell signalling (Ohvo-Rekila et al., 2002; Geiger et al.,
2012).
(2-{[3-(icosa-5,8,11,14,17-pentaenoyloxy)-2-
PC
PC (20:5(5Z,8Z,11Z,14Z,17Z)/P-18:1(11Z)) is a phosphatidylcholine, a
(octadeca-1,11-dien-1-yloxy)propyl
(20:5(5Z,8Z,11Z,14
component of the lipid bilayer and as a phospholipid, generally involved in
Z,17Z)/P-
membrane structure, metabolism and cell signalling (Ohvo-Rekila et al.,
18:1(11Z))
2002; Geiger et al., 2012).
(2-{[3-(icosa-5,8,11,14,17-pentaenoyloxy)-2-
PC
PC (20:5(5Z,8Z,11Z,14Z,17Z)/P-18:1(11Z)) is a phosphatidylcholine, a
(octadeca-1,11-dien-1-yloxy)propyl
(20:5(5Z,8Z,11Z,14
component of the lipid bilayer and as a phospholipid, generally involved in
Z,17Z)/P-
membrane structure, metabolism and cell signalling (Ohvo-Rekila et al.,
18:1(11Z))
2002; Geiger et al., 2012).
74
IUPAC name
Synonym
Characteristics
{[(2S,3S,4R)-3,4-dihydroxy-2-[(9R)-9-
IPC 18:0;3/16:0; i1
IPC 18:0;3/16:0; i1is
an inositol-phosphoceramide (sphingolipid).
hydroxyhexadecanamido]octadecyl]oxy}({[(1s,
Sphingolipids act as signalling molecules that affect cancer, diabetes, heart
3R)-2,3,4,5,6-pentahydroxy
disease, microbial infection, neurological disorder and immune dysfunction
cyclohexyl]oxy})phosphinic acid
as well as structural role in actin cytoskeleton (Dickson, 2008).
(NA)
cyclohex-2,5-diene-
Cyclohex-2,5-diene-1-carboxyl-CoA is involved in benzoate metabolism
1-carboxyl-CoA
with links to chlorocyclohexane, chlorobenzene degradation, citrate cycle
and glycolysis (KEGG: Benzoate degradation).
75
IUPAC name
Synonym
Characteristics
(NA)
6-ketoxycyclo hex-
6-ketoxycyclo
1-ene-1-carboxyl-
metabolism with links to chlorocyclohexane, chlorobenzene degradation,
CoA
citrate cycle and glycolysis (KEGG: Benzoate degradation).
2-succinyl benzoyl-
O-succinyl benzoyl-CoA is involved in the metabolism of ubiquinone, other
CoA;
terpenoid-quinones and secondary metabolites with links to alph-tocopherol
o-succinyl benzoyl-
(vitamin E) metabolism and oxidative phosphorylation (KEGG: Ubiquinone
CoA;
and other terpenoid-quinone biosynthesis)
(NA)
succinylbenzoylCoA
hex-1-ene-1-carboxyl-CoA
is
involved
in
benzoate
76
From the LC-MS/MS analysis of the 4-day fermented PDB of P. kudriavzevii
M12, a total of 44 metabolites were identified and this is as shown in Table 4.1. To
the best of knowledge, this is the first time that these metabolites are reported from
P. kudriavzevii and associated with cockroach attraction. The metabolites shown in
Table 4.1 are mostly secondary metabolites which were not found in the 1-day
fermented PDB of P. kudriavzevii M12. There were 12 phospholipids, 9 carboxylic
acids, 5 steroids, 3 nucleosides, 3 sphingolipids, 3 coenzymes, 2 monosaccharides, as
well as 1 ester, 1 purine, 1 cycloalkene, 1 polyketide, 1 glycerolipid, 1 indane and 1
tetrapyrrole. The ratio of these metabolites is shown in Figure 4.4. According to the
manufacturer (Conda Pronadisa), the PDB used in this study contains dextrose (20
g/L) and infusions from potatoes (6.5 g/L), which makes it a carbohydrate-rich
medium. The type of metabolites identified in this case is unique, which is mostly
lipids. According to Frisvad et al. (2007), the secreted fungal exometabolites consist
mainly of secondary metabolites, including lipids and overproduced organic acids. In
the fungal kingdom, organic compounds such as lipids, alcohols and volatile
compounds are messengers for intraspecies fungal communication which is critical
to address basic biological functions including growth, mating, morphological
switching and expression of virulence (Cottier and Muhlschlegel, 2012).
Interestingly, in the attraction study of German cockroach reported by Nalyanya and
Schal (2001), peanut butter was found to be among the attractants that were
significantly attractive. This could possibly due to the presence of lipids and
corroborates the present metabolite profile obtained for study on the 4-day fermented
PDB of P. kudriavzevii M12. In addition, both highly volatile and some less volatile
components of carboxylic acids could elicit the attractant-aggregation response of
German cockroach (Scherkenbeck et al., 1999).
In this study, the 4-day fermented PDB of P. kudriavzevii M12 was found to
be a better cockroach attractant than the 1-day fermented broth, which suggests the
appropriate ratio and type of the attractant could be derived after 4 days of aerobic
growth of the yeast in PDB. Change in growth phase has a profound effect on the
lipidome of yeast cells (Klose et al., 2012). As noted by Nout and Bartelt (1998), the
appropriate mixture of metabolites is crucial for attraction of sap bettles
(Carpophilus humeralis), though the complete profile may not always be necessary
for full attractiveness. As the 4-day fermented broth neither had a sharp fruity smell
77
nor revealed any particular flavor note, the broth may contain more metabolites of
higher molecular weight, hence profiling with LC-MS/MS was appropriate.
Additionally, ESI-MS analysis in either positive or negative ion mode is ideal for
most of the charged exometabolites such as carboxylic acids and lipid species
(Johnson, 2005).
From Figure 4.4, approximately 48% of the exometabolites found in the 4day fermented PDB of P. kudriavzevii M12 are membrane lipids, mainly derivatives
of phospholipids, steroids, sphingolipids and glycerolipid. Most of the phospholipids
identified are glycerolphospholipids of phosphatidylethanolamine (m/z: 453.609,
634.132) and phosphatidylcholine (m/z: 741.053, 749.136, 762.060, 790.030,
790.051). These phosphatidylethanolamines and phosphatidylcholines from P.
kudriavzevii M12 may function as pheromonal attractant of cockroaches. The
phospholipids coupled with the oligosaccharides originated from PDB may attract
the American cockroach for nuptial feeding. This corroborates with a study on the
sequential courtship behavior of German cockroach by Kugimiya et al. (2002) that
revealed a synergistic action between phospholipids (phosphatidylethanolamine and
phosphatidylcholine) and oligosaccharides in eliciting nuptial feeding response of
virgin females on the pheromonal secretion from male cockroaches. Earlier research
by Tsuji and Ono (1970) also showed that some lipids such as glycerol and related
compounds could function as dietary phagostimulants for cockroaches including
American cockroach.
Another possibility of cockroach attraction is due to the nature of
phospholipids which are hydrolysable lipids that contain an ester. The C-O bond of
the ester could be hydrolyzed by phospholipases from P. kudriavzevii M12 to form
carboxylic acids and alcohol, which could possibly elicit cockroach response. An
earlier Blast2GO annotation of P. kudriavzevii M2 genome revealed the presence of
genes coding for phospholipases.
Among the metabolite produced in the 4-day fermented PDB of P.
kudriavzevii M12 was isoamyl acetate (m/z: 129.062; IUPAC name: 3- methylbutyl
acetate). The ester compound confers banana aroma and this could be derived from
the metabolism of leucine amino acid into 3- methylbutanol, which is further
esterified by alcoholacetyltransferase to the corresponding volatile-branched 3-
78
methylbutyl acetate (Vandamme, 2003). It is an essential alarm pheromone that
could induce defensive behavior of honeybee (Apis mellifera) (Trhlin and Rajchard,
2011). Isoamyl acetate also attracts larvae of Drosophila melanogaster (Oppliger et
al., 2000).
The compound 6-acetamido-2-oxohexanoate (m/z: 188.119) is a derivative of
oxocarboxylic acid found in the 4-day fermented PDB of P. kudriavzevii M12.
Interestingly, derivatives of oxocarboxylic acid including 2-oxobutanoic, 2-oxo-3methylbutanoic,
2-oxopentanoic,
2-oxo-3- methylpentanoic,
2-oxo-4-
methylpentanoic and 2-oxohexanoic have been found to be an attractant that could
elicit significant landing response of mosquito Anopheles gambiae (Healy et al.,
2002).
In addition, a heme compound (m/z: 617.713) was also found in the 4-day
fermented PDB of P. kudriavzevii M12. In yeast, heme acts as internal barometer of
oxygen tension, functioning in oxygen sensing and utilization (Zhang et al., 1998).
It is possible for cockroach to be lured by phospholipids and sphingolipid.
Three
monosaccharide
derivatives,
D-glucosaminate
(m/z 195.181),
beta-
pseudouridine (m/z 243.137) and 1D-myo- inositol 1,3,4,5-tetrakisphosphate (m/z
501.371) were found in the 4-day fermented PDB of P. kudriavzevii M12. They
could attract cockroaches as monosaccharide derivatives acting as volatile
aggregation pheromones, attracted the Caribbean fruit fly (Anastrepha suspensa)
(Walse et al., 2008).
Cyclohex-2,5-diene-1-carboxyl-CoA (m/z 874.807), 6-ketoxycyclohex-1ene-1-carboxyl-CoA (m/z 890.445) and 2-succinyl benzoyl-CoA(m/z 972.253) are
three coenzyme and benzoic acid derivatives found in the 4-day fermented PDB of
P. kudriavzevii M12. While Coenzyme A is synthesized from pantothenate and
cysteine, benzoic acid is a precursor to many compounds such as benzoates. Volatile
derivatives of Coenzyme A and benzoic acid are involved in insect-plant interactions
(Gaid et al., 2012).
Two exometabolites, 3,7-dimethyluric acid (m/z 197.182) and xanthylic acid
(m/z 365.245) are purine base derivatives found in the 4-day fermented PDB of P.
kudriavzevii M12. They are related to purine, caffeine and alkaloid metabolism.
79
Some insects use plant alkaloids as a source of attractants, pheromones and defense
substances. Arctiid moth (Utetheisa ornatrix) use alkaloids as attractants (Meinwald,
1990; Conner et al., 1981).
One of the exometabolites found in the 4-day fermented PDB of P.
kudriavzevii M12 is 3-oxo-7,8-dihydro-alphaionol (m/z 211.206), a cycloalkene that
is produced by yeast fermentation of carbohydrates. Male fruitflies ( Bactrocera
latifrons) were attracted to cut eggplant and one of the attractants was 3-oxo-7,8dihydro-alphaionol (Nishida et al., 2009). In addition, Enomoto and co-workers
reported that 3-oxo-7,8-dihydro-alphaionol is an insect attractant for the male fruit
fly (Bactrocera latifrons) (Enomoto et al., 2010).
Isorhamnetin (m/z 315.112), a polyketide, and one of the exometabolites
found in the 4-day fermented PDB of P. kudriavzevii M12 is in the flavonol
subgroup of polyphenols. Isorhamnetin as a polyphenol is present in beer and wine
(Arranz et al., 2012).Polyphenols as secondary metabolites contribute to the
organoleptic properties of wines and beers. According to Wand and Bennett, beer is
a powerful attractant for cockroaches (Wang and Bennett, 2006).Isorhamnetin could
attract insects as isorhamnetin 3-O-neohesperidoside along with sugars and amino
acids acted as strong phagostimulants for the western corn root worm (Diabrotica
virgifera virgifera LeConte) (Kim and Mullin, 2007). In a related work by
Takahashi,
isorhamnetin
3-7-O-di-β-D-glucopyranoside,
a
derivative
of
isorhamnetin, was shown to be the insect attractant in the corolla of turnip (Brassica
rapa) (Sasaki andTakahashi, 2002).
Pallidol (m/z 455.382) is another exometabolite found in the 4-day fermented
PDB of P. kudriavzevii M12. It is a resveratrol dimer present in wine. El-Sayed and
co-workers used wine along with other baits and wine was effective in attracting
lepidopteran species (El-Sayed et al., 2005).
UDP-galactose (m/z 566.743 ) is a pyrimidine nucleoside diphosphate and a
derivative of uracil. It is an important component of carbohydrate metabolism. Being
one of the exometabolite found in the 4-day fermented PDB of P. kudriavzevii M12,
UDP-galactose may attract cockroaches. Uracil was among the metabolites that
80
attracted Coquillettidia mosquito species when the semiochemicals from aquatic
plant roots were identified (Serandour et al., 2008).
81
CHAPTER 5
CONCLUSIONS AND RECOMMENDATION
5.1
Conclusions
The 4-day fermented PDB of P. kudriavzevii M12 was found to be a better
cockroach attractant, which trapped the highest number of both nymphs and adult
cockroaches (an average of 48 cockroaches per catch). Ssuccessive attraction was
done consecutively at the same location after two weeks, which resulted in a
decrease of almost 80% of the cockroach population at the studied location. The
metabolites in the 4-day fermented PDB of P. kudriavzevii M12 were profiled by
liquid chromatography-mass spectrometry (LC-MS/MS), thus revealing the presence
of secondary metabolites from the yeast strain for cockroach attraction. A total of 44
exometabolites with diverse properties and structures were identified and many were
intermediates and products of the central metabolic pathway with most being lipids
and carboxylic acids. In conclusion, P. kudriavzevii M12 showed a great potential as
eco-friendly cockroach attractant and the attraction could be resulted from the
metabolites produced.
5.2
Recommendation
Future research on cockroach attraction should focus on determining the
optimum concentration of the known metabolites required to elicit responses needed,
as well as to up-scale the production of yeast metabolites and to preserve the
metabolites for long-term storage. Attractants from yeast which are eco- friendly and
efficient, could be potentially used in control of cockroaches and even other pests.
82
Future research on downstream processing of the yeast metabolites should
also focus on the use of freeze dried powder sprayed with some water. In order to
preserve the metabolites, attention should also be focused on the use of supercritical
carbon dioxide extraction which can utilize the carbon dioxide waste product and
may qualify for carbon credits for cost reduction (Ghafoori et al., 2007).
83
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APPENDIX A
Mass spectra for positive ion detection of one day fermentated PDB of P.
kudriavzevii M12.
109
APPENDIX B
Mass spectra for positive ion detection of four days fermentated PDB of P.
kudriavzevii M12.
110
APPENDIX C
Mass spectra for positive ion detection of PDB (control).
111
APPENDIX D
Mass spectra for negative ion detection of one day fermentated PDB of P.
kudriavzevii M12.
112
APPENDIX E
Mass spectra for negative ion detection of four days fermentated PDB of P.
kudriavzevii M12.
113
APPENDIX F
Mass spectra for negative ion detection of PDB (control)

- Faculty of Biosciences and Medical Engineering

Transcription

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